MOLECULAR MECHANISMS OF THE INNATE IMMUNE …rq941zv2693...The ability of F. tularensis to escape phagosomal degradation and replicate in the macrophage cytosol is central to its pathogenesis

MOLECULAR MECHANISMS OF THE INNATE IMMUNE RESPONSE TO

FRANCISELLA TULARENSIS

A DISSERTATION

SUBMITTED TO

THE DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY

AND THE COMMITTEE ON GRADUATE STUDIES

OF STANFORD UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Jonathan Wiley Jones

August 2010

http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/rq941zv2693

© 2010 by Jonathan Wiley Jones. All Rights Reserved.

Re-distributed by Stanford University under license with the author.

This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 United States License.

ii



http://purl.stanford.edu/rq941zv2693

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Denise Monack, Primary Adviser


Manuel Amieva


Stanley Falkow


William Nelson


David Schneider

Approved for the Stanford University Committee on Graduate Studies.

Patricia J. Gumport, Vice Provost Graduate Education

This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.

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iv

Abstract

Francisella tularensis is a facultative intracellular pathogen that causes the

disease tularemia. The ability of F. tularensis to escape phagosomal degradation and

replicate in the macrophage cytosol is central to its pathogenesis. The macrophage

responds to the presence of cytosolic F. tularensis with the production of type-I

interferons (IFN) and subsequent activation of the inflammasome, which leads to host

cell death that eliminates the bacterium’s replicative niche. Very little is known about

the molecular mechanisms that lead to cytosolic recognition of F. tularensis or

bacterial factors that modulate this process and allow the bacterium to establish a

niche in the macrophage. The bacterial ligand(s) that trigger the cytosolic response as

well as the host pattern recognition receptors that lead to type-I IFN production and

inflammasome activation are unknown. By investigating the molecular mechanisms of

F. tularensis recognition in the cytosol we hypothesized that we would uncover novel

bacterial PAMPs and novel host PRRs and greatly broaden our understanding of

innate immunity.

To this end we conducted a forward genetic screen of a F. novicida transposon

library to identify mutants that resulted in an increased or decreased cytosolic response

in macrophages. We identified 164 F. novicida mutants that lead to increased type-I

IFN production and inflammasome activation in macrophages. These included a

major outer membrane protein, fopA, as well as genes involved in LPS/capsule/cell

wall biosynthesis, FTN_1212, lpcC, wbtA, kdsA, and lpxH. We also identified 74

mutants that resulted in decreased type-I IFN and inflammasome responses in

macrophages. These included 17 of the 19 genes in the Francisella Pathogenicity

v

Island, and several purine and pyrimidine biosynthesis genes. These mutants

demonstrated a correlation between intracellular replication and induction of the

cytosolic responses. The fact that we did not identify a single mutant that replicated

intracellularly as efficiently as wild-type F. novicida but did not activate the cytosolic

responses suggested that the presence of the bacterial ligand was associated with

bacterial replication and possibly an essential gene.

Finally, we identified AIM2 as the host receptor responsible for inflammasome

activation in response to cytosolic F. novicida. We showed that lysing cytosolic F.

novicida leads to release of bacterial DNA that triggers type-IFN through a pathway

involving the adaptor STING. STING-dependent type-I IFN production increases the

expression of AIM2, which complexes with the bacterial DNA and initiates

inflammasome activation. We further demonstrate that AIM2 is critical for innate

immunity to F. novicida infection in vivo. Thus we identified a novel bacterial ligand

and novel cytosolic sensing components that play a role in the host defense to bacterial

infections.

vi

Acknowledgements

First and foremost I have to thank God for all of His blessings. I know that

everything I have comes from Him. I want to thank my family for their continued

love and support throughout my life. My mom, Carolyn Jones, instilled in me the

importance of education, and encouraged me to pursue my dreams no matter how far

from home they took me. My father, Wiley Jones, gave me strength, and continues to

be my inspiration even after his passing. I love you both very much. My

grandparents, sisters, aunts, uncles, nieces and nephews remind me that life is bigger

than graduate school, and no matter where I go I will always have a place to call

home. I want to thank my friends from Detroit, Troy Walls, Tarik & Crystal Green,

Blair Parkman, Nathan Hood, Cordelia Ziraldo, and Konadu & Kim Addai for never

letting me forget my roots.

I have had an amazing time at Stanford. I have to thank Denise Monack for

guiding me through this crazy process of grad school. You are a great mentor,

colleague, and friend. I’d like to thank the Falkow, Amieva, and Monack labs for

making lab fun, even when science wasn’t. Elizabeth Joyce knows everything and is

an amazing teacher. To my classmates, you guys are wonderful. Jeff & Lacey

Margolis helped me keep my head on straight, and Beth Ponder helped make Cookies

& Cream a success every year. You all are the best. It’s been a wild ride. Peace and

I’m out!!!

vii

Table of Contents Section Page

Abstract ................................................................................................................... iv Table of Contents ................................................................................................... vii Table of Figures ...................................................................................................... ix List of Tables.............................................................................................................x Chapter 1: General Introduction .............................................................................1

Innate immunity.......................................................................................................................................... 1 Pattern Recognition Receptors (PRRs) ..................................................................................................... 3 Type-I IFNs................................................................................................................................................. 6 Inflammasomes........................................................................................................................................... 8 Tularemia .................................................................................................................................................. 14 Intracellular lifestyle of F. tularensis ...................................................................................................... 17 Innate immunity to F. tularensis ............................................................................................................. 19

Chapter 2: A genetic screen identifies novel F. novicida genes that modulate the cytosolic innate immune responses in macrophages..............................................22

2.1 CHAPTER 2 SUMMARY ................................................................................................................ 23 2.2 INTRODUCTION.............................................................................................................................. 25 2.3 RESULTS ........................................................................................................................................... 28

2.3.1 A transposon screen identifies fopA as a suppressor of the macrophage cytosolic innate immune response. ................................................................................................................................ 28 2.3.2 ΔfopA stimulates the same cytosolic surveillance pathway as wild-type F. novicida. .......... 31 2.3.3 A ΔfopA mutant has reduced fitness in mice............................................................................ 36 2.3.4 Identification of F. tularensis mutants that differentially induce the cytosolic response in macrophages by a genome-wide forward genetic screen.................................................................. 37 2.3.5 F. novicida LPS mutants hyper-induce the cytosolic responses. ............................................ 56 2.3.6 F. novicida LPS mutants stimulate increased TLR2-depedent signaling............................... 60 2.3.7 F. novicida LPS mutants hyper stimulate the inflammasome................................................. 64 2.3.8 LPS mutants have reduced fitness in vivo................................................................................ 66 2.3.9 Phagosomal escape is required for induction of the cytosolic response by LPS mutants. ... 67 2.3.10 LPS mutants induce increased proinflammatory cytokine signaling in the phagosome. ... 70 2.3.11 Surface-exposed PAMPs mediate recognition of LPS mutants. ........................................... 72 2.3.12 Cytosolic localization is necessary but not sufficient to induce the cytosolic responses. .. 75 2.3.13 Bacterial DNA and protein synthesis are required to induce the cytosolic responses........ 79 2.3.14 The cytosolic response to F. novicida shares characteristics with the response to transfected dsDNA. ............................................................................................................................. 85

2.4 DISCUSSION..................................................................................................................................... 88 Chapter 3: AIM2 is required for innate immune recognition of Francisella tularensis..................................................................................................................90

3.1 CHAPTER 3 SUMMARY ................................................................................................................ 91 3.2 INTRODUCTION.............................................................................................................................. 92 3.3 RESULTS ........................................................................................................................................... 94

viii

3.3.1 AIM2 is essential for inflammasome activation in response to cytosolic dsDNA. ............... 94 3.3.2 AIM2 is required for inflammasome activation in response to F. tularensis....................... 101 3.3.3 AIM2 and ASC form a complex with F. tularensis DNA. ................................................... 104 3.3.4 AIM2 is required for the formation of an ASC focus............................................................ 107 3.3.5 Type I IFN increases AIM2 protein levels and inflammasome activity............................... 109

3.4 Discussion......................................................................................................................................... 115 Chapter 4: Discussion ...........................................................................................119 Chapter 5: Materials and Methods ......................................................................129

5.1 BEIR “two-allele” transposon library screen. ................................................................................ 129 5.2 Bacterial strains and growth conditions.......................................................................................... 130 5.3 Bacterial Mutagenesis. ..................................................................................................................... 130 5.4 Bone marrow-derived macrophage culture and infections............................................................ 131 5.5 Macrophage gene expression analysis. ........................................................................................... 132 5.6 ISRE-L929 assays. ........................................................................................................................... 133 5.7 NF-kB reporter cell assays............................................................................................................... 133 5.8 Mice, bacteria, and reagents ............................................................................................................ 133 5.9 Immunofluorescence Microscopy ................................................................................................... 134

REFERENCES .....................................................................................................137

ix

Table of Figures Figure Page

Figure 1 - An F. novicida ΔfopA mutant hyper-induces the cytosolic innate immune response in macrophages. ....................................................................................................................................... 30

Figure 2 - The cytosolic response to ΔfopA is IRF3- and IFNAR- dependent but TLR- and NOD- independent. ......................................................................................................................................... 33

Figure 3- The hyper-cytotoxicity ofΔfopA is ASC- and caspase-1 dependent but NLRP3-independent............................................................................................................................................................... 35

Figure 4 - ΔfopA is less fit in vivo than wild-type F.novicida. .................................................................. 37 Figure 5 - LPS mutants hyper-induce the type-I IFN response in macrophages....................................... 58 Figure 6 - LPS mutants hyper-induce IL-1β release and host cell death. .................................................. 59 Figure 7 - TLR signaling contributes to the early but not late responses to LPS mutants........................ 61 Figure 8- TLR2 contributes to the early but not late cytosolic responses to LPS mutants....................... 63 Figure 9 - ASC, caspase-1, and IFNAR are required for LPS mutant inflammasome activation............ 65 Figure 10 – LPS mutants have reduced fitness in vivo. .............................................................................. 67 Figure 11 - Phagosomal escape is required for LPS mutants to hyper-induce the cytosolic responses. . 69 Figure 12 - LPS mutants hyper-induce NF-κB-dependent cytokines in the phagosome.......................... 71 Figure 13 - Surface exposed PAMPs mediate recognition of LPS mutants. ............................................. 74 Figure 14 - Cytosolic localization is necessary but insufficient to induce the cytosolic responses. ........ 78 Figure 15 - Bacterial protein synthesis and DNA synthesis are required to induce the cytosolic

responses. ............................................................................................................................................. 82 Figure 16 - Replication is not required to induce the cytosolic response with high bacterial load.......... 84 Figure 17 - The cytoslic response to F. novicida shares characteristics with that of dsDNA.................. 86 Figure 18 - Generation of Aim2-/- mice. ....................................................................................................... 96 Figure 19 - AIM2 is essential for inflammasome activation in response to cytosolic dsDNA. ............... 97 Figure 20 - AIM2 is essential for inflammasome activation in response to dsDNA in peritoneal

macrophages. ....................................................................................................................................... 98 Figure 21 - AIM2 is dispensable for IFN-β and TNF-α production in response to dsDNA. ................. 101 Figure 22 - AIM2 is required for inflammasome activation in response to F. tularensis. ..................... 103 Figure 23 - AIM2 and ASC form a complex with F. tularensis DNA. ................................................... 105 Figure 24 - F. tularensis DNA triggers IL-1β secretion. .......................................................................... 107 Figure 25 - AIM2 is required for the formation of an ASC focus............................................................ 109 Figure 26 - Type-I IFN increases AIM2 protein levels and inflammasome activity. ............................. 111 Figure 27 - Type-I IFN facilitates formation of the AIM2-containing inflammasome. ......................... 113 Figure 28 - AIM2 is required for host defense against F. tularensis ....................................................... 115

x

List of Tables Table Page

- Table 1 – Mutants that hyper-induce the cytosolic responses ............................................................... 39 - Table 2 – Mutants that hypo-induce the cytosolic responses ................................................................ 51 - Table 3 – Mutants attenuated for intracellular replication are hypo-stimlate the cytosolic responses76 - Table 4 – Primers for F. novicida cloning and mutagenesis ............................................................... 135

1

Chapter 1: General Introduction

Innate immunity

As scientists studying microbial pathogenesis we are uncovering an

evolutionary war that has been going on since the first time a single-celled organism

ate a microbe. Since then there has been an enormous selective pressure for microbes

to evolve strategies to resist detection and killing by host cells. From the microbe’s

perspective, it either needs the host cell in order to multiply (such is the case with

viruses, and obligate intracellular bacteria/parasites), or it simply is trying to take

advantage of a niche free of competition for nutrients from other microbes, and avoid

detection and killing by other cells. Few microbes have the ability to survive inside of

a host, but those that can are termed pathogens or symbionts. Interacting with another

organism provides a significant number of challenges: you have to be able to attach to

the surface or enter a perspective host, you have to be able to maintain your location,

and you must be able to obtain nutrients to either grow or persist in the face of an

immune system bent on your destruction.

Two arms of the immune system, the innate and adaptive responses, work

together to detect and clear pathogens. Innate immunity is the first line of defense

against invading microbes, and usually clears most infections without a problem.

However if the innate responses prove insufficient, the orgasnism activates adaptive

immunity to specifically target the pathogen. In contrast to adaptive immune

mechanisms, which are only observed in vertebrates, innate immune defenses are

found in all multi-cellular organisms. Therefore, it is evolutionarily ancient. When

2

observing the characteristics of the innate and adaptive immune responses it is easy to

see how the two work in concert. First, unlike adaptive immunity, which takes time to

mount an antimicrobial response, innate immune defenses are constitutively in place

and react rapidly upon infection. Second, the adaptive immune system responds to a

specific microbial challenge, with the ability to generate T and B cell responses to

specific pathogens, while the innate immune system is non-specific and instead

recognizes “patterns” or “themes” in molecules unique to microbes. Finally, the

adaptive immune system displays immunological memory, in that it “remembers” the

microbe that it eliminated and responds even faster upon subsequent challenge with

the same microbe. In contrast, the innate immune system has no memory, and reacts

with the same speed and efficacy after repeated challenge with a microbe. The innate

immune system works to fight off the initial infection and prime the adaptive immune

system should the infection persist.

The innate immune system includes anatomical barriers to infection, secretory

components, and cellular mediators of pathogen detection and clearance. Anatomical

barriers include the skin, which prevents penetration of pathogens, the movement of

cilia that clears pathogens from air passages, the flushing action of tears and saliva,

and mucous that lines the lungs and gastrointestinal tract, which traps and clears

invading pathogens. Tears and saliva also contain antimicrobial factors such as

lysozyme, which can dissolve microbial cell walls. If these anatomical barriers are

breached, serum also contains secreted factors like lactoferrin that can sequester

essential iron, eliminating an important co-factor for many microbial processes. The

serum also contains complement, which can lyse infectious organisms, leading to their

3

phagocytosis, and lead to inflammation and recruitment of other cellular responses.

Cellular mediators of innate immunity are derived from the bone marrow and include

macrophages, dendritic cells, neutrophils, natural killer cells, mast cells, basophils and

eosinophils. These cells can kill microbes by phagocytosis and degradation, or

through secretion of toxic molecules.

Pattern Recognition Receptors (PRRs)

The critical function of the immune system is the ability to discriminate

between self and non-self. Being the first line of defense to infection, innate immune

mechanisms must be able to protect against a diverse array of potentially threatening

microbes, like viruses, bacteria, and fungi, all having different microbial factors to

promote survival and virulence. With such a monumental task, the innate immune

system takes a broad approach to identifying and eliminating all manner of microbes,

using a relatively small set of germline-encoded sensors called pattern recognition

receptors (PRRs). These PRRs are expressed on antigen presenting cells (APCs) like

macrophages and dendritic cells, and take advantage of conserved molecules that are

unique to microbes and often essential to microbial existence. Thus they are difficult

for the microbes to alter significantly to avoid detection. The microbial molecules

sensed by PRRs have been termed pathogen associated molecular patterns or PAMPs.

Discovery of the Toll-like receptors (TLRs) was a breakthrough in the field of host-

pathogen interaction.

4

In 1996, studies in the fruit fly Drosophila, which only has an innate immune

system, led to the identification of Toll as the central receptor in the host defense to

fungal infection (92). One year later a homologous human Toll-like receptor was

identified (109). At present there are 11 TLRs that have been identified and

implicated in innate immunity (162). TLRs 1,2,4,5 and 6 recognize components of

microbial cell wall while TLRs 3,7,8, and 9 recognize microbial nucleic acids. TLR4

is probably the best characterized of the TLRs and is essential for recognizing

bacterial lipopolysaccharide (LPS) (136). TLR1, TLR2, and TLR6 recognize

lipoproteins from microbial cell wall (163, 164) and TLR5 recognizes bacterial

flagellin (60). Of the nucleic acid sensors TLR9 recognizes unmethylated CpG motifs

of bacterial DNA (63). TLR3 responds to viral double stranded-RNA (5), while TLR7

and TLR8 respond to single stranded RNA from viruses (61). The TLRs can also be

grouped according to their cellular location with TLR1, 2, 4, 5, and 6 located at the

cell surface and TLR7, 8, and 9 localized to endosomes. The differential localization

of TLRs allows cells of the innate immune system to survey multiple environments for

potentially harmful pathogens.

TLR signaling leads to the production of proinflammatory cytokines like

Tumor necrosis factor-α (TNF-α), interferon-α/β (IFN)-α/β, IFN-γ, and interleukin-

12 (IL-12). All TLRs except TLR3 use an adaptor protein myeloid differentiation

factor 88 (MyD88), which leads to the activation of nuclear factor kappa-light-chain-

enhancer of activated B cells (NF-κB) (2). TLR3 and TLR4 use the adaptor TIR-

domain-containing adaptor protein-inducing IFN-β (TRIF) to activate interferon

regulatory factor 3 (IRF3) and NF-κB in a MyD88-independent manner (3, 162, 182).

5

Whether a pathogen’s niche is on the surface of a cell, inside a phagosome, or

in the cell cytosol, it must be able to manipulate the host cell in order to establish an

environment to promote survival and replication. To this end, pathogenic bacteria

have acquired/evolved secretion systems and bacterial toxins that allow the pathogen

to penetrate cell membranes and deliver effector molecules directly into the cell

cytosol. As an evolutionary counterpart to bacterial secretion systems, host cells have

evolved cytosolic surveillance mechanisms to detect danger signals in this fragile

compartment of the cell. The nucleotide-binding oligomerization domain (NOD)

proteins recognize components of the microbial cell wall in the cytosol (7, 24). NOD1

senses γ-D-glutamyl-meso-diaminopimelic acid, and NOD2 senses muramyl dipeptide

(MDP), both fragments of peptidoglycan in bacterial cell wall. The NODs activate

NF-κB through the adaptor Rip-like interacting caspase-like apoptosis-regulatory

protein kinase (RICK, also known as RIP2) to stimulate pro-inflammatory cytokine

production (71).

Additionally, two RNA helicases, retinoic acid inducible gene I (RIG-I) and

melanoma differentiation-associated gene 5 (MDA5) detect viral dsRNA in the

cytosol independent of TLR3 detection (184, 185). These two helicases uses the

adaptor molecule mitochondrial antiviral signaling (MAVS) (also called IFN-β

stimulator 1 (IPS-1), virus induced signaling adaptor (VISA), or Cardif) to stimulate

production of type-I IFNs (IFN-α/β) through NF-κB and IRF-3 activation. There also

appears to be an intracellular sensor(s) for dsDNA that acts independently from RIG-I

and MDA5 to produce type-I IFNs (74, 127, 153). In this case, the production of type-

I IFNs is still IRF3 dependent but is independent of MAVS signaling. One such

6

dsDNA sensor, DNA-dependent activator of interferon regulatory factors (DAI) (also

called DLM-1 or ZBP-1) was recently identified though lack of in vivo relevance

suggests that redundant sensors must exist (75, 161). Other cytosolic dsDNA sensors

that trigger type-I IFN production remain unknown.

Type-I IFNs

The type-I IFNs were the first cytokines discovered and represent an

interesting class of cytokines in host defense. All vertebrates examined to date have a

gene that encodes IFN-β and at least two that encode IFN-α, together with natural

killer (NK) cells, T cells, and B cells (154). With the exception of LPS induced type-I

IFN production through TLR4, type-I IFN is produced exclusively in response to

nucleic acids. Furthermore, whereas the TLRs are only present on a subset of

specialized immune cells, all nucleated cells can produce type-I IFNs and all nucleated

cells express the type-I IFN receptor (IFNAR), which allows them to respond to type-I

IFNs in the environment. Thus, we can surmise that all nucleated cells express one or

more cytosolic PRRs that can signal the presence of intracellular infections through

the detection of nucleic acids and lead to the production of type-I IFNs.

Type-I IFNs were first described over 50 years ago as a soluble factor

produced by cells treated with inactivated virus that blocked subsequent infection with

live virus (72, 73, 120). It was later discovered that type-I IFNs signal through

IFNAR in an autocrine and paracrine fashion to induce expression of hundreds of

genes that establish an “antiviral state” in the cell (152, 172). We do not really

7

understand the mechanisms behind this antiviral state, as most of the IFN-induced

genes have not been ascribed a function. However, it is thought that many of these

proteins cooperate with other signaling pathways to fully potentiate innate immunity.

For example, treating cells with type-IFNs sensitizes them to apoptosis upon

subsequent viral infection (152) or treatment with pore-forming bacterial toxins (188).

This mechanism of cell suicide would effectively eliminate the replicative niche for

intracellular infections. Perhaps for this reason several viruses have evolved

mechanisms to block the apoptotic process (151).

Although mainly studied as an antiviral mechanism, a number of recent reports

demonstrate that type-I IFN is induced by a number of intracellular bacteria such as

Mycobaterium tuberculosis (52, 176), Listeria monocytogenes (124), Legionella

pneumophila (129), and Francisella tularensis (64). The bacterial PAMP that

stimulates type-IFN production in these infections is unknown, though in the case of

Listeria monocytogenes infection and Francisella tularensis infection it is suspected

that a cytosolic DNA sensor could be involved (64, 153). Similar to its effects on viral

infections, type-I IFN can acts to in concert with other cytosolic sensing pathways like

the inflammasome to trigger a protective host cell death (32, 64). The mechanism

linking the type-I IFN pathway and the inflammasome is currently unknown and is a

central question of this thesis work.

The existence of a host cell mediated death pathway represents an interesting

paradox in host defense. The host cell effectively sacrifices itself for the survival of

the whole organism. Theoretically this would only benefit an organism when tissue

renewal can preserve the hosts physiological functions in the face of irreplaceable cell

8

loss. Perhaps this is why only vertebrates have evolved type-I IFN signaling. Most

cells in vertebrates are renewable, while most cells in adult invertebrates are

postmitotic (154). Therefore loss of even a few key cells in invertebrates could be

deleterious for the organism.

Inflammasomes

Protection from invading microbes and eliminating and replacing dying cells

are two of the greatest challenges that multicellular organisms must have faced during

evolution. Our innate immune system has learned to cope with these challenges by

evolving alarm systems that can detect invading microbes and injured tissue, and

initiate inflammation through the activity of cytokines and chemokines. Inflammation

is one of the first responses of the innate immune system to infection and is

characterized by redness, heat, swelling, and pain at the site of the infection. While

inflammation is essential for fighting off infections and repairing damaged tissue, it

can lead to further tissue damage if not tightly regulated. The same immune cells,

molecules, and mechanisms involved in fighting pathogens can be harmful to

uninfected tissue is not controlled. Tissue damage sustained during an infection can

result from either direct action of a microbe or microbial on tissues (such as factors

used for colonization/ toxins produced) or it can be the indirect result of an

inflammatory response aimed at eliminating the pathogen, or both. Thus, intricate

coordination of the immune response is required to strike a delicate balance between

9

eliminating invading microbes and limiting inflammation to maintain tissue integrity.

Too little of a response can lead to proliferation of the pathogen and the onset of

disease, while too much of a response can lead to local and systemic tissue damage

and even death in severe cases. An aberrant inflammatory response is the cause of

several autoimmune diseases. Only an appropriate response eliminates the threat and

goes undetected by the host.

The key regulators of inflammation are enzymes known as inflammatory

caspases. These inflammatory caspases are activated in complexes known as

inflammasomes, which are cellular sensors of danger signals located in the cytosol of

certain immune cells. Once the inflammasome complex activates caspases, they

cleave pro-inflammatory cytokines into their mature, active forms, and direct their

unconventional secretion from the cell. Additionally, the caspases control a form of

cellular suicide. As this cell death is accompanied by the aforementioned released of

pro-inflammatory cytokines, it has been termed “pyroptosis”, from the Greek roots

“pyro” for fire denoting the inflammation, and “ptosis” meaning falling, denoting the

death of the cell (89). For the purposes of this thesis, caspase-dependent cell death

will just be referred to as cell death.

Caspase-1 is a member of a family of intracellular aspartate-specific cysteine

proteases. One of the subfamily of “inflammatory caspases”, its subfamily members

include caspase-4, -5, -11 (which exists in rodents), and -12(149). Caspase-1 exists as

an inactive 45kDa precursor, but once activated it undergoes autocatalytic cleavage

into 20kDa and 10kDa subunits (p20 and p10) (9, 169, 180). In the cytosol, active

caspase-1 processes pro-IL-1β(13, 87), pro-IL-18 (51), and pro IL-33 (147), into their

10

mature, active forms that are secreted and regulate inflammation (in the case of IL-1β

and IL-18), and generation of TH2 responses (in the case of IL-33). When regulated

properly, IL-1β is critical for the host response to infection, but excessive levels of Il-

1β are associated with several inflammatory diseases such as rheumatoid arthritis,

inflammatory bowel disease, and septic shock to name a few (40). Clearly

inflammation can be a double-edged sword but it is essential to combat infection and

restore tissue homeostasis after infection.

In 2002 it was discovered that a multiprotein complex termed the

“inflammasome” was responsible for activating caspase-1(101). The inflammasome is

composed of a NOD-like receptor (NLR), and the adaptor protein apoptosis associated

spec-like protein containing a caspase recruitment domain (ASC). The NLRs are a

family of cytosolic pattern recognition receptors (PRRs) that activate inflammatory

and antimicrobial responses by sensing “danger signals” or danger-associated

molecular patterns (DAMPs) (105) and conserved microbial products termed pathogen

associated molecular patterns (PAMPs) (70, 171). There are 14 members of the NLRs

subfamily, which are characterized by a C-terminal leucine rich repeat (LRR) domain,

a central oligomerization domain (NACHT), and an N-terminus that is either a caspase

recruitment domain (CARD) (as in NOD1, NOD2, and the NLRC family), three

Baculovirus IAP Repeats (BIR) (as in the NAIPs), or a pyrin domain (PYD) (as in the

NLRP family). The adaptor ASC (also known as PYCARD) has an N-terminal PYD

that facilitates interactions with the PYD domain of NLRs, and a C-terminal CARD

domain that recruits caspase-1 through CARD-CARD interactions (102). LRRs are

also found in the Toll-like receptors (TLRs), which sense danger signals and microbial

11

patterns on the cell surface and in endosomes (78, 162). Interestingly, the TLRs

cannot distinguish between pathogenic and non-pathogenic microbes because they

sense conserved microbial patterns that are present in both and they are located

extracellularly and in endosomes, where both pathogenic and non-pathogenic

microbes reside. The NLRs also sense conserved microbial patterns, but the only way

these microbial patterns can reach the cytosol is if they are delivered there by

disruption of cell membranes by toxins, specialized secretion systems of pathogenic

microbes, and/or cytosolic pathogens. Therefore the location of the sensors and not

the ligands they sense makes the NLRs specific for detecting pathogens (16).

The vast number of NLR proteins allows the inflammasome to respond to

numerous pathogens and danger signals. The cytosolic DAMP or PAMP sensed

determines which NLR forms the complex. An inflammasome is named after the

NLR that forms it (i.e. the NLRP3 inflammasome, the NLRC4 inflammasome, etc.),

and so it can come in many different flavors. The inflammasome has been implicated

in the host response to numerous pathogens and danger signals, as well as a number of

autoimmune and auto-inflammatory diseases. For more a more thorough discussion of

inflammasome regulation and its role in autoimmunity I suggest the reviews by

Brodsky et al. (16), McIntire et al. (108), and Martinon, et al. (103). For my thesis

work I have focused on the role of inflammasomes in pathogen recognition.

Conserved microbial structures, such as bacterial cell wall components are

potent activators of innate immunity. Muramyl dipeptide (MDP) is a breakdown

product of bacterial peptidoglycan that is recognized by NOD2 and induces

transcriptional activation of proinflammatory cytokines through the adaptor RIP2.

12

MDP is also a potent activator of the inflammasome, which results in release of

mature IL-1β. Inflammasome activation by MDP involves both NOD2 and NLRP3,

suggesting that NLRP3 is an additional sensor of MDP (99, 100, 131). NOD2 has

also been shown to cooperate with NLRP1b in inflammasome activation in response

to MDP and anthrax lethal toxin (19, 69). These results suggest that inflammasome

complexes may contain of multiple NLRs that act synergistically to activate caspase-1

in response to PAMPs.

Many bacteria employ pore-forming toxins in their pathogenic arsenal to

establish infections. NLRP3 has been implicated in the inflammasome response to

listeriolysin O form Listeria monocytogenes, α-toxin from Staphylococcus aureus and

aerolysin from Aeromonas hydrophila (49, 98). The precise mechanism by which

NLRP3 recognizes these toxins is unknown. NLRP3 also activates the inflammasome

in response to high extracellular concentrations of ATP (98), various crystalline

compounds (23, 41, 59, 104), as well as viral and bacterial nucleic acids (81, 119).

With such a diverse array of stimuli, the hypothesis is that these ligands potentiate a

common terminal signal that activates NLRP3. Potassium efflux, membrane damage,

and stimulation of reactive oxygen species have all been implicated as the terminal

signal for NLRP3, but each has caveats. Therefore, the precise mechanism of NLRP3

activation remains a mystery.

NLRC4 was the first NLR shown to activate caspase-1 in response to bacterial

infection with Salmonella typhimurium in a type-III secretion system (T3SS)-

dependent manner (96). It was later revealed that NLRC4 sensed bacterial flagellin

that was secreted into the host cytosol through the T3SS, likely due to the evolutional

13

similarity of the T3SS with the flagellar biosynthesis machinery (50, 113). Detection

of cytosolic flagellin was later linked to the inflammasome response to Pseudomonas

aeruginosa, Listeria monocytogenes, and Legionella pneumophila (114, 116, 141,

174), though in the case of Legionella the type-IV secretion system (T4SS) was

required and Naip5 was required in addition to NLRC4 (141, 186). NLRC4 was also

implicated in recognition of Shigella flexneri (159), which encodes a T3SS but does

not express flagellin (170), as well as non-flagellated strains of Pseudomonas (157),

suggesting that NLRC4 must recognize more than just flagellin. The answer was

recently provided when it was demonstrated that NLRC4 could also detect a basal

body rod component of the T3SS, which shares a sequence motif with flagellin (115).

This motif is essential to NLRC4-mediated recognition and explains how NLRC4 can

respond to diverse pathogens that express either a T3SS or flagellin.

Studies of NLRP1, NLRP3, and NLRC4 have expanded our understanding of

the role of inflammasomes in the host response to pathogens. However, many

pathogenic signals for inflammasome activation have yet to be elucidated.

Transfection of bacterial, viral, or mammalian double-stranded DNA (dsDNA)

triggers caspase-1 activation in a manner that requires ASC, but not any of the known

NLRs (119, 139). Similarly, infection with Francisella tularensis activates an ASC-

dependent inflammasome that does not require NLRP1, NLRP3, or NLRC4 (97, 98).

Moreover, inflammasome activation by Francisella requires type-I IFN signaling,

establishing a novel mechanism of inflammasome regulation (64). Thus, Francisella

tularensis is a useful tool to expand our understanding of inflammasome activation

14

and will allow us to uncover new NLRs and hence new stimuli involved in the host

response to pathogens.

Tularemia

Tularemia is a zoonotic disease caused by the bacterium Francisella tularensis.

The bacterium was first isolated from ground squirrels in 1911 in Tulare County

California (107) after it was associated with an outbreak of a plague-like disease in

rodents following the San Francisco earthquake. Further characterization of

Francisella revealed that it is a pathogen with one of the widest host ranges of any

known bacteria. It can infect rodents, hares, rabbits, muskrats, beavers, and voles

among mammals, and can be transmitted by ticks, mosquitoes, and biting flies (123).

Disease outbreaks in human populations will often parallel outbreaks in wild animals.

Reports from Sweden have demonstrated a correlation between outbreaks of tularemia

in vole and hare populations and outbreaks in humans (168).

Although rabbits, hares, and rodents are important sources for human infection,

tularemia is an acute disease in these animals and therefore they are unlikely to be a

reservoir. Thus, the true environmental reservoir for Francisella remains a mystery.

Outbreaks of tularemia in Europe have often been associated with fresh water (12, 37,

62) where it may exist in biofilms (95) or associated with amoebae (1). Whatever the

reservoir, it is clear that Francisella have evolved mechanisms to survive in a wide

range of environments, with limited nutrient sources, and in the face of immune

mechanisms.

15

Francisella are pleomorphic, gram-negative coccobacilli ranging from 0.5-

2um in size. Francisella tularensis is subdivided into four subspecies (ssp.):

tularensis, holarctica, mediasiatica, and novicida. An additional Francisella species,

Francisella philomiragia has also been identified. Tularemia seems to be

geographically restricted to the Northern hemisphere. Subspecies tularensis, also

known as type A, is found almost exclusively in North America, with a single reported

case in Europe (56). F. tularensis is the most virulent subspecies, with an infectious

dose in humans as low as 10 organisms (44, 112). Subspecies holarctica, or type B, is

found mainly in North America, Europe, and Asia (117, 128). The Live Vaccine

Strain (LVS) is derived from an isolate of subspecies holarctica and causes a

tularemia like disease in mice although it is non-pathogenic to humans. The specific

cause of the attenuation of LVS is unknown. Subspecies mediasiatica is found mainly

in central Asia and subspecies novicida is found primarily in North America, although

is has recently been isolated in Australia (178). Subspecies novicida is genetically

tractable and causes a tularemia like disease in mice making it an ideal laboratory

strain for studies of Francisella biology.

F. tularensis garnered much interest in the 1950s when the United States and

U.S.S.R began evaluating the organism’s potential as a biological weapon. The ability

to aerosolize the organism, the low infectious dose, and the high incidence of mortality

if left untreated make F. tularensis an ideal candidate for biological warfare. As a

result, the CDC has categorized Francisella as a Category A bioterrorism agent. In

the host, tularemia has an average incubation period of 3-5 days, but can range from 1-

14 days. The clinical manifestations of tularemia may vary depending on the route of

16

infection, the bacterial subspecies, and host genetics. The most severe form of the

disease is pneumonic tularemia where the bacteria are inhaled directly into the lungs

by aerosol. This usually occurs during farming activities where dust is generating

moving hay that had been inhabited by infected rodents (66, 155, 160, 168). Cases of

pneumonic tularemia have also recently been reported when a lawnmower ran over the

carcass of a rabbit that had been infected with Francisella (46). This results in a very

acute infection whose symptoms include high fever, chills, malaise, and cough (45).

Pneumonic tularemia is extremely severe and has a mortality rate of 30% if untreated

(38).

Direct contact of Francisella with the conjunctiva can result in the

oculoglandular form of the disease. This presentation is rare, accounting for 1-4% of

cases (130). Other rare presentations of the disease are oropharyngeal or

gastrointestinal tularemia, which is contracted by ingestion of contaminated food or

water (62). Symptoms can include sore throat, diarrhea, and even laceration of the

bowel.

The most common presentation of tularemia is classified as ulceroglandular,

where the bacterium is introduced through the skin, usually from a tick bite (126, 167)

or from an abrasion during skinning of an infected animal. This accounts for 90% of

tularemia cases (166). A characteristic ulcer forms at the site of infection. The

bacteria then move to the draining lymph nodes, which can lead to lymphadenopathy,

similar to the bubo observed in Plague patients. From the lymph nodes the bacteria

can spread systemically to the reticuloendothelial organs, colonizing the lung, liver,

spleen, bone marrow, and blood. Symptoms of tularemia may include fever, chills,

17

headaches, diarrhea, muscle aches, joint pain, dry cough, and progressive weakness

(29, 45). This form of the disease is rarely fatal, with a mortality rate of less than 3%

of cases (45), although it may take time to resolve without treatment. However, an

acute form of the disease, typhoidal tularemia, can occur with ssp. tularensis infection,

which is characterized by septicemia without the appearance of an ulcer or bubo and

carries a mortality rate between 30% and 60% (45, 53). Although tularemia can be a

severe and debilitating disease, in most cases it can be effectively treated with

antibiotics. Streptomycin and gentamicin are the first drugs of choice, though

chloramphenicol, tetracycline, and ciprofloxacin have also been used to effectively

treat tularemia. Antibiotic treatment has reduced the overall case fatality to

approximately 2% (45, 142).

Intracellular lifestyle of F. tularensis

Tularemia is characterized by both significant extracellular and intracellular

bacterial phases of F. tularensis. F. tularensis is a facultative intracellular pathogen,

whose primary intracellular niche in the mammalian host is the macrophage, though it

has been shown to replicate inside dendritic cells, hepatocytes, neutrophils, and type II

alveolar epithelial cells in the host (20, 58, 93, 106, 179). Intracellular replication is

crucial to F. tularensis pathogenesis as mutants that fail to replicate intracellularly are

avirulent in mice. Because F. tularensis is found primarily in macrophages in vivo

and because the macrophage represents an important mediator of host defense, the

18

macrophage serves as the most widely used in vitro model to study F. tularensis

infection.

F. tularensis is engulfed by the macrophage via a spacious psuedopod loop

mechanism (30). This mechanism is visually and mechanistically distinct from

conventional, coiling, or ruffling phagocytosis that is observed with other pathogens

and inert particles. Efficient bacterial uptake depends on complement factor C3 and

the corresponding complement receptor (CR3) (30, 148). The mannose receptor (MR)

and scavenger receptor class A (SRA) also play a minor role in bacterial uptake (10,

135, 148). The PI3K pathway does not seem to play a significant role in uptake, but

the Syk/Erk axis appears to play a role through an undefined mechanism (132, 133).

Lipid rafts also play a role in bacterial entry into macrophages and may determine the

biogenesis of the initial phagosome that contains F. tularensis (165).

After uptake, F. tularensis initially resides in a membrane bound vacuole

termed the Francisella-containing Phagosome (FCP). The FCP acquires the early

endosomal antigen 1 (EEA1) within 5 minutes of uptake, but this marker rapidly

dissociates from the phagosome followed by the acquisition of late endosomal markers

Lamp1, Lamp2, and the Rab7 GTPase within 15-30 minutes (26, 31, 145). The FCP

does not significantly fuse with lysosomes and though not absolutely required,

acidification of the phagosomal acts as a cue for F. tularensis to escape the phagosome

into the host cell cytosol (28, 143). Phagosomal escape is rapid, occurring within 60

minutes of macrophage infection. Once in the cytosol F. tularensis replicates to high

numbers. Both phagosomal escape and intracellular replication are mediated by a

locus of F. tularensis genes known as the Francisella Pathogenicity Island (FPI) (122).

19

FPI mutants remain in the initial phagosome, which progresses to lysosomes (14). FPI

mutants are also avirulent in vivo (18, 177). At late stages of infection F. tularensis

can be found again inside a membrane bound vacuole that exhibits characteristics of

autophagosomes (26). The role of autophagy in F. tularensis pathogenesis is

unknown, but F. tularensis downregulates several host proteins required for the

formation of autophagosomes (22).

Innate immunity to F. tularensis

The intracellular lifestyle of F. tularensis brings it in contact with distinct

environments of the macrophage, namely the surface during uptake, the initial

phagosome, and the host cell cytosol. As discussed earlier, the macrophage is armed

with innate immune defenses in each of these compartments that can respond to the

presence of F. tularensis. Furthermore, there is crosstalk between signaling pathways

that link sensing in one compartment to innate responses in other compartments.

Although complement factors are able to kill a number of bacteria, F.

tularensis species are resistant to direct killing by complement. It was initially

thought that complement protection was derived from the presence of a bacterial

capsule (67), although the existence of capsule has been difficult to demonstrate

biochemically. Moreover, F. novicida and F. philomiragia, which lack an apparent

capsule, are also resistant to serum killing (43).

At the macrophage surface, host TLRs engage F. tularensis. Unlike LPS from

enteric bacteria, F. tularensis LPS is only mildly inflammatory and stimulates a low

20

level of proinflammatory cytokine production (57). This likely is due to the unique

structure of F. tularensis lipid A. Unlike the hexa-acylated lipid A from E. coli and

other gram-negative enterics, lipid A from F. tularensis is tetra-acylated (139). It is

thought that this altered structure makes it unrecognizable to LPS-binding protein, and

therefore subverts TLR4 recognition (11, 33, 57). Consistent with this observation,

TLR4 deficient mice are not more susceptible to infection with F. tularensis than

WILD-TYPE mice (27, 36). F. tularensis does significantly stimulate TLR2 signaling

resulting in proinflammatory cytokine production, and intracellular bacteria colocalize

with TLR2 and MyD88 (35, 83, 94). In vivo, TLR2 does not seem to play a role in

host protection during intradermal challenge, but is important in an intranasal model of

infection (36, 94). However, Myd88 deficient mice are completely susceptible to

sublethal doses of LVS when given intradermally (36).

After phagosomal escape F. tularensis is subject to cytosolic innate immune

recognition. In the cytosol, F. tularensis activates a cytosolic surveillance pathway

that is characterized by the production of type-I IFNs (64). The cytosolic sensor

responsible for type-I IFN production is unknown. Autocrine and paracrine signaling

through IFNAR leads to inflammasome activation, resulting in release of mature IL-

1β and IL-18, and host cell death (64). Inflammasome activation is critical to host

defense in vivo as mice lacking inflammasome components have increased bacterial

burden and succumb to infection much faster than wild-type mice (97). The NLR

involved in sensing cytosolic F. tularensis also remains a mystery. Interestingly, an in

vivo genetic screen identified two proteins FTT0584 and FTT0748 that modulate

inflammasome activation by an unknown mechanism (177). Mutants in both of these

21

genes have reduced fitness in vivo. Further studies on the cytosolic stage of F.

tularensis are required to elucidate mechanisms of bacterial virulence and host

defense.

Several features make tularemia an ideal model for studies of innate immunity

and host pathogen interactions. First, tularemia is an acute disease, with the host

either resolving the infection or succumbing to the infection before significant onset of

the adaptive response. Second, F. tularensis is one of only five currently described

bacterial pathogens that reside in the host cell cytosol, and the only one that does not

use actin-based motility for cell-to-cell spread. Lastly, F. tularensis lacks homologs of

a number of canonical toxins, secretion systems, virulence factors, and even PAMPs

possessed by other mammalian pathogens. This provides the opportunity to discover

new bacterial PAMPs, and new mechanisms of virulence and pathogen recognition.

In my thesis work I set out to elucidate the molecular mechanisms of the cytosolic

response to F. tularensis.

22

Chapter 2: A genetic screen identifies novel F. novicida genes that modulate the cytosolic innate immune responses in macrophages Jonathan W. Jones1and Denise M. Monack1

1 Department of Microbiology and Immunology, Stanford University School of

Medicine, Stanford, CA 94305, USA.

23

2.1 CHAPTER 2 SUMMARY

Francisella tularensis is a facultative intracellular pathogen that replicates in

the macrophage cytosol. Macrophages respond to the presence of cytosolic F.

tularensis with the production of type-I IFN, and activation of the inflammasome,

which leads to release of proinflammatory cytokines and host cell death. Little is

known about the bacterial ligands that induce the macrophage cytosolic reponse, or

virulence mechanisms the bacteria use to subvert this response. We wanted to identify

bacterial factors that modulate the cytosolic response in macrophages.

We conducted a forward genetic screen to identify F. novicida genes that

resulted in increased or decreased type-I IFN production and host cell death in

macrophages. In total, we identified 236 genes with an altered cytosolic response.

Deletions of the outer membrane protein fopA as well as several genes involved in

LPS/capsule/cell wall resulted in hyper-stimulation of the cytosolic responses. This

phenotype was partially due to an altered membrane surface that exposed TLR2-

stimulating PAMPs. Deletions of genes in the Francisella Pathogenicity Island, as

well as those involved in purine and pyrimidine biosynthesis were crucial for

intracellular replication and induction of the cytosolic response in macrophages.

We further demonstrate that cytosolic localization is necessary but not

sufficient to induce the cytsolic response. Bacterial protein and DNA synthesis are

required to induce the host response, but bacterial replication is dispensible. Finally,

we show that the host response to cytosolic F. novicida shares characteristics with the

response to transfected dsDNA and we hypothesize that recognition of cytosolic F.

24

novicida DNA mediates type-I IFN production and inflammasome activation in

response to infection. Bacterial mutant that hyper-stimulate this pathway may release

more dsDNA through intracellular lysis.

25

2.2 INTRODUCTION

The innate immune system is the first line of defense against invading

microbes. As part of their arsenal, immune effector cells have evolved a number of

sensors, termed pattern recognition receptors (PRRs) that can detect conserved

microbial patterns or pathogen associated molecular patterns (PAMPs) derived form

microbes. These PAMPs are not unique to any given pathogen, but are usually

structural components of microbial cell wall, and nucleic acids that are evolutionarily

constrained and difficult for microbes to significantly alter to avoid detection. Instead,

many host-adapted pathogens have evolved strategies to modulate host sensing, either

by using bacterial proteins to mask PAMPs or by secreting effector proteins that target

and disrupt host-signaling pathways. This constant interplay of host sensors and

microbial PAMPs and effectors determines the outcome of the infection

As pathogens have evolved to survive in various locations in the cell, so too

have PRRs evolved that reside in and survey diverse cellular compartments. The Toll-

like receptors (TLRs) are located are located on the macrophage surface and in

endosomes, and respond to conserved molecules like bacterial LPS, lipoproteins,

nucleic acids, and flagellin. The TLRs mediate the production of proinflammatory

cytokines through the adaptors MyD88 and Trif. In the cytosol there are several

groups of sensors that mediate pathogen recognition. The RNA helicases, RIG-I and

MDA5, respond to viral RNA and trigger the “antiviral state” characterized by the

production of type-I IFNs through the adaptor MAVS. NOD1 and NOD2 detect

bacterial peptidoglycan fragments and initiate proinflammatory cytokine production

26

through the adaptor RIP2. The cytosol is also equipped with several Nod-like

receptors (NLRs) that initiate the formation of a multiprotein complex containing ASC

and caspase-1, termed the inflammasome. Various stimuli such as pore-forming

toxins, peptidoglycan fragments, and flagellin trigger inflammasome activation.

Inflammasome activation leads to autocatalytic cleavage of capsase-1, which leads to

processing and release of mature IL-1β and IL-18, and host cell death termed

pyroptosis.

Francisella tularensis is an intracellular pathogen that parasitizes host

macrophages. Upon uptake into host macrophages the bacterium initially resides in a

membrane bound vacuole. However, it rapidly escapes the phagosome and resides in

the macrophage cytosol where it can replicate to high numbers. Lipid A from many

enteric pathogens stimulates TLR4 signaling at the host surface. F. tularensis

possesses a unique lipid A that is not recognized by TLR4. Instead TLR2 is involved

in sensing F. tularensis at the surface and in the vacuole, and leads to the production

of proinflammatory cytokines like proIL-1β and TNF-α. Macrophages respond to the

presence of cytosolic F. tularensis with the production of type-I IFNs in an IRF3

dependent but TLR-, RIG-I, MDA-5, and NOD-independent manner. This response is

reminiscent of the host response to cytosolic nucleic acids. Autocrine and paracrine

signaling of type-I IFN through the type-I IFN receptor (IFNAR) is crucial to

subsequent activation of an ASC-containing inflammasome, which leads to release of

mature IL-1b and IL-18 and host cell death. We refer to the combination of the type-I

IFN response and the macrophage cell death response as the cytosolic responses.

None of the known inflammasome Nod-like receptors (NLRs) play a role in

27

inflammasome activation in response to F. tularensis. The bacterial ligand(s) and host

receptor(s) responsible for recognizing cytosolic F. tularensis are unknown.

Furthermore, there is evidence that F. tularensis has mechanisms to dampen the host

response and delay the activation of the inflammasome (177).

In order to elucidate the mechanisms by which F. tularensis modulates the

cytosolic responses we performed a forward genetic screen of two F. novicida

transposon libraries to identify mutants that induced increased to decreased type-I IFN

and cell death responses from macrophages. We found that a gene encoding a major

F. tularensis outer membrane protein, fopA, as well mutants in genes involved in

LPS/capsule/cell wall synthesis were responsible for dampening the cytosolic response

to F. tularensis. We also replicated previous findings that mutants that failed to

escape the phagosome do not trigger the cytosolic responses. Further study

demonstrated that cytosolic localization is necessary but not sufficient to induce the

cytosolic responses. Finally, we show that induction of the cytosolic response by F.

novicida does not require bacterial replication, but is dose dependent and shares many

characteristics with the cytosolic response dsDNA.

28

2.3 RESULTS

2.3.1 A transposon screen identifies fopA as a suppressor of the macrophage

cytosolic innate immune response.

To identify bacterial genes involved in modulating the cytosolic response to F.

tularensis, we screened a F. novicida transposon library (177) for mutants that

exhibited enhanced or diminished type-I IFN and cell death responses upon infection

in macrophages. Approximately 10,000 individual transposon insertion mutants were

used to infect Pam3CSK pre-stimulated bone marrow-derived macrophages (BMDMs)

in 96-well plates. The amount of type-IFN in macrophage supernatant was measured

by a type-I IFN reporter cell line that produces luciferase in response to type-I IFN

(ISRE-L929) (79). To confirm the initial results from the ISRE screen we infected

Pam3CSK pre-stimulated BMDMs with individual transposon insertion mutants from

the F. novicida transposon library (177) and measured the macrophage IFN-β

response by quantitative RT-PCR. Transposon insertions in the pdpA and clpB genes

were attenuated in inducing and IFN-β response in macrophages similar to the control

ΔFPI mutant (Fig. 1A). These results are consistent with previous reports that

demonstrate that the ΔpdpA mutant is attenuated for phagosomal escape and therefore

would not induce the cytosolic response. A ΔclpB mutant is attenuated for intracellular

replication in BMDMs and virulence in mice (55, 111). Furthermore, ClpB displays

homology to ClpV proteins from type VI secretion systems, and we speculate that it

may play a role in the function of the FPI, which would make a ΔclpB mutant

attenuated for phagosomal escape, though this has not been formally shown. These

29

results were proof of principle that our screen would identify F. novicida genes that

modulate the cytosolic responses.

Surprisingly, library mutant Tn96G6, which was identified as a transposon

insertion in the fopA gene, induced a 17-fold higher induction of IFN-β in

macrophages than wild-type F. novicida (Fig. 1A), similar to a control ΔFTT0584

mutant that hyper-induces the cytosolic response (177). This result was confirmed

with an isogenic deletion of fopA (Fig. 1B). Consistent with the link between the

type-I IFN response and the host cell death response (34, 64), a ΔfopA mutant killed

77% of infected macrophages by 6hrs PI, while wild-type F. novicida induced

negligible killing (Fig. 1C). We were able to complement these phenotypes by re-

introducing a wild-type copy of the fopA gene in trans (Fig. 1B,C). This hyper-

induction of the cytosolic responses was not due to increased replication of the

Tn96G6 or ΔfopA mutants (Fig.1D). These results indicate that the fopA gene

suppresses the cytosolic response to F. novicida.

30

A B

C D

- Figure 1 - An F. novicida ΔfopA mutant hyper-induces the cytosolic innate immune response

in macrophages.

IFN-β mRNA was quantified by quantitative RT-PCR at 5hrs post-infection (PI) in wild-type BMDMs

that were either Pam3CSK4 pre-stimulated (A) or unstimulated (B) and infected with the indicated

strains of F. novicida at an MOI of 10:1. (C) Host cell death was quantified by lactate dehydrogenase

(LDH) release at 6hrs PI in unstimulated BMDMs infected at an MOI of 10:1. (D) Intracellular

replication was assayed by gentamicin protection assay over 8hrs in unstimulated BMDMs infected at

an MOI of 1:10. Error bars for quantitative RT-PCR represent standard deviation of 3 technical

31

replicates. Error bars for cell death assay represent standard deviation of infections done in triplicate.

Graphs are representative of three independent experiments.

2.3.2 ΔfopA stimulates the same cytosolic surveillance pathway as wild-type F.

novicida.

FopA is an abundant outer membrane protein that may interact with LPS and

peptidoglycan, which are considered PAMPs for TLR and NOD receptors

respectively. Previous studies demonstrated that the IFN-β response to wild-type F.

novicida was independent of TLR and NOD signaling, (64). However, we were

curious to know if deletion of fopA would lead to an increased release of TLR and

NOD ligands and a contribution of these PRRs to the cytosolic response. We

investigated the roles of TLR and NOD signaling to the cytosolic response

respectively by infecting myd88/trif-/- and rip2-/- macrophages with wild-type F.

novicida and ΔfopA, and measuring the IFN-β and host cell death responses. We

observed only a slight decrease in IFN-β mRNA levels in rip2-/- macrophages and no

difference in myd88/trif-/- macrophages compared to wt (Fig. 2A, C). We also saw no

effect on the cell death response in the absence of TLR or NOD signaling (Fig. 2D, E).

This indicated that deletion of fopA did not lead to increased cytosolic recognition

through release of LPS or peptidoglycan fragments.

32

A B

C D

E

33

- Figure 2 - The cytosolic response to ΔfopA is IRF3- and IFNAR- dependent but TLR- and

NOD- independent.

Unstimulated wt, irf3-/-, myd88/trif-/-, ifnar-/- or rip2-/- BMDM were infected with the indicated

strains of F. novicida at an MOI of 10:1 and IFN-β mRNA was quantified by quantitative RT-PCR at

5hrs PI (A, B, and C) and cell death was measured by LDH assay at 7hrs PI (D and E). Error bars for

quantitative RT-PCR represent standard deviation of 3 technical replicates, while error bars for cell

death represent standard deviation of triplicate infections. Graphs are representative of three

independent experiments.

The IFN-β response to wild-type F. novicida is dependent on IRF3 and

autocrine and paracrine signaling through IFNAR acts to amplify IFN-β production

(64). While the IFN-β response to ΔfopA was completely IRF3 dependent (Fig. 2A)

we saw only a modest reduction in IFN-β mRNA in the absence of IFNAR signaling

(Fig. 2B). Additionally, type-I IFN signaling was required for the full host cell death

response to ΔfopA, although the mutant also induced higher levels of type-I IFN-

independent cell death than wild-type F. novicida (Fig. 2D). Therefore we conclude

that the IFNAR-dependent amplification of IFN-β observed in response to wild-type

F. novicida is not required for the response to ΔfopA, possibly because fopA either

suppresses the amplification loop, or because ΔfopA releases more of the IFN-β

stimulating ligand, and thus amplification of the signal is not required. This leads to

an increase in type-IFN dependent and independent host cell death responses by the

macrophage.

Cytosolic F. novicida induces an inflammasome-dependent host cell death that

is triggered by recognition from an unknown NLR (97, 98). To determine if ΔfopA

34

induced cell death through the same pathway as wild-type F. novicida we infected wt,

asc-/-, and casp-1-/- BMDM with wild-type F. novicida or ΔfopA and compared the

early and late cell death responses. The ΔfopA mutant induced host cell death in an

ASC- and caspase-1-depedent manner, just like wild-type F. novicida (Fig. 3A).

Furthermore, ΔfopA hyper-stimulated the ASC-dependent-caspase-1-independent late

cell death observed in response to wild-type F. novicida (Fig. 3B). Finally, since

NLRP3 has been implicated in the inflammasome response to peptidoglycan

fragments (100) and FopA could interact with peptidoglycan in the outer membrane,

we investigated if NLR3 contributed to the cell death response to ΔfopA comparing the

cell death responses of wt and nlrp3-/- BMDMs to infection with ΔfopA. Again, like

wild-type F. novicida host cell death to ΔfopA was completely NLRP3-independent

(Fig. 3C). These results further suggest cytosolic recognition of ΔfopA occurs through

the same mechanism as wild-type F. novicida. Thus, fopA could suppress the

cytosolic sensor leading to IFN-β or it could suppress release of the ligand from the

bacteria.

35

A B

C

- Figure 3- The hyper-cytotoxicity ofΔfopA is ASC- and caspase-1 dependent but NLRP3-

independent.

BMDM from wt, asc-/-, casp-1-/-, or nlrp3-/- mice were Pam3CSK4 pre-stimulated (A and B) or

unstimulated (C) and infected with the indicated strains of F. novicida for 3.5hrs (A), 5.5hrs (B), or 8hrs

(C) and cell death was measured by LDH assay. Means and standard deviations are shown for

infections done in triplicate. Representative graphs of three independent experiments are shown.

36

2.3.3 A ΔfopA mutant has reduced fitness in mice.

To investigate the role of fopA in virulence we performed a competitive index

experiment where we infected wt C57/BL6J mice intradermally (i.d.) with an equal

ratio of wild-type F. novicida and ΔfopA mutant for two days and harvested skin and

spleen to enumerate CFU. If the mutant and wild-type are equally fit in vivo we

would expect a competitive index of 1. However, we observed a competitive index

for ΔfopA that was significantly less than 1 (Fig. 4), indicating that this mutant has

reduced fitness in mouse infections relative to wild-type F. novicida. The hyper-

stimulation of the cytosolic immune responses in macrophages could contribute to the

attenuation of the mutant, though we cannot rule out the possibility that a mutant in an

outer membrane protein could be more susceptible to killing by complement or other

antimicrobial defenses as well.

37

- Figure 4 - ΔfopA is less fit in vivo than wild-type F.novicida.

wt C57BL6/J mice were infected intradermally (i.d.) with 105 cfu of an equal ratio of wt F. novicida

and ΔfopA. At 2 days post infection skin and spleen was harvested and bacterial loads were determined

by plating serial dilutions of homogenized tissue on selective and non-selective MH agar.

2.3.4 Identification of F. tularensis mutants that differentially induce the

cytosolic response in macrophages by a genome-wide forward genetic screen.

Although we had identified fopA in a screen of our transposon library, we

experienced difficulty in identifying other transposon insertions by genomic

sequencing. Therefore, we decided to repeat the forward genetic screen using a

commercially available “two-allele” F. novicida transposon library from BEI

Resources to search for mutants that induced an increased or decreased type-I IFN

response and/or an increased or decreased host cell death response in bone marrow-

derived macrophages. This simplified the identification of screen hits as the position

38

of transposon insertion in each mutant was provided. Furthermore, we were confident

that this screen gave us complete coverage of the F. novicida genome. The amount of

type-I IFN in macrophage supernatants was measured by incubating culture

supernatant from infected macrophages with a reporter cell line that produces

luciferase in response to type-I IFNs (79). Macrophage cell death was measured by

lactate dehydrogenase release assay (LDH). In total we identified 278 transposon

insertions representing 236 F. novicida genes that differentially induced the cytosolic

response in macrophages relative to the wild-type strain (Table 1 and 2). This

represents approximately 17% of the non-essential F. novicida genome.

39

- Table 1 – Mutants that hyper-induce the cytosolic responses

FTN # Plate Well Gene Description Functional class FTN_0007 9 D10 hypothetical protein (predicted

secretion signal, transmembrane domain)

hypothetical - novel

FTN_0018 26 C7 sdaC serine permease transport - amino-acid

FTN_0073 6 B10 yidC conserved membrane protein of unknown function

unknown function - conserved

FTN_0074 2 B8 conserved hypothetical protein (signal peptide)

hypothetical - conserved

FTN_0088 27 C4 ybgL protein of unknown function, LamB/YcsF family


FTN_0137 3 H12 protein of unknown function (predicted secretion signal)

unknown function - novel

FTN_0139 22 F2 hypothetical protein hypothetical - novel

FTN_0148 6 A7 hypothetical membrane protein (predicted signal sequence)


FTN_0157 8 C2 membrane protein of unknown function


FTN_0193 5 B5 cydA cytochrome bd-I terminal oxidase subunit I

energy metabolism

FTN_0203 19 D10 protein of unknown function (predicted secretion signal)


FTN_0215 9 E8 hypothetical protein hypothetical - novel

FTN_0234 6 B1 pgsA phosphatidylglycerophosphate synthetase

fatty acids and lipids metabolism

FTN_0276 26 B2 mviN multidrug/oligosaccharidyl-lipid/polysaccharide (MOP)

transporter

transport - drugs / antibacterial compounds

FTN_0280 3 H10 hypothetical protein hypothetical - novel

40

FTN_0284 5 D9 prophage maintenance system killer protein (DOC)

mobile and extrachromosomal element functions - phage or plasmid related proteins

FTN_0292 10 E5 protein of unknown function unknown function - novel

FTN_0311 32 F2 hemK modification methylase, HemK family

translation, ribosomal structure and biogenesis

FTN_0330 9 C9 minD septum formation inhibitor-activating ATPase

cell cycle

FTN_0340 16 H7 protein of unknown function unknown function - novel

FTN_0341 1 B7 protein of unknown function (predicted secretion signal)

unknown function novel

FTN_0345 8 B3 DNA uptake protein, SMF family transport

FTN_0388 3 C7 protein of unknown function unknown function - novel

FTN_0391 13 D11 LemA-like protein putative enzymes

FTN_0409 17 C4 adhC Zn-dependent alcohol dehydrogenase

energy metabolism

FTN_0415 19 C3 pilA Type IV pili, pilus assembly protein

motility, attachment and secretion structure

FTN_0416 9 E11 lpxE lipid A 1-phosphatase fatty acids and lipids metabolism

FTN_0444 27 C10 membrane protein of unknown function


FTN_0449 11 C8 conserved protein of unknown function (predicted secretion

signal)


41

FTN_0463 9 F4 hypothetical protein hypothetical - novel

FTN_0487 4 A1 30S ribosomal protein S21 translation, ribosomal structure and biogenesis

FTN_0528 12 F4 lpxH UDP-2,3-diacylglucosamine hydrolase


FTN_0530 8 C8 mpl UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-

diaminopimelate ligase

cell wall / LPS / capsule

FTN_0533 31 B6 drug:H+ antiporter-1 (DHA1) family protein


FTN_0546 16 D9 flmK dolichyl-phosphate-mannose-protein mannosyltransferase

family protein


FTN_0550 19 C9 sohB peptidase family S49 protein (predicted secretion signal, periplasmic) stalk mutant?

post-translational modification, protein

turnover, chaperones - protein modification

FTN_0558 25 C3 ostA1 organic solvent tolerance protein, OstA


FTN_0568 22 B3 birA birA-like protein post-translational modification, protein


FTN_0576 21 H8 conserved protein of unknown function (predicted secretion

signal)


FTN_0582 9 G12 gph phosphoglycolate phosphatase putative enzymes

FTN_0595 18 F1 outer membrane protein of unknown function


42

FTN_0611 17 D6 kdsA 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase


FTN_0633 18 D4 katG peroxidase/catalase other metabolism - degradation, utilization,

assimilation

FTN_0638 24 C8 sulfate permease family protein transport

FTN_0656 12 D6 Zn-dependent peptidase, M16 family



FTN_0657 31 B5 metallopeptidase, M16 family post-translational modification, protein


FTN_0660 29 F1 pepA cytosol aminopeptidase amino acid metabolism

FTN_0672 12 G3 secA preprotein translocase, subunit A (ATPase, RNA helicase)


FTN_0709 14 E11 hypothetical protein (predicted secretion signal, transmembrane

domain)


FTN_0714 12 G1 protein of unknown function (predicted secretion signal)


FTN_0742 27 D11 serB phosphoserine phosphatase amino acid metabolism - biosynthesis

43

FTN_0746 29 D12 alr alanine racemase amino acid metabolism - degradation,

utilization, assimilation

FTN_0747 13 A2 amino acid-polyamine-organocation (APC) superfamily

protein

transport - amino-acid

FTN_0756 4 G11 fopA OmpA family protein cell wall / LPS / capsule

FTN_0757 22 D11 membrane protein of unknown function


FTN_0772 22 C2 conserved protein of unknown function


FTN_0785 5 B6 isochorismatase family protein putative enzymes

FTN_0812 17 C10 bioD dethiobiotin synthetase cofactors, prosthetic groups, electron

carriers metabolism

FTN_0814 7 E11 bioF 8-amino-7-oxononanoate synthase cofactors, prosthetic groups, electron

carriers metabolism

FTN_0828 13 B4 protein of unknown function (predicted secretion signal)

pseudogene in Schu4


FTN_0839 32 E10 conserved protein of unknown function


FTN_0841 8 D10 ThiJ/PfpI family protein putative enzymes

FTN_0842 7 G10 aroG phospho-2-dehydro-3-deoxyheptonate aldolase

amino acid metabolism - biosynthesis

FTN_0863 7 B2 hypothetical membrane protein hypothetical - novel

FTN_0877 26 C10 cls; ybhO cardiolipin synthetase fatty acids and lipids metabolism

44

FTN_0901 10 C8 isomerase putative enzymes

FTN_0905 4 B10 yrbI 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase

fatty acids and lipids metabolism (lnvolved

in LPS synthesis)

FTN_0907 16 E6 dacD D-alanyl-D-alanine carboxypeptidase


FTN_0939 8 B10 hypothetical protein hypothetical - novel

FTN_0943 8 C12 rimI ribosomal-protein-alanine acetyltransferase


FTN_0967 29 G11 vanY D-alanyl-D-alanine carboxypeptidase


FTN_1007 2 H8 rplY 50S ribosomal protein L25 translation, ribosomal structure and biogenesis

FTN_1027 12 H7 ruvC holliday junction endodeoxyribonuclease

DNA replication, recombination,

modification and repair -

restriction/modification

FTN_1029 4 F11 elbB conserved protein of unknown function


FTN_1030 3 B5 lipA lipoic acid synthetase cofactors, prosthetic groups, electron

carriers metabolism

FTN_1036 4 H11 protein of unknown function unknown function - conserved

FTN_1037 16 E4 hypothetical membrane protein hypothetical - novel

45

FTN_1046 2 D9 wzb low molecular weight (LMW) phosphotyrosine protein

phosphatase (exopolysaccharide production)



FTN_1058 6 A6 tig trigger factor (TF) protein (peptidyl-prolyl cis/trans

isomerase



FTN_1064 1 A4 PhoH family protein, putative ATPase

signal transduction and regulation

FTN_1066 11 B5 transporter-associated protein, HlyC/CorC family

transport

FTN_1070 7 D7 ospD2 protein of unknown function unknown function - novel

FTN_1073 30 F2 DNA/RNA endonuclease G nucleotides and nucleosides metabolism

FTN_1074 4 H10 X-prolyl aminopeptidase 2 post-translational modification, protein


FTN_1087 8 G1 cynT carbonic anhydrase other metabolism - degradation, utilization,

assimilation

FTN_1093 18 A5 protein of unknown function unknown function - novel

FTN_1104 11 H2 hypothetical protein (predicted secretion signal)


FTN_1109 16 C7 rhodanese-like family protein putative enzymes

FTN_1123 13 B6 conserved hypothetical protein hypothetical - conserved


46

FTN_1135 10 B10 aroB 3-dehydroquinate synthetase amino acid metabolism - biosynthesis

FTN_1146 2 B7 aspC2 aspartate aminotransferase other metabolism - degradation, utilization,

assimilation FTN_1148 4 B11 glycoprotease family protein post-translational

modification, protein turnover, chaperones

FTN_1149 5 A9 nagA N-acetylglucosamine-6-phosphate deacetylase

carbohydrate metabolism - degradation, utilization,

assimilation

FTN_1154 13 B3 type I restriction-modification system, subunit S

DNA replication, recombination,

modification and repair - repair

FTN_1187 27 E6 protein of unknown function unknown function - conserved

FTN_1212 28 A1 glycosyl transferase, group 1 cell wall / LPS / capsule

FTN_1213 3 D7 glycosyl transferase, family 2 cell wall / LPS / capsule

FTN_1214 19 B7 glycosyl transferase, family 2 cell wall / LPS / capsule

FTN_1215 27 C6 kpsC capsule polysaccharide export protein KpsC


FTN_1217 19 G1 ATP-binding cassette (ABC) superfamily protein

transport

FTN_1218 7 C4 glycosyl transferase, group 1 cell wall / LPS / capsule

FTN_1219 30 C10 galE UDP-glucose 4-epimerase carbohydrate metabolism - degradation, utilization,

assimilation

47

FTN_1221 8 E6 rpe D-ribulose-phosphate 3-epimerase energy metabolism

FTN_1223 10 C4 conserved hypothetical membrane protein DoxD like family protein


FTN_1239 10 D7 5-formyltetrahydrofolate cycloligase

putative enzymes

FTN_1240 24 C11 BolA family protein cofactors, prosthetic groups, electron

carriers metabolism

FTN_1241 14 F10 DedA family protein (predicted secretion signal)

putative enzymes

FTN_1242 18 B12 DedA family protein putative enzymes

FTN_1253 25 C10 lpcC glycosyl transferase, group 1 cell wall / LPS / capsule

FTN_1254 2 G7 protein of unknown function unknown function - novel

FTN_1255 5 C11 glycosyl transferase, family 8 cell wall / LPS / capsule

FTN_1256 7 B6 membrane protein of unknown function


FTN_1273 32 A7 fadD1 long chain fatty acid CoA ligase other metabolism - degradation, utilization,

assimilation

FTN_1296 2 F3 yhbH sigma54 modulation protein translation, ribosomal structure and biogenesis

FTN_1330 10 C5 pyk pyruvate kinase carbohydrate metabolism - degradation, utilization,

assimilation FTN_1368 14 E7 feoA Fe2+ transport system protein A transport

FTN_1369 3 F7 protein of unknown function (cell division protein)


48

FTN_1404 11 B6 yadH ATP-binding cassette (ABC) superfamily protein

transport

FTN_1417 31 F11 manB phosphomannomutase carbohydrate metabolism - biosynthesis

FTN_1418 5 C3 manC mannose-1-phosphate guanylyltransferase


FTN_1421 6 C3 wbtH glutamine amidotransferase/asparagine

synthase

amino acid metabolism - biosynthesis

FTN_1423 3 A12 wbtG glycosyl transferase, group 1 cell wall / LPS / capsule

FTN_1426 20 H10 wbtE UDP-glucose/GDP-mannose dehydrogenase family protein


FTN_1427 1 B5 wbtD glycosyl tranferase, group 1 cell wall/ LPS/ capsule

FTN_1428 3 B11 wbtO transferase cell wall / LPS / capsule

FTN_1429 21 B1 wbtP galactosyl transferase cell wall / LPS / capsule

FTN_1431 6 G3 wbtA dTDP-glucose 4,6-dehydratase cell wall / LPS / capsule

FTN_1452 12 E7 two-component response regulator


FTN_1470 12 B3 ispA geranyl diphosphate synthase/farnesyl diphosphate

synthase

cofactors, prosthetic groups, electron

carriers metabolism

FTN_1472 17 D5 conserved protein of unknown function


FTN_1476 26 A3 protein of unknown function unknown function - novel

49

FTN_1496 8 F10 coaE dephospho-CoA kinase cofactors, prosthetic groups, electron

carriers metabolism


FTN_1504 20 D8 glucokinase regulatory protein signal transduction and regulation

FTN_1515 3 F5 hypothetical membrane protein hypothetical - novel

FTN_1533 21 A7 conserved protein of unknown function


FTN_1537 16 H9 hypothetical protein hypothetical - novel

FTN_1548 9 A1 yfgL conserved protein of unknown function


FTN_1602 4 E5 deoB phosphopentomutase other metabolism - degradation, utilization,

assimilation

FTN_1603 10 E6 regulatory factor, Bvg accessory factor family


FTN_1613 12 D11 peptidase, U61 family (predicted secretion signal, peptidoglycan

recycling)


turnover, chaperones - protein degradation


FTN_1617 20 A1 qseC two-component regulator, sensor histidine kinase


FTN_1630 21 C1 secG preprotein translocase, subunit G, membrane protein


FTN_1653 13 A11 hypothetical membrane protein hypothetical - novel

FTN_1656 16 B11 conserved hypothetical protein hypothetical - conserved

50

FTN_1665 6 C4 magnesium chelatase cofactors, prosthetic groups, electron

carriers metabolism

FTN_1686 26 D10 hypothetical membrane protein hypothetical - novel

FTN_1704 20 B4 pcm protein-L-isoaspartate O-methyltransferase



FTN_1732 11 D10 Mg-dependent Dnase DNA replication, recombination,

modification and repair - degradation

FTN_1734 13 E11 protein of unknown function (predicted secretion signal)



FTN_1757 4 D5 D-isomer specific 2-hydroxyacid dehydrogenase

energy metabolism

FTN_1766 19 C6 drug/metabolite transporter (DMT) superfamily protein


intergenic 19 B4 intergenic 21 A1 intergenic 25 G11 intergenic 26 H7 intergenic 3 H11

13 B5 intergenic 3 B3 isftu3 isftu3 IS element 16 D10 isftu2 isftu2 IS element 30 B7 isftu6 isftu6 IS element

51

- Table 2 – Mutants that hypo-induce the cytosolic responses

FTN # Plate Well Gene Description Functional class FTN_0019 29 A2 pyrB aspartate carbamoyltransferase post-translational

modification, protein turnover, chaperones -

chaperones

FTN_0020 8 D3 carB carbamoyl-phosphate synthase large chain


FTN_0021 30 D8 carA carbamoyl-phosphate synthase small chain


FTN_0024 14 E2 pyrC dihydroorotase amino acid metabolism FTN_0035 29 E4 pyrF orotidine-5'-phosphate

decarboxylase transport - drugs /

antibacterial compounds

FTN_0036 32 G12 pyrD dihydroorotate oxidase nucleotides and nucleosides metabolism

FTN_0085 16 F12 uspA universal stress protein nucleotides and nucleosides metabolism

FTN_0108 16 B12 trmU tRNA(5-methylaminomethyl-2-thiouridylate) methyltransferase

nucleotides and nucleosides metabolism

FTN_0124 11 G7 ssb single-strand DNA binding protein

amino acid metabolism - degradation,

utilization, assimilation

FTN_0140 23 C7 ABC-type anion transport system, duplicated permease component

nucleotides and nucleosides metabolism

FTN_0156 1 G2 plsC 1-acylglycerol-3-phosphate acyltransferase


FTN_0266 16 F2 htpG chaperone Hsp90, heat shock protein HtpG

energy metabolism

FTN_0289 2 D1 proQ activator of osmoprotectant transporter ProP

transport

FTN_0336 21 C9 hypothetical protein (predicted secretion signal, transmembrane

domain)

transport - amino-acid

FTN_0343 32 E7 aminotransferase putative enzymes

52

FTN_0357 25 G7 pal peptidoglycan-associated lipoprotein, OmpA family


FTN_0419 8 E12 purM phosphoribosylformylglycinamide cyclo-ligase

putative enzymes

FTN_0420 6 E2 SAICAR synthetase/phosphoribosylamine-

glycine


FTN_0422 32 H9 purE N5-carboxyaminoimidazole ribonucleotide mutase

transport

FTN_0504 5 B11 cadA lysine decarboxylase transport FTN_0529 29 C4 pyrE orotate phosphoribosyltransferase transport

FTN_0535 4 B4 drug:H+ antiporter-1 (DHA1) family protein


FTN_0540 5 E6 pckA phosphoenolpyruvate carboxykinase


FTN_0570 7 A9 perM PerM family protein motility, attachment and secretion structure

FTN_0571 12 C12 xasA amino acid-polyamine-organocation (APC) superfamily

protein

transport

FTN_0604 25 G6 AMP-binding protein unknown function - novel

FTN_0616 30 H2 rumA RNA methyltransferase, trmA family

putative enzymes

FTN_0618 21 F11 glk1 ROK family protein translation, ribosomal structure and biogenesis

FTN_0654 29 D6 conserved hypothetical membrane protein


FTN_0728 14 H4 predicted Co/Zn/Cd cation transporter


FTN_0737 22 A12 potI ATP-binding cassette putrescine uptake system, membrane protein,

subunit I


FTN_0738 27 G12 potH ATP-binding cassette putrescine uptake system, membrane protein,

subunit H


FTN_0818 12 H9 lipase/esterase translation, ribosomal structure and biogenesis

FTN_0915 4 E1 yqeY conserved protein of unknown function (cytosolic)


53

FTN_0946 27 D6 pilF Type IV pili, pilus assembly protein


FTN_1013 22 G3 monovalent cation:proton antiporter family protein

putative enzymes

FTN_1022 27 D4 protein of unknown function (possible transcriptional regulator

arsR)


FTN_1034 7 B1 rnfB iron-sulfur cluster-binding protein unknown function - novel

FTN_1063 9 H6 yleA tRNA-methylthiotransferase MiaB protein


FTN_1112 11 H4 cphA cyanophycin synthetase hypothetical - conserved

FTN_1137 3 A2 pilQ type IV pili secretion component hypothetical - novel

FTN_1199 20 G10 conserved protein of unknown function (predited secretion

signal, transmembrane domain)


FTN_1201 10 C1 capB capsule biosynthesis protein CapB hypothetical - novel

FTN_1264 31 C11 rluD ribosomal large subunit pseudouridine synthase D


FTN_1275 32 D6 emrB drug:H+ antiporter-1 (DHA2) family protein


FTN_1290 24 E4 mglA macrophage growth locus, protein A


FTN_1298 17 A7 trmE GTPase of unknown function hypothetical - novel

FTN_1309 5 F4 pdpA protein of unknown function hypothetical novel

FTN_1310 18 A9 pdpB protein of unknown function unknown function - novel

FTN_1311 9 F12 iglE protein of unknown function unknown function - novel

FTN_1312 23 H4 vgrG conserved hypothetical protein unknown function - conserved

FTN_1313 9 D6 iglF hypothetical protein unknown function - conserved

FTN_1314 20 A6 iglG conserved hypothetical protein unknown function - novel

FTN_1314 23 F2 iglF hypothetical protein cell wall / LPS / capsule

54

FTN_1315 14 G8 iglH protein of unknown function signal transduction and regulation

FTN_1316 21 A4 dotU protein of unknown function other metabolism - biosynthesis

FTN_1317 2 B6 iglI protein of unknown function transport - drugs / antibacterial compounds

FTN_1318 20 F8 iglJ hypothetical protein translation, ribosomal structure and biogenesis

FTN_1319 1 G9 pdpC hypothetical protein transport - carbohydrates (sugars,

polysaccharides) FTN_1321 27 E5 iglD intracellular growth locus protein

D nucleotides and

nucleosides metabolism

FTN_1322 14 G2 iglC intracellular growth locus protein C

post-translational modification, protein turnover, chaperones

FTN_1323 10 G2 iglB intracellular growth locus protein B

FTN_1324 5 C8 iglA intracellular growth locus protein A

intergenic

FTN_1325 2 F2 pdpD protein of unknown function

FTN_1422 28 B6 wbtN glycosyl transferase, group 1

FTN_1465 24 H5 pmrA two-component response regulator

FTN_1518 21 F10 relA GDP pyrophosphokinase/GTP pyrophosphokinase

FTN_1521 17 F8 10 TMS drug/metabolite exporter protein

FTN_1559 4 A10 rplS 50S ribosomal protein L19

FTN_1586 9 B2 sugar transporter, MFS superfamily

FTN_1699 6 D2 purL phosphoribosylformylglycinamide synthase

FTN_1743 11 F3 clpB chaperone clpB intergenic 28 E5

8 H2

55

We identified 72 F. novicida mutants that resulted in a decreased macrophage

cytosolic response (Table 2). The 72 gene insertions included several known F.

tularensis virulence factors such as the transcriptional regulators mglA and pmrA and

17 of the genes in the FPI. Mutants in the FPI and its transcriptional regulators fail to

escape the phagosome and therefore fail to induce the cytosolic response in

macrophages and are attenuated in a mouse model of tularemia (Brotcke et al., 2006,

Henry et al., 2007, Weiss et al. 2007). Interestingly, two genes located in the FPI,

pdpE and anmK, were not identified in our screen. Further analysis of these mutants

showed that neither ΔpdpE nor ΔanmK were attenuated for intracellular replication in

RAW267.4 macrophages, thus we surmise that these mutants escape from the

phagosome as efficiently as wild-type. Furthermore, these mutants induced

macrophage cell death as efficiently as wild type. In total, these results validated our

screening method. We also identified 164 mutants that hyper-induced the macrophage

cytosolic response, resulting in increased kinetics of type-I IFN secretion and

macrophage cytotoxicity. These mutants fell into several functional categories

including proteins of unknown function, transport, post-translational modification,

fatty acid and lipid metabolism, and cell wall/LPS/capsule. This result suggests that

either the induction of the cytosolic response is the result of the interaction of multiple

bacterial factors with the host cytosolic surveillance pathway, or that an essential

bacterial molecule is responsible for induction of the cytosolic response. Furthermore,

we observed a positive correlation of the type-I IFN response and macrophage

cytotoxicity with all mutants identified except one. A ΔkdsA mutant induced higher

56

levels of type-I IFN relative to wild type but induced lower levels of macrophage

cytotoxicity. Taken together, these results suggest that several F. tularensis gene

products modulate the host cytosolic response and these genes fall into several

functional categories. Moreover, the macrophage type-I IFN response and cell death

is tightly linked and positively correlated upon infection with F. tularensis such that

the kinetics of type-I IFN production determine the kinetics of host cell death.

2.3.5 F. novicida LPS mutants hyper-induce the cytosolic responses.

We chose to focus on genes characterized as LPS/capsule/cell wall because

several clusters of these genes were identified in our screen. Furthermore, wild-type

F. novicida LPS does not activate TLR4-dependent signaling, so we hypothesized that

characterizing mutants with altered LPS/capsule/cell wall that hyper stimulate immune

responses might lead us to the ligand(s) and the molecular mechanism of the cytosolic

response. Therefore we generated clean deletion mutants in FTN_1212, lpcC, wbtA,

kdsA, and lpxH and measured type-I IFN induction and host cell death during

macrophage infections. ΔFTN_1212, ΔlpcC, ΔwbtA, ΔkdsA, and ΔlpxH induced IFN-

β transcript and protein secretion in macrophages with increased magnitude and

kinetics relative to wild-type F. novicida (Fig. 5A-D). Additionally, all of these

mutants induced increased secretion of IL-1β in Pam3CSK4 pre-stimulated

macrophages relative to wild-type F. novicida (Fig. 6A). We observed a similar

increase in IL-1β secretion in unstimulated macrophages. Increased IL-1β secretion

was accompanied by increased kinetics of host cell death with all mutants tested save

57

ΔkdsA, which induced a higher level of cell death than wild-type F. novicida at 7hrs

post-infection, but only killed 20% of the macrophages by 10hrs post-infection while

50% of the macrophages were killed by wild-type over the same time (Fig. 6B, C).

58

A B

C D

- Figure 5 - LPS mutants hyper-induce the type-I IFN response in macrophages.

Unstimulated wild-type BMDMs were infected with the indicated strains of F. novicida at a multiplicity

of infection of 10 bacteria per macrophage. At the indicated timepoints (A and B) IFN-β mRNA levels

were determined by quantitative RT-PCR and normalized to the level of IFN-β mRNA in uninfected

BMDM or (C and D) total type-I IFN in macrophage supernatant was determined ISRE-L929 reporter

cell assay. Luciferase levels were normalized to the level from uninfected macrophage supernatant.

Means and standard deviations are plotted for experiments done in triplicate. Graphs are representative

of three independent experiments.

59

A

B

C

- Figure 6 - LPS mutants hyper-induce IL-1β release and host cell death.

Pam3CSK4 pre-stimulated wt BMDM were infected with the indicated strains of F. novicida and IL-1β

release was measured at 5.5hrs PI (A). The kinetics of host cell death was measured by LDH release

assay (B and C) in unstimulated wt BMDM. Means and standard deviations of triplicate infections are

shown. Graphs are representative of three independent experiments.

60

2.3.6 F. novicida LPS mutants stimulate increased TLR2-depedent signaling.

Type-I IFN can be induced by TLR signaling. F. novicida induces TLR2

dependent signaling, resulting in the production of proinflammatory cytokines. We

posited that mutants with altered LPS may lead to increased TLR recognition,

accounting for the increased cytosolic responses. To this end we infected wt and

myd88/trif-/- BMDM with wild-type F. novicida or the LPS mutants and measured

type-I IFN secretion and host cell death. We found that the increased type-I IFN

production observed with the LPS mutants at 2hrs post-infection was completely

MyD88/Trif dependent (Fig. 7A). However, at 8hr post-infection, there was a

significant production of type-I IFN from the LPS mutants in the absence of TLR

signaling (Fig. 7B). Furthermore, we observed very little contribution of TLR

signaling to the host cell death response (Fig. 7C). This suggests that TLR signaling

contributes to the early hyper-induction of type-I IFN, but cytosolic signaling is

responsible for later type-I IFN production and host cell death.

61

A B

C

- Figure 7 - TLR signaling contributes to the early but not late responses to LPS mutants.

wt or myd88/trif-/- BMDM were infected with the indicated strains of F. novicida at an MOI of 10:1.

Type-I IFN in macrophage supernatant was measured by ISRE-L929 reporter cell assay at 2hrs post-

infection (A) or 8hrs PI (B). Host cell death was measured by LDH assay at 8hr PI (C). Means and

standard deviations of triplicate infections are shown. Graphs are representative of three independent

experiments.

62

TLR2-dependent signaling contributes to proinflammatory cytokine production

in response to wild-type F. tularensis ssp. To see if the MyD88/Trif-depedent hyper-

IFN phenotype of the LPS mutants was TLR2-dependent we infected wt or tlr2-/-

BMDM with wild-type F. novicida or the LPS mutants an measured type-I IFN

secretion and host cell death. We found that the hyper-induction of type-I IFN

observed with the LPS mutants at 2hrs post-infection was TLR2-dependent (Fig. 8A).

However, in agreement with the results from the myd88/trif-/- BMDM, the hyper-

simulation of type-I IFN induction and host cell death at 8hrs post-infection was

TLR2-independent (Fig. 8B, C). Thus, we conclude that F. novicida LPS mutants

hyper-stimulate TLR2 dependent type-IFN production, but this is only partially

contributes to the induction of the cytosolic responses.

63

A B

C

- Figure 8- TLR2 contributes to the early but not late cytosolic responses to LPS mutants.

wt or tlr2-/- BMDM were infected with the indicated strains of F. novicida at an MOI of 10:1. Type-I

IFN in macrophage supernatant was measured by ISRE-L929 reporter cell assay at 2hrs (A) or 8hrs (B)

post-infection. Host cell death was measured by LDH assay at 8hr PI (C). Means and standard

deviations of triplicate infections are shown. Graphs are representative of three independent

experiments.

64

2.3.7 F. novicida LPS mutants hyper stimulate the inflammasome.

F. novicida infection induces ASC and caspase-1 inflammasome-dependent

IL-1β secretion. Furthermore, this inflammasome activation requires type-I IFN

signaling. To determine if the hyper-inducing mutants were hyper-stimulating an

inflammasome-dependent pathway we infected wt, ifnar-/-, asc-/-, and caspase-1-/-

macrophages with each of the mutants and measured IL-1β release and host cell death.

Similar to wild-type F. novicida, all mutants induced IL-1β release and host cell death

in an ifnar-, asc-, and caspase-1-dependent manner (Fig. 9A-C). Unlike previous

findings with ΔoppB and ΔFTT_1209c hyper-cytotoxic mutants (18), ΔFTN_1212,

ΔlpcC, ΔwbtA, ΔkdsA, and ΔlpxH did not exhibit increased intracellular replication in

macrophages compared to wild-type F. novicida (Fig. 9D). The hyper-IFN inducing

phenotype of these mutants remained intact in asc-/- and caspase-1-/- macrophages

(Fig. 9E). A decrease in type-I IFN was observed in ifnar-/- macrophages infected

with wild-type F. novicida or ΔFTN_1212 relative to wild-type macrophages (Fig.

9E), consistent with previously published reports (34, 64). Surprisingly, ΔlpcC and

ΔwbtA induced a type-I IFN response in an ifnar-independent manner (Fig. 9E).

Together these results suggest that ΔFTN_1212, ΔlpcC, ΔwbtA, ΔkdsA, and ΔlpxH

hyper-stimulate inflammasome activation by a mechanism independent of bacterial

replication.

65

A B

C D

E

- Figure 9 - ASC, caspase-1, and IFNAR are required for LPS mutant inflammasome

activation.

BMDM from wt, asc-/-, casp-1-/-, or ifnar-/- were infected with the indicated strains of F. novicida at an

MOI of 10:1 (A, B, C, and D) or 100:1 (E). IL-1β release was measured by ELISA in Pam3CSK4 pre-

66

stimulated at 5.5 hrs (A) or in unstimulated BMDM at 7hrs post-infection (B) . To avoid complications

with host cell death intracellular replication was measured in asc-/- BMDM by gentamicin protection

assay (D). Replication was normalized to cfu counts at 30min post-infection. Type-I IFN secretion was

measured by ISRE-L929 reporter cell assay at 5hrs post-infection (E). Means and standard deviations of

triplicate infections are shown. Graphs are representative of three independent experiments.

2.3.8 LPS mutants have reduced fitness in vivo.

Lipid A is an essential molecule for bacterial viability and many F. novicida

mutants with alterations in LPS have reduced fitness in mouse infection models (80,

150, 173, 177). Furthermore, other F. novicida mutants that display hyper-

cytotoxicity in macrophages are attenuated in vivo (Weiss et al, 2007). We determined

the relative fitness of each of our hyper-inducing mutants in a mouse model of

infection by measuring the competitive index after intradermal challenge. At 48-hours

post-infection all hyper-inducing mutants displayed a competitive index significantly

less than 1 in the skin and spleen of infected mice, indicating that these mutants are

less fit than wild-type F. novicida in vivo (Fig. 10). The competitive index for each

mutant was significantly lower in the spleen than in the skin, suggesting a defect in

either dissemination from the site of infection or an inability to colonize systemic

sites. These results confirm that FTN_1212, lpcC, wbtA, kdsA, and lpxH are F.

novicida virulence factors and further demonstrate the correlation between the ability

to limit the macrophage cytosolic responses and survival in vivo.

67

- Figure 10 – LPS mutants have reduced fitness in vivo.

wt C57BL6/J mice were infected intradermally (i.d.) with 105 cfu of an equal ratio of wild-type F.

novicida and individual LPS mutants. At 2 days post-infection skin and spleen was harvested and

bacterial loads were determined by plating serial dilutions of homogenized tissue on selective and non-

selective MH agar.

2.3.9 Phagosomal escape is required for induction of the cytosolic response by

LPS mutants.

Cytosolic localization is required for induction of the type-I IFN and cell death

responses in F. novicida, as mutants in FPI genes are restricted to the phagosome and

thus do not induce the cytosolic responses. To determine if the hyper-inducing

mutants still required phagosomal escape to induce the cytosolic response we

constructed FPI deletions in the ΔFTN_1212, ΔlpcC, ΔwbtA, and ΔkdsA backgrounds

68

and measured the kinetics of the type-I IFN response and inflammasome activation in

bone marrow-derived macrophages. Similar to a ΔFPI deletion mutant

ΔFTN_1212ΔFPI, ΔlpcCΔFPI, and ΔwbtAΔFPI double mutants were attenuated in

their ability to elicit type-I IFN production in macrophages (Fig. 11C). The double

mutants were also failed to activate the inflammasome characterized by attenuated

induction of IL-1β secretion and host cell death (Fig. 11A, B). From these results we

conclude that FPI-dependent phagosomal escape is required for induction of the

cytosolic response by the hyper-inducing mutants.

69

A B

C D

- Figure 11 - Phagosomal escape is required for LPS mutants to hyper-induce the cytosolic

responses.

Unstimulated wt BMDM were infected with the indicated strains of F. novicida at an MOI of 10:1. At

8hrs post-infection IL-1β release was measured by ELISA at (A) and cell death was measured by LDH

assay (B). Kinetics of type-I IFN secretion was measured by ISRE-L929 reporter cell assay (C). (D) is

a bar graph representing the luciferase levels at 2hrs post-infection in (C). Means and standard

deviations of triplicate infections are shown. Graphs are representative of three independent

experiments.

70

2.3.10 LPS mutants induce increased proinflammatory cytokine signaling in the

phagosome.

Although the hyper-inducing FPI double mutants were unable to stimulate an

increase in macrophage type-I IFN production over time, we observed an initial

increase in the magnitude of type-I IFN produced at 2hrs post-infection relative to

wild-type F. novicida and a single ΔFPI mutant (Fig. 11C, D). Interestingly, at 2hrs

post-infection hyper-inducing single mutants and their ΔFPI-double mutant

counterparts induced a similar level of type-I IFN in macrophages (Fig. 11D). This

result suggests that the type-I IFN response has a vacuolar component that is FPI

independent at 2hrs post-infection and that mutants that hyper-induce the cytosolic

response also hyper-induce this vacuolar response.

Previous reports demonstrated that retention of F. novicida in the vacuole is

characterized by production of pro-inflammatory cytokines such as TNF-α and IL-1β

(35). To extend our study of the vacuolar responses induced by the hyper-inducing

mutants we investigated macrophage production of these pro-inflammatory cytokines

in response to the hyper-inducing ΔFPI double mutants. Macrophages secreted

increased amounts of TNF-α and produced higher levels of pro-IL-1β transcript in

response to ΔFTN_1212ΔFPI, ΔlpcCΔFPI, ΔwbtAΔFPI, and ΔkdsAΔFPI double

mutants relative to a ΔFPI single mutant (Fig . 12A, B). Thus we have demonstrated

that in addition to an increased IFN-β response, these mutants hyper-induce the

production of TNF-α and pro IL-1β.

71

A B

C

- Figure 12 - LPS mutants hyper-induce NF-κB-dependent cytokines in the phagosome.

wt BMDM were infected with the indicated strains of F. novicida at an MOI of 10:1 for 8hrs. Pro-IL-

1β mRNA levels were measured by quantitative RT-PCR and normalized to the level in uninfected

BMDM (A). TNF-α in supernatants was measured by ELISA (B). (C) RAW-κB luciferase reporter

cells were infected with the indicated strains of F. novicida for 8hrs at an MOI of 10:1. Means and

standard deviations of triplicate infections are shown. Graphs are representative of three independent

experiments.

72

Since IL-1β and TNF-α production both require nuclear translocation of NF-

κB, we hypothesized that the hyper-inducing ΔFPI double mutants stimulated higher

levels of NF-κB. To test our hypothesis we used our hyper-inducing single mutants

and hyper-inducing ΔFPI double mutants to infect a RAW 264.7 macrophage cell line

containing a luciferase-linked NF-κB reporter. At 8hrs post-infection we observed a

significant increase in luciferase production from RAW macrophages infected with

ΔlpcC, ΔwbtA, ΔkdsA, and their ΔFPI double mutant counterparts relative to wild-type

F. novicida and a ΔFPI single mutant (Fig. 12C). The ΔFTN_1212 and

ΔFTN_1212ΔFPI mutants did not induce more luciferase production than wild-type F.

novicida. These results suggest that ΔlpcC, ΔwbtA, ΔkdsA, and their ΔFPI double

mutant counterparts stimulate increased translocation of NF-κB relative to wild-type

and ΔFPI single mutant F. novicida, resulting in increased magnitude and kinetics of

production of several NF-κB-dependent pro-inflammatory cytokines.

2.3.11 Surface-exposed PAMPs mediate recognition of LPS mutants.

Deletion of genes involved in LPS/cell wall/capsule production could lead to

an altered outer membrane surface and expose PAMPs not detectable in wild-type F.

novicida. To determine if the increased cytokine production in response to the LPS

mutants could be due to exposure of PAMPs on the bacterial surface we exposed wt

BMDM to UV-killed wild-type F. novicida or LPS mutants and measured TNF-α and

73

type-I IFN secretion in macrophage supernatants at 2hrs post-infection. We observed

increased stimulation of TNF-α and type-I IFN with UV killed LPS mutants compared

to wild-type. F. novicida (Fig 13A, C). Furthermore, we saw no difference in TNF-α

or type-I IFN production from macrophages infected with wild-type F. novicida or

LPS mutant strains that had been heat-killed (HK) (Fig. 13B, D). These results

suggest that hyper-induction of cytokines by the LPS mutants is not dependent on

bacterial viability but is dependent on an intact outer membrane. Therefore we

conclude that an altered outer membrane in the LPS mutants exposes PAMPs that are

not accessible on wild-type F. novicida.

74

A B

C D

- Figure 13 - Surface exposed PAMPs mediate recognition of LPS mutants.

wt BMDMs were infected for 2hrs at an MOI of 10:1 with the indicated strains of UV-killed (A and C)

or heat-killed (B and D) F. novicida. TNF-α in macrophage supernatant was measured by ELISA (A

and B). Type-I IFN in macrophage supernatant was measured by ISRE-L929 reporter cell assay (C and

D). Means and standard deviations of triplicate infections are shown. Graphs are representative of three

independent experiments.

75

2.3.12 Cytosolic localization is necessary but not sufficient to induce the cytosolic

responses.

Mutants that hypo-induced the cytosolic response fell into several broad

predicted functional categories such as chaperones, transporters, amino acid

metabolism, and nucleotide metabolism. Importantly, we did not identify a F. novicida

mutant that replicated as efficiently as wild-type but did not induce the cytosolic

response. Additional screening of selected transposon insertions as well as review of

the literature revealed a positive correlation between intracellular replication and

induction of the cytosolic response (Table 3); mutants that failed to replicate

intracellularly did not induce a measurable cytosolic response and mutants that were

able to replicate but to a lower extent than wild-type also hypo-induced the cytosolic

response. No mutant tested replicated to higher levels than wild-type, though it should

be noted that ΔoppB and ΔFTT_1209c mutants display increased intracellular

replication and increased macrophage cytotoxicity relative to a wild-type F. novicida

strain (18). These genes were not identified in our screen.

76

- Table 3 – Mutants attenuated for intracellular replication are hypo-stimlate the cytosolic

responses

Gene IFN Cell Death Intracellular replication in macrophages

Reference

Wild-type F. novicida

+ + + Henry et al., 2007

pdpA - - - Brotcke et al, 2006 pdpB - - - Brotcke et al., 2006 pdpD +/- +/- +/- Ludu et al., 2008

caiC/migR +/- +/- +/- Buchan et al., 2009, Brotcke unpublished

pmrA +/- +/- +/- Mohapatra et al., 2007 htpG - - - Weiss et al., 2007 carA - - - Schulert et al., 2009 carB - - - Schulert et al., 2009 proQ +/- +/- +/- This study

pal ? +/- +/- This study purMCD - - - Pechous et al., 2006

perM +/- +/- +/- This study emrB +/- +/- +/- This study clpB - - - Gray et al., 2002

cphA +/- +/- +/- Brotcke, unpublished

We further investigated the correlation between bacterial escape, cytosolic

replication, and induction of the cytosolic response. Previous work demonstrated that

bacterial localization in the cytosol was required to induce the cytosolic response (64,

97). To determine is cytosolic localization is sufficient to induce the cytosolic

response we took advantage of a purine auxotroph, ΔpurMCD, which has been

reported to escape the phagosome as efficiently as wild-type but fails to replicate in

the macrophage cytosol in the absence of exogenous purines (134). In total, 10 purine

or pyrimidine biosynthesis mutants were identified in our screen that hypo-induced the

cytosolic responses. We infected bone marrow derived macrophages with wild-type

F. novicida, ΔpurMCD, or ΔiglC and measured the kinetics of the type-I IFN and cell

77

death responses. Whereas wild-type F. novicida induced robust type-I IFN and cell

death responses from the macrophage a both the ΔpurMCD and ΔiglC mutants failed

to induce either of these responses (Fig. 14A, B). This data suggests that bacterial

localization in the macrophage cytosol is necessary but not sufficient to induce the

cytosolic responses.

78

A

B

- Figure 14 - Cytosolic localization is necessary but insufficient to induce the cytosolic

responses.

Unstimulated BMDM were infected with the indicated strains of F. novicida at an MOI of 10:1.

Kinetics of type-I IFN secretion in supernatant was measured by ISRE-L929 assay (A). Kinetics of

host cell death was measured by LDH release assay (B). Means and standard deviations are shown for


79

2.3.13 Bacterial DNA and protein synthesis are required to induce the cytosolic

responses.

During a macrophage infection with F. novicida greater than 60% of bacteria

are present in the cytosol within 2hrs post-infection (28). However, we do not observe

intracellular replication until 4hrs post-infection, consistent with published findings

(175). During this lag phase in intracellular replication the bacterium exhibits

transcriptional changes that may be required in order to establish a replicative niche in

the cytosol (175). Since bacterial localization in the cytosol was insufficient to induce

the cytosolic responses and the results of the F. novicida transposon screen

demonstrated a correlation between intracellular replication and induction of the

cytosolic responses we sought to determine if intracellular replication, protein

synthesis, and/or DNA synthesis were necessary to induce the cytosolic responses.

We infected macrophages with wild-type F. novicida at a multiplicity of infection

(MOI) of 10 and at 1, 2, or 3hrs post-infection we treated the macrophages with

bacteriostatic antibiotics to block intracellular replication and measured the kinetics of

type-I IFN induction and macrophage cell death. Both chloramphenicol and nalidixic

acid were bacteriostatic at the concentrations used and no intracellular replication was

observed in the presence of antibiotics over 8hrs of infection (Fig. 15I). Antibiotic

treatment at 0, 1, and 2hrs post-infection led to attenuated induction of both the type-I

IFN and cell death responses (Fig. 15A-F). Interestingly, treatment at 3hrs post-

infection resulted in induction of the type-I IFN response with similar magnitude and

kinetics as untreated samples (Fig. 15G). However, treatment at 3hrs post-infection

led to a significant attenuation of the host cell death response (Fig. 15H). These

80

results suggest that a minimum of 3 hours of protein and DNA synthesis are required

by the bacterium to induce the cytosolic response. Furthermore, intracellular

replication was not required for induction of type-I IFN, but host cell death was

attenuated at this MOI in the absence of replication. This was the first time we were

able to observe a type-I IFN response that did not result in a cell death response.

81

A B

C D

E F

82

G H

I

- Figure 15 - Bacterial protein synthesis and DNA synthesis are required to induce the cytosolic

responses.

wt BMDM were infected with wt F. novicida at an MOI of 10:1 and bacteriostatic antibiotics were

added at 0hr (A and B), 1hr (C and D), 2hr (E and F), or 3hr post-infection (G and H). Type-I IFN in

macrophage supernatant was measured by ISRE-L929 reporter assay (A,C,E, and G). Host cell death

was measured by LDH assay (B,D,F and H). Intracellular replication was measured by gentamicin

protection assay (I). Means and standard deviations are shown for infections done in triplicate.

Representative graphs of three independent experiments are shown.

Intracellular replication is dispensable for induction of type-I IFN, but required

for the induction of cell death at an MOI of 10. This could suggest that the ligand

responsible for the type-I IFN response is expressed during the lag phase, while the

83

ligand responsible for host cell death is expressed during the replicative phase.

Alternatively, the threshold for induction of type-I IFN may be lower than the

threshold for induction of host cell death, and bacterial replication may serve as a

means to increase the concentration of the ligand(s). To test this hypothesis we

infected macrophages at an MOI of 100 to increase the initial concentration of all

bacterial ligands and treated the macrophages with bacteriostatic antibiotics at 3hrs

post-infection to block replication. Similar to the results at an MOI of 10, treatment

with bacteriostatic antibiotics had no effect on the magnitude and kinetics of type-I

IFN production (Fig. 16A). Additionally at this higher MOI, the bacteria induced host

cell death in the absence of replication, though there was a slight decrease in kinetics

(Fig. 16B). These results suggest that the ligand(s) for induction of both type-I IFN

and host cell death are present in the cytosol by 3hrs post-infection, and bacterial

replication increases the concentration of the ligand(s) such that it can be recognized

by the inflammasome.

84

A

B

- Figure 16 - Replication is not required to induce the cytosolic response with high bacterial

load.

wt BMDM were infected with wild-type F. novicida at an MOI of 100:1. Bacteriostatic antibiotics

were added at 3hr PI. Type-I IFN in macrophage supernatant was measured by ISRE-L929 reporter

asay (A). Host cell death was measured by LDH assay (B). Means and standard deviations are shown

for infections done in triplicate. Representative graphs of three independent experiments are shown.

85

2.3.14 The cytosolic response to F. novicida shares characteristics with the

response to transfected dsDNA.

Type-I IFN induction and host cell death are both induced by transfection of

dsDNA into the macrophage cytosol (119, 153). We wanted to know if we observed

the same dose dependent activation of the two responses with dsDNA transfection as

we observed with F. novicida infection. To this end we infected wt BMDM with

increasing MOIs of F. novicida or transfected increasing doses of poly(dA:dT) and

measure type-I IFN secretion and host cell death. The characteristics of the responses

were very similar between the different stimuli. An 8 hr infection with an initial dose

of less that 30 bacteria per macrophage resulted in induction of the type-I IFN

response without induction of the cell death response, similar to what we had observed

at an MOI of 10 in the presence of bacteriostatic antibiotics (Fig. 17A). At an MOI of

30 or higher, we observed induction of the type-I IFN response and host cell death

(Fig. 17A). Similarly, transfection of less than 15ng/well of poly(dA:dT) induced

type-I IFN secretion from macrophage with no host cell death (Fig. 17B). However

transfection with poly(dA:dT) at concentrations of 15ng/well or higher induced a

robust type-I IFN response and host cell death (Fig. 17B). Importantly, we did not

observed induction of host cell death without accompanying type-I IFN induction.

86

A B

C

- Figure 17 - The cytoslic response to F. novicida shares characteristics with that of dsDNA.

wt BMDM were infected with an increasing MOI of F. novicida for 8hrs(A) or transfected with

increasing concentrations of poly(dA:dT) for 5 hrs (B) and type-I IFN in macrophage supernatant was

measured by luciferase reporter assay and cell death was measured by LDH assay. (C) wt or ifnar-/-

BMDM were infected with F. novicida or transfected with poly(dA:dT) at the indicated concentrations

and host cell death was measured by LDH assay. Means and standard deviations are shown for


87

Since host cell death in response to F. novicida requires type-I IFN signaling,

we wanted to know if transfection of dsDNA displayed a similar dependence on type-I

IFN signaling. Therefore we infected wild-type and ifnar-/- macrophages with F.

novicida at MOIs of 62:1 and 125:1 or transfected the same strains of macrophages

with poly(dA:dT) at concentration of 62ng/well and 125ng/well. We observed a

significant decrease in host cell death in ifnar-/- macrophages compared to wt

macrophages that were infected with F. novicida or transfected with poly(dA:dT) (Fig.

17C). These results, combined with the results from our screen, support the idea that

F. novicida dsDNA triggers the cytosolic responses in macrophages.

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2.4 DISCUSSION

In this report we identified several F. novicida genes that modulate

macrophage innate immune responses. Mutants in a gene encoding a major outer

membrane protein, fopA, and the LPS genes FTN_1212, lpcC, wbtA, kdsA, and lpxH

induce increased type-I IFN and inflammasome activation in macrophages. Further

study revealed that these mutants induce the cytosolic responses through an identical

pathway as wild-type F. novicida, dependent on type-I IFN signaling and

inflammasome components ASC and caspase-1. Alterations in the outer membrane

and LPS led to increased stimulation of TLRs, and resulted increased proinflammatory

cytokine production. This increased TLR signaling is likely due to detection of surface

exposed PAMPs, and not due to the production of a new PAMP that is not present in

wild-type F. novicida. The increased TLR2-dependent type-I IFN production could

synergize with the type-I IFN produced from the unknown cytosolic receptor and lead

to the increased inflammasome activation observed with these mutants. However, our

results also indicate that the fopA and LPS mutants hyper-stimulate the cytosolic

receptor as well.

The mutants that alter the cytosolic response in macrophages represent 17% of

the F. novicida genome and fall into several broad functional categories. This would

represent and extremely large number of genes dedicated to target a specific host

pathway and since F. novicida is not host adapted, we surmise that the mutants that

induce an increase in the cytosolic response relative to wild-type F. novicida do so by

converging on a specific pathway or release of the same PAMP. Therefore we

89

conclude that fopA and F. novicida LPS act to dampen the cytosolic response by

limiting the release or exposure of a common F. novicida PAMP.

We observed a positive correlation between intracellular replication and

induction of the cytosolic responses. We did not identify an F. novicida mutant that

replicates intracellularly as efficient as wild-type F. novicida but does not induce the

cytosolic response. However, it appears that the macrophage does not sense

intracellular replication, as we were able to induce the cytosolic response in the

presence of bacteriostatic antibiotics. On the other hand, replication provided a link

between type-I IFN and host cell death at low infectious doses. We propose that the

same PAMP induces the type-I IFN response and cell death responses in a dose

dependent manner, and replication increases the cytosolic concentration of this PAMP.

In support of this hypothesis we observed a similar dose dependent and type-I IFN

receptor-dependent response to transfected dsDNA. The concentration of cytosolic

dsDNA would increase during bacterial replication. However, release of bacterial

DNA during intracellular replication has not been demonstrated previously. DNA

release could result from bacterial lysis induced by a host response, or it could be part

of the intracellular replications cycle that had not been previously described. Mutants

in outer membrane proteins or LPS could have an unstable outer membrane and be

more susceptible to intracellular lysis, resulting in increased release of dsDNA. We

will not be able to definitely say that bacterial DNA is the PAMP that triggers the

cytosolic response until we identify the host receptors for type-I IFN or inflammasome

activation.

90

Chapter 3: AIM2 is required for innate immune recognition of Francisella tularensis Jonathan W. Jones1*, Nobuhiko Kayagaki2*, Petr Broz1, Thomas Henry1, Kim

Newton2, Karen O'Rourke2, Salina Chan2, Jennifer Dong2, Yan Qu2, Meron

Roose-Girma3, Vishva M. Dixit2, Denise M. Monack1

1Department of Microbiology and Immunology, Stanford School of Medicine, Stanford

University, California, USA 2Department of Physiological Chemistry, 3 Department of Molecular Biology,

Genentech Inc., South San Francisco, California, USA.

This chapter has been accepted for publication in the Proceedings of the National

Academy of Sciences.

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3.1 CHAPTER 3 SUMMARY

Macrophages respond to cytosolic nucleic acids by activating cysteine protease

caspase-1 within a complex called the inflammasome. Subsequent cleavage and

secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18 is critical for

innate immunity. Here we show that macrophages from mice lacking absent in

melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger

inflammasome assembly. Caspase-1 activation in response to intracellular pathogen

Francisella tularensis also required AIM2. Immunofluorescence microscopy of

macrophages infected with F. tularensis revealed striking co-localization of bacterial

DNA with endogenous AIM2, and inflammasome adaptor ASC. By contrast, type I

interferon (Type-I IFN; IFN-α and -β) secretion in response to F. tularensis did not

require AIM2. Type-I IFN did, however, boost AIM2-dependent caspase-1 activation

by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in

infected macrophages lacking either the Type-I IFN receptor (IFNAR) or stimulator of

interferon genes (STING). Finally, AIM2-deficient mice displayed increased

susceptibility to F. tularensis infection compared to wild-type mice. Their increased

bacterial burden in vivo confirmed that AIM2 is essential for an effective innate

immune response.

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3.2 INTRODUCTION

The innate immune system reacts to diverse molecules, which are collectively

termed pathogen-associated molecular patterns (PAMPs) and damage-associated

molecular patterns (DAMPs) (4, 86). These molecules include nucleic acids. RNA,

for example, is recognized by several toll-like receptors, as well as the RNA helicases

retinoic acid inducible gene-I (RIG-I, also called DDX58), melanoma differentiation-

associated gene-5 (MDA5, also called IFIH1), and laboratory of genetics and

physiology 2 (LGP2) (4). DNA recognition mechanisms have proved more elusive.

Toll-like receptor (TLR)9 is located in phagosomes and recognizes DNA with CpG

motifs, leading to NF-κB dependent inflammatory responses (63). DNA-dependent

activator of IFN-regulatory factors (DAI, also known as DLM-1 and ZBP1), the first

identified cytosolic DNA sensor, binds cytosolic dsDNA and leads to the production

of type-I IFN, although the lack of demonstrated relevance in vivo has lead to the

hypothesis that redundant cytosolic DNA sensors exist (75, 161). Additionally, the

recently identified adapter stimulator of interferon genes (STING) or mediator of IRF3

activation (MITA) [hereafter referred to as STING] mediates type-I IFN production in

response to DNA transfection, as well bacterial and viral infection (76, 77, 137, 187).

DNA is also a potent activator of a multiprotein complex known as the

inflammasome, which contains a nucleotide-binding oligomerization domain (NOD)-

like receptor (NLR), the adapter apoptosis-associated speck-like protein containing a

CARD (ASC, also known as PYCARD) and the cysteine protease caspase-1 (119).

Recent overexpression and knockdown studies in cell lines suggested that DNA

engages AIM2, a novel NLR, which then interacts with ASC to promote

93

inflammasome assembly and caspase-1 activation (21, 68). AIM2 is a type-I IFN-

inducible cytosolic protein containing PYRIN and HIN200 domains (21, 90)

(Supplementary Fig. 1C). The HIN domain facilitates binding of DNA, while the

PYRIN domain allows for the association with ASC and formation of a caspase-1

activating inflammasome, leading to the processing and release of mature IL-1β and

IL-18, and host cell death (21, 47, 68). The role of AIM2 in innate immunity is

unknown.

The causative agent of tularemia, Francisella tularensis is a facultative

intracellular gram-negative pathogen that escapes phagosomal degradation in

macrophages and replicates in the host cell cytosol. Cytosolic replication is required

for bacterial virulence, as F. tularensis mutants that fail to escape the vacuole cannot

replicate in macrophages and are avirulent in mice (17, 54, 91, 122, 177). Moreover,

cytosolic F. tularensis sequentially activates pro-inflammatory host responses,

characterized by the initial production of type-I IFN, such as IFN-β, which is required

for the subsequent activation of an ASC inflammasome (34, 64). Inflammasome

activation is critical to host defense against F. tularensis, as mice lacking

inflammasome components are more susceptible to infection (97). The PAMPs

produced during F. tularensis infection and the host pattern recognition receptors

(PRRs) required for pathogen recognition remain a mystery.

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3.3 RESULTS

3.3.1 AIM2 is essential for inflammasome activation in response to cytosolic

dsDNA.

We investigated the role of AIM2 in vivo with gene-targeted aim2-/- mice (Fig.

18A, B). Western blotting with an antibody raised against amino acids 2-274 of

mouse AIM2 confirmed that aim2-/- bone marrow-derived macrophages (BMDMs)

lacked AIM2 protein (Fig. 18C). First, we compared double-stranded DNA (dsDNA)-

induced IL-1β secretion from aim2-/-, asc-/-, nalp3-/-ipaf-/-, and wild-type BMDMs.

Cells were primed with lipopolysaccharide (LPS) to induce pro-IL-1β expression and

then transfected with either poly(dA-dT) • poly(dA-dT) [hereafter referred to as

poly(dA:dT)], poly(dG-dC) • poly(dG-dC) [hereafter referred to as poly(dG:dC)],

pcDNA3 plasmid DNA, calf thymus DNA, or Listeria monocytogenes DNA. All of

these dsDNAs induced AIM2- and ASC-dependent IL-1β secretion (Fig. 19A). In

contrast, Nalp3 (98, 104) (also called NLRP3), which engages the caspase-1 adaptor

protein ASC in response to a variety of PAMPs and DAMPs, and Ipaf (96) (also called

NLRC4), which engages ASC in response to Salmonella typhimurium (S.

typhimurium), were dispensable for dsDNA-induced IL-1β secretion. Loss of AIM2,

unlike ASC deficiency, did not cause a general defect in IL-1β secretion since aim2-/-

and wild-type BMDMs secreted equivalent amounts of IL-1β after infection with S.

typhimurium or treatment with LPS plus ATP (Fig. 19A). Similar results were

obtained with peritoneal macrophages (Fig. 20). AIM2 deficiency also blocked IL-18

95

secretion in response to dsDNA but not ATP (Fig. 19B). Therefore, AIM2 is essential

for IL-18 and IL-1β secretion in response to dsDNA.

96

- Figure 18 - Generation of Aim2-/- mice.

A, Strategy for deleting exon 5 of mouse aim2, which encodes the initiating methionine (ATG) and the

entire PYRIN domain. Gene targeting was performed in C57BL/6 C2 embryonic stem cells. B, PCR

genotyping of aim2-/- mice. Primers 5’CCA GTG TTT CTC AAC TGT ACT GCT AT, 5’TAG GAG

TGC CCT CCC TTA ATG, and 5’TTG GAG ACA GAC TCT GGT GAA G yield a 197 bp DNA

fragment for the wild-type allele and a 397 bp DNA fragment for the knockout allele. C, BMDMs were

incubated with 1000 U/mL IFN-b for 5 h. AIM2 was western blotted with 4G9.1.4 rat anti-mouse

AIM2 monoclonal antibody.

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- Figure 19 - AIM2 is essential for inflammasome activation in response to cytosolic dsDNA.

= asc-/-, = nalp3-/- ipaf-/-, = aim2-/-, = wt. A, IL-1b secretion by LPS-primed BMDMs treated

with 5 mM ATP or transfected with 1 ug/mL of the indicated dsDNAs for 16 h. BMDMs were infected

with S. typhimurium (multiplicity of infection = 100) without LPS priming. B, IL-18 secretion by

BMDMs treated as in (A). LPS priming was used ATP stimulation only. C, Upper panels show mature

IL-1b and cleaved caspase-1 secreted from LPS-primed BMDMs after stimulation with ATP or

transfection with dsDNA for 5 h. Lower panels show pro-caspase-1 and pro-IL-1β in the cell lysate. D,

IFN-β secretion by BMDMs treated as in (A), but without LPS priming. Graphs show the mean ±

standard deviation of triplicate wells and are representative of 3 independent experiments.

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- Figure 20 - AIM2 is essential for inflammasome activation in response to dsDNA in peritoneal

macrophages.

= asc-/-, = nalp3-/- ipaf-/-, = aim2-/-, = wt. Peritoneal macrophages were harvested 5 days after

intraperitoneal injection of 4% thioglycollate (DIFCO). These macrophages were primed with 500

ng/mL LPS for 5 h, then transfected with 1 ug/mL of the dsDNAs indicated for 16 h. As controls, LPS-

primed cells were cultured in medium alone (cont) or stimulated with 5 mM ATP. Additional

macrophages were not primed (cont) or were infected with S. typhimurium (multiplicity of infection,

100). IL-1β secreted into the culture supernatant was measured by ELISA. Graphs show the mean ±


Processing of pro-IL-1β and pro-IL-18 by caspase-1 is necessary for secretion

of biologically active IL-1β and IL-18 (39, 88) and so we compared caspase-1

activation in wild-type and aim2-/- BMDMs by western blotting for the p20 and p10

caspase-1 subunits that are generated by autocatalytic cleavage and released from the

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cell by a poorly defined mechanism. Consistent with AIM2 promoting caspase-1

activation in response to dsDNA, culture supernatants from LPS-primed wild-type

BMDMs contained mature IL-1β plus the caspase-1 p10 and p20 subunits after

transfection with poly(dA:dT) or poly(dG:dC), but supernatants from aim2-/- BMDMs

did not (Fig. 19C). aim2-/- BMDMs expressed wild-type levels of pro-caspase-1 and

pro-IL-1β, and they released IL-1β and processed caspase-1 normally in response to

LPS plus ATP. These data indicate a specific requirement for AIM2 in caspase-1

activation by dsDNA.

Next, we determined whether aim2-/- BMDMs produce inflammasome-

independent proinflammatory cytokines such as type-I IFN and TNF-α in response to

dsDNA. TANK-binding kinase 1 (TBK1) and the transcription factors IRF3 and IRF7

signal type-I IFN synthesis in response to dsDNA (74, 153), but how dsDNA engages

this pathway is unclear. Unexpectedly, aim2-/-, asc-/-, and caspase-1-/- BMDMs

produced significantly more TNF-α (Fig. 21A) and IFN-β (Fig. 19D, Fig. 21C) than

wild-type or nalp3-/-ipaf-/- BMDMs after transfection with poly(dA:dT) or pcDNA3.

There was little or no difference in IFN-β and TNF-α production, however, when the

cells were transfected with poly(dG:dC) or a 45 base pair IFN-stimulatory DNA (ISD)

(153). In addition, wild-type, aim2-/-, asc-/-, and nalp3-/-ipaf-/- BMDMs produced

equivalent TNF-α in response to LPS (Fig. 21A). Therefore, AIM2 is dispensable for

IFN-β and TNF-α secretion. Increased IFN-β and TNF-α production by aim2-/- and

caspase-1-/- BMDMs in response to poly(dA:dT) or pcDNA3 correlated with enhanced

cell survival. Between 35-45% of wild-type BMDMs had released lactate

dehydrogenase (LDH) at 5 hours after transfection with poly(dA:dT) or pcDNA3,

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whereas most caspase-1-/- and aim2-/- BMDMs remained viable (Fig. 21B).

Poly(dG:dC) was less cytotoxic, killing ~15% of wild-type BMDMs. Consistent with

AIM2 engaging ASC and caspase-1 in response to dsDNA, but not all stimuli, aim2-/-

BMDMs were as sensitive as wild-type BMDMs to caspase-1-dependent death after

infection with S. typhimurium.

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- Figure 21 - AIM2 is dispensable for IFN-β and TNF-α production in response to dsDNA.

BMDMs were unstimulated (cont), treated with 1 mg/mL LPS, or transfected with 1 ug/mL of the

dsDNAs indicated for 16 h. A, = asc-/-, = nalp3-/- ipaf-/-, = aim2-/-, = wt. TNF-α secreted into

the culture supernatant was measured by Bio-Plex Cytokine Assay (Bio-Rad). B, = casp-1-/-, =

aim2-/-, = wt. Cytotoxicity was measured by LDH release. C, = casp-1-/-, = wt. IFN-β secreted

into the culture supernatant was measured by ELISA. Graphs show the mean ± standard deviation of

triplicate wells and are representative of 3 independent experiments.

3.3.2 AIM2 is required for inflammasome activation in response to F. tularensis.

We then explored the contribution of AIM2 to innate immunity to bacterial

infection. We focused on inflammasome activation in response to F. tularensis (84,

110) because this intracellular pathogen escapes phagosomal degradation, replicates in

the cytosol, and triggers ASC-dependent, but Nalp3- and Ipaf-independent, caspase-1

activation (97). When wild-type, aim2-/-, asc-/-, and caspase-1-/- BMDMs were

infected with F. tularensis subspecies novicida, only wild-type cells secreted IL-1β

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(Fig. 22A) and died (Fig. 22B), indicating that AIM2, like ASC and caspase-1, is

essential for inflammasome activity. We propose that AIM2 recognizes cytosolic F.

tularensis because the avirulent mutant ΔFPI (177), which cannot escape phagocytic

vacuoles, failed to stimulate IL-1β secretion (Fig. 22A) or macrophage death (Fig.

22B).

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- Figure 22 - AIM2 is required for inflammasome activation in response to F. tularensis.

= asc-/-, = casp-1-/-, = aim2-/-, = wt. A, IL-1β secretion by BMDMs infected with F.

tularensis ssp. novidica strain U112 or isogenic mutant ΔFPI for 5 h. (multiplicity of infection, moi)

BMDMs treated with 5 mM ATP for 4 h were primed with 500 ng/mL Pam3CSK4 for 16 h.

multiplicity of infection, moi. B, Cytotoxicity as measured by LDH release. Graphs show the mean ±


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3.3.3 AIM2 and ASC form a complex with F. tularensis DNA.

The HIN200 domain of AIM2 recognizes dsDNA, and its PYRIN domain can

engage ASC (47, 68). We visualized inflammasome assembly by

immunofluorescence confocal microscopy of BMDMs infected with F. tularensis and

stained with antibodies detecting endogenous AIM2, ASC, and F. tularensis. At 5.5

hours post-infection, wild-type, asc-/-, and caspase-1-/- BMDMs contained multiple

AIM2 specks tightly associated with bright DAPI-staining material, likely reflecting

leaked bacterial DNA due to its proximity to irregular-shaped bacterial remnants (Fig.

23A). Regular shaped bacteria stained dimly with DAPI. Infections with F. tularensis

pre-labeled with Hoechst 33342 nucleic acid stain confirmed that AIM2 was recruited

to bacterial DNA (Fig. 23B). Merged images revealed that AIM2 overlapped almost

completely with the bacterial DNA (Fig. 23A, 23B, Fig. 25A), indicating that DNA

leaked from F. tularensis likely is the PAMP recognized by AIM2 during an infection.

Consistent with this notion, IL-1b secretion from wild-type BMDMs primed with

Pam3CSK4 and then transfected with F. tularensis extract was abolished when the

extract was pre-incubated with DNase I (Fig. 24A). Purified F. tularensis DNA

transfected into Pam3CSK4-primed BMDMs also stimulated AIM2-dependent IL-1β

secretion (Fig. 24B).

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- Figure 23 - AIM2 and ASC form a complex with F. tularensis DNA.

A, Immunofluorescence microscopy of F. novidica U112-infected BMDMs at 5.5 h post-infection.

Scale bars, 10 mm. Differential interference contrast, DIC. Arrows indicate co-localization of DNA,

degraded bacteria, AIM2, and ASC. Asterisks label diffuse AIM2 accumulation with DNA. Images are

representative of at least 3 independent biological replicates. B, BMDMs were infected with F.

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novidica U112 pre-labeled with Hoechst 33342 nucleic acid stain. Upper panel scale bar, 10 mm; lower

panel, 2 mm. Arrows and asterisks indicate co-localization of bacterial DNA and AIM2.

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- Figure 24 - F. tularensis DNA triggers IL-1β secretion.

A, Extract from F. tularensis ssp. novicida strain U112 was prepared in 10 mM Tris-HCl pH 7.5 with a

French press. Extract corresponding to 2x106 cfu was treated with nothing (cont), proteinase K

(Qiagen) for 16 h at 55°C, or DNase I (Qiagen) for 2h at 25°C, and then transfected into BMDMs

primed with 500 ng/mL Pam3CSK4 for 5 h. IL-1β secreted into the culture supernatant was assayed 3

h later. B, = aim2-/-, = wt. Pam3CSK4-primed BMDMs were transfected with 1 mg/mL F.

tularensis DNA, which was isolated from bacteria with a Qiagen DNeasy Blood and Tissue Kit. IL-1β

secreted into the culture supernatant was assayed 16 h later. Graphs show the mean ± standard

deviation of triplicate wells and are representative of 3 independent experiments.

3.3.4 AIM2 is required for the formation of an ASC focus.

Despite multiple AIM2 specks forming in an infected cell adjacent to bacterial

remnants, ASC was recruited to a single AIM2 speck in ~15-22% of wild-type or

caspase-1-/- BMDMs (Fig. 23A, Fig. 25B). The vacuole-restricted F. tularensis

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mutant ΔFPI did not stimulate ASC focus formation, consistent with its inability to

stimulate IL-1β secretion (Fig. 22A). Importantly, ASC focus formation required

AIM2 because no foci were detected in aim2-/- BMDMs. We, and others, have shown

that similar ASC foci are formed in macrophages infected with S. typhimurium upon

activation of NOD-like receptors ((49); P.B., K.N., M. Lamkanfi, S. Mariathasan,

V.M.D., and D.M.M., manuscript in preparation). Our data suggests that although

AIM2 recognizes cytosolic DNA at multiple sites, only one of these sites will form the

platform on which the ASC-containing inflammasome is built.

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- Figure 25 - AIM2 is required for the formation of an ASC focus.

A, Three-dimensional reconstruction of a confocal image taken of a wild-type BMDM from (c). Scale

bar, 0.5 mm. B, = F. tularensis, = ΔFPI. Graph showing the percentage of infected BMDMs

containing an ASC focus in (Fig. 23A). Bars represent the mean ± standard deviation of 2 independent

experiments. At least 300 cells of each genotype were examined per infection.

3.3.5 Type I IFN increases AIM2 protein levels and inflammasome activity.

Given that AIM2 protein expression is increased in BMDMs treated with IFN-

β (Fig. 18C) or infected with F. tularensis (Fig. 26A) and that type-I IFN signaling is

required for efficient inflammasome signaling in response to F. tularensis (64), we

sought to delineate the signaling pathway(s) driving type-I IFN and AIM2 synthesis

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after F. tularensis infection. We investigated the contribution of STING because it

complexes with TBK1 and mediates type-I IFN production in response to DNA (77).

Unlike wild-type BMDMs, which synthesized IFN-β mRNA in response to F.

tularensis, but not the avirulent mutant ΔFPI, sting-/- BMDMs did not upregulate IFN-

β gene expression after infection (Fig. 26B). sting-/- BMDMs synthesized IFN-β

mRNA normally in response to transfected poly I:C, which engages RIG-I and MDA5

(82), excluding a general defect in IFN-β transcription. We speculate that bacterial

DNA leaked from lysing F. tularensis leads to STING-dependent Type-I IFN

production, although the sensor that recognizes the DNA remains unknown. Just as

AIM2 was not required for IFN-β secretion from BMDMs transfected with dsDNA

(Fig. 19D), AIM2 deficiency did not compromise IFN-β secretion from BMDMs

infected with F. tularensis (Fig. 27A).

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- Figure 26 - Type-I IFN increases AIM2 protein levels and inflammasome activity.

= ifnar-/-, = sting-/-, = wt. BMDMs were infected with S. typhimurium (moi = 100), F. tularensis

ssp. novidica strain U112, or isogenic mutant ΔFPI for 5 h. (multiplicity of infection, moi). A, Western

blot of AIM2 protein expression. B, IFN-β mRNA expression quantified by RT-PCR. As a control,

BMDMs were transfected with 1 mg/mL polyI:C. C, IL-1β secretion into the culture supernatant. D,

Cytotoxicity as measured by LDH release. Where indicated, BMDMs were treated with 1000 U/mL

recombinant IFN-β at 1 h post-infection. Graphs show the mean ± standard deviation of triplicate wells

and are representative of 3 independent experiments.

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The increased AIM2 expression observed in F. tularensis-infected wild-type

BMDMs was not observed in sting-/- or type-I IFN receptor-deficient ifnar-/- BMDMs

(Fig. 26A). In addition, failure to upregulate AIM2 correlated with abrogated IL-1b

secretion (Fig. 26C) and reduced cell death (Fig. 26D). Forced expression of AIM2 in

ifnar-/- BMDMs restored IL-1β secretion in response to F. tularensis (Fig. 27B) and

exogenous IFN-β restored cell death in infected sting-/-, but not ifnar-/- BMDM

cultures (Fig. 26D). We conclude that STING-dependent type-I IFN production

boosts inflammasome activity during F. tularensis infection by increasing AIM2

expression.

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- Figure 27 - Type-I IFN facilitates formation of the AIM2-containing inflammasome.

A, = asc-/-, = caspase-1-/-, = aim2-/-, = wt. IFN-β secretion by BMDMs that were untreated

(cont) or infected with F. tularensis ssp. novicida strain U112 and isogenic mutant ΔFPI for 5 h.

multiplicity of infection, moi. B, = ifnar-/- + AIM2/GFP, = ifnar-/- + GFP. ifnar-/- bone marrow

was transduced with pMSCV2.2-IRES-GFP encoding mouse AIM2 or the empty parental vector, after

retroviral particles were generated with the Phoenix.Eco packaging cell line. Three days later,

macrophages were differentiated with M-CSF-containing medium, and GFP-positive cells were sorted

in a FACS Aria (Becton Dickinson). These BMDMs were primed with 500 ng/mL Pam3CSK4 for 16 h

and then infected with F. tularensis or treated with 5 mM ATP. IL-1β secreted into the supernatant was

assayed after 5 h. Graphs show the mean ± standard deviation of triplicate wells and are representative

of 3 independent experiments.

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3.3.6 AIM2 is required for host defense against F. tularensis.

Finally, to extend our findings on the role of AIM2 in cultured macrophages to

an in vivo setting, we challenged wild-type, aim2-/-, and caspase-1-/- mice with F.

tularensis. caspase-1-/- mice fail to control F. tularensis infections (97), and aim2-/-

mice were equally impaired at limiting F. tularensis replication (Fig. 28). Average

bacterial loads in liver, lung, and spleen of aim2-/- or caspase-1-/- mice at 36 hours

post-infection were 120- to 19,000-fold higher than in wild-type mice. These data

demonstrate that AIM2 is essential for innate immunity to F. tularensis in vivo.

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- Figure 28 - AIM2 is required for host defense against F. tularensis

Mice were infected intradermally with 1x105 colony forming units (cfu) of F. tularensis ssp. novidica

strain U112F. Organs were harvested after 36 h, homogenized, and cfu were determined by plating

serially diluted tissue extracts on modified MH agar. Bars indicate the geometric mean cfu per

genotype.

3.4 Discussion

Collectively, our results provide genetic evidence that AIM2 is an essential

DNA sensor of the innate immune system. Furthermore, AIM2 plays a critical role in

defense against F. tularensis. To our knowledge we also provide the first visualization

of an endogenous inflammasome NLR complexed with its ligand in the context of an

infection. In our model, F. tularensis escapes phagosomal degradation into the

macrophage cytosol where some bacteria lyse, releasing DNA into the cytosol. We

support this model with visualization of Hoechst pre-labeled bacterial DNA observed

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outside of aberrantly shaped F. tularensis. An unknown sensor(s) recognizes cytosolic

bacterial DNA and signals through the adapter STING to produce Type-I IFN.

Autocrine and paracrine signaling through IFNAR leads to an increase in AIM2

protein levels, which accelerates recognition of bacterial DNA by AIM2. We observe

colocalization of AIM2 specks with bacterial DNA, one of which acts as a nucleus for

formation of an ASC focus. We believe this complex represents a mature

inflammasome that leads to secretion of mature IL-1β and host cell death. This model

represents coordination of two independent DNA sensing pathways to produce a

complete host response to a bacterial infection.

Previous DNA transfection studies have demonstrated inflammasome

activation in the absence of type-I IFN signaling, suggesting that endogenous levels of

AIM2 are sufficient for recognition of transfected DNA (119). Although we observe a

dependence on type-I IFN signaling for F. tularensis inflammasome activation, we

demonstrate that we can restore inflammasome activation in the absence of type-I IFN

signaling through exogenous expression of AIM2, suggesting that AIM2 is sufficient

for recognition of F. tularensis. Considering these results we hypothesize that either

the mechanism of DNA delivery or concentration of DNA delivered during F.

tularensis infection is insufficient to be recognized by the endogenous levels of AIM2

present in macrophages. The sequential activation of the type-I IFN and

inflammasome responses also lead us to speculate that the threshold of DNA required

to induce type-I IFN signaling is less than that required to induce AIM2

inflammasome activation.

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Bacterial pathogens can exploit a wide range of niches within a host yet very

few bacteria replicate inside the host cytosol, namely L. monocytogenes, Shigella

flexneri, and F. tularensis. The inflammasome has been implicated in innate

immunity to all of the aforementioned pathogens, although different NLRs, and

different bacterial ligands mediate these events. Inflammasome activation by L.

monocytogenes has been attributed to production of the pore-forming toxin LLO,

which is recognized by the Nalp3 inflammasome, as well as bacterial flagellin, which

is engaged by the IPAF inflammasome (158, 174). Recent studies have elucidated a

shared motif between bacterial flagellin and components of the type III secretion

system (T3SS) (115), which explains the ability of IPAF to recognize S. flexneri,

which contains a T3SS and lacks flagellin. It is not clear whether the AIM2

inflammasome senses either of these pathogens. On the other hand F. tularensis is

non-flagellated, lacks a T3SS, and has not been shown to produce pore-forming

toxins. Thus, DNA seems to be the only known inflammasome ligand possessed by F.

tularensis.

Cross-talk between the type-I IFN, NF-κB, and inflammasome pathways is

poorly understood. We observed enhanced TNF-α and IFN-β secretions in asc-/-

compared to wild-type BMDMs when transfected with poly(dA:dT) or pcDNA3 but

not when transfected with poly(dG:dC) or ISD. One possible explanation for this

difference is that there are at least two DNA sensors for type-I IFN (and TNF-α),

which recognize different types of DNAs. One such receptor-mediated pathway may

receive negative feedback from the inflammasome, while the other does not.

Additionally, type-I IFN positively regulates the AIM2 inflammasome in response to

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F. tularensis infection. Although a role for the AIM2 inflammasome has not been

demonstrated during Listeria monocytogenes infection it should be noted that type-I

IFN signaling also accelerates inflammasome activation in response to this pathogen

(156). AIM2 is also likely required for recognition of dsDNA viruses, such as

Vaccinia virus (68). Interestingly, Vaccinia actively inhibits the antiviral effects of

type-I IFN with the viral E3L protein, which may delay inflammasome activation and

hence the innate immune response to the pathogen (25). In addition, AIM2 may

contribute to aberrant IL-1β production in response to host DNA, leading to arthritis-

like autoimmune disease pathology. The coordination between the type-I IFN

response and the AIM2 inflammasome in the context of pathogen infection and auto-

immunity warrants further investigation and is likely to have broad significance in our

understanding of innate immunity.

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Chapter 4: Discussion

Francisella is able to infect several cell types in vitro, and has been

associated with epithelial cells and hepatocytes in vivo, as well as its well-

characterized infection of immune cells like macrophages and dendritic cells. Only

certain subsets of host cells express inflammasome components, namely macrophages,

dendritic cells, natural killer cells, and epithelial cells. The observation that mice

lacking inflammasome components are unable to control the bacterial burden

emphasizes the importance of the inflammasome in promoting host defense against

bacterial challenge (97). Inflammasome activation has been observed upon infection

with F. novicida, and F. tularensis LVS, but not with the most virulent F. tularensis

type A. Instead of activation and inflammasome dependent cell death, type A

activates a caspase-3 dependent apoptosis. Furthermore, F. tularensis type A actively

suppresses pulmonary dendritic cells and macrophages in the lung following aerosol

challenge (15). This leads to a suppression of early proinflammatory cytokines such

as TNF-α, IL-1β, and IL-12. Furthermore, this strain seems to promote an anti-

inflammatory environment by inducing cytokines like TGF-β, which may aid the

bacterium in subverting host defenses. It is interesting to speculate that type A strains

are more virulent as a result of their ability to either suppress or avoid inflammasome

activation. Given the high degree of genome sequence identity across all Francisella

strains we set out to identify bacterial genes that might play a role in subverting host

defenses, as well as identify the bacterial ligand(s) responsible for activating host

responses by screening an F. novicida library. We decided to use a bacterial genetic

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screen because similar methods had proved useful in identifying flagellin as the

inflammasome activating ligand of Salmonella species (113). Since flagellin is not an

essential molecule of Salmonella, and does not affect its ability to replicate in

macrophages we hypothesized that we would be able to identify a Francisella mutant

that would replicate intracellularly as efficiently as wild-type, but fail to activate the

immune responses. This turned out to be not the case. To our surprise, our screen in

chapter 2 identified over 200 bacterial genes that modulated the host response, from

several different functional classes. In reviewing the list it became apparent that there

was a connection between the bacterium ability to reach the cytosol and induction of

the host response. Also, we noticed that an abundance of mutants with potential outer

membrane/LPS defects hyper-stimulated immune responses. The characterization of

these mutants in chapter 2 was difficult to interpret due to the pleotropic effects

resulting from deleting of outer membrane components. In retrospect, isolation of

theses mutants was giving us a clue that an unstable outer membrane would lead to

increased release of DNA, which we would later identify as the stimulator of the

cytosolic responses. Therefore, the suppression of the cytosolic response that we

attributed to these genes was likely due to their role in maintaining cell wall integrity

and not likely due to direct inhibition of host pathways. These results suggest that the

most virulent type-A strain must have other mechanisms to avoid inflammasome

activation, either with a tougher cell wall, or with other genes that block DNA

recognition.

F. tularensis is a water pathogen and not adapted to the mammalian host

therefore it lacks genes that specifically target mammalian innate immune pathways.

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In contrast, viruses like Vaccinia virus actively block type-I IFN production. Also,

host adapted bacteria like Shigella flexneri, and Salmonella typhi secrete effector

proteins that target and subvert host pathway to establish a replicative niche. Many F.

tularensis genes are annotated as hypothetical, with no homology to proteins of known

function. The ability of F. tularensis to infect mammalian macrophages likely arises

from its ability to replicate inside of amoebae. A similar notion could be applied to

studies of Legionella species, which are also natural parasite of amoeba and only

infect humans accidentally. It may be that to truly understand the pathogenic

functions of several F. tularensis genes we may need to look at it’s interactions with

fresh water amoeba, which share a few characteristics of macrophages but will present

distinct survival challenges for the bacterium. Though some work has been done in

Acanthamoeba castellanii, Dictostylium may serve as a better tool because of it

genetic tractability. Studies on the interaction of F. tularensis with amoeba could

greatly enhance our understanding of its pathogenic strategies and mechanisms by

which it persists in environmental reservoirs.

The findings in chapter 3 suggest that bacterial lysis in the cytosol leads to

release of F. tularensis DNA, induction of the type-I IFN pathway, and activation of

the AIM2 inflammasome. The molecular mechanism that causes bacterial lysis is

unclear but several hypotheses emerge from this work. First, the observation that

induction of the cytosolic responses requires bacterial protein and DNA synthesis

suggests that lysis in the macrophage cytosol is a bacterial mediated process. In broth

culture in rich media, bacteria exhibit a life cycle characterized by a lag phase of no

bacterial replication, a log phase with a net increase in bacterial multiplication, a

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stationary phase of limited nutrient availability where replication plateaus, and a death

phase with a net decrease in bacterial numbers. During this death phase many bacteria

lyse, releasing their contents into the culture media. Therefore, bacterial lysis is a

natural part of the bacterial life cycle and this could be the mechanism of DNA release

in the host cytosol. Additionally, the macrophage phagosome is a professional

microbe-killing machine, and although F. tularensis is well equipped to escape with

its life that does not mean that it is not wounded in the battle. Studies to date on the

intracellular trafficking of the Francisella containing vacuole suggest that the bacteria

escape before acquiring markers of lysosomes or degradative enzymes (26, 144, 145).

However, slight perturbations in the bacterial envelope during the vacuolar stage may

be enough to induce lysis once the bacteria reach the cytosol. This hypothesis is

supported by the large number genes involved in outer membrane and LPS synthesis

identified in Chapter 2 that led to an increased cytosolic response in the macrophage.

If these mutants have an unstable outer membrane they may lyse at a higher frequency

than wild-type F. novicida and lead to increased cytosolic sensing by the DNA

pathway. In support o this idea, recent reports show that Listeria monocytogenes lyses

at a low frequency in the macrophage cytosol and induces AIM2-dependent

inflammasome activation (146). Furthermore, L. monocytogenes mutants that induced

higher levels of inflammasome activation were shown to lyse with increased

frequency than wild-type L. monocytogenes.

Yet another hypothesis is the existence of antimicrobial defenses in the cytosol

itself. Little is known about the cytosolic environment except that it is pH neutral.

Also we known little about changes to this environment after macrophages are

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stimulated with proinflammatory cytokines such as interferons. The interferons were

first described for their ability to induce and antiviral state in cells, but whether or not

this includes defenses in the cytosol is unknown. IFN-γ induces bacterial killing in the

cytosol (42), and INF-β induces a similar transcriptional response as IFN-γ in

macrophages so we hypothesize that type-I IFNs may induce bacterial lysis itself.

These hypotheses are not mutually exclusive and it’s likely that multiple mechanisms

contribute to bacterial lysis.

Although much work has been focused on the antimicrobial defenses of

macrophages much of that work has focused on the killing mechanisms of the

phagosome, which is highly efficient at eliminating the majority of invading

organisms. However, for bacteria that escape phagosomal degradation little is known

about potential antimicrobial mechanisms in the host cell cytosol. One mechanism

that has gained interest in recent years is autophagy, which has the potential to

reintroduce cytosolic bacteria into a degradative environment. Autophagy has been

implicated in host defense against Group A Streptococcus (121), Shigella flexneri

(125), and L. monocytogenes (138). The case of Shigella is interesting in that an

autophagy protein, Atg5, is thought to target a specific Shigella protein VirG and

target the bacteria to an autophagosome. Francisella does in fact enter into an

autophagous vacuole during the late stages of macrophage infection (26), though the

molecular mechanism and the impact of this finding on the outcome of the infection

are unclear.

DNA release is detrimental to the bacterium in this infection model but it may

be beneficial to the bacterium in other environments. Francisella is a competent

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organism so release of bacterial DNA may serve as a way to exchange genetic

information and drive evolution. Furthermore, recent work has demonstrated the

ability of Francisella to establish biofilms (95), which are bacterial communities

characterized by subpopulations in heterogeneous metabolic states. Biofilms often

contain dead bacteria, which can provide a source of nutrients for living populations.

Furthermore, Pseudomonas aeruginosa release bacterial DNA, which forms an

adhesive network to promote biofilm structure (6). Additionally, type-IFN signaling

resulting from the release of bacterial DNA is beneficial to the bacterium in vivo (65).

Therefore, lysis of a small percentage of bacteria may benefit the overall bacterial

community, much like pyroptosis of individual macrophages is important to survival

of the host during infections.

In this thesis work we have identified AIM2 as a critical component of the

innate immune response to F. tularensis infection. The Alnemri group (48)

independently came to the same conclusion, thus supporting our results. Recent repots

have also shown that AIM2 is critical for host defense against viral infection, and

plays a role in detecting intracellular L. monocytogenes (85, 140, 181). Clearly, our

understanding of the role of AIM2 in innate immunity, as well as innate immune

mechanisms for sensing intracellular pathogens is just beginning. It’s interesting that

the AIM2 inflammasome does not recognize a molecule that is unique to microbes,

but instead recognizes any dsDNA with a minimum length of 45 base pairs. It is the

aberrant localization of dsDNA in the cytosol that acts as the danger signal rather than

the molecule itself. It is interesting that IPAF, which recognizes the bacterial specific

flagellin monomers, is able to recruit caspase-1 directly, while other NLRs (AIM2,

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NLRP3, and NLRP1), which responds to host and microbial molecules (DNA, ATP,

gout crystals, and LLO share NLRP3) require the adapter protein ASC. This may

provide another level of regulation to prevent aberrant inflammation from the

recognition of self. In any case, the host innate immune response has used the

compartmentalization of signals as additional method to distinguish safety from

danger.

Innate immune responses have mostly been studied with purified ligands such

as LSP, MDP, or DNA, but studies of immune recognition in the context of an

infection is lacking. In this thesis work we demonstrate innate immune recognition of

bacterial DNA in the context of an intracellular infection. This brings out several

differences from previous reports using DNA transfection. First, in chapter 3 and in

previous reports (34, 64) we see a strong dependence on type-I IFN signaling for

inflammasome activation in response to cytosolic F. tularensis infection. In contrast,

type-I IFN signaling is dispensable for inflammasome activation in response to

transfected dsDNA (119). The difference may be in the concentration of DNA

delivered by the two methods. During a F. tularensis infection, we observe

logarithmic intracellular replication by the bacteria, and thus we only observe a small

percentage of bacterial lysis events leading to DNA release. Each rare lysis event will

release approximately 4-5 femtograms of bacterial DNA, based on the genome size of

F. tularensis. This is a relatively small intracellular concentration of DNA compared

to studies with transfected dsDNA, where concentrations of 1µg/mL are typically

used. In chapter 2, we demonstrate that lower concentration of transfected dsDNA

lead to a type-I IFN dependent host cell death. But we also show that mutants that

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hyper-induce the cytosolic response can activate a type-I IFN-independent cell death,

possible because they release more DNA than wild-type F. tularensis. Thus, the

coordination between the type-I IFN response and the inflammasome may be a factor

of the concentration of cytosolic DNA. We cannot however rule out the importance of

other differences between the two stimuli, such as DNA size and charge. We do, in

fact, observe different degrees of type-I IFN and inflammasome activation with

poly(dG:dG) and poly(dA:dT). Thus, the nature of the DNA may dictate the host

receptor that produces type-I IFN, and different receptors may assert different effects

on the inflammasome.

We observed a positive feedback loop between type-I IFN signaling and

inflammasome activation in macrophages, but the link in vivo seems to be less clear.

Shown in both previous reports (97) and in chapter 3, mice deficient in inflammasome

components are more susceptible to infection with F. tularensis. However, mice

deficient in type-I IFN signaling are more resistant to infection (65). Similar results

were also obtained for L. monocytogenes infections. This apparent discrepancy

between our in vitro results and the in vivo results is partially explained by the control

of type-I IFN signaling on Il-17 production (65). Mice deficient in type-I IFN

signaling produce more IL-17, which leads to a greater influx of neutrophils that can

better control bacterial infections (65). Additionally, IFN-γ can restore inflammasome

activation in vivo in an IFNAR-deficient mouse by signaling through the IFN-γ

receptor, which would result in increased expression of AIM2 and subsequent

inflammasome activation. Thus an interesting paradox exists, where type-I IFN is

beneficial to the host in vitro and detrimental in vivo during bacterial infections.

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There is significant crosstalk between the type-I IFN and host cell dearth

pathways. In both chapter 2 and chapter 3 we observed that lack of type-I IFN

signaling results in decreased inflammasome activation, with STING serving as a

critical adaptor for the production of type-I IFN downstream of DNA sensing.

Additionally, we observe an increase in type-I IFN production in macrophages

deficient for inflammasome components that are stimulated with dsDNA. These

results suggest not only positive feedback but also negative feedback loops between

the two pathways. To better understand the molecular mechanisms of the coordination

between these two pathways we need to identify the host receptors that lead to type-I

IFN production. One such receptor, DAI (161), is either not active in macrophages, or

there are redundant receptors since DAI-deficient macrophages and mice respond

normally to stimulation with dsDNA (75). However, a new cytosolic DNA sensor has

been identified, LRRFIP1 (183), that mediates type-I IFN production in macrophages

in response to L. monocytogenes and vesicular stomatitus virus. If LRRFIP1 were

also involved in the macrophage type-I IFN response to F. tularensis we would have a

system to study the coordination of the IFN pathway and inflammasome pathway in a

biologically relevant model.

Recent work has clearly demonstrated the importance of inflammasome in

pathogen detection and innate immunity to infection. However, we can use these

pathogens as tools to better understand aberrant inflammation associated with

autoimmunity and auto-inflammatory diseases. Future studies on AIM2 could help us

understand the mechanisms behind systemic lupus erythematosus (SLE). Thus F.

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tularensis may a useful tool for probing innate immune responses and better

understanding host biology.

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Chapter 5: Materials and Methods

5.1 BEIR “two-allele” transposon library screen.

A sequenced two-allele transposon mutant library was used to test for F. novicida

transposon mutants that elicited increased or decreased type-I IFN and macrophage

cell death responses (the following reagent was obtained through the NIH Biodefense

and Emerging Infections Research Resources Repository, NIAID, NIH: F. tularensis

subsp. novicida, “Two-Allele” Transposon Mutant Library Plates 1-14, 16-32). The

library represents two or more transposon insertions in all non-essential genes. At the

time of screening, Plate 15 of the library was unavailable due to quality control issues,

resulting in a library size of 2,954 mutants. The two-allele library was received frozen

in 96-well format. The two-allele library was grown overnight in 96-well plates in

TSB supplemented with 0.2% L-cysteine at 37°C with aeration. Bone marrow-derived

macrophages from C57BL/6 mice were seeded in 96-well plates at a density of 105

macrophages per well and cultured overnight at 37°C with 5% CO2. Individual

transposon mutants were diluted into complete macrophage media and used to infect

BMDM at an MOI of ~500:1. Infected macrophages were centrifuged for 15min at

730 x g and incubated for 30min at 37°C. The infected media was then removed and

replaced with fresh complete macrophage media containing 10ug/mL gentamicin and

further incubated at 37°C with 5%CO2 for the duration of the experiment. At 4.5hrs

post infection 75uL of macrophage supernatant was collected and frozen at -80°C.

The amount of type-I interferon in the supernatant was later determined using the

reporter cell line ISRE-L929. At 6.5 hrs post-infection 50uL of supernatant was

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collected and assayed for macrophage cell death by CytoTox96 non-radioactive

cytotoxicity assay (Promega) according to the manufacturers instructions.

5.2 Bacterial strains and growth conditions.

Francisella novicida U112 and all mutant strains were grown to stationary phase in

TSB supplemented with 0.2% cysteine at 37°C with aeration.

5.3 Bacterial Mutagenesis.

Targeted deletions were generated in the U112 strain by splicing by overlap extension

(SOE) PCR (Liu, 2007), as previously described (Brotcke,, 2008) using the primers in

Table A1. Briefly, SOE PCR was used to generate a construct containing a

kanamycin resistance cassette expressed by the groEL promoter flanked by ~600bp

regions of the chromosome 5' and 3' to the gene of interest. The resulting PCR

product was transformed into U112 by chemical transformation and transformants

were selected on MMH agar with 30µg/ml kanamycin. Gene deletions were

confirmed by sequencing. Bacterial mutants were complemented in trans by deletion

mutants were complemented in trans by introducing the wild-type gene, as well as the

CAT cassette, into gro-gfp pFNLTP6 (Maier, 2004). Complementation constructs

were generated by digestion of the pFNLTP6 at the NotI and BamHI sites, removing

the gfp gene, and ligation of the vector with the gene of interest. The resulting

plasmid expressed the complementing gene under the regulation of the constitutive

groEL promoter. Complemented strains were selected for growth on 5µg/ml

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chloramphenicol and also confirmed by sequencing. All complementation primers are

listed in Table A1.

5.4 Bone marrow-derived macrophage culture and infections.

Bone marrow-derived macrophages were prepared from mouse femurs and cultured in

DMEM supplemented with 10% heat-inactivated fetal calf serum and 10%

conditioned L929-MCSF supernatant at 37°C with 5% CO2. For cytokine

measurements and cytotoxicity assays 105 macrophages per well were seeded into 96-

well TC plates (BD) and bacteria were diluted into macrophage media to reach an

MOI of 10 bacteria per macrophage. Macrophages were centrifuged at 730 x g for 15

minutes and incubated at 37°C with 5% CO2 (Time zero). After 30 minutes infected

media was removed and replaced with macrophage media containing10ug/mL

gentamicin for the duration of the experiment. For intracellular replication assays 2.5

x 105 macrophages were seeded in 24-well TC plates and infected as described above.

At the indicated timepoints macrophages were washed 3 times with PBS, lysed in 1%

saponin solution, and serial dilutions were plated on MMH agar for determination of

cfu. Bone marrow cells were stimulated or infected with S. typhimurium or F.

tularensis as described (97). dsDNAs and polyI:C were transfected with

Lipofectamine 2000 (Invitrogen). For priming, BMDMs were cultured with 500

ng/mL ultra-pure LPS or Pam3CSK4 (Invivogen) for 5 h. Priming was used to induce

production of pro-IL-1β in macrophages. Priming with either LPS or Pam3CSK4

induces similar levels of pro-IL-1β, however, Pam3CSK4 does not induce Trif-

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dependent IFN-I production as observed with LPS. IL-1β (Meso), IFN-β (PBL), and

IL-18 (MBL) were measured by ELISA, TNF-a and IL-6 by Bioplex-23 cytokine

assay (Bio-Rad). Cytotoxicity was measured by LDH release (Promega). Caspase-1

was immunoblotted with 4B4 rat anti-mouse caspase p20 (Genentech) or rabbit anti-

caspase p10 (Santa Cruz, sc-514), IL-1b with a rabbit polyclonal (GeneTex).

5.5 Macrophage gene expression analysis.

Bone marrow-derived macrophages were seeded at 106 macrophages per well in a 6-

well dish and infected at an MOI of 10:1 with the appropriate strain. At the timepoints

indicated infected macrophages were incubated with 1mL of TRIzol reagent

(Invitrogen) for 5 minutes with shaking and frozen at -80°C. RNA was isolated using

the RNEasy mini kit (Quiagen) as per the manufacturers instructions. IFN-β, pro IL-

1β, and β-actin gene expression was determined by real time qRT-PCR analysis. Real-

time Quantitative RT-PCR was performed on a 7300 Real Time PCR system (Applied

Biosystems) using rTth enzyme (Applied Biosystems), SYBR green, and primers for

IFN-β, pro IL-1β, and β-actin. Gene specific transcript levels were normalized to the

level of β-actin mRNA. Primers used for IFN-β mRNA quantification are described

(8). Experiments were performed with an iCycler (Bio-Rad) using SYBR green

(Applied Biosystems).

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5.6 ISRE-L929 assays.

ISRE-L929 cells were seeded at a density of 105 cells per well in 96-well viewplate 96

TC plates (Perkin Elmer) and cultured in DMEM supplemented with 10% heat-

inactivated fetal calf serum overnight at 37°C with 5% CO2. ISRE cells were

incubated with 60uL of macrophage supernatant for 4 hours at 37°C with 5% CO2.

After 4 hours the supernatant was removed and the ISRE cells were lysed with 40uL

per well of Glo lysis buffer (Promega) for 7 minutes with shaking. Bright Glow

luciferase reagent (Promega) was added at 40uL per well and the plates were read

immediately on a luminometer using the Veritas software.

5.7 NF-kB reporter cell assays.

RAW 264.7 macrophages containing a NF-kb luciferase reporter were seeded at a

density of 105 cells per well in 96-well luminometer plates (Nunc). RAW cells were

infected at an MOI of 10:1 as described above and luciferase was measured at 2hrs

post-infection using the Bright Glow luciferase assay (Promega).

5.8 Mice, bacteria, and reagents

asc-/-, caspase-1-/-, ifnar-/-, ipaf-/-, nalp3-/-, and sting-/- mice have been described (77,

96, 98, 118). nalp3-/-ipaf-/- mice were generated by nalp3-/- and ipaf-/- intercrosses. All

mice were backcrossed to C57BL/6 for at least 10 generations. Competitive index

experiments were conducted by mixing a 1:1 ratio of wild-type F. novicida strain

U112 and mutant bacteria in PBS, and inoculating the mice intradermally with a total

134

of 105 cfu in 50uL of bacterial suspension. After 48 hours mice were sacrificed, skin

and spleen samples were homogenized in sterile PBS, and serial dilutions were plated

on MMH agar and MMH agar supplemented with 30ug/mL kanamycin. The

competitive index was calculated as the ratio of mutant to U112 after 48 hours relative

to the ratio of mutant to U112 in the input. Intradermal infections with F. tularensis

ssp. novicida strain U112 and isogenic mutant ΔFPI (177) were performed as

described (97). The Genentech and Stanford University animal care and use

committees approved all mouse studies. S. typhimurium was from ATCC. Reagents

included poly(dA:dT), poly(dG:dC), calf thymus DNA, polyI:C, and ATP (Sigma),

pcDNA3.1(+) (Invitrogen), IFN-b (R&D Systems), and Pam3CSK4 (Invivogen).

Sense (5’TAC AGA TCT ACT AGT GAT CTA TGA CTG ATC TGT ACA TGA

TCT ACA) and anti-sense ISD (5’TGT AGA TCA TGT ACA GAT CAG TCA TAG

ATC ACT AGT AGA TCT GTA) (153) were synthesized and annealed at Genentech.

5.9 Immunofluorescence Microscopy

BMDMs (1.25x105) were seeded onto glass cover slips in 24-well plates for infection.

Where indicated, F. tularensis was incubated for 30 min at 37°C with 50 mg/mL

Hoechst 33342, then washed 7 times prior to infection. Cells were washed twice with

PBS, fixed for 15 min at 37°C with 4% paraformaldehyde in PBS, washed 3 times

with PBS, and stained with rabbit anti-mouse AIM2 at 1/500 (Genentech), 8E4.1 rat

anti-mouse ASC at 1/2000 (Genentech), and chicken anti-Francisella at 1/2000

(Monack Laboratory) for 30 min in blocking buffer (3% BSA, 0.1% Saponin in PBS).

135

Cells were washed 3 times with PBS and incubated 30 min with Alexa488 anti-rat,

Alexa594 anti-rabbit, and Alexa647 anti-chicken antibodies (Invitrogen). Cells

washed 4 times with PBS and stained with DAPI were imaged with a Zeiss LSM700

confocal microscope. DAPI was omitted in samples with Hoechst-stained bacteria.

- Table 4 – Primers for F. novicida cloning and mutagenesis

Primer Sequence ftn_1212 checkF TAT TGA TAG TGA TGA TTG GG ftn_1212 F1 TAA GCA AAT AAA AGC TGC TG

ftn_1212 inv1 GCT TAT CGA TAC CGT CGA CCT CAA TTA ACT TCT AGT AAT TCT TTT

ftn_1212 inv2 GAT ATC GAT CCT GCA GCT ATG CAA AAT TTT AAG GAA TGA AAT GAA

ftn_1212 R1 AAT GAC TCA ACA TCT GCT AC ftn_1212 checkR AGC ATT AGC AAT GAC TAT AC lpcC checkF TTA ATT GGA ACT GTG ATA GC lpcC F1 TAT CAA TTT CAT GTT CAA CG

lpcC inv1 GCT TAT CGA TAC CGT CGA CCT CGA TTT ATT TAT ATT AAA ATA TTC

lpcC inv2 GAT ATC GAT CCT GCA GCT ATG CCT AGG TTA TAA GAT TAG CCG lpcC R1 AAA TGG TAA AGG GCT AGT TG lpcC checkR AGA GCA AGT CAA ACA AGC TC wbtA checkF TCG ATT AGA TAA GGC AAA AC wbtA F1 AAA GCT TGT TGC TAA ACA CC

wbtA inv1 CGC TTA TCG ATA CCG TCG ACC TCT TGT TAA TTT TTA GAA AAT ATC

wbtA inv2 GAT ATC GAT CCT GCA GCT ATG CAA TAT ATG AAA GAC AGA ATT TAT

wbtA R1 TAA AAC CTT GCC TTA TCT GC wbtA checkR AGC ACA AAC ATT ACT CAT CC FPI checkF AAT CAG CTA TGG ATC GTA GC FPI f1 TAA TCC ACA GAT ATT ATG CG

FPI cm inv1 AAA TAC GAT GAG TGA CAA CCT GTC TAC TTA ATT AGA ACA TAA C

FPI cm inv2 AGT GGC AGG GCG GGG CGT AAA CTT ACT ACT CTT ACA AGT AAA C

FPI r1 TAT GGA AGT TCT GTT TAA CC FPI checkR AGC AAA CAC TAC AAT TAT TCC kdsA checkF TGG TGA AGT TAA GGT TTT TG kdsA f1 ATG ATA GAA GAA ATC GTT GC

kdsA inv1 GCT TAT CGA TAC CGT CGA CCT CTT GTG ATA ATT ATA CAG AAA AAG

kdsA inv2 GAT ATC GAT CCT GCA GCT ATG CGG CAT AAA TAA TGG CTG GTA kdsA r1 TTT ATT TTG CGC ACC ATC TG

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kdsA checkR ACT CTA GCC ATT TTT TAC TC purMCD checkF TTA TCA TGG GTA GTC ATA CC purMCD f1 TAT ACT AAT GGG TCA AAT CG purMCD inv1 cm AAA TAC GAT GAG TGA CAA CCT TTA TTT CCT TTT AAT CAA T purMCD inv2 cm AGT GGC AGG GCG GGG CGT AAA TAA TGT CTA AGC TAA ATC T purMCD r1 ATT CTA CGC TCA AAT CGT AG purMCD checkR ACC CTA TGC TTA AAC TAT AG purMCD inv1 kanfrt GCT TAT CGA TAC CGT CGA CCT CTT TAT TTC CTT TTA ATC AAT purMCD inv2 kanfrt GAT ATC GAT CCT GCA GCT ATG CAT AAT GTC TAA GCT AAA TCT lpxH checkF ATA GAT ATC CTA ACT TAA CC lpxH f1 TGT ATG TTT ATA GAG TTT GC lpxH inv1 GCT TAT CGA TAC CGT CGA CCT CAT TTT TGA CGG TAC TGT TTA lpxH inv2 GAT ATC GAT CCT GCA GCT ATG CTT TAA CTT CAG AGC TGA ATT lpxH r1 ACT ATA TAG TTC CAT CTG GC lpxH checkR TTA TGC TTA TAC ATC GTG GC

137

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MOLECULAR MECHANISMS OF THE INNATE IMMUNE …rq941zv2693...The ability of F. tularensis to escape phagosomal degradation and replicate in the macrophage cytosol is central to its pathogenesis