Role  of  Rho  small  GTPases  in  the  invasion  of  Lung  Epithelial  Cells  by  Legionella  pneumophila    

               By                

Elizabeth  Barker                

A  thesis  submitted  in  conformity  with  the  requirements    for  the  degree  of  Master  of  Science  

Graduate  Department  of  Cell  and  Systems  Biology  University  of  Toronto  

             

©  Copyright  by  Elizabeth  Barker,  2014            

Role  of  Rho  small  GTPases  in  the  invasion  of  Lung  Epithelial  Cells  by  Legionella  pneumophila    

Elizabeth  Barker  Degree  of  Master  of  Science  

Graduate  Department  of  Cell  and  Systems  Biology  University  of  Toronto  

2014  Abstract    Legionella  pneumophila  is  the  main  cause  of  Legionnaires’  Disease,  a  fatal  

pneumonia.  The  filamentous  form  of  Legionella  (FLp)  is  capable  of  invading  human  

lung  epithelial  cells  through  unique  cell  surface  rearrangements.  These  unique  

structures  curve  around  the  bacteria,  entrapping  it  at  the  host  cell  surface,  favoring  

subsequent  invasion.  This  requires  cytoskeleton  rearrangements  mediated  by  

bacterial  activation  of  E-­‐cadherin  and  𝛽1-­‐integrin  receptors.  We  investigated  the  

roles  of  the  small  GTPases  Rac1,  RhoA,  and  Cdc42  (actin  regulators)  in  the  

attachment  and  internalization  of  FLp  to  lung  epithelium.  We  found  Rac1,  Cdc42,  

RhoA,  and  their  downstream  effectors,  are  involved  in  attachment,  but  not  in  

internalization  of  FLp.  Internalization  was  instead  dependent  on  a  T4SS-­‐delivered  

toxin.  This  indicates  that  bacterial  attachment  is  dependent  on  host  cell  receptors  

activating  downstream  small  GTPases,  causing  actin  reorganization  and  bacterial  

entrapment,  but  internalization  is  dependent  on  a  bacterial  toxin  that  polymerizes  

actin  independently  of  Rho  small  GTPases.  

   

Acknowledgements    Writing  the  final  parts  of  my  thesis  has  been  a  surreal  experience,  one  that  I  didn’t  always  think  I  would  get  to.  The  past  two  years  have  been  a  long  road,  and  one  that  has  taught  me  much  more  than  what  I  expected  at  the  outset.  I  have  only  been  able  to  complete  this  project  thanks  to  unwavering  support  of  family  and  friends,  to  the  support  and  expertise  of  my  lab  mates,  and  of  course,  to  my  supervisor,  Dr.  Mauricio  Terebiznik,  who  has,  by  turns,  made  me  laugh  (often),  cry  (only  once),  and  break  pipettes  (only  once,  by  accident!).  He  has  persistently  pushed  me  to  do  things  that  I  never  expected  to  be  able  to  do,  provided  a  unique  learning  environment  in  which  to  grow  as  a  scientist  and  person,  and  been  an  invaluable  resource  to  me  in  times  of  stress  and  confusion.  And  so  to  you,  Dr.  Terebiznik,  I  want  to  offer  my  sincere  thanks  for  never  giving  up  on  me!    I  would  also  like  to  thank  Dr.  Cyril  Guyard  and  his  lab  for  generously  providing  many  of  the  reagents  and  materials  that  were  used  in  this  study.  Also,  for  teaching  me  how  to  teach,  and  for  important  advice  on  experimental  planning,  and  career  choices,  I  would  like  to  thank  Dr.  Shelley  Brunt.  Without  these  resources  my  graduate  career  would  have  been  much  more  difficult.      To  the  undeniable  doyenne  of  our  lab,  Akriti  Prashar  –  you  are  doubtless  one  of  the  hardest  working  people  I’ve  ever  met,  and  I  would  have  been  lost  countless  times  without  your  expertise.  Thank  you  for  your  timely  help,  advice,  and  listening  ear.  And  thank  you  for  always  answering  my  sometimes  silly  questions  -­‐  even  if  your  answer  was  just  an  incredulous  look,  it  seemed  to  convey  all  the  information  I  needed!  To  my  fellow  lab  mates  –  Arujun,  I  don’t  know  what  I  would  have  done  without  another  Masters  student  to  talk  to!  Thank  you  for  always  having  a  smile  on  your  face,  and  for  your  consistently  helpful  nature  and  advice.  (Also  thanks  for  thinking  those  New  Orleans  beignets  were  worth  a  second...and  third…trip  to  the  French  Quarter)!  To  Sahara,  Amriya,  and  Iram  –  the  laughs  we  have  shared  truly  made  long  days  seem  shorter.  It  was  a  pleasure  to  share  an  office  and  lab  with  all  of  you  –  and  thanks  for  always  being  ready  to  offer  helpful  advice  and  assistance.  I  also  need  to  thank  our  neighbours  down  the  hall  –  Dr.  Rene  Harrison  and  her  students  were  an  invaluable  resource  in  countless  ways.    Thanks  need  also  to  be  directed  to  my  Grandpa  Barker,  Grandpa  Sherk  (whose  PhD  inspired  me  to  begin  graduate  studies),  and  Grandma  Sherk.  Your  generosity  has  enabled  me  the  freedom  to  pursue  a  higher  education,  as  well  as  emphasizing  the  importance  of  learning,  and  for  that  I  will  be  forever  grateful.  Thanks  also  for  providing  a  wonderful  example  of  hard  work  in  the  face  of  adversity,  and  of  thankfulness  in  all  situations.      Of  course,  it  goes  without  question  that  the  completion  of  my  project  was  in  part  due  to  the  support  of  my  incredible  family.  To  Dad,  Mom,  and  Jeff  –  thank  you  for  everything.  For  the  intangibles  -­‐  unwavering  support  and  love,  and  for  the  practical  financial  support  and  advice.  Lastly,  I  would  like  to  thank  Joel  Westacott.  I  am  so  blessed  that  you  are  in  my  life,  and  I  would  not  have  been  able  to  do  this  without  you.  Thanks  for  patiently  listening,  for  reminding  me  what  is  truly  important,  for  making  even  mundane  things  fun,  and  for  providing  a  safe  place  to  fall  in  those  awesome  arms  of  yours!    

     

 .  

Table  of  Contents  Abstract                      Acknowledgements                  Table  of  Contents                  List  of  Figures                    List  of  Abbreviations                  1.0 Introduction                 1  

1.1 Legionella:  From  the  pond  to  the  lung           1  1.2 L.  pneumophila:  A  Complex  life  cycle       2  1.3 Legionella:  An  Intracellular  Pathogen       6  1.4 Legionella  pneumophila  And  Infection  of  Epithelial  Cells    10  1.5 Filamentous  Legionella  invade  Lung  epithelial  cells   11  1.6 Receptor-­‐Mediated  Entry  of  FLp  in  LEC       15  1.7 Actin  Reorganization:  The  Common  Denominator   17  1.8 Aims                 23  

 2.0 Materials and Methods 25

2.1 Bacterial Strains and Cultivation 25 2.2 Cell Culture, Constructs and Transfections 25 2.3 Bacterial Infections 27 2.4 Pharmacological Inhibitions 27 2.5 Antibodies and Immunostaining 28 2.6 Attachment and Internalization Assays 30 2.7 Localization Assays 30 2.8 Wide field Fluorescence Microscopy 31 2.9 Scanning Electron Microscopy 31 2.10 Quantification and Statistical Analysis 32

3.0 Results 34

3.1 Small GTPases accumulate at FLp invasion sites 34 3.2 Cdc42 and myosin X mediate FLp attachment through

filopodia formation 34 3.3 Role of Rac1 in FLp attachment 41 3.4 Role of RhoA in FLp attachment 44 3.5 Role of actin nucleators mDia and Arp2/3 FLp attachment 50 3.6 Role  of  small  GTPases  in  FLp  internalization     55 3.7 Role of cdc42 in FLp internalization 55 3.8 Role of Myosin X in internalization of FLp 57 3.9 Role of Rac1 in FLp internalization 58 3.10 Role of RhoA in FLp internalization 60

   

3.11 Role of ROCK, MLCK, and myosin II in FLp Internalization 62

3.12 Role of mDia in FLp internalization 63 3.13 Role of arp2/3 in FLp internalization 64 3.14 Attachment and internalization dependency on Type IVSS 65

4.0 Discussion 68

4.1 Roles For FLp Receptors in Signal Transduction 71 4.2 Filopodia, Hooks, and Invasion 73 4.3 Lamellipodia, Wraps, and Invasion 77 4.4 RhoA and Invasion 79 4.5 Downstream Effectors: Contribution to Invasion 81 4.6 Future Directions and Implications 84 4.7 Conclusions 86

References 87                                                          

   

List  of  Figures    Figure  1.  Intracellular  life  cycle  of  Legionella  pneumophila.       4    Figure  2.  Hooks  are  similar  to  filopodia  both  morphologically     13    and  molecularly.    Figure  3.  Small  GTPase  activation  cycle.           20  

 Figure  4.  Possible  mechanism  for  the  attachment  of  FLp  to         24  NCI-­‐H292  and  its  eventual  internalization.    Figure  5.  Experimental  methodology.             29    Figure  6.  Small  GTPases  accumulate  at  the  site  of  FLp  invasion.     35-­‐36    Figure  7.  Inhibition  of  Cdc42  affects  FLp  attachment.         38    Figure  8.  Internalization  of  FLp  is  not  significantly  affected         56  by  inhibition  of  Cdc42.    Figure  9.  Pretreatment  of  cells  with  bradykinin  increases       40    attachment  of  FLp  and  increases  number  of  filopodia.    Figure  10.  Myosin  X  affects  attachment  of  FLp  to  host  cells,  but  not   42  internalization.    Figure  11.  Inhibition  of  Rac1  affects  FLp  attachment.         43    Figure  12.  Inhibition  of  RhoA  does  not  affect  FLp  attachment.     45-­‐46    Figure  13.  Attachment  of  FLp  at  1  hour,  but  not  internalization,       48  is  affected  by  ROCK  inhibition.    Figure  14.  Attachment  of  FLp  at  1  hour,  but  not  internalization,       49  is  affected  by  MLCK  inhibition.    Figure  15.  Attachment  of  FLp  at  1  hour,  but  not  internalization,       51  is  affected  by  myosin  II  inhibition.    Figure  16.  Attachment  of  FLp  at  1  hour,  but  not  internalization,     53    is  affected  by  mDia  inhibition.    Figure  17.  Attachment  of  FLp  at  1  hour,  but  not  internalization,     54    is  affected  by  arp2/3  inhibition.  

 

 Figure  18.  Internalization  of  FLp  at  6  hours  is  not  significantly       59  affected  by  inhibition  of  Rac1.    Figure  19.  Internalization  of  FLp  is  significantly  affected  by       61    inhibition  of  RhoA  at  6  hours.    Figure  20.  T4SS  mutant  FLp  exhibit  similar  binding,  but  decreased   66  internalization  compared  to  WT  FLp.    Figure  21.  Model  of  the  molecular  mechanism  involved  in  the     69-­‐70    early  invasion  steps  of  filamentous  Legionella  pneumophila  into  human  lung  epithelial  cells.                                                                    

   

List  of  Abbreviations    FLp       Filamentous  Legionella  pneumophila  LEC       Lung  epithelial  cells  LCV       Legionella-­‐containing  vacuole  MIF       Mature  intracellular  form  MLCK Myosin light chain kinase MW Membrane wrap RF Replicative form  ROCK       Rho associated coiled-coil containing protein kinase                                                                  

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1.0!INTRODUCTION!

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1.1!Legionella:!From!the!pond!to!the!lung!!!

Legionnaires’ disease is a severe form of pneumonia characterized by a non-productive

cough, fever, chills, headache, and myalgia!1. The mortality rate associated with

Legionnaires’ disease is relatively high (15-25%), especially among those who are

elderly or immunocompromised (where the rate can be as high as 50%)2!3!4!1.!Most cases

of the diseases are caused by the gram negative, aerobic bacterium, Legionella

pneumophila serogroup 1, although several other Legionella species have also been found

to cause disease (including L. bozemanae, L. micdadei, and L. longbeachae)!4!5!1. A mild

form of legionellosis, Pontiac fever, is characterized by mild, flu-like symptoms!11. No

deaths have been reported due to Pontiac fever!1.!Both types of infections have been

reported world wide, including several fatal outbreaks of Legionnaires’ disease in Canada

within the last several years!6. Human-to-human spread of the bacteria has not been

documented, and this plus the fact that the disease mainly affects individuals with

previous conditions indicates that this is an opportunistic pathogen!7!4!1.

L. pneumophila is naturally found nearly ubiquitously in fresh water bodies, where their

natural hosts are different species of amoeboid protozoans (such as Acanthamoeba spp.,

Hartmannella spp., Tetrahymena spp., Dictyostelium spp.), although they do exist in a

free-living form!5!8!2. L. pneumophila have also been reported in moist soil environments,

and within biofilms!8!5!1. The bacteria are phagocytized by amoeba!7 but evade the normal

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phagosomal pathway, converting its phagosome into a specialized niche for replication

called the legionella containing vacuole, or LCV!5. The cellular processes in these

protozoan species are conserved among many eukaryotes; thus when L. pneumophila

developed the means to infect amoeboid cells, it was also able to infect human

macrophages.

Most human infections occur when the bacteria contaminate man-made water systems,

such as cooling towers, hot tubs, grocery store produce misters, and sprinkler systems!5!4!7. The increased incidence of L. pneumonphila infection in the late 20th century can be

attributed to the increasing prevalence of such systems!5. These environments tend to lead

to the aerosolization of water droplets. Water droplets containing the bacterium can then

be inhaled into the lung!5!2. Inhalation of amoeba which have been infected with L.

pneumophila can also result in infection!9.

1.2 L. pneumophila: A Complex life cycle

L. pneumophila is polymorphic species, in that they exhibit different morphologies with

different levels of virulence based on their growth phase!5!4,!10. This is a useful adaptation

for a species that has such a wide range of potential environments. In ideal, nutrient-rich

environments, L. pneumophila replicate exponentially, and do not display the typical

infectious characteristics that they acquire later on!4!5. They exhibit an increased

transcription of genes involved in the metabolism of carbohydrates and amino acids, and

are referred to as the replicative form (RF)!5!11. As nutrients are consumed by the growing

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bacterial population, the bacteria transition to “MIFs” – mature, infectious forms, which

express traits encouraging escape from the spent host cell and transmission into a new

one!4!5!10. MIFs tend to be found as short, thick, motile flagellate rods!4!5. This form has

proven more resistant to antibiotics and osmotic shock in addition to being more invasive

to cells!4. Recently, a third morphotype has been reported called the intermediate form,

which, as the name suggests, exhibits intermediary characteristics between the RF and the

MIF!11.

As the name suggests, the MIF is the morphotype that most often infects phagocytic cells!11. Once the LCV has been established inside the cell, the transition to the RF begins,

with the bacteria first passing through the intermediate form!11. As replication progresses,

nutrients within the LCV and the host cell are consumed, slowing bacterial reproduction.

After replication, the bacteria pass back through an intermediary form, and back into the

MIF, which escape and invade surrounding cells (Figure 1)!11.

The transition between the two main forms appears to be prompted by a decrease in

amino acid concentration that often follows exponential growth!4!5. Uncharged tRNAs

binding to ribosomes stimulate the production of ppGpp (3’,5’-bispyrophosphate) via

RelA and SpoT!4!5. This triggers signals from RpoS and FliA, as well as LetA/LetS!4!5. In

turn, this leads to the transcription of genes encoding the infectious, transmissive

phenotype, such as the flaA gene involved in flagellar synthesis, and those for some

Dot/Icm substrates!4!5. Specifically, RpoS can regulate bacterial motility, sodium

sensitivity, and evasion of the endocytic pathway!5. It can also regulate the expression of

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MIF

T4SS

E!ector Toxin

Nucleus

Lysosome/ Endocytic Pathway

Vesicle

ER

Golgi

Intermediate form

Ribosome

Mitochondria

RF

LCV FLp

Figure 1. Intracellular life cycle of Legionella pneumophila. (A) The MIF (mature intracellular form) of L. pneumophila is initially phagocytosed via coiling or zipper phagocytosis. (B) Once inside the host cell, the bacteria injects toxins via a T4SS. These toxins allow it to evade the normal endocytic degradation pathway, and to hijack vesicles from the ER/Golgi system, which help to establish the LCV (Legionella containing vacuole). (C) Mitochondria and ribosomes become associated with the LCV, which now contains an intermediate morphotype of L. pneumophila. (D) After extensive fusion with ER/Golgi vesicles, the LCV membrane looks similar to vesicular membranes, and remains associated with ribosomes and mitochondria. Inside the LCV, the transition to the RF (replicative form) is completed, and the bacteria start to replicate. (E) As nutrient levels decline, the bacteria begin the transition to the MIF through the intermediate phase. (F) The intracellular life cycle is completed when the MIFs are released from the host cell, either by lysis or pore formation. (G) The "lamentous form of Legionella pneumophila (FLp) is also capable of infecting host cells, and follows a similar (although somewhat delayed) pathway as the rod-shaped morphotype.

A

B

C

D

E

F

G

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FliA, which regulates infectivity, lysosomal avoidance, and biofilm formation!5. The

LetA/LetS system increases production of small RNAs that de-repress CsrA, allowing

transcription of genes leading to MIFs!5. This is called the stringent-response pathway,

and is also found in other bacterial species!4!11. It is important for promoting long-term

survival in harsh conditions!4.

In addition to this biphasic life cycle, L. pneumophila can be found as rods (up to 5!m in

length), but are also capable of filamentation!12!10!8. Several other pathogenic bacterial

species, including uropathogenic Escherichia coli, Salmonella enterica, Shigella flexneri

and Mycobacterium tuberculosis, are capable of forming filaments!13. Filamentation is

thought to allow bacteria to survive adverse environmental conditions, such as low

nutrient levels, host effectors, predation, and even antimicrobial therapies!13. Proteus

mirabilis is also capable of forming filamentous swarm cells, which have a more potent

invasive ability than the normal rod cells!14. Similarly, the bacillary daughters of

filamentous B. pseudomallei cells were found to have a comparable fitness to filaments

(that is, they were antibiotic-resistant), suggesting that the proteomic changes that are

triggered during filamentation can persist in the population!13. Thus filamentation is a

relatively wide-spread phenomenon.

Filaments tend to be between 10-50 times longer than the normal rod-shaped phenotype!13. Cell growth in the absence of septation and cell division results in the formation of

filaments, which tend to have multiple copies of the organism’s chromosome!13. Genes

collectively referred to as “filamentous-temperature sensitive,” or Fts, are generally

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responsible for generating the septum in dividing bacterial cells!15. The best characterized

of these is the FtsZ gene, encoding the FtsZ protein, which forms the scaffold for the

developing septum, and provides some contractile force for division!15. Accordingly,

mutation or manipulation of these genes or proteins may result in filamentation!13!15.

L. pneumophila filaments can be longer than 50!m in length, are multinucleate, and have

been found in patient samples as well as amoeba, macrophages, and epithelial cells!13!12!10!8. Legionella pneumophila filaments (FLp) have more recently been shown to have an

invasive ability!10. In fact, it was found that FLp may actually outcompete the rod-shaped

form of the bacteria in attachment assays in epithelial cells, and is able to invade and

replicate inside macrophages!10!16.

1.3 Legionella: An Intracellular Pathogen

Once inside the lungs, the bacteria is engulfed by alveolar macrophages via coiling

phagocytosis!5!4!7. Inside alveolar macrophages, their intracellular life cycle mimics that

of the cycle within amoeba!7. Following scission from the plasma membrane, the

intracellular pathway followed by the nascent phagosome containing the bacteria (the

LCV) differs significantly from the normal degradative pathway, as mediated by the

bacteria itself.

Following the canonical pathway for maturation, nascent phagosomes have a similar

membrane to the plasma membrane, and initially fuse with sorting/early endosomes,

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which tend to display Rab5 (involved in membrane fusion) and EEA1 (early endosome

antigen 1, involved in membrane trafficking), endowing the phagosome with mildly

acidic (pH ~6.0) properties, and a few proteases!5!17. Late endosomes, displaying Rab7,

Rab9, and LAMPs (lysosomal-associated membrane proteins) are next associated with

the phagosome (now termed a ‘late phagosome’), which further lowers the pH of the

organelle (pH ~5.5), and contribute a relatively high proportion of hydrolytic enzymes to

its lumen!17. Lastly, the late phagosome interacts with lysosomes, which contain a high

concentration of active proteases and lipases, and are extremely acidic, lowering the pH

in the organelle, now termed a phagolysosome, to pH~5.5!5. Lysosomes are typically

marked by high concentrations of LAMPs and cathepsin D!17!5. Non-pathogenic bacteria

or other particles are killed and degraded in such compartments!17!5.

Instead of being degraded through this system, Legionella are able not only to survive,

but also to replicate, by manipulating the normal pathway and forming the Legionella

containing vacuole, or the LCV!4. The LCV does not fuse with endosomes, instead

recruiting vesicles and membranes from the secretory pathway, creating a specialized

intracellular niche!4!5. Little is known about the mechanisms through which the bacteria

avoids the acidification of the LCV!7. Soon after phagocytosis, ER vesicles are recruited

to the LCV, and the LCV appears associated with mitochondria4!5!7. The LCV membrane

begins to appear more similar to that of the ER vesicles than the plasma membrane, and

contains proteins from vesicles that normally circulate between the ER and Golgi

apparatus!5. The hijacked resources are used to support the growth of the bacteria in the

LCV!7.

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Most of the typical endosomal markers are absent from the LCV, but it does contain

Rab7, which normally regulates vesicle traffick from early to late endosomal stages, and

Rab1, which helps control the fusion of ER-derived vesicles with Golgi!5. Sec22b, a

SNARE normally found on ER-derived vesicles which binds to pre-Golgi intermediate

compartments, is also delivered to the LCV membrane!5.

Following these early events, the LCV continues to mature, associating with calnexin

(resident ER protein), and then almost fully resembling the rough ER (with many

ribosomes studding the membrane) after 4-6 hours!5!4. The pH neutral LCV then serves as

a reservoir for L. pneumophila replication (the bacteria replicate by binary fission)

beginning at around 4-10 hours after phagocytosis, and lasting for around 20 hours!5!4!18.

Bacterial progeny release from the host cell is currently a poorly understood process!7.

Late in the replication process, the bacteria can be found free in the cytosol, suggesting

that at some point they are released from the LCV, possibly by pore formation or

membrane lysis!5. L. pneumophila then escapes the host cell via pores and lysis of the

plasma membrane!5!4!9. Released bacterial cells can then infect surrounding host cells!5!4.

In order to manipulate host cell machinery, L. pneumophila has a variety of toxin

effectors. The delivery of these proteins to the cell is achieved through two main

secretion systems – a type II secretion system (T2SS), and a type IV secretion system

(T4SS), which is known to translocate about 200 effector proteins!9!5!4. Invasion of host

cells, survival of the host cells (in order to support bacterial life), growth of the bacteria

8

!

inside host cells, and bacterial egress are all dependent on these toxins, and the

mechanism through which they are delivered!9!5. Indeed, mutations in the genes encoding

the proteins of the T4SS lead to severe defects in L. pneumophila pathogenesis

(interestingly, mutations in the individual toxins cause much less of an effective,

suggesting functional redundancy between them)!9!5!4. Many other pathogenic species use

varieties of the T4SS in the course of their infection!5.

The Lp T4SS is also known as the Dot/Icm system because of its simultaneous discovery

by two research groups, who found that defects in certain genes lead to problems in

organelle trafficking (Dot – defective in organelle trafficking) and intracellular

replication (Icm – intracellular multiplication)!4!5!9. Although it is capable of mobilizing

plasmids, its most important function appears to be the mobilization of proteins from the

bacteria to the host cell – both through the host cell plasma membrane at the onset of

infection, and through the LCV membrane, into the host cell cytoplasm further on into

the infection!4!5!9. These effector protein target several important host cell processes,

although identifying the exact mechanism and function is difficult due to the paralogous

nature of many of the toxins!5!9. Major host cell processes that are hijacked by L.

pneumophila include: vesicular trafficking pathways, small GTPase regulation,

translation and the induction of stress response, and inhibition of apoptosis!5.

The T2SS, which in L. pneumophila is also known as the Lsp (Legionella secretion

pathway) system, is required on some level for pathogenesis, as well as environmental

persistence (especially at low temperatures)!5. Unlike the T4SS, substrates of the T2SS

9

!

are secreted into the culture media!5. Mutations in the genes encoding the proteins for the

Lsp T2SS lead to more moderate defects in pathogenesis than mutations in the T4SS!5.

1.4 Legionella pneumophila and Infection of Epithelial Cells

Pathogens are able to invade and replicate inside nonprofessional phagocytes such as

epithelial cells!5. E. coli can be taken up by epithelial cells in a phagocytosis-like manner

involving membrane ruffles, and can then multiply intracellularly!19. Listeria

monocytogenes cells can also invade epithelium following formation of phagocytic cup-

like structures, and then can replicate and cause septicaemia, central nervous system

infections, and other disorders!20. Similarly, Salmonella is able to invade nonphagocytic

cells following the formation of host cell membrane ruffles or stress fibres!21. Shigella

flexneri can also invade epithelial cells using host cell membrane folds, followed by

colonization and subsequent spread to other cells!22!23.

Most previous studies on L. pneumophila have focused on their intracellular life cycle

using macrophages!10. However, the vast majority of the lung surface, where the inhaled

bacteria would make first contact, is composed of alveolar epithelial cells!24, and various

Legionella species are capable of infecting these cells!25!26!27!28!29. It has been speculated

that epithelial cells represent an alternative, potentially safer site of replication within

hosts compared to macrophages!28. L. pneumophila is reportedly able to attach to various

epithelial cell lines, in some cases regardless of its virulence!28!25!27!29. Although in some

cases epithelial cell invasion reportedly relied on intact virulence genes!29!27, one study

10

!

suggested that strains incapable of invading macrophage cell lines are capable of

invading epithelial cells!25, highlighting the importance of understanding the systems

involved in invasion of epithelial cells. The mechanisms behind these systems, however,

are unclear!28!29. Once the bacteria is intracellular, several studies have showed that it is

capable of replicating and spreading!29!28.

The exact mechanisms by which rod-shaped L. pneumophila attach to and are taken up

by epithelial cells have not been specified, as previous studies have had difficulty

distinguishing structures involves in the attachment and internalization of the bacteria, or

have focused on the intracellular replication aspect of the bacterial life cycle.

1.5 Filamentous Legionella invade Lung epithelial cells

Recently, Prashar et al have shown that the filamentous form of Legionella pneumophila

(FLp) are capable of invading and replicating inside human lung epithelial cells (LEC)!10.

They do so using structures that look similar to filopodia and lamellipodia!10.

In order to examine their environment, cells use long (10!m), thin (0.1-0.3!m),

cylindrical extensions of the plasma membrane called filopodia!30!31. Filopodia are

underlain by actin filaments, extend outward from the cell body, and play roles in many

cellular processes!30 31. Additionally, filopodia/protrusive structures have been known to

play some role in bacterial infection for several decades, as invading bacteria (including

Yersinia, Shigella, Neisseria, and enteropathogenic E.coli species) may initially be bound

11

!

to filopodia!31!32!33!34!35. Lamellipodia (sheet like, spreading structures underlain by actin),

which have roles in motility, the development of adhesions, macropinocytosis, and

phagocytosis, may develop from ingrowth between filopodia!36.

FLp invade human lung epithelial cells following the development of actin-based

structures termed hooks and membrane wraps (MW), which are exclusive to the

filamentous form of the bacteria!10. At the onset of FLp infection, slender (less than

1.0!m) cell surface filopodia curve around the short axis of the bacteria, apparently

trapping the FLp at the surface!10. These structures, which are rich in actin and plasma

membrane, are called hooks (Figure 2)!10. Hooks precede the onset of the second FLp

structure, which are lamellipodia-like in nature, and are called membrane wraps!10.

Membrane wraps are broader sheets, also enriched in actin and plasma membrane, which

entrap segments of FLp at the host cell surface!10.

Several proteins localize strongly to filopodia, and some are considered markers of these

structures!31. Most are regulators of the actin cytoskeleton, such as ENA/VASP proteins,

formins, fascins, mDias, integrins, and myosin X (Figure 2)!31. Myosin X is an

unconventional myosin motor protein found at low levels in most tissue types in

vertebrates, where it localizes primarily in puncta at the tips of filopodia!37!38!39. It is also

found at the leading edge of lamellipodia!40. It associates with actin filaments of the

cellular cytoskeleton, and exhibits intrafilopodial motility (forward and retrograde) using

an ATP-dependent mechanism!39!38!41. In addition to moving along filaments, myosin X

can carry other proteins along the filaments, and deliver the cargo to the ends of filopodia!

12

Prashar, A., et al (2012). Cellular Microbiology. 14(10): 1632-1655.

Bohil, A. B., et al. (2006). PNAS. 103: 12411-12416.

integrin

cadherin

f-actinCdc42

myosin X

VASP

plasma membrane

External FLp

Figure 2. Hooks are similar to !lopodia both morphologically and molecularly. Both !lopodia and hooks are underlain by a dense, !lamentous actin cytoskeleton and enriched in plasma membrane. Additionally, both contain integrins, cadherins, Cdc42, myosin X, and VASP. Filopodia are likely the !rst attachment point of FLp to host cells, and develop into hooks which help secure the bacteria to the host cell surface.

PIP3

13

!

41!38. Two of the known cargos of myosin X are integrins and cadherins!38. Thus, it is

possible it plays a role in localizing FLp receptors within the host cell.

Additionally, overexpression of myosin X increases the number and length of filopodia!42!40, and has been shown to have a role in the infection processes of both Shigella flexneri

and Listeria!42. Our lab has also found that myosin X localizes to the tips of both

filopodia, and hooks (unpublished data). Due to its ability to stimulate filopodia

production, and its ability to localize known FLp receptors, it likely plays a role in FLp

invasion of LECs.

The structures associated with FLp infection of LEC change as the infection progresses.

Over the course of the infection, the frequency of hooks does not change, but the number

of membrane wraps increases!10. Both structures could be found associated with the same

FLp!10. The length of the MWs increase, entrapping larger sections of the bacterial

filaments, until about six hours after infection, when the FLp are no longer entrapped at

the host cell surface, but instead are fully internalized into the body of the host cell!10.

Even the longest FLp can be found to be fully internalized by 16 hours post infection!10.

Following entry into lung epithelial cells, FLp filaments elongate, and then fragment

asymmetrically into short rods!10. In turn, these replicate by binary fission!10. Due to the

combined efforts of fragmentation and replication, host cells quickly became fully

populated with rod-shaped L. pneumophila, which are capable of infecting new host cells!10. It is possible that FLp are actually more efficient intracellular reproducers than the rod

14

!

shaped form, as the time required for a filamentous cell to fragment into several shorter

filaments was less than the time it took for one rod-shaped cell to divide!10.

Superficially, membrane wraps are similar to the well-characterized zipper mode of

phagocytosis!10!43. Zipper phagocytosis involves the formation of two protrusions of the

plasma membrane around the foreign particle following its attachment to a phagocytic

receptor!43. The protrusions/pseudopods are fairly symmetrical, and grow following the

contour of the particle they are engulfing!43!44. Once they meet, they fuse, forming a

sealed vacuole, which buds off of the cell membrane!43. The nascent phagosome can then

follow the typical intracellular processing pathway (non-pathogens), or evade the

pathway, forming a vacuole for infection (pathogens).

Other modes of bacterial entry involve various other types of structures. Salmonella

typhimurium and Shigella flexneri trigger abundant membrane ruffling at the entry site,

with disorganized structures eventually engulfing the pathogen, as well as any material

close by, as the engulfing ruffles do not strictly follow the contours of the bacterial cell as

with more conventional phagocytosis modes!43!44!45!21!22!23. This is generally termed a

“trigger” mechanism!44.

1.6 Receptor-Mediated Entry of Filamentous Legionella pneumophila in Lung Epithelial

Cells

15

!

The zipper mechanism of invasion requires the continued engagement and activation of

receptors at the host cell surface!44!10!43. The host cell receptors involved in the invasion

of LEC by FLp are !1-integrin and E-cadherin!10. Both are enriched in membrane wraps,

and blocking antibodies against them cause a deficit in FLp attachment (blocking a single

receptor at a time caused a 50% decrease in attachment, while blocking both almost fully

abrogated attachment)!10.

!1-integrin is a member of the integrin family of surface glycoproteins, which mediate

cell-extracellular matrix and some cell-cell interactions!46!47. All integrins are composed

of an ! and a ! subunit, each of which contain an extracellular domain, a transmembrane

domain, and a cytoplasmic tail!46. Integrins bind strongly to the fibronectin and

vitronectin found in the ECM, but can also bind a variety of other proteins, such as

complement factors!46. Due to the transmembrane nature of their structure, integrins have

proven to be important bidirectional signal transducers, passing mechanical and

biochemical signals across the plasma membrane!46. Several species of pathogenic

bacteria have evolved ways in which to hijack these integrin signaling systems to initiate

their uptake!46!48. Several bacterial adhesins (bacterial surface components that facilitate

adhesion) can bind to integrin, including Yersinia’s invasin, and Listeria monocytogenes’

internalins A and B!46!48!44. E. coli, Salmonella typhimuirium, Staphylococcus aureus, and

some Shigella species are amoung the pathogens that also engage host cell integrins to

facilitate entry!46!49. Integrin binding causes receptor clustering, initiating a cascade of

signals including the activation of cytoskeletal re-arrangers, leading to engulfment of the

pathogen!46!47.

16

!

Cadherins mediate cell-cell interactions, and are components of adherens junctions!50.

They have a similar structure to integrins, with an extracellular domain, a transmembrane

domain, and a cytoplasmic domain!50. There are multiple types of cadherins, usually

named due to the cell types in which they can be found: E-cadherin is a component of

epithelial cell junctions, VE-cadherin is found in juctions of endothelial cells, and

fibroblasts contain N-cadherin!50. Cadherins are also well-structured to participate in

signaling events, and play a particular role in modifying the actin cytoskeleton!50. Several

known pathogens bind to E-cadherin to promote their uptake into host cells by modifying

the host cell cytoskeleton, including Listeria monocytogenes, which expresses the E-

cadherin ligand internalin A, and Streptococcus pneumonia, which has adhesion A!20!51!52!53.

1.7 Actin Reorganization: The Common Denominator

Although the structures involved in bacterial entry are diverse, they share a common

denominator: all of them are rich in actin, and/or involve actin reorganization!10!44!20!49!21!22!48. Filopodia and lamellipodia, which play a role in some phagocytosis processes, are

both enriched in actin!36!31. Hooks and wraps, the structures unique to FLp infection of

LECs are similarly enriched in actin and plasma membrane!10. Thus actin is a key player

in pathogen invasion generally, and FLp invasion specifically.

17

!

In fact, inhibiting actin polymerization with cytochalasin-D negatively affected the

morphology of wraps entrapping FLp, and caused their retraction!10.

The actin cytoskeleton of cells has a role in some of the most fundamental cellular

processes, such as cytokinesis, motility, and intracellular transport!54. Many of the

proteins involved in maintaining this integral system are controlled by small GTPases!54!55!56. The Rho family of small GTPases act as molecular switches in eukaryotic cells!55!56!57. They are active in their GTP-bound state, at which point they interact very specifically

(due to their changed conformation) with their targets/effectors!55. When bound to GDP,

they are inactive!55!56!57. The conversions between the GTP and GDP bound states are

controlled by three groups of regulators: guanine nucleotide exchange factors (GEFs),

GTPase activation proteins (GAPs), and guanine nucleotide dissociation inhibitors

(GDIs)!55!57. GEFs activate small GTPases by catalyzing the exchange of GDP for GTP!55. GAPs inactivate them by stimulating the proteins’ intrinsic GTPase activity (cleaving

GTP to GDP)!55. GDIs maintain the small GTPases in their inactive state, blocking

spontaneous activation!55. The small GTPases are very tightly controlled in this manner;

there are over 80 GEFs, and 70 GAPs for Rho family GTPases alone!58.

In their inactive form, the small GTPases are sequestered in the cell cytoplasm by GDIs!56!57!59. An extra cellular signal begins the activation process!56!57!59. After translation,

GTPases are posttranslationally modified at their COOH end with the addition of a lipid

(farnesyl, palmitoyl, etc)!60. When this moiety is exposed by the removal of the GDI, the

18

!

protein can localize in the plasma membrane!57!59!60. The activation can then be

completed by a GEF (Figure 3)!56!57!59.

The small GTPases are divided into the Ras, Rho, Ran, Arf/Sar and Rab families!57. The

Rho family, responsible for cytoskeletal rearrangements, is divided into subfamilies, the

best characterized members being the Rho, Rac, and Cdc42 subtypes!56!57. Over 50

effectors have been identified for these three main subtypes, which appear to convert to

an active conformation when they bind to their small GTPase!55. Rho has three isoforms

(A, B, and C), as does Rac (1, 2, and 3)!55. The best characterized of these isoforms are

RhoA, and Rac1 (Cdc42 is also well described)!58.

RhoA activation induces contractile actin-myosin filaments called stress fibres!58!54!55!61.

Stress fibres are linked to the cytoplasmic tails of integrins found in focal adhesions, and

play roles in migration, cell contraction, and would healing, which requires a coordinated

response between neighbouring cells!62!54. The production of stress fibres follows a fairly

well-defined pathway: GTP bound RhoA activates rho associated coiled-coil containing

protein kinase (ROCK), which phosphorylates several substrates, notably myosin II, and

myosin light chain phosphatase (MLCP)!62!63!58. Myosin II can also be activated by the

action of myosin light chain kinase (MLCK) 62 63 58. Thus myosin II is activated in three

ways: (1) phosphorylation of MLCK relieves MLCK’s inhibitory effect on myosin II and

causes MLCK to phosphorylate myosin II, (2) phosphorylation of MLCP, which inhibits

the phaosphatase activity of MLCP, allowing myosin II to remain active, and (3) the

direct phosphorylation of myosin II by ROCK!63. Phosphorylation of myosin II stimulates

19

Cytoplasm

ECM

small GTPase

GDP

GDI

GDI

small GTPase

GDP

small GTPase

GTP

GDI

GEF

GAP

Figure 3. Small GTPase activation cycle. Upon an external stimulus, inactive small GTPases (bound to GDP), which are maintained in an inactive con!guration by speci!c GDIs (guanine nucleotide dissociation inhibitors) and sequestered in the cytosplasm, are released from their GDI and targeted to the membrane through the exposed lipid moiety. After localization in the membrane, the activation process is completed when the GDP is exchanged for GTP, which is governed by specifc GEFs (guanine nucelotide exchange factors). The reverse process is guided by speci!c GAPs (GTPase activating proteins), and then the re-addition of GDIs.

E"ectors

Cytoskeleton Rearrangement

MotilityWould healing

Phagocytosis

Mitosis

Bacterial invasion

20

!

it to bind to filamentous actin, which enhances the ATPase activity of the motor, leading

to the bundling of actin fibres (this motor is an excellent cross-linker of actin), and

contraction!63!64!21!65. Myosin II has been implicated in macropinocytosis and

phagocytosis, and we know that it is involved in FLp invasion!63!21!10. Active myosin II is

recruited to membrane wraps, and inhibiting it prevents the elongation of wraps without

causing a retraction!10. The force generated by stress fibres may facilitate invasion by

driving membrane rearrangement, or could help to “close” membrane wrap structures in

the same way that it helps to close wounds in the epithelium!62!21.

Several other proteins downstream of RhoA also participate in actin reorganization. mDia

are members of the formin family, and are actin filament nucleators and elongators which

are activated by both RhoA, and ROCK, and may cooperate with ROCK in the formation

of stress fibres!63!66.

The activation of Rac1 leads to the production of lamellipodia!54!55!61. Cdc42 activation

leads to expression of filopodia at the cell surface!54!55!61. One protein which contributes

to these structures is Arp2/3!31!67. Rac1 and Cdc42 can both activate Arp2/3, through

different intermediate effectors!21. Arp2/3 is an actin filament nucleator which forms new

branches of filamentous actin off of existing f-actin!67. It is also involved in actin

polymerization and reorganization, including the production of membrane ruffles, and

thus possibly hooks and MWs!67!21. Cdc42 is also able to activate formins!39. Due to their

ability to produce structures (filopodia and lamellipodia) that look similar to structures

we known are associated with FLp invasion, we postulate a role for them in FLp invasion.

21

!

The interplay between the small GTPases is complex; an abundance of crosstalk between

them tends to make their study difficult!68. The proteins may cooperate or up-regulate one

another, or they may antagonize one another!68. There are also temporal and spatial

relationships with regard to their activation and affects!68. For example, many GEFs can

activate multiple small GTPases!68. RhoA can inhibit Rac1, and Rac1 can inhibit RhoA,

but they are also capable of activating each other under certain conditions!68. This

illustrates the intricacies involved in these systems, as well as the need for further study

in this area.

All of these small GTPases have been reported as to having a role in bacterial invasion!61.

They may either be activated directly or indirectly by bacterial toxins, or through a more

passive activation through the binding of receptors such as integrins or cadherins at the

host cell surface!58!69!20!34!45!22!48!23!56. Enteropathogenic E. coli inject effectors that

activate a GEF which activates RhoA, as well as effectors that stimulate cdc42 activation

and filopodia production!34!69. Salmonella also mediate their own uptake by activating

Rho-family GTPases, as do Shigella!45!21!22!23. L. monocytogenes is able to invade

following binding to E-cadherin, which triggers a signal cascade including both Rac1 and

Arp2/3!20. Similarly, the binding of Y. enterocolitica to !1-integrin on the host cell

triggers uptake via Cdc42 and downstream effectors!48. The binding of !1-integrin and E-

cadherin by FLp may likewise cause changes to the actin cytoskeleton resulting in

bacterial uptake!50!70!71!72. While it is clear that L. pneumophila is capable of manipulating

some host cell small GTPases once it is internalized (particularly those involved in

22

!

vesicle trafficking, no toxins have been identified to date that affect RhoA, Rac1, or

Cdc42!57.

1.8 Aims

Most of the surface of the lung is composed of epithelial cells, and previous research in

our lab has proven that FLp can invade human LECs, and may in fact be more efficient

intracellular reproducers!10. Additionally, many other bacterial species are capable of

filamentation, so studying the mechanisms by which bacteria of this morphology are

taken up could prove relevant to other pathogens and diseases.

Given the role that actin plays in FLp invasion, and the ability of small GTPases to

modify actin structures, the goal of the project was to investigate the role of the small

GTPases RhoA, Rac1, and Cdc42 in the invasion of LECs by FLps. Specifically we

sought to understand the underlying molecular mechanisms behind the formation of both

hooks and wraps.

We know that the initial attachment of FLp occurs via !1-integrin and E-cadherin on the

host cell filopodia. We postulate that binding to these receptors activates rho family small

GTPases such as Cdc42, RhoA and Rac1, which activate downstream actin nucleators.

These effectors stimulate actin rearrangement and actin polymerization, triggering wrap

formation. Wraps entrap FLp at the host cell surface, and internalization of the entrapped

FLp eventually occurs, also possibly through the action of small GTPases (Figure 4).

23

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!

2.0 MATERIALS AND METHODS

2.1 Bacterial Strains and cultivation

Legionella pneumophila strain Lp02 constitutively expressing RFP (Lp02-RFP) or Lp02

∆dotA (T4SS mutant) were used in this study, originally obtained from M.S. Swanson

and A. Ensminger respectively. Bacteria from frozen glycerol stocks were streaked onto

buffered charcoal-yeast extract (BCYE) agar and grown at 37°C and 5% CO2. To obtain

inocula enriched in the filamentous form of L. pneumophila, bacteria were harvested

from plates after growth for 3-4 days and cultured in buffered yeast extract (BYE) broth

under low agitation conditions (100 r.p.m) at 37°C. After 24 hours these pre-cultures

were sub-cultured at OD600 of 0.05 and incubated in fresh BYE media under the same

conditions for about 16 hours to get post-exponential phase bacteria with an OD600 of

between 2-3. Throughout this study, bacteria longer than 5.0!m were considered

filamentous.

2.2 Cell Culture, Constructs and Transfections

Human alveolar epithelial cells, NCI-H292 (type II pneumocyte)(CRL-1848), were

purchased from American Tissue Culture Collection (ATCC). Cells were maintained in

RPMI-1640 media supplemented with 10% FBS (Wisent, Quebec) at 37°C in 5% CO2

and passed at around 80-90% confluency. Around 36 hours before infection, cells were

seeded onto glass coverslips in 24-well tissue culture treated plates at a density of around

25

!

1-1.5x105 cells/mL. If necessary, approximately 18-24 hours before Legionella infection

(epithelial cells at around 50% confluency), transfections were performed as per the

manufacturer’s instructions using Mirus TransIT 2020 transfection reagent (Mirus Bio

LLC), or FuGENE HD Transfection Reagent (Roche Diagnostics). Briefly, the following

were added per well: 25!L RMPI-1640 (FBS free), 0.75!L of transfection reagent, and

1!g of plasmid DNA. The transfection reaction was incubated for 20 minutes at room

temperature before being added to cells (25!L/well). Cells were then returned to normal

growth conditions.

The constructs used were as follows: PM-GFP encoding the

myristoylation/palmitoylation sequence from Lyn fused to GFP in pcDNA3 vector

(generously provided by Dr. Sergio Grinstein, The Hospital for Sick Children, Toronto,

ON, Canada), Rac1WT-GFP and Rac1DN-GFP (N17) in peGFP-N3 (from Dr. Sergio

Grinstein, The Hospital for Sick Children, Toronto, ON, Canada), cdc42WT-GFP and

cdc42DN-GFP (generously provided by Dr. Katalin Szaszi, St. Michael’s Hospital,

Toronto, ON, Canada), RhoAWT-GFP and RhoADN-GFP (from Dr. Sergio Grinstein,

The Hospital for Sick Children, Toronto, ON, Canada), GFP-rGBD in pCS2+ (purchased

from Addgene, cloned by Dr. K. Burridge, University of North Carolina at Chapel Hill,

North Carolina, USA), YFP-PAK-PBD (Dr. Katalin Szaszi, St. Michael’s Hospital,

Toronto, ON, Canada) . Myosin X-GFP, Myosin X FERM-GFP, Myosin X TAIL-GFP

(generously provided by Dr. Richard E. Cheney, University of North Carolina, Chapel

Hill, North Carolina, USA).

26

!

2.3 Bacterial Infections

Aliquots of Lp02-RFP from the 16 hour (post exponential) BYE culture were obtained,

and pelleted down at 6000g for 5 minutes at room temperature. The bacterial pellet was

then washed with PBS and pelleted two more times. Lastly, the pellet was resuspended in

PBS so that the post exponential phase bacteria were used at OD600 2. Before infection,

the prepared NCI-H292 cells (confluency 70-90%) were washed three times with PBS,

then fresh RPMI-1640 (without 10% FBS) was added back to the wells. Following the

addition of any necessary inhibitors, the bacterial cells were then added to the NCI-H292

cells at an MOI of between 125-500 (depending on the experiment). The experiment was

then incubated for the required time at 37°C in 5% CO2. Following completion of the

experiment, cells were washed three times with PBS to remove any FLp that were not

attached to the host cells, then fixed with 4% paraformaldehyde diluted in PBS for 20

minutes, in preparation for antibody staining.

2.4 Pharmacological Inhibitions

Experiments using pharmacological inhibitors required pre-treatments for some assays

but not for others. If relevant, pre-treatments have been indicated in results. Control

experiments were carried out using reagent vehicles. Treatments were performed in the

following conditions: bradykinin (100ng/mL, serum-free media)(Tocris), ML 141

(20!M, serum-free media)(Tocris), NSC 23766 (50!M, serum-free media)(Tocris), C3

transferase (0.5!g/mL, serum-free media, 2 hour pre-treatment)(Cytoskeleton), ML 7

27

!

(50!M, serum-free media)(Millipore), wiskostatin (100!M, serum-free media)(Tocris),

blebbistatin (100!M, serum-free media)(Sigma), SMIFH2 (25!M, serum-free

media)(Millipore), rho kinase inhibitor (1!M, serum-free media)(Millipore).

2.5 Antibodies and Immunostaining

Application of all antibodies and phalloidin occurred at room temperature, with gentle

shaking. Differential immunostaining was used to identify external and wrap

entrapped/internal FLp segments, which are inaccessible to antibodies without

permeabilization (Figure 5). External staining was performed as follows: All antibodies

were diluted in 5% non-fat milk in PBS. Following fixation of experimental cells with

4% PFA, cells were blocked with 5% non-fat milk in PBS. Cells were washed three times

with PBS before external staining. Anti Legionella pneumophila antibody (anti Lp01)

(Ontario Agency for Health Promotion and Protection (OAHPP)) was applied to cells at

1:5000. Cells were then washed again three times with PBS before the application of a

secondary antibody, Alexa Fluor –conjugated 647 (Life Technologies), used at 1:5000.

Internal staining was performed as follows: Cells were washed 3 times with PBS, then

permeablized with 0.1% Triton X-100 in PBS. Anti-Lp01 was then applied as for

external staining, but with secondary antibody Alexa Fluor-555 (Life Technologies).

Additionally phalloidin 488 (Life Technologies) was used to label actin (1:500). After

completion of staining, cells were mounted on microscopy slides using Dako Fluorescent

Mounting Medium (Dako).

28

Attachment Assay (1 hour infection)

Count FLp per host cell

Internalization Assay (6 hour infection)

Measurement of internalized portion

Measurement of two separate internalized portions

Figure 5. Experimental methodology. (A) For attach-ment assays, FLp were incubated with LECs for 1 hour to allow attachment, followed by immunostaining with primary antibody against L. pneumophila, then labelled (Alexa-Fluor 647) secondary antibody. FLp/cell was then assessed via !uorescence microscopy. (B) For internal-ization assays, FLp were incubated with LECs for 1 hour to allow attachment, then washed to remove excess FLp. The infection was then allowed to proceed for a further 5 hours. Primary and secondary antibody were then applied as in (A). Following this, which labelled external segments of FLp, cells were permeablized, and the primary antibody was applied again. Secondary antibody with a di"erent tag (Alexa-Fluor 555) was then applied, labelling internalized portions (either wrap entrapped, or in the cel body) of the FLp. The length of internalized FLp segments could then be measured following !uorescent microsocpy. Note that multiple FLp segments could be internalized along the same #lament. In such cases, the segments were measured as two separate events.

29

!

For small GFP-GTPase-expressing cells, cells were mounted following the external

secondary antibody (no internal staining), as application of a detergent to permeabilize

cells could potentially alter small GTPase localization.

2.6 Attachment and internalization Assays

For experiments assessing attachment, cells were transfected with the proteins of interest

and/or treated with inhibitors in RPMI-1640 (without serum) for the indicated periods at

37°C in 5% CO2 . Following this treatment cells were exposed to FLp inocula at 37°C in

5% CO2 for 1 hour, washed 2 times to remove free bacteria, fixed with 4% PFA and

stained as described above.

To assess the effect of inhibitors on the lengths of bacteria internalized, we allowed FLp

to attach for 1 hour prior to applying the inhibitors, then incubated for an additional 5

hours to allow the formation of wraps. Wrap progression was assessed via differential

immunostaining (as described above).

2.7 Localization Assays

To characterize the localization of the small GTPases in infected cells, we used LECs

transfected with small GTPase constructs, and then infected them with FLp. Cells were

30

!

placed at 37°C in 5% CO2 for 2 hours, followed by gently washing with PBS (two times),

and fixation and staining as described above.

2.8 Spinning disk fluorescence microscopy

Confocal images were acquired using a spinning disc confocal microscope (Quorum

Technologies) consisting of an inverted fluorescent microscope (DMI6000B; Leica)

equipped with an EM-CCD camera (Hamamatsu Photonics), ORCA-R2 cameras and

spinning disc confocal scan head, as well as an ASI motorized XY stage, and a Piezo

Focus Drive (Quorum Technologies). The equipment was controlled by MetaMorph

acquisition software (Molecular Devices). Imaging was either with 40x or 64x oil

immersion objective, 1.3 NA. Image processing and analysis were performed using

Volocity software (PerkinElmer). Unless indicated otherwise, images shown are merged

z-planes.

2.9 Scanning electron microscopy

Cells were fixed after 6 hour timepoints as in the internalization inhibitors assay. Cells

were fixed using 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2),

followed by post-fixation in 1% OsO4 for 1 hour. Cells were dehydrated in ethanol and

sputter-coated with gold. Images were acquired using a scanning electron microscope

(JSM 820; JEOL Ltd). Detailed descriptions of SEM procedures can be found elsewhere!73.

31

!

2.10 Quantification and Statistical Analysis

The attachment efficiency of FLp to treated and untreated cells was determined by

assessing the numbers of attached bacteria divided by the total number of host cells found

on microscopy fields (40X) acquired randomly from 1mm coverslips with host cells

labeled with phalloidin. Approximately 42 fields, representing at least 800 host cells,

were counted for each treatment or control in each repetition of the experiments. Data

was processed utilizing the software GraphPad Prism, with data shown being mean

±SEM from three independent experiments unless stated otherwise. Statistical analysis

was performed using a two-tailed, Student’s t test. A 90% confidence interval was used to

determine statistical significance and P≤0.05 was considered to be statistically

significant.

To assess attachment in transfected cells, transfected cells (expressing GFP) were found

at random (without looking at FLp wavelengths), and then the number of FLp attached to

the cell was counted. At least 100 transfected host cells were counted per coverslip in the

experiment.

For the assessment of internalization, random images of FLp invading inhibited, vehicle-

treated, or transfected host cells were acquired, and each internalized portion of the FLp

(as indicated via differential immunostaining) was measured using Volocity software.

Each internalization event was considered independent, even if several internalized

32

!

portions could be found along the length of a single bacterial filament (Figure 5). The

internalized lengths in treated versus control cells were graphed using GraphPad Prism.

Data shown represent mean from three independent experiments unless otherwise stated.

Statistical analysis was performed using a two-tailed, Student’s t test, using GraphPad

Prism software. A 90% confidence interval was used to determine statistical significance

and P≤0.05 was considered to be statistically significant.

33

!

3.0 RESULTS

3.1 Small GTPases accumulate at FLp invasion sites

Although we knew that actin rearrangements are required for FLp invasion, the particular

small GTPases involved in the process had not yet been determined19. Therefore, to

determine which small GTPases were involved, NCI-H292 cells expressing either RhoA-

GFP, Rac1-GFP, Cdc42-GFP or the plasma membrane marker PM-GFP were infected

with cultures of FLp for two hours to examine the distribution of the small GTPases

around the internalized regions of the bacteria. The expression of PM-GFP allowed the

detection of membrane wraps. Through the analysis of confocal imaging we observed

that 80% of the FLp attached to the epithelial cells form wraps, which is consistent with

previous findings in our lab and therefore validates our experimental methodologies and

conditions (Figure 6A,B). Importantly, RhoA Rac1 and Cdc42 were recruited to the site

of bacterial attachment and internalization (Figure 6A). Utilizing GFP-rGBD, which

binds to active RhoA , and YFP-PAK-PBD which labels active Cdc42 and Rac1, we

determined that the small GTPases recruited to the site of entry of the bacteria are in

active states (Figure 6A,D,F). The recruitment of all the constructs occurred either as

discrete tubes surrounding the internalized portion of the FLp, or, more commonly, as a

patch of fluorescence in the region of the internalized portion (Figure 6B-G).

3.2 Cdc42 and myosin X mediate Filamentous Legionella pneumophila attachment

through filopodia formation

34

PM-GFPExternal FLpInternal FLp

RhoAWT-GFPExternal FLpInternal FLp

A

B

C

Figure 6.

35

Cdc42WT-GFPExternal FLpInternal FLp

Rac1WT-GFPExternal FLpInternal FLp

YFP-PAK-PBDExternal FLpInternal FLp

GFP-rGBDExternal FLpInternal FLp

Figure 6. Small GTPases accumulate at the site of FLp invasion. (A) Transfected cells were infected with FLp and analyzed for accumulation of the indicated proteins. NCI-H292 cells were !xed 2 hours after infection with Lp02-RFP to allow membrane wrap formation to occur, followed by staining for external L. pneumophila. Internalized portion of !lamentous bacterial cells was analyzed for an increased presence of small GTPases compared to the surrounding cell body. Numbers indicate the proportion of events that had an accumulation. (B-G) Fluorescence micrographs showing the accumulation of PM-GFP, RhoAWT-GFP, GFP-rGBD (localizes to active RhoA), Cdc42WT-GFP, YFP-PAK-PBD (localizes to active Cdc42 and Rac1), and Rac1WT-GFP respectively around attached FLp in NCI-H292 cells. Accumulations occured either as di"use clouds above the internalized portion of FLp, or as more de!ned tubes.

DE

F G

36

!

Since active Cdc42 localizes to FLp invasion sites, we investigated its role in the initial

attachment of the bacteria to NCI-H292 cells. Following a 1 hour pre-treatment of lung

cells with the allosteric inhibitor of Cdc42!74 , ML 141, there was a significant reduction

in the attachment of FLp to host cells, as compared to vehicle-treated NCI-H292 cells

(Figure 7A).

The initial attachment of FLp is mediated by hooks!10. Morphological and molecular

mechanisms indicate that hooks are filopodia attached to the bacteria (probably via

receptors expressed in this structure). Cdc42 stimulates the formation of filopodia, and

ML141 treatment is reported to reduce filopodia!74. The SEM image from Figure 8D

confirmed this finding, as cells treated with the inhibitor appeared much smoother, with

considerably fewer filopodia. Cells treated for 2 and 5 hours with ML 141 and stained for

actin also qualitatively appeared to exhibit fewer, shorter filopodia (Figure 7B, 8C).

Altogether this indicates that the reduction in FLp attachment by ML141 could be caused

by impairing filopodia formation.

In an attempt to confirm this results using alternative methods, we transfected NCI-H292

cells with Cdc42-GFP and Cdc42DN-GFP constructs, and compared attachment of FLp.

As in the case of ML 141, cells transfected with dominant-negative Cdc42 have fewer

dorsal filopodia (Figure 7D) and showed a decrease in attachment in the DN transfected

cells compared to the wild-type cdc42 cells (Figure 7C).

37

Control ML 1410.0

10

20

30

40

50 *

cdc42WT cdc42DN0

10

20

30

40

50

60

control

ActinPlasma MembraneDAPI

ML 141

ActinPlasma MembraneDAPI

ActinCdc42WT-GFPDAPI

ActinCdc42DN-GFPDAPI

A B

C

D

Figure 7. Inhibition of Cdc42 affects FLp attachment. (A) NCI-H292 cells were pre-treated with

ML 141 to inhbit Cdc42 function, then infected with FLp for one hour, followed by fixation, staining,

and assessment of attachment. Data shown represent mean ± SEM (B) NCI-H292 cells were treated

with a vehicle control or ML 141 for 2 hours (to mimic pre-treatment and infection time) without being

infected, followed by fixation and staining with phalloidin to visualize actin structures (including filopo-

dia). Cells appear slightly collapsed, and the number and/or structure of stress fibres and filopodia

are abnormal. (C) NCI-H292 cells were transfected with wild type (WT) or dominant negative (DN)

constructs of Cdc42 and infected with FLp for 1 hour, followed by fixation, staining, and assessment

of attachment. (D) NCI-H292 cells expressing the DN Cdc42 construct appeared to have fewer

dorsal filopodia than those expressing the WT construct following fixation, staining, and microscopic

analysis.

FLp/

100

Hos

t Cel

ls

38

!

Considering that filopodia are possibly the initial attachment sites of FLp, due to the fact

that they protrude upwards from the cell surface, and as they possess FLp receptors, it

makes sense that reducing the number of filopodia by knocking down Cdc42 would

reduce the number of attachment sites, leading to less FLp binding. Conversely, we

postulated that increasing filopodial number might provide more binding sites, and thus

greater FLp attachment. To test this hypothesis, we utilized bradykinin, which is known

to activate Cdc42 and stimulate filopodia production in a variety of cell types!75. Upon

treatment with bradykinin, lung cells exhibited increased numbers of filopodia compared

to vehicle-treated controls (Figure 9B) and significantly higher binding of FLp than cells

treated with a vehicle control (Figure 9A). Together, this supported our hypothesis that

Cdc42 mediates binding of FLp though the formation of filopodia, and subsequently

hooks at the FLp attachment site.

Myosin X is an unconventional motor protein that localizes to filopodial tips, and can

potentially carry FLp receptors to those tips!39!38!37. It has also been shown to induce

filopodia formation!42!40. Since our previous results suggest that filopodia could be the

initial attachment site of FLp, we postulated a role for myosin X in the attachment of FLp

to NCI-H292 cells. In order to evaluate its role, we transfected cells with either Myosin

X-GFP (Figure 10C), a ∆FERM myosin X (missing part of the FERM domain, unable to

carry receptors), or a ∆TAIL myosin X (missing the PH domain, the MyTH4 domain, and

the FERM domain, unable to induce filopodia or carry receptors)!39!33. We found that

cells transfected with MyoX-GFP had significantly higher binding than control, MyoX-

39

Control Bradykinin 0

5

10

15

20

25

30

35*

control Bradykinin

A

B

Actin Actin

Figure 9. Pretreatment of cells with bradykinin increases attachment of FLp and increases number of filopodia. (A) NCI-H292 cells were pre-treated with bradykinin, then infected with

aliquots of FLp for one hour, followed by fixation, staining, and assessment of attachment. Data

shown represent mean ± SEM. (B) NCI-H292 cells were treated with a vehicle control or bradyki-

nin for 2 hours (to mimic pre-treatment and infection time) without being infected, followed by

fixation and staining with phalloidin to visualize actin.

FLp/

100

Hos

t Cel

ls

40

!

FERM-GFP, or MyoX-TAIL-GFP. The MyoX-TAIL mutant had significantly lower

binding than control (Figure 10A).

Taken together, these results suggest that it is both the presence of additional filopodia,

and the presence of receptors, that help mediate FLp attachment. The inability of the

TAIL mutant to facilitate attachment could be due to its lack of filopodia promotion, or

the lack of receptors. The FERM mutant essentially creates filopodia without carrying

receptors, but has similar levels of attachment to the control, which would suggest that

the receptors are not as important as the physical presence of filopodia. However, the WT

protein, which is fully functional, has a higher level of attachment than the control (and

the FERM mutant), suggesting that in addition to just the presence of filopodia, the

presence of receptors carried by the protein are also important.

3.3 Role of Rac1 in Filamentous Legionella pneumophila attachment

Since active Rac1 localizes to FLp invasion sites, we investigated its role in the initial

attachment of the bacteria to NCI-H292 cells. Following a 2 hour pre-treatment of lung

cells with NSC 23766 (Figure 11B), which inhibits Rac1 by preventing GEF binding!76,

there was a significant reduction in the attachment of FLp to host cells, as compared to

vehicle-treated NCI-H292 cells (Figure 11A).

Attachment of FLp is mediated initially by hooks, but also by membrane wraps, which

appear similar to lamellipodia!10. Rac1 is involved primarily in lamellipodia formation in

41

MyoX-WT-GFPActin

Figure 10. Myosin X affects attachment of FLp to host cells, but not internalization. (A-B) NCI-H292 cells were transfected with GFP

(control), MyoX-WT, or a MyoX-mutant (TAIL or

FERM) construct and infected with aliquots of

FLp for 1 (A) or 6 (B) hours. Following fixation

and staining, the attachment to or internalization

in transfected host cells was measured. Data

shown in (A) are mean ± SEM. (C) Localization

of Myosin-X at the tips of actin rich filopodia

(red). Enlarged images of framed areas are

VKRZQ�WR�WKH�ULJKW��6FDOH�EDU����ȝP��

C

A

B

42

Control NSC0.00

2

4

6

8

10

12

14

16

18

20

22

24

26*

Rac1WT Rac1DN0

10

20

30

40

control

ActinPlasma MembraneDAPI

ActinPlasma MembraneDAPI

NSC

A B

C

D

Figure 11. Inhibition of Rac1 affects FLp attachment. (A) NCI-H292 cells were pre-treated with

NSC 23766 to inhbit Rac1 function, then infected with FLp for 1 hour, followed by fixation, stain-

ing, and assessment of attachment. Data shown represent mean ± SEM. (B) NCI-H292 cells

were treated with a vehicle control or NSC for 3 hours (to mimic pre-treatment and infection time)

without being infected, followed by fixation and staining with phalloidin to visualize actin struc-

tures. Actin structures do not differ appreciably from control cells. (C) NCI-H292 cells were trans-

fected with wild type (WT) or dominant negative (DN) constructs of Rac1 and infected with FLp for 1 hour, followed by fixation, staining, and assessment of attachment. (D) NCI-H292 cells

expressing the DN Rac1 construct appeared similar to those expressing the WT construct follow-

ing fixation, staining, and microscopic analysis, although actin structures appeared less defined.

ActinRac1WT-GFPDAPI

ActinRac1DN-GFPDAPI

FLp/

100

Hos

t Cel

ls

43

!

many cells types, although it can also play a role in filopodia formation!61!55!54. Since

lamellipodia can also form between adjacent filopodia, it is also possible that wraps

forms between hooks!36. Thus Rac1 has several possible roles in FLp attachment. When

Rac1 is inactivated by NSC 23766, treatment it has been shown that there are less

lamellipodia!76. Thus the binding impairment that we see when Rac1 is inhibited could be

due to lack of appropriate wrap formation.

We also transfected NCI-H292 cells with Rac1DN-GFP (Figure 11D) and measured

attachment. Rac1DN-GFP cells appeared to have lower binding of FLp than those

transfected with the wild-type version of the protein (Figure 11C), echoing the results

with the pharmacological inhibitor. Thus, Rac1 appears to be involved in the attachment

of FLp to host cells, although whether this is due to hook or membrane wrap formation is

unclear.

3.4 Role of RhoA in Filamentous Legionella pneumophila attachment

Since active RhoA localizes to sites of FLp invasion, we investigated its role in bacterial

attachment. Following a 2 hour pre-treatment of host cells with C3 transferase, which

inhibits RhoA by ADP-ribosylation in the effector binding region of the small GTPase!77,

there was not a significant difference in attachment of FLp compared to vehicle-treated

control cells (Figure 12A).

44

Control C30.00

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

RhoAWT RhoADN0

5

10

15

20

25

ActinPlasma MembraneDAPI

control

ActinPlasma MembraneDAPI

C3

ActinRhoAWTDAPI

ActinRhoADNDAPI

A B

C

D

FLp/

100

Hos

t Cel

ls

Figure 12.

45

Figure 12. Inhibition of RhoA function does not affect FLp attachment. (A)

NCI-H292 cells were pre-treated with C3 transferase to inhibit RhoA function, then

infected with FLp for 1 hour, followed by fixation, staining, and assessment of attach-

ment. Data shown are mean ± SEM. (B) NCI-H292 cells were treated with a vehicle

control or C3 for 3 hours (to mimic pre-treatment and infection time) without being

infected, followed by fixation and staining with phalloidin to visualize actin structures

(including stress fibres). Cells appear collapsed, and the number and structure of

stress fibres appears abnormal. (C) NCI-H292 cells were transfected with wild type

(WT) or dominant negative (DN) constructs of RhoA and infected with FLp for 1 hour,

followed by fixation, staining, and assessment of attachment. (D) NCI-H292 cells

expressing the DN RhoA construct appeared to have fewer stress fibres than those

expressing the WT construct following fixation, staining, and microscopic analysis.

46

!

We found that NCI-H292 cells treated for 3 hours with the RhoA inhibitor C3 transferase

had fewer stress fibers and tended to be rounder than control cells (Figure 12B). Thus it

was surprising that our results suggest that RhoA does not have a role in FLp attachment.

Consistent with this, however, we found that NCI-H292 cells transfected with a dominant

negative form of RhoA (Figure 12D) exhibited similar binding of FLp to cells transfected

with a wild-type form of RhoA, (Figure 12C). This was unexpected considering that we

saw active RhoA recruited to the FLp invasion site. Since we knew that RhoA was active,

we sought to determine if the downstream effectors of RhoA were active, and if they

contributed to attachment.

Specifically, we explored Rho kinase (ROCK), Myosin light chain kinase (MLCK), and

myosin II. Rho-associated protein kinase (ROCK) is a downstream effector of RhoA, and

helps to form stress fibres!58!62!63. We inhibited ROCK with an ATP-competitive Rho

kinase inhibitor!78. Pre-treatment of host cells for 1 hour with this inhibitor significantly

abrogated FLp attachment to cells (Figure 13A-B).

Myosin light chain kinase is downstream of ROCK (and thus downstream of RhoA)!63!62!58. It is involved in contraction of stress fibres!64, and thus is probably involved in

bacterial attachment. We inhibited MLCK with the ATP-competitor ML 7!79. Pre-

treatment of host cells for 1 hour with ML 7 resulted in a severe defect in FLp attachment

(Figure 14A).

47

Control ROCK Inhibitor

0.00

2

4

6

8

10

12

14

16

18*

Control ROCK Inhibitor

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

ActinPlasma MembraneDAPI

ROCK Inhibitorcontrol

ActinPlasma MembraneDAPI

ROCK Inhibitor

ActinPlasma MembraneDAPI

control

ActinPlasma MembraneDAPI

A

B

C

D

Figure 13. Attachment of FLp at 1 hour, but not internalization, is affected by ROCK inhibition. (A) NCI-H292 cells were pre-treated with ROCK inhibitor, then infected with FLp for one hour, followed by fixation, staining, and assessment of attachment. Data shown represent mean ± SEM. (B) NCI-H292 cells were treated with a vehicle control or inhibitor for 2 hours (to mimic pre-treatment and infection time) without being infected, followed by fixation and staining with phalloidin to visualize actin structures. Cells have significantly fewer stress fibres. (C) NCI-H292 cells were exposed to aliquots of FLp for one hour, then washed and exposed to inhibitor, then incubated for a further 5 hours, followed by fixation, staining, and microscope analysis. The length of FLp fragments internalized was not significantly different. Red lines indicate means. (D) NCI-H292 cells were treated with inhibitor for 5 hours, but left uninfected. Following fixation and staining, actin structures within the cell were observed to be perturbed. Cells have fewer stress fibres, and exhibited less spreading, and appeared more disorganized.

FLp/

100

Hos

t Cel

ls

48

Control ML70.00

2

4

6

8

10

12

14

16

18 *

Control ML70

5

10

15

20

25

30

35

40

45

Figure 14. Attachment of FLp at 1, but not internalization, is affected by MLCK inhibition. (A) NCI-H292 cells were pre-treated with ML7 to inhibit MLCK, then infect-

ed with FLp, followed by fixation, staining, and assessment of attachment. Data

shown represent mean ± SEM. (B) NCI-H292 cells were exposed to aliquots of FLp for one hour, then washed and exposed to inhibitor, then incubated for a further 5

hours, followed by fixation, staining, and microscope analysis. The length of FLp

fragments internalized was not significantly different between vehicle treated controls

and ML 7 treated cells. Red lines indicate means.

A B

FLp/

100

Hos

t Cel

ls

49

!

Myosin II is a downstream target of RhoA acting through the preliminary effectors

ROCK and MLCK!63!62!58. It is involved in the formation (via bundling of actin filaments)

and contraction of stress fibres, as well as being involved in the remodeling of cell-cell

contacts!63!80!21!64!65. We inhibited myosin II with blebbistatin, which inhibits stress fibre

contraction. Pre-treatment of host cells for 30 minutes resulted in significantly decreased

attachment of FLp at 1 hour (less than half of control) (Figure 15A).

Together these results suggest that the proteins downstream of RhoA are involved in

promoting FLp attachment, perhaps through the formation and contraction of stress fibres

which help manipulate actin to form wraps, or perhaps through the action of filopodia,

which we also found to be absent in inhibited cells. The fact that C3 treatment did not

affect attachment could imply that the bacteria is releasing an effector toxin that activates

the downstream proteins independently of RhoA, or is activating RhoA despite the

presence of the inhibitor (thus making RhoA insensitive to C3).

3.5 Role of actin nucleators mDia and Arp2/3 Filamentous Legionella pneumophila

attachment

Some of the main actin nucleators in cells are members of the mDia family, which

elongate actin filaments in various structures such as filopodia, and participate in stress

fibre formation, among other roles!39!63!81!82. Formins such as mDia are downstream of

Cdc42 (as well as RhoA)!39!63!66. Since we found Cdc42 at FLp invasion sites, we

postulated that active formins might also accumulate there, and play a role in FLp

50

Control Blebbistatin0.00

2

4

6

8

10

12

14

16*

Control Blebbistatin0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5

40.0

42.5

control

ActinPlasma MembraneDAPI

ActinPlasma MembraneDAPI

Blebbistatin

control

ActinPlasma MembraneDAPI

ActinPlasma MembraneDAPI

Blebbistatin

Figure 15. Attachment of FLp at 1 hour, but not internalization, is affected by myosin II inhibition. (A)

NCI-H292 cells were pre-treated with blebbistatin to inhibit myosin II, then infected with FLp, followed by

fixation, staining, and assessment of attachment. Data shown represent mean ± SEM. (B) NCI-H292 cells were

treated with a vehicle control or inhibitor for 1.5 hours (to mimic pre-treatment and infection time) without being

infected, followed by fixation and staining with phalloidin to visualize actin structures. Cells appeared collapsed,

with thick, disorganized actin-based protrusions throughout the cells. (C) NCI-H292 cells were exposed to

aliquots of FLp for one hour, then washed and exposed to inhibitor, then incubated for a further 5 hours,

followed by fixation, staining, and microscope analysis. The length of FLp fragments internalized was not

significantly different in control versus treated cells. Red lines indicate means. (D) NCI-H292 cells were treated

with inhibitor for 5 hours, but left uninfected. Following fixation and staining, actin structures within the cell were

observed. Cells were characterized by decreased cell spreading and shorter stress fibres.

A

B

C D

FLp/

100

Hos

t Cel

ls

51

!

invasion. We were able to inhibit mDia1 and mDia2 with SMIFH2 (small molecule

inhibitor of formin homology 2), which has been shown to inhibit the actin

polymerization activity of the mDias!81!82.

Following pretreatment of host cells with SMIFH2 for 2 hours, the attachment of FLp to

SMIFH2-treated cells was much less (about one third) as that of control cells (Figure

16A). This suggests that the actin-polymerization activity of the mDias is required for

FLp attachment.

Arp2/3 helps create filopodia and lamellipodia!67!21, which are related to hooks and wraps

respectively, and thus likely to FLp attachment!10. Various studies have reported Arp2/3

to be downstream of both Cdc42 and Rac1!67!21. Since we found the active form of both

of these small GTPases at the invasion site of FLp, we reasoned that Arp2/3 could also

contribute to hook and wrap formation. Thus we wished to study its role in FLp invasion.

To inhibit Arp2/3, we used wiskostatin, which has been shown to inhibit actin assembly!83!84.

Cells treated with wiskostatin for 1.5 hours appeared to have fewer and shorter filopodia,

and cells had a more stretched appearance. Cells also appeared to have an under-

developed actin network compared to vehicle-treated controls (Figure 17B). Pre-

treatment with wiskostatin for 30 minutes almost fully prevented the attachment of FLp

to host cells (Figure 17A). Together this suggests that the inhibition of binding upon

52

Control SMIFH2

0.00

2

4

6

8

10

12

14

16*

Control SMIFH2

0

3

6

9

12

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24

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control

ActinPlasma MembraneDAPI

ActinPlasma MembraneDAPI

SMIFH2

ActinPlasma MembraneDAPI

SMIFH2

ActinPlasma MembraneDAPI

CD

B

A

Figure 16. Attachment of FLp at 1 hour, but not internalization, is affected by mDia

inhibition. (A) NCI-H292 cells were pre-treated with SMIFH2 to inhibit mDia, then infected

with FLp, followed by fixation, staining, and assessment of attachment. Data shown repre-

sent mean ± SEM. (B) NCI-H292 cells were treated with a vehicle control or inhibitor for 3

hours (to mimic pre-treatment and infection time) without being infected, followed by fixa-

tion and staining with phalloidin to visualize actin structures. Cells looked similar to control

except for occasional blebbing. (C) NCI-H292 cells were exposed to aliquots of FLp for

one hour, then washed and exposed to inhibitor, then incubated for a further 5 hours,

followed by fixation, staining, and microscope analysis. The length of FLp fragments inter-

nalized was not significantly different in vehicle control versus inhibited cells. Red lines

indicate means. (D) NCI-H292 cells were treated with inhibitor for 5 hours, but left uninfect-

ed. Following fixation and staining, actin structures within the cell were observed.

FLp/

100

Hos

t Cel

ls

control

53

Control Wiskostatin0.00

2

4

6

8

10

12

14

16*

Control Wiskostatin0.0

2.5

5.0

7.5

10.0

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20.0

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25.0

27.5

30.0

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35.0

37.5

40.0

42.5

control

ActinPlasma MembraneDAPI

Wiskostatin

ActinPlasma MembraneDAPI

control

ActinPlasma MembraneDAPI

Wiskostatin

ActinPlasma MembraneDAPI

Figure 17. Attachment of FLp at 1 hour, but not internalization, is affected by arp2/3 inhibition. (A) NCI-H292 cells were pre-treated with wiskostatin to inhibit arp2/3,

then infected with FLp for 1 hour, followed by fixation, staining, and assessment of

attachment. Data shown represent mean ± SEM. (B) NCI-H292 cells were treated with a

vehicle control or inhibitor for 1.5 hours (to mimic pre-treatment and infection time)

without being infected, followed by fixation and staining with phalloidin to visualize actin

structures. Cells appear to have fewer filopodia, and a disorganized actin network. (C)

NCI-H292 cells were exposed to aliquots of FLp for one hour, then washed and

exposed to inhibitor, then incubated for a further 5 hours, followed by fixation, staining,

and microscope analysis. The length of FLp fragments internalized was not significantly

different in vehicle control versus inhibited cells. Red lines indicate means. (D)

NCI-H292 cells were treated with inhibitor for 5 hours, but left uninfected. Following

fixation and staining, actin structures within the cell were observed.

AB

C

D

FLp/

100

Hos

t Cel

ls

54

!

Arp2/3 disruption could be due to disruption of the actin-based structures filopodia/hooks

and lamellipodia/wraps.

3.6 Role of small GTPases in Filamentous Legionella pneumophila internalization

In addition to attachment, we wanted to study the role of the small GTPases in bacterial

internalization. We were able to do this by performing longer infections, and then fixing

cells and performing differential immunostaining (Figure 5). This allowed us to

quantitatively measure the lengths of entrapped FLp segments. We know that wrap

maintenance and elongation requires actin polymerization 10, and so we reasoned that this

process could be governed by the small GTPases that we were studying.

3.7 Role of Cdc42 in Filamentous Legionella pneumophila internalization

In order to study the role of Cdc42 in bacterial internalization, we allowed FLp to attach

for one hour, then applied ML 141 and allowed the infection to proceed for a further 5

hours. We found that there was no significant difference between the lengths of FLp

internalized in control versus treated cells after a total of 6 hours of infection (Figure 8A).

In order to study wrap morphology, we also acquired SEM micrographs of infected host

cells at the 6 hour timepoint. ML 141 treated cells had very few “normal” filopodia

compared to control cells, and thus appeared much smoother (Figure 8D). The wraps

entrapping FLp had fewer filopodia/hooks associated with them, and appeared less

55

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141

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A B

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ML

141

cont

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56

!

protrusive than control wraps (they did not extend upwards from the cell surface as much

as in vehicle-treated controls) (Figure 8D).

In an attempt to replicate results using alternative methods, we also transfected NCI-

H292 cells with Cdc42-GFP and Cdc42DN-GFP constructs, and then assessed

internalization of FLp with regards to transfected cells. There was no significant

difference in the internalized lengths of FLp sections in the dominant negative Cdc42

cells compared to the wild type expressing cells (Figure 8B). Additionally, those cells

transfected with the DN construct appeared to have fewer dorsal filopodia (on top of the

cell) (Figure 7D).

Taken together, this suggests that internalization does not depend on Cdc42.

Additionally, although wrap morphology alters slightly upon cdc42 inhibition, it appears

that MW maintenance does not depend on the action of Cdc42.

3.8 Role of Myosin X in internalization of Filamentous Legionella pneumophila

For membrane wrap elongation to occur, FLp receptors must be trafficked to the

membrane!10. The mechanism by which !1-integrin is localized to the developing wrap is

unknown!10. Since myosin X is capable of carrying such receptors!38, and we found that it

has a role in bacterial attachment, we postulated a role for the protein in the elongation of

membrane wraps, and thus bacterial internalization. In order to assess a potential role for

the unconventional motor protein myosin X in internalization of FLp, we infected host

57

!

cells transfected with myosin X or mutant myosin X with FLp. Neither MyoX-WT or

MyoX-FERM affected the lengths of FLp internalized after 6 hours, but MyoX-TAIL

seemed to slightly increase internalization (although this was not confirmed through

multiple trials)(Figure 10B). Thus although myosin X helps mediate bacterial attachment,

it is not clear if it plays a role in membrane wrap elongation or internalization.

3.9 Role of Rac1 in Filamentous Legionella pneumophila internalization

We next wanted to assess the role of Rac1 in FLp internalization by infecting host cells

with bacteria for 1 hour to allow normal attachment, then adding the inhibitor NSC 23766

for a further 5 hours. We found that there was no significant difference in the lengths of

FLp internalized in host cells treated with control versus NSC 23766 at 6 hours (Figure

18A), although we found slight morphological alterations in treated cells (Figure 18C).

In agreement, we found that there was almost no difference in the lengths of the

internalized portions of FLp in dominant negative versus wild-type Rac1 transfected cells

(Figure 18B), similar to results with the pharmacological inhibitor. Additionally we did

not observe significant differences in actin morphology between DN and WT-transfected

host cells (Figure 11D).

Together these results suggest that Rac1 does not play a role in the elongation of wraps or

true internalization of FLp, although it does play a role in attachment despite insignificant

differences in host cell morphology.

58

Control NSC0

2

46

8

1012

14

1618

20

2224

26

2830

32

3436

38

4042

44

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Rac1WT Rac1DN0

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35

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ActinPlasma MembraneDAPI

ActinPlasma MembraneDAPI

NSC

A B

C

Figure 18. Internalization of FLp at 6 hours is not significantly affected by inhibition of Rac1. (A) NCI-H292 cells were exposed to FLp for one hour, then

washed and exposed to NSC, then incubated for a further 5 hours, followed by

fixation, staining, and microscope analysis. The length of FLp fragments internal-

ized was not significantly different between NSC and vehicle treated controls. Red

lines indicate mean. (B) NCI-H292 cells were transfected with either a WT or DN

construct of Rac1, then infected with FLp for a total of 6 hours. Fixation, staining,

and microscopic analysis followed. Red lines indicate mean. (C) NCI-H292 cells

were treated with NSC for 5 hours, but left uninfected. Following fixation and stain-

ing, actin structures within the cell were observed. Cells appear collapsed and the

actin cytoskeleton appears perturbed. Cells also appear elongated compared to

vehicle-treated controls.

59

!

3.10 Role of RhoA in Filamentous Legionella pneumophila internalization

Previous studies have found that RhoA activity is required for the invasion of some

bacterial pathogens, such as E.coli, into human epithelial cells!58!54!69!19. We also know

that myosin II, which is downstream of RhoA, plays a role in FLp invasion, particularly

in elongating wrap structure!10!63!62!58.

In order to assess the role of RhoA in FLp internalization, we allowed bacterial

attachment for 1 hour before adding the inhibitor C3 transferase for 5 hours. We found

that inhibition of RhoA significantly decreased the lengths of FLp internalized at 6 hours

post infection, although the actual difference in length was quite small (approximately

3!m)(Figure 19A). Contrary to results obtained with the pharmacological inhibitor,

internalization did not appear different between RhoADN and RhoAWT host cells

(Figure 19B).

However, C3 inhibits 3 isoforms of Rho – A, B, and C, whereas the DN is a RhoA.

Although several studies have postulated slightly different roles for each of the isoforms,

in over-expression studies, they have all been found to form stress fibres, and another

study found that all three isoforms localize to sites of Shigella invasion, suggesting each

plays a role!58!23. Therefore, if we only knocked down RhoA, but RhoB and RhoC are

also playing a role in invasion of FLp, one would expect to see a less robust result.

Therefore we postulate the RhoA does have a role in internalization.

60

Control C30

5

10

15

20

25

30

35

40 *

RhoAWT RhoADN0

5

10

15

20

25

30

35

C3

ActinPlasma MembraneDAPI

control

ActinPlasma MembraneDAPI

Figure 19. Internalization of FLp is significantly affected by inhibi-tion of RhoA at 6 hours. (A) NCI-H292 cells were exposed to FLp for

one hour, then washed and exposed to C3, then incubated for a further 5

hours, followed by fixation, staining, and microscope analysis. The length

of FLp fragments internalized was significantly different between C3 and

vehicle treated controls. Red lines indicate means. (B) NCI-H292 cells

were transfected with either a WT or DN construct of RhoA, then infected

with FLp for a total of 6 hours. Fixation, staining, and microscopic analy-

sis followed. Red lines indicate means. (C) NCI-H292 cells were treated

with C3 for 5 hours, but left uninfected. Following fixation and staining,

actin structures within the cell were observed to be pertrubed, and cells

appeared to be collapsing.

A

B

C

61

!

Lung cells treated for 5 hours looked collapsed, with much less spreading, many sharp

edges, and notably, very few stress fibres (Figure 19C). Interestingly, many filopodia-like

projections could be observed in some areas.

3.11 Role of ROCK, MLCK, and myosin II in Filamentous Legionella pneumophila

internalization

We found that inhibition of ROCK, MLCK, and myosin II, the proteins downstream of

RhoA, decreased FLp attachment to host cells, and so we were interested in their role in

bacterial internalization. As previously, we allowed bacterial attachment without the

inhibitor, and then added inhibitor and allowed the infection to proceed for a further 5

hours. We inhibited ROCK with a rho kinase inhibitor, MLCK with ML 7, and myosin II

with blebbistatin.

We found that none of the inhibitors used significantly decreased the lengths of FLp

segments that were internalized (Figuress 13C, 14B, 15C), although a small (non-

significant) decrease can be seen when myosin II is inhibited. Cells treated for 5 hours

with rho kinase inhibitor appeared to have much less filamentous actin than controls,

were less spread, and tended to grow overlapping one another. Additionally, some

blebbing was observed (Figure 13D).

62

!

Cells treated with blebbistatin for 5 hours showed significantly less cell spreading than

vehicle-treated controls, had disorganized actin protrusions extending from cells, and had

stress fibres that appeared to be shorter than those in vehicle-treated controls (Figure

15D). They also had fewer filopodia in some areas.

Together, these results suggest that ROCK, MLCK, and myosin II do not independently

contribute significantly to FLp internalization, despite their inhibition causing obvious

morphological changes in host cell actin. This suggests that there is an actin-independent

mechanism aiding in bacterial internalization. Taking into account the result that RhoA

does play at least a minor role in internalization, this suggests that RhoA is either acting

through a completely different pathway to mediate internalization, or that it is acting

through multiple routes (ie, the ROCK/MLCK/myosin II pathway as well as activating

other actin nucleators and polymerizers), and inhibiting just one route at a time does not

result in a significant reduction in internalized lengths. Alternatively, it suggests that an

FLp effector toxin is aiding bacterial internalization.

3.12 Role of mDia in Filamentous Legionella pneumophila internalization

Since we found that mDia was involved in allowing the attachment of FLp to host cells,

possibly by providing the actin polymerization necessary for hook or wrap formation, we

were also interested in its role in the internalization of FLp. In order to assess

internalization, we allowed the bacteria to attach for 1 hour, then applied the SMIFH2

63

!

inhibitor for a further 5 hours. The length of internalization portions of FLp at 6 hours

was not significantly different between inhibitor and control treated cells (Figure 16C).

Cells treated with the mDia inhibitor for 5 hours showed blebbing and some cell

elongation. They also appeared to have fewer, shorter, and thinner stress fibres. The actin

in many cells appeared more diffuse than in vehicle-treated controls (Figure 16D).

Together these results suggest that although mDias are involved in the actin

polymerization necessary for FLp attachment, they are not involved in wrap elongation or

true internalization.

3.13 Role of Arp2/3 in Filamentous Legionella pneumophila internalization

Similarly to mDia, we found that Arp2/3 plays a role in FLp attachment, probably by

mediating the formation of the hooks and wraps downstream of either Cdc42 or Rac1!21!67. We were also interested in discovering its role in bacterial internalization. Again, we

allowed bacteria to attach normally for 1 hour, then added wiskostatin for 5 hours.

Internalized lengths of FLp were unaffected at 6 hours (Figure 17C).

Cells treated with wiskostatin for 5 hours appeared to have fewer and shorter filopodia,

and a more stretched appearance as well as an under-developed actin network compared

to vehicle-treated controls (Figure 17D).

64

!

Together, the results suggest that while Arp2/3 plays a role in FLp attachment, it does not

play a role in internalization. The fact that internalization was not affected, even though

wiskostatin perturbed many aspects of the cellular actin cytoskeleton would suggest that

internalization could be governed at least in part by an actin-independent mechanism.

Additionally, we know that MWs are actin-based structures, and that they entrap FLp

segments, contributing to internalization. Therefore it is surprising that inhibiting actin

nucleators does not result in a decrease in internalization. This could be due to an

exogenous actin nucleator secreted into the host cell cytoplasm that is mediating actin

rearrangement and facilitating wrap elongation.

3.14 Attachment and internalization dependency on Type IVSS effectors

Since our results suggested that there were many mechanisms involved in both the

attachment and internalization of FLp, and since we did not find a major contributor to

internalization among the main actin polymerizing proteins that we studied, we suspected

that an FLp toxin could be affecting the process. Many bacterial species are capable of

injecting effectors that mediate their uptake by hijacking host cell actin!85!56!57.

Accordingly, we performed attachment and internalization assays on host cells using a

T4SS mutant strain of Legionella pneumophila compared to our regular strain. The T4SS

mutant is unable to inject effector toxins due to a defect in the type 4 secretion system.

We found that the attachment of filamentous T4SS mutants to host cells did not differ

significantly from the wild type FLp attachment (Figure 20). However, there was a

65

10

0

20

30

Figure 20. T4SS mutant FLp exhibit similar binding, but decreased internalization compared to WT FLp. (A) NCI-H292 cells were infected with T4SS mutant FLp, or with WT FLp, and binding was assessed after 1 hour (cells were !xed, stained, and counted). Data shown are mean ± SEM from three independent experiments. (B) NCI-H292 cells were infected with T4SS mutant FLp, or with WT FLp for 1 hour, followed by washing to remove unattached bacteria. The infection was then allowed to proceed for a further 5 hours, followed by !xation, di"erential staining, and measurement of internalized FLp segments. Red lines indicate mean lengths.

66

!

significant decrease in the internalized lengths of the T4SS mutants compared to the WT

FLp strain. This suggests that while attachment may be a passive process driven by

activation of host cell receptors and small GTPase activation, internalization is, at least in

part, mediated by the bacterial itself.

67

!

4.0 DISCUSSION

The filamentous morphotype of Legionella pneumophila (FLp) can be found in patients

and environmental samples, and has been proven capable of entering and replicating

inside lung epithelial cells (LECs) such that it may even out-compete and out-replicate

the rod-shape form!10. The invasion of epithelial cells by FLp depends on actin-based

structures that look similar to filopodia and lamellipodia, called hooks and wraps,

respectively. The ultimate goal of this work was to determine the role of the small

GTPases Cdc42, Rac1, and RhoA (as well as several downstream effectors), in the initial

invasive steps of FLp in human LECs.

We found that Cdc42 and Rac1 are involved in bacterial attachment. Cdc42 mediates the

production of filopodia, which likely develop into hooks that entrap FLp at the host cell

surface. Myosin X also plays a role in attachment, probably by the production of

filopodia, and partially by the localization of the host cell receptors !1-integrin and E-

cadherin. The role of Rac1 in attachment is slightly less clear, although it could assist in

the formation of MWs, which also help entrap bacteria at the cell surface (Figure 21).

We did not find a role for Cdc42 or Rac1 in bacterial internalization. We did, however,

find that RhoA contributes to internalization. However, the effect was small. Since we

found that internalization was partially T4SS dependent, it is likely that the action of

RhoA overlaps with the action of an FLp effector that is causing actin nucleation and

allowing efficient uptake (Figure 21).

68

myo

sin X

actin

E-ca

dher

inß1

-inte

grin

Exte

rnal

FLp

Inte

rnal

FLp

Poss

ible

FLp

Toxi

n

AB

CD

E

F

Cdc4

2m

yosin

XCd

c42

Rac1

actin

po

lym

eriz

atio

n

Filo

podi

a fo

rmat

ion,

re

cept

or

loca

lizat

ion

Filo

podi

aFL

p bi

ndin

gH

ook

form

atio

n

Cdc4

2Ra

c1?

actin

po

lym

eriz

atio

nW

rap

form

atio

n

Wra

pel

onga

tion

RhoA

E!ec

tor?

actin

po

lym

eriz

atio

n

Rece

ptor

bi

ndin

g

RhoA

E!ec

tor?

actin

po

lym

eriz

atio

n

69

Figure 21. Model of the molecular mechanisms involved in the early invasion steps of !lamentous Legionella pneumophila into human lung epithelial cells. (A) The actions of Cdc42 and myosin X stimulate !lopodia production. Host cell receptors are localized to the cell surface or to !lopodial tips by myosin X. (B) The action of myosin X stabilizes existing !lopodia and helps form more of the protru-sive structures. (C) Attachment of FLp to receptors at the tips of !lopodia triggers a signal cascade that results in activation of Cdc42 and Rac1, causing actin polymer-ization and nucleation via the e"ectors mDia and arp2/3, and possibly myosin II. The actin polymerization triggered by Cdc42 and Rac1 results in the production of additional !lopodia attachment sites, and hooks. (D) Receptor binding in (C) may also cause !lopodia retraction, and the actin reorganization triggered by the active small GTPases helps form hooks, which attach FLp to the cell surface, increasing contact area. Addtionally, the bacteria may present the host cell with a toxin the aids in actin polymerization. (E) The combined action of the mechanisms in (D) result in the formation of wraps at the host cell surface, and activation of RhoA, leading to stress !bre production (via the myosin II pathway) and actin polymeriza-tion (via arp2/3 and mDia). The possible toxin may also contribute to actin polym-erization here. (F) Wraps continue to elongate.

70

!

4.1 Roles For FLp Receptors in Signal Transduction

E-cadherin and !1-integrin are the receptors engaged by FLp, and are responsible for

their attachment and internalization. The activation of these receptors and downstream

signaling including myosin II activation lead to actin polymerization and

host cell surface rearrangement resulting in the entrapment and eventual internalization of

the filamentous bacterial cell!10. My results show that Rac1, RhoA, and Cdc42 are

recruited to regions of the host cells associated with internalized portions of FLp around

the same time as wrap formation. It likely that their recruitment is triggered by the

binding and activation of E-cadherin and !1-integrin by the FLp.

Bound integrins can activate Rac1and Cdc42 through multiple pathways such as

activation of GEFs (like Vav2) and activation of paxilin which recruits GEFs, leading to

actin polymerization!59!71. Engagement of cadherins activates Rac1 and Cdc42!50!86. In

fact, as MDCK cells contact one another (through E-cadherin) , Rac1-GFP accumulates

in the regions of contact, and lamellipodia develop!50. Remarkably, molecular actin

organization appears similar during cellular migration and spreading, and the processes

involved in phagocytosis!48. Indeed, several of the same pathways involved in GTPase

activation during migration and spreading can be triggered by the binding of pathogens to

E-cadherin or !1-integrin.

71

!

Y. enterocolitica binds to host cells via !1-integrin, following which WASp and active

Arp2/3 were recruited to invasive area, resulting in cytoskeletal rearrangement and

bacterial invasion!48. Additionally, blockage of !1-integrin inhibited formation of the

actin-rich invasive structure (in this case, a phagocytic cup) and bacterial uptake!48.

Microinjection of dominant negative forms of Cdc42 and Rac1 achieved the same

inhibition, as did the inhibiter C3 transferase (against RhoA)!48. This suggests that

binding of !1-integrin activates actin polymerization via Cdc42, and that Rac1 and RhoA

are also important!48. Similarly, we know that blocking !1-integrin decreases FLp

attachment!10, and our study found that blocking Cdc42 and Rac1 inhibited the bacterial

attachment that precedes entry, and that blocking RhoA retarded uptake. Their study did

not distinguish between attachment and internalization, but both still suggest that receptor

signaling through small GTPases is necessary for bacterial invasion.

Y. pseudotuberculosis also binds !1-integrin via invasin, causing receptor clustering, and

leading to Rac1 activation!59. S. aureus uses integrins as binding sites, and its

internalization requires localized accumulation of polymerized actin, similarly to FLp!49.

This study suggested that engaged integrins cause the activation of host cell protein-

tyrosine kinases, which were key for internalization of the bacteria!49. PTKs have been

known to work through small GTPases to promote actin cytoskeleton rearrangement,

linking the signal cascade of receptor binding to small GTPase activation!59!71. Similarly,

L. monocytogenes is able to invade non-phagocytic cells by binding to E-cadherin,

triggering actin reorganization through a signal cascade involving Rac1, and finally

Arp2/3!20.

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Our results seem complimentary to these studies, as the same activation systems seem

likely to have been at work in our epithelial cells – binding of !1-integrin and E-cadherin

at the cell surface activates small GTPases which cause actin cytoskeletal rearrangement

and the development of actin-based structures that allow attachment, and eventual

bacterial internalization.

4.2 Filopodia, Hooks, and Invasion

Our results show quite clearly that Cdc42 is required for the attachment of FLp to NCI-

H292 cells though the formation of hooks and wraps. Pharmacological activation of it

(via bradykinin treatment) led to an increase in binding, while pharmacological inhibition

(via ML 141) lowered it. Expression of Cdc42DN also lowered attachment. We also

know that Cdc42 is generally responsible for the production of filopodia within

eukaryotic cells!54!55!61. Therefore, it would appear that Cdc42 contributes to the

attachment of FLp to host cells through filopodia, or hooks.

The role of filopodia in bacterial attachment to host cells has been well documented.

Enteropathogenic E. coli subvert host-cell small GTPases, particularly Cdc42, in order to

stimulate filopodia and/or pedestal formation!34. These “extra” filopodia are proposed to

help maintain bacterial adherence to the host cells, although the specifics of this

advantage are not discussed!34. The formation of filopodia also precedes Shigella invasion

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of host cells, with the structures acting as the first contact point between the bacterial and

host cells!32. Y. enterocolitica also bind initially to !1-integrin at filopodial tips!35.

In the case of our system, we found that filopodia are a limiting factor for binding, and

that the presence of receptors can enhance binding. Thus, in our system, as in the EPEC

and Shigella studies, the structural characteristics of filopodia are a major contributor to

FLp binding, but as in the Yersinia study, they are also important due to the fact that they

contain the FLp receptors !1-integrin and E-cadherin (having additional filopodia

enhances binding by presenting more potential receptors to filamentous bacterial cells).

Additionally, it may be possible that simply having a greater surface area of host cell

could promote binding. Since filopodia protrude from the cell surface, it is possible that

they may entangle the long bacterial cells, resulting in additional binding.

Filopodia formation can be induced by myosin X in a number of ways: through the

transport of integrins (or other proteins) to tips, where these proteins bind to substrate,

thus stabilizing filopodia, by bundling of nascent actin filaments, by interacting with

formins, or by functioning as a member of the tip complex!39!40.

We found that myosin X probably plays two roles in FLp infection. The first is through

the formation of filopodia (and possible their transition to hooks), and the second is

through the localization of host cell receptors. This was because expression of the wild-

type construct of myosin X (stimulates filopodia and localizes receptors) significantly

increased binding of FLp compared to controls, but expression of the FERM mutant,

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which retains the ability to stimulate filopodia but cannot localize receptors) resulted in

attachment levels similar to that of the control.

A two-fold role (creating filopodia and transporting receptors) for myosin X in FLp

invasion fits well with other results about FLp invasion. We have shown through other

means that filopodia are important for infection (by manipulating Cdc42), and previous

studies have highlighted the role of the receptors (knocking down receptors severely

hinders binding)!10. Several other studies have looked into the role of myosin X in cell

morphogenesis and phagocytosis. Knockdown of the protein delays recruitment of E-

cadherin to cell-cell contacts, and impairs integrin funciton!37, which would presumably

decrease binding of FLp.

One proposal for the involvement of myosin X in membrane protrusions that facilitate

Shigella spread could also explain myosin X’s role in attachment independently from its

receptor localization role. The tail of myosin X is able to bind via its PH regions to PIP3

in the membrane, while its head is bound to actin filaments!42. As the head “walks” along

the filaments, it “transports” the membrane to which its tail is attached toward the tip,

elongating the membrane, and allowing space for the actin filament to grow in the

filopodium!42. Myosin X works in a similar way to extend pseudopods in phagocytosis!87.

In our model, this type of behavior could allow myosin X to elongate filopodia around

FLp to facilitate the formation of hooks and wraps. PIP3, as well as myosin X, can be

found in filopodia and lamellipodia, and our lab has also found it in hooks and wraps

(unpublished data)!87!37!40!36!88. Interestingly, this effect could be limited to the

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filamentous form of L. pneumophila, as inhibition of PIP3 or myosin X activity inhibits

the ingestion of large (6!m), but not small (2!m), particles during phagocytosis!87!89.

Filopodia are dynamic structures, exhibiting growth and retraction behaviors!90!31. As

they retract, they are capable of “pulling” attached particles, including pathogens, back

toward the cell body, even across large distances!90!32!91!92. This retraction process can be

induced after the binding of integrins stimulates Cdc42!91, and could possibly be a

mechanism through with FLp cells make their way to the host cell surface, where wraps

could subsequently form. The signaling process for wraps could possibly be the same as

that for the formation of hooks/filopodia, but on a broader scale; increased binding of

receptors due to a larger area of contact between the host cell surface (not just filopodia)

and the long bacterial cell could induce signaling cascades involving more of the small

GTPases leading to larger perturbations in the actin cytoskeleton. Indeed, receptor

clustering has been proven to be necessary for FLp attachment and wrap elongation, and

clusters of !1-integrin show greater signaling potential with regards to the small GTPases!10!59. Similarly, a study involving the invasion process of Y. enterocolitica noted that

multiple contact points between the bacteria and the cell surface (where the pathogen

binds to !1-integrin) led to the internalization of the bacterial cell, and that blocking the

receptors prevented attachment and internalization!35.

Interestingly, the internalization of FLp appeared unaffected by Cdc42 manipulation. It is

therefore likely that its prime contribution to FLp infection is through the development of

filopodia and hooks.

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4.3 Lamellipodia, Wraps, and Invasion

My results show that Rac1 accumulates at sites of FLp invasion for a significant

proportion of events, suggesting it is important to the invasion process. Since we found

that inhibiting Rac1 affects attachment, its role is likely in the production of an

attachment structure. Rac1 is responsible for producing lamellipodia!54!55!61. Since

membrane wraps are lamellipodial protrusions, it seems likely that Rac1 could be

responsible for the production of wraps, which help secure FLp to the host cell.

Many invasion mechanisms involve the production of host cell ruffles, which are often

due to the action of Rac1, and allow efficient bacterial internalization!45!21!22!93. S. flexneri

invasion involves membrane ruffles that require action of Rac1!22. Membrane ruffles

engulf bacteria into the host cells!22. S. typhimurium also produces membrane ruffles

during invasion due to activation of Rac1!45!21!93. Although the above studies elucidating

the importance of Rac1 to bacterial invasion involved bacterial effectors, engagement of

receptors can result in production of membrane ruffles/lamellipodia mediated by the

recruitment and activation of Rac1!70!59!71!86. Engagement of E-cadherin by antibodies has

been shown to cause Rac1 recruitment and activation!86!70, and although they did not

report on the behavior of actin following this, it is likely that lamellipodia were produced

as a result, since engagement of cadherin by other cadherins has been shown to result in

Rac1 activation and localized lamellipodia formation!94.

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Similarly, integrin binding is reportedly characterized by two phases, one of which results

in Rac1 (and Cdc42) activation, and actin polymerization!59!71. Integrins are also

proposed to be signal transducers that promote Rac1 activation, which in turn promotes

the formation of ventral lamellipodia to restore micro-wounds in endothelium cells (it is

interesting that this study specifically states ventral lamellipodia, which occur on the cell

body, similar to where wraps form, as opposed to conventional lamellipodia which

generally occur on the edges of cells)!95. The engagement of surface receptors by bacteria

leading to Rac1 activation could also lead to cytoskeleton rearrangements. In fact, the

invasion of L. monocytogenes, which initially binds to the host cell receptor E-cadherin,

requires Rac1 and actin remodeling, which echoes our results!20. Thus, our results that

activated Rac1 seems to contribute to actin remodeling and FLp attachment fit well with

what is known about such systems.

It is curious that we don’t see a reduction in the lengths of internalized FLp considering

our proposed model, in which Rac1 contributes to wrap structure, as one would expect

that inhibiting it would decrease wrap length, which would be reflected in our

internalization assays. Like Cdc42, it seems that bacterial cells that manage to remain

attached to the host cell undergo internalization normally, as inhibition of Rac1 did not

significantly affect internalization.

Thus, it is likely other proteins that affect internalization. Possibly, the bacteria is

bypassing endogenous host cell systems, and affecting its own uptake. However, it is also

possible that although the initiation of wrap structure requires Rac1, maintenance and

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elongation of the structures do not. Overall, our results suggest that both Rac1 and Cdc42

act right after the attachment of the bacteria to the host cell, but as the bacteria releases

effectors, these small GTPases contribute less to invasion as the effectors stimulate

bacterial uptake.

!

4.4 RhoA and Invasion

Contrary to all the other inhibitors used, C3 transferase, the inhibitor of RhoA, B, and C,

resulted in a small but significant decrease in internalization, but not one in attachment.

This appears odd considering that inhibiting ROCK/MLCK/myosin II does not affect

internalization.

Although the decrease in internalized lengths is significant when host cells were treated

with C3, it is very small, suggesting that some other mechanism is playing a key role in

the internalization of FLp. One (actin-independent) candidate for this role is clathrin.

Clathrin is a protein that forms a lattice-like structure around the cytosolic face of the

invaginating membrane during endocytosis, providing a structural role, and acting as a

scaffold for recruitment of transmembrane proteins!96!97. When the final endosome

pinches off from the membrane, the clathrin quickly disassociates from the vesicle!97.

Although the natural function of this process is for uptake of macromolecules and

internalization of transmembrane proteins, many viruses are known to co-opt this system

to facilitate their entry!97!96. Invasive bacteria including Listeria monocytogenes have also

been reported to use this system to invade non-phagocytic cells!96!97. This clathrin-

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dependent uptake was of particular importance to bacteria that enter cells by binding to

cellular receptors (using the zipper mechanism), which is the case for FLp. Clathrin

accumulation at bacterial invasion sites occurrs independently of actin polymerization!97,

which would explain why internalization occurred even when we inhibited Arp2/3- and

mDia. Preliminary data from our lab has found that clathrin accumulates at sites of FLp

invasion, lending credibility to this mechanism.

In addition, taking into account our T4SS results (no inhibition of attachment, but

inhibition of internalization), it is likely that the internalization difference is small

because alternative, pathogen-driven mechanisms are guiding uptake. L. pneumophila

releases hundreds of effectors and toxins, the effects of which are mostly unknown!5!4!9!7.

Recently, a L. pneumophila toxin that directly interacts with actin has been identified –

VipA binds actin in the host cell and directly polymerizes filaments!98. Although to date

this particular actin manipulation seems to be related to organelle trafficking!98, it

suggests that the bacteria is capable of manipulating actin on some level. VipA is

translocated through the T4SS!98. We found that T4SS L. pneumophila exhibits delayed

internalization, while attachment is unaffected. It could be the absence of VipA in host

cells that is causing the T4SS mutant’s slow internalization. A role for VipA could

explain how internalization proceeded even when known actin manipulators (mDia,

Arp2/3, myosin II) were inhibited. If FLp relies heavily on VipA for internalization,

inhibition of endogenous nucleators would not be a problem for the bacteria, and the role

of RhoA in internalization would be lessened.

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Another curious aspect of our study is the incongruity of results between RhoA, and the

proteins of the ROCK/MLCK/myosin II pathway. One would expect that if inhibiting

ROCK, MLCK, or myosin II affects attachment, inhibiting RhoA would, too. Because we

do not see this pattern, it is possible that the ROCK/MLCK/myosin II pathway can be

activated by a protein other than RhoA. One phagocytosis study reported that upon

activation by PIP3, Rac1 can activate PAK (p21 activated kinase), which phosphorylates

myosin light chains, thus activating the myosin and leading to contractility!99!61. Some

lipids, especially arachidonic acid, can also activate ROCK independently of RhoA!63.

Intriguingly, it has been reported that bacterial lipopolysaccharides can cause an increase

in arachidonic acid secretion in some cell types!100. Therefore it is a possibility that FLp

invasion stimulates arachidonic acid release, and ROCK activation.

Additionally, myosin II activity can be regulated by Ca2+ levels!101. When Ca2+ is present,

it modulates the calcium-binding messenger protein calmodulin, which can then bind to

MLCK, which contains a Ca2+/calmodulin-binding sequence!101. This can lead to

activation of MLCK, which then phosphorylates and activates myosin II!101. These or

other, unknown, activation systems could be working to activate the

ROCK/MLCK/myosin II pathway independently of RhoA. Thus, when RhoA is inhibited

by C3, attachment is not inhibited, as it is when ROCK, MLCK, or myosin II is inhibited,

because the downstream effectors can be alternatively activated.

4.5 Downstream Effectors: Contribution to Invasion

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All of the downstream inhibitors used exhibited the same pattern of results – attachment

was decreased in the presence of inhibitor compared to vehicle control, while

internalization was not significantly affected. This suggests that they are involved in

initiating attachment-based structures (carrying out the signals given to them by the small

GTPases), but if FLp cells can attach despite the presence of inhibitor, the internalization

occurs normally.

With regards to the downstream effectors, most can be activated by multiple pathways,

and regulated by multiple mechanisms. Arp2/3, for example, has variously been reported

to be activated by all three of the studied small GTPases!67!61. Thus, unless you are

directly inhibiting Arp2/3, it can always be activated by at least one pathway, which

could be why there is such a large affect on attachment with wiskostatin, but not as much

with either ML 141 or NSC (Cdc42 inhibited or Rac1 inhibited). Many other pathways

involving the small GTPases and their downstream effectors also exist, illustrating the

complexity of the systems that we studied.

It seems that endogenous, intact, host cell proteins are important for attachment, but that

internalization is, at least in part, directed by the pathogen, based on our T4SS data. Thus,

when assessing attachment, we see deficits when actin polymerizers are inhibited (either

directly or by inhibition of small GTPases). However, when assessing internalization, we

do not see such a situation. As noted above, a bacterial nucleator (such as VipA) could be

driving internalization independently of host cell processes, such that inhibition of

endogenous nucleators is unimportant to the invasion process. Alternatively, such an

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effector could be manipulating host cell nucleators, activating them such that they are

“immune” to pharmacological inhibition.

Aside from contributions to internalization by FLp effectors, it is likely we do not see a

defect in internalization because there is a “functional redundancy” involved – multiple

proteins are probably contributing to internalization, such that inhibiting one of them has

a small, but non-significant effect on results. Inhibiting more than one at a time could

result in a significant decrease in internalized lengths. In addition to functional

redundancy between our studied downstream proteins, other proteins could be involved

in actin nucleation or reorganization. Profilin forms a complex with globular (non

filamentous) actin, and then associates with the growing end of an actin filament, where

ATP-hydrolysis enhances the lengthening of the filament!102. Although profilin enhances

Cdc42-induced polymerization, it does not appear to be directly downstream of this small

GPTase!103. Thus, this protein was intact in all of our assays, and could have contributed

to internalization processes. Gelsolin is also a polymerizer of actin filaments, and acts by

binding to two globular actin monomers, then associating with an existing filament to

elongate it!104. Additionally, it can sever existing filaments (by insertion between actin

subunits), creating two shorter filaments, each of which can then be elongated!104.

Gelsolin is downstream of Rac1, but is also dependent on Ca2+ levels!61. Thus, even when

Rac1 was inhibited, it could potentially be active. Finally, cofilin regulates actin

polymerization and can even help form cellular protrusions!61!105!106. Signal cascades have

linked Cdc42 and Rac1 to cofilin activity!61. Since it is downstream of two small

GTPases, it could become activated even if Cdc42 or Rac1 was inhibited.

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4.6 Future Directions and Implications

Elucidating the roles of several ubiquitous eukaryotic small GTPases can help us

understand the mechanisms guiding the formation of hooks and wraps, and potentially

develop ways to block attachment of FLp to host cells, preventing infection and the need

for antibiotics. It is also possible that some of the same pathways that are activated by the

binding of the FLp to host cell receptors are common to other bacteria that are

internalized via variations of zipper phagocytosis, or other filamentous species. Thus

findings from studies such as this have the potential to be generalized to other pathogenic

species.

The study of the invasiveness of filamentous bacterial cells is not only important in and

of itself (the study of potentially novel infection mechanisms), but it also represents a

unique infection model for translation to non-filamentous cells. Many cellular processes

occur too fast for proper, in-depth study, including things like membrane fusion during

phagocytosis of small particles. However, longer cells can potentially “slow” these

processes down, allowing the study of the particular mechanisms guiding their

occurrence. Indeed, a recent study has shown that the FLp morphology delays

phagosomal maturation events in macrophages!10. Additionally, several studies have

indicated that all three small GTPases we studied control entry of bacterial species into

epithelial cells!22, but do not specify their exact roles, because internalization occurs

quickly, and within a relatively small area of the host cell. In our case, we were able to

more specifically pinpoint the potential roles of each small GTPase, and found that

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although they are all important for the full invasion process, two contribute mostly to

actin polymerization for the creation of attachment structures, while the last contributes at

least partially to internalization. It is possible that this is the case for a broad category of

bacterial species. This specificity of function could potentially lead to more effective

treatment methods by identifying specific target molecules that affect entry.

In order to more fully understand the pathways governing FLp invasion, future studies

should focus on the upstream receptors from the small GTPases, and the extent to which

either !1-integrin or E-cadherin recruit each specific small GTPase, as well as the

intermediary proteins which guide small GTPase accumulation and activation.

Interestingly, we only found a small affect on internalization upon inhibition of the small

GTPases, which suggests alternative modes of internalization into the host cell body.

Elucidation of these alternative modes will be important for future studies. One of these

alternative modes could be a FLp effector, delivered through the T4SS, so further study

on the timing of effector injection, the identity of actin nucleation toxins (potentially

VipA), and host cell effects are key to fully understanding internalization dynamics

specifically. Alternatively, results from our lab have found that clathrin localizes to sites

of FLp invasion, which could also be a mechanism through which FLp makes its way into

the host cell body. Further analysis of clathrin dynamics would be beneficial.

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4.7 Conclusions

In this study we set out to analyze the roles of Cdc42, Rac1, and RhoA, as well as their

downstream effectors (ROCK, MLCK, myosin II, Arp2/3, mDia), in the invasion of lung

epithelial cells by filamentous Legionella pneumophila. We found that all but RhoA were

involved in attachment, presumably by stimulating actin polymerization. Additionally,

none except for RhoA played a role in internalization, although the role of RhoA

appeared to be small. We also studied the role of myosin X, and found that it seems to

assist in attachment, but not internalization.

!!!!!!!!!!!!!!!!!!!!!!

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