Structure and function of the Borrelia

burgdorferi porins P13 and P66

Mari Bonde

Department of Molecular Biology

Umeå Center for Microbial Research (UCMR)

Laboratory for Molecular Infection and Medicine Sweden (MIMS)

Umeå University Umeå 2015

Responsible publisher under Swedish law: the Dean of the Medical Faculty

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

Copyright © Mari Bonde

ISBN: 978-91-7601-264-2

ISSN: 0346-6612

Elektronisk version tillgänglig på http://umu.diva-portal.org/

Cover picture: Borrelia burgdorferi

Printed by: Department of Chemistry Printing Service

Umeå, Sweden 2015

Det har jag aldrig provat tidigare

så det klarar jag helt säkert

-Pippi Långstrump

Table of contents

ABSTRACT ........................................................................................ I

LIST OF ABBREVIATIONS AND TERMS ...........................................II

KORT SAMMANFATTNING PÅ SVENSKA ....................................... III

PAPERS IN THIS THESIS: ................................................................ V

PAPERS NOT INCLUDED IN THE THESIS: ...................................... VI

1. INTRODUCTION........................................................................... 1

1.1. LYME DISEASE – CAUSED BY THE BACTERIUM B. BURGDORFERI .......... 1

1.1.1. History of Lyme disease .......................................................................1

1.1.2. Classification ........................................................................................ 2

1.1.3. Symptoms ............................................................................................. 2

1.1.4. Detection ............................................................................................... 3

1.1.5. Treatment ............................................................................................. 3

1.1.6. Prevention ............................................................................................ 4

1.2. GENERAL CHARACTERISTICS OF BORRELIA .................................... 5

1.2.1. Life cycle ............................................................................................... 6

1.2.2. Genome organization .......................................................................... 7

1.2.3. Paralogous genes and sequence similarities on plasmids................. 7

1.2.4. Importance of plasmids ....................................................................... 8

1.3. LABORATORY MIMICRY OF THE B. BURGDORFERI HOSTS .................. 10 1.4. B. BURGDORFERI METABOLISM AND CARBON UTILIZATION. .............. 12

1.5. GENE REGULATION IN B. BURGDORFERI ....................................... 13

1.6. BORRELIA MEMBRANE COMPOSITION .......................................... 14

1.6.1. Transport systems in Borrelia ...........................................................15

1.6.2. Outer membrane proteins ................................................................. 16

1.7. LIPOPROTEINS INVOLVED IN INFECTION AND PATHOGENICITY .......... 17

1.7.1. Osps ...................................................................................................... 17

1.7.2. The vls locus ........................................................................................ 18

1.7.3. OspE/F-like proteins .......................................................................... 18

1.7.4. Mlp ...................................................................................................... 19

1.7.5. DbpA/B ............................................................................................... 19

1.7.6. BBK32 ................................................................................................. 19

1.8. INTEGRAL OUTER MEMBRANE PROTEINS, PORINS .......................... 20

1.8.1. General characteristics of porins ...................................................... 20

1.9. PORINS IN BORRELIA .............................................................. 22

1.9.1. P13 and its paralog, BBA01 ............................................................... 22

1.9.2. P66, with dual function ..................................................................... 23

1.9.3. DipA .................................................................................................... 24

2. METHODS ..................................................................................25

2.1. BLACK LIPID BILAYER ASSAY ......................................................25

2.2. NANODISCS ............................................................................ 27

3. AIMS OF THE THESIS ............................................................... 29

4. RESULTS AND DISCUSSION ..................................................... 30

4.1. PAPER I AND II ...................................................................... 30

4.1.1. Characterizations of the actual pore sizes and sizes of the protein

complexes formed by the P13 and P66 porins. ................................. 30

4.1.2. Pore-forming activity of P13 ............................................................. 30

4.1.3. Size of the pores formed by P13 and P66 .......................................... 31

4.1.4. Oligomeric constitution of the P13 and P66 complexes ................... 32

4.2. PAPER III ............................................................................. 34

4.2.1. Structural analysis of P13 in Nanodisc ............................................ 34

4.2.2. P13 is a unique channel-forming protein ......................................... 34

4.2.3. P13 in Nanodiscs ................................................................................ 35

4.3. PAPER IV ............................................................................. 36

4.3.1. Integrin binding and porin function of P66 during B. burgdorferi infection. ............................................................................................. 36

4.3.2. Pore formation and integrin binding analysis of P66 mutants ...... 37

4.3.3. Importance of the integrin-binding region of P66 in the

establishment of a B. burgdorferi infection and endothelial

transmigration ................................................................................... 37

4.4. PAPER V ............................................................................... 39

4.4.1. Function of P13 and P66 in B. burgdorferi during osmotic stress

adaptation induced by glycerol. ....................................................... 39

4.4.2. Porin transcription and expression in B. burgdorferi bacteria

cultured under osmotic stress ........................................................... 39

4.4.3. Increased transcription of an extracellular matrix binding protein

and membrane localization of a heat shock protein in response to

p66 deficiency. .................................................................................... 40

4.4.4. Similarities in transcriptional regulation of putative transporters

and stress factors in B31-A and ∆p13∆p66 in response to osmotic

stress. .................................................................................................. 41

5. CONCLUSIONS .......................................................................... 42

6. ACKNOWLEDGEMENTS............................................................ 43

7. REFERENCES ............................................................................ 45

8. PAPERS I-V

i

Abstract Borrelia burgdorferi is an elongated and helically shaped bacterium that is the

causal agent of the tick-borne illness Lyme disease. The disease manifests with

initial flu-like symptoms and, in many cases, the appearance of a skin rash called

erythema migrans at the site of the tick bite. If left untreated the disease might

cause impairment of various organs such as the skin, heart, joints and the nervous

system. The bacteria have a parasitic lifestyle and are always present within a host.

Hosts are usually ticks or different animals and birds that serve as reservoirs for

infection. B. burgdorferi are unable to synthesize building blocks for many vital

cellular processes and are therefore highly dependent on their surroundings to

obtain nutrients. Because of this, porins situated in the outer membrane, involved

in nutrient uptake, are believed to be very important for B. burgdorferi. Except for

a role in nutrient acquisition, porins can also have a function in binding

extracellular matrix proteins, such as integrins, and have also been implicated in

bacterial adaptation to new environments with variations in osmotic pressure.

P13 and P66 are two integral outer membrane proteins in B. burgdorferi

previously shown to have porin activities. In addition to its porin function, P66

also has integrin binding activity. In this thesis, oligomeric structures formed by

the P13 and P66 protein complexes were studied using the Black lipid bilayer

technique in combination with nonelectrolytes. Initial attempts were also made to

study the structure of P13 in Nanodiscs, whereby membrane proteins can insert

into artificial lipid bilayers in their native state and the structure can be analyzed

by electron microscopy. In addition, the role of P13 and P66 in B. burgdorferi

osmotic stress adaptation was examined and also the importance and role of the

integrin-binding activity of P66 in B. burgdorferi infections in mice.

Using Black lipid bilayer studies, the pore forming activity of P13 was shown to

be much smaller than previously thought, exhibiting activity at 0.6 nS. The

complex formed by P13 was approximately 300 kDa and solely composed of P13

monomers. The channel size was calculated to be roughly 1.4 nm. Initial Nanodisc

experiments showed a pore size of 1.3 nm, confirming the pore size determined by

Black lipid bilayer experiments. P66 form pores with a single channel conductance

of 11 nS and a channel size of 1.9 nm. The porin assembles in the outer membrane

into a large protein complex of 420 kDa, containing exclusively P66 monomers.

The integrin-binding function of P66 was found to be important for efficient

bacterial dissemination in the murine host but was not essential for B. burgdorferi

infectivity. Neither P13 nor P66 had an active role in osmotic stress adaptation.

Instead, two p13 paralogs were up-regulated at the transcript level in B.

burgdorferi cultured under glycerol-induced osmotic stress.

ii

List of abbreviations and terms a.a. Amino acid

α-MGalDAG α-monogalactosyl diacylglycerol

ApoA-1 Apolipoprotein A-1

BLB assay Black lipid bilayer assay

BN-PAGE Blue native polyacrylamide gel electrophoresis

CFH Complement inhibiting factor H

CFHL-1 Factor H-like protein 1

CFHR Factor H-related proteins

CM Cytoplasmic membrane

Cp Circular plasmid

CRASP Complement regulatory acquiring surface protein DPhPC Diphytanoyl phosphatidylcholine

DMC Dialysis membrane chamber

Elp OspE/F-like leader peptide proteins

EM Electron microscopy

Erp OspE/F-related proteins

GlcNAc N-acetylglucosamine

HDL High density lipoprotein

kbp kilobase pairs

Lp Linear plasmid

Mab Monoclonal antibody

Mlp Multicopy lipoprotein family

MSP Membrane Scaffold Protein

ND Nanodisc

NE Nonelectrolyte

OM Outer membrane

OMP Outer Membrane Proteins

Opp Oligopeptide permease

Osp Outer surface protein

PAGE Polyacrylamide gel electrophoresis

PC Phosphatidylcholine

PCR Polymerase chain reaction

PG Phosphatidylglycerol

PEG Polyethylene glycol

POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

PTS Phosphotransferase

RND Resistance-nodulation-division

SCC Single channel conductance

SDS-PAGE Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis s.l. sensu lato

s.s. sensu stricto

TCA cycle Tricarboxylic acid cycle

TEM Transmission electron microscopy

iii

Kort sammanfattning på svenska Borrelia burgdorferi är en bakterie med många unika egenskaper som

orsakar sjukdomen Lyme borrelios. Borrelia kan idag lätt behandlas med

antibiotika om sjukdomen upptäcks i ett tidigt stadium. Det är först om

sjukdomen tillåts fortgå som symptom som nervsmärta och ansiktsförlamning

kan uppstå och dessutom vara svåra att koppla till en Borrelia-infektion.

Multiresistenta bakterier har blivit en stor del av vår vardag och även om

Borrelia-bakterierna idag inte är resistenta mot flertalet antibiotika är det

kanske speciellt viktigt, innan det är för sent, med forskning som kan leda till

upptäckter av unika angreppsställen för nya läkemedel.

Målet med denna avhandling var att studera hur två Borrelia proteiner, P13

och P66, ser ut, är uppbyggda och även vilken funktion de har. Dessa proteiner

är tänkbara vaccinkandidater eftersom de sitter i yttre membranet hos

bakterierna och sticker ut på ytan mot våra värdceller, vilket gör att vi reagerar

mot dem vid en infektion. P13 och P66 är också viktiga kanaler för bakterierna

vid upptag av näringsämnen och byggstenar från omgivningen. Ämnen som

bakterierna inte kan producera själva. Pga. denna funktion är P13 och P66

tänkbara proteiner för blockering med ett läkemedel som skulle förhindra

bakterien från att föröka sig i och med att de förlorar möjligheten att

tillgodogöra sig näring. Detta i sin tur skulle leda till att vårt eget

immunförsvar hinner rensa undan bakterierna innan infektionen blivit för

stor och vi blivit sjuka. P66 har förutom porin funktionen även en adhesions

funktion när proteinet kan binda integriner som sitter på olika typer av celler

i vår kropp, bl. a. immunceller och epitelceller i våra blodkärl och vävnader.

Den integrin bindande funktionen är viktig för bakterierna vid en infektion

eftersom det gör det möjligt för bakterierna att binda till våra celler. Ett steg

som är viktigt för att de senare ska kunna ta sig ut från blodkärlen till våra

vävnader.

P13 och P66 visade sig kunna bilda stora proteinkomplex i ytter membranet

hos bakterierna med en storlek på 300 kDa respektive 420 kDa. De är inga

specifika poriner som bara kan transportera en viss typ av molekyl med t.ex.

en viss laddning, utan kan ombesörja upptaget av många olika typer av

ämnen. Eliminering av p66 orsakade att ett annat adhesionsprotein,

uppreglerades. En omplacering av ett normalt cytoplasmatiskt lokaliserat

chaperon-protein till ytter-membranet hos bakterierna kunde också ses i

frånvaro av P66. Chaperonet GroEL har i andra bakterier, bl. a. Helicobacter

pylori, bakterien som orsakar magsår, beskrivits som ett protein som kan

förflytta sig till ytan av bakterierna och där ha en liknande funktion som P66,

dvs. att binda extracellulära matrisprotein. Förändringen i uttryck av

iv

adhesionsproteinet och förflyttningen av chaperonet till membranet var en

följd av p66-eliminering och mest troligt ett sätt för bakterierna att

komplettera den förlorade integrinbindande funktionen av P66.

Det har tidigare visats att poriner är involverade i skyddet mot osmotisk

stress i andra bakterier. Denna funktion hos P13 och P66 i Borrelia kunde inte

ses när bakterier utsattes för osmotisk stress med glycerol, som orsakar en

form av membranstress. Däremot kunde vi med hjälp av transkriptomanalys

se att Borrelia-bakterier uppreglerade transkriptionen av två paraloger till

P13 vid hyper-osmotisk stress. Borrelia bakteriens användning av dessa

paraloga proteiner har tidigare trotts ske enbart i frånvaro av ett funktionellt

P13 protein. Nu visade det sig att P13-paraloger har en egen funktion även i

närvaro av P13, nämligen att vara involverade i regleringen av hyperosmotisk

stress och därmed skydda bakterierna i denna stressituation. Andra gener som

påverkades av osmotisk stress med glycerol var gener för stressfaktorer och

pumpar i inre membranet hos bakterien.

v

Papers in this thesis:

I. Barcena-Uribarri, I.*, Thein, M.*, Maier, E., Bonde, M., Bergström, S.,

Benz, R. (2013) Use of nonelectrotytes reveals the channel size and the

oligomeric constitution of the Borrelia burgdorferi P66 porin. PLoS

One 8(11):e78272.

II. Bárcena-Uribarri, I., Thein, M., Barbot, M., Sans-Serramitjana, E.,

Bonde, M., Mentele, R., Lottspeich, F., Bergström, S., Benz, R. (2014)

Study of the protein complex, pore diameter and pore forming activity

of the Borrelia burgdorferi P13 porin. J Biol Chem 289 (27):18614-

24.

III. Bonde, M.*, Olofsson, A.*, Frost, M., Jegerschöld, C., Bergström, S.,

Sandblad, L. Structural analysis of the B. burgdorferi integral outer

membrane protein, P13, in lipid bilayer Nanodiscs. (Manuscript)

IV. Ristow, L. C., Bonde, M., Lin, Y-P., Sato, H., Curtis, M., Geissler, E.,

Hahn, B. L., Fang, J., Wilcox, D. A., Leong, J. M., Bergström, S.,

Coburn, J. (2015) Integrin binding by Borrelia burgdorferi P66

facilitates dissemination but is not required for infectivity. Cell

Microbiol. doi: 10.1111/cmi.12418

V. Bonde, M., Östberg, Y., Bunikis, I., Nyunt Wai, S., Bergström, S.

Effects of osmotic stress in P13 and P66 porin deficient Borrelia

burgdorferi mutants. (Submitted manuscript)

*These authors contributed equally to this work

vi

Papers not included in the thesis:

Bárcena-Uribarri, I.*, Thein, M.*, Sacher, A., Bunikis, I., Bonde, M.,

Bergström, S., Benz, R. (2010) P66 porins are present in both Lyme disease

and relapsing fever spirochetes: a comparison of the biophysical properties

of P66 porins from six Borrelia species. BBA 1798(6):1197-203.

Bunikis, I., Kutchan-Bunikis, S., Bonde, M., Bergström, S. (2011) Multiplex

PCR as a tool for validating plasmid content of Borrelia burgdorferi. J

Microbiol Methods 86(2):243-7

Thein, M., Bonde, M.*, Bunikis, I.*, Denker, K., Sickmann, A., Bergström,

S., Benz R. (2012) DipA, a pore-forming protein in the outer membrane of

Lyme disease spirochetes exhibits specificity for the permeation of

dicarboxylates. PLoS One 7(5): e36523.

Bárcena-Uribarri, I., Thein, M., Bonde, M., Bergström, S., Benz, R. (2012)

Porins in the Genus Borrelia, p 139-160. In Ali Karami (Ed.), Lyme disease.

InTech. (Review).

Surowiec, I., Orikiiriza, J., Karlsson, E., Nelson, M., Bonde, M., Kyamanwa,

P., Karenzi, B., Bergström, S., Trygg, J., Normark, J. Metabolic signature

profiling as a diagnostic and prognostic tool in paediatric Plasmodium

falciparum malaria. (2015) (Open Forum Infectious Diseases. Under

revision.)

*These authors contributed equally to this work

1

1. Introduction In 1982, the bacterium Borrelia burgdorferi was found to be associated with

Lyme disease. Thus, the microorganism causing Lyme disease has not been

known for a long period of time, although symptoms of the disease had already

been described in the 19th century. Borrelia bacteria have complex nutritional

requirements and a generation time of 5-12 hours. These characteristics make

cultivation and research of the bacteria a challenging mission. Many virulence

factors of Borrelia have been found and studied but still the functions of many

proteins in this bacterium are unknown. The stage at which Borrelia

transcribes a certain group of genes or expresses specific proteins in its

complex life cycle has been studied excessively, but often the function and

actual role of these proteins has not been revealed. The goal of this thesis was

to address how two pore-forming protein complexes situated in the outer

membrane of B. burgdorferi, P13 and P66, are formed and if the proteins have

a function in adaptation to osmotic stress.

1.1. Lyme disease – caused by the bacterium

B. burgdorferi

1.1.1. History of Lyme disease

Lyme arthritis was first reported and described in 1977 by Steere and co-

workers (1). An epidemic of oligoarthritis occurred in the town of Old Lyme

and areas close by, in Connecticut, USA. Mainly children were affected and

many of them were misdiagnosed as having juvenile rheumatoid arthritis. In

many cases the patients developed an expanding skin rash prior to onset of

arthritis and this led to suspicions that something was incorrect with the

diagnosis. The skin rash, now called erythema migrans, was reported by a

Swedish dermatologist, Arvid Afzelius, in the year 1909 (2). Afzelius suspected

that a tick bite was the cause of the skin lesion. Because of the wooded areas

in Old Lyme and peak occurrence of the skin rash in summer, Steere et al. also

believed that ticks were involved in the transmission and postulated that the

symptoms arose from transmission of an infectious agent by an arthropod

vector. Thereafter, a treponema-like spirochete could be found in the

Northern American deer tick, Ixodes scapularis and the spirochete could later

be isolated from blood and cerebrospinal fluid from patients with erythema

2

migrans (3-6). The isolated spirochete belonged to the genus Borrelia (7) and

was named Borrelia burgdorferi after its co-discoverer, Willy Burgdorfer.

1.1.2. Classification

Treponema pallidum (syphilis), Treponema denticola (periodontal disease),

Leptospira interrogans (leptospirosis), and Brachyspira hyodysenteria

(human diarrheal disease, swine dysentery) are all human pathogens

belonging to the same phylogenetic group, the Spirochetes, as Lyme disease

Borrelia. Spirochetes are only remotely related to Gram-positive and Gram-

negative bacteria, possessing many characteristics separate from most other

bacteria (8). The human pathogens Lyme disease Borrelia and relapsing fever

Borrelia belong to the genus Borrelia, as do the avian pathogen B. anserina

and the bovine pathogen B. coriaceae. Lyme disease Borrelia spp. can further

be divided into several species, collectively called B. burgdorferi sensu lato

(s.l.). Three genospecies of B. burgdorferi s.l. predominate as human

pathogens, Borrelia burgdorferi sensu stricto (s.s.), Borrelia garinii (9) and

Borrelia afzelii (10). In the US, B. burgdorferi s.s. is the main cause of Lyme

disease while B. afzelii and B. garinii are more frequent in Europe.

1.1.3. Symptoms

B. burgdorferi s.l., is the causal agent of Lyme disease, a disease with initial

flu-like symptoms and, in many cases, the appearance of a skin rash called

erythema migrans at the site of the tick bite. Most infected humans in Europe

and North America develop this red skin rash, which is the most apparent and

easily noticeable sign and symptom of the disease (11, 12). Without this

dermatological sign, the disease can be difficult to recognize and hence be

difficult to treat at an early stage. In as many as 10% of the Lyme disease

patients, no symptoms of the infection can be seen at all. Early, the disease is

easily treated with antibiotics but can, if left untreated, impair various organs

such as the skin, heart, joints and the nervous system (13). Three Borrelia

species are the dominating pathogens of Lyme disease borreliae. B.

burgdorferi s.s. is the causal agent of Lyme disease in the USA and is

associated with clinical manifestations like carditis and arthritis. Isolates of B.

burgdorferi have also been noted to be differentially infectious (14). B. afzelii

and B. garinii are the two predominant species causing Lyme disease in

Europe, although B. burgdorferi s.s. also exists. Dermatological

3

manifestations like acrodermatis chronica atrophicans are attributed to B.

afzelii infections while neuroborreliosis is most frequently caused by B.

garinii (15, 16).

1.1.4. Detection

A B. burgdorferi infection is most easily recognized if the skin rash, erythema

migrans, has evolved and the patient knows that he or she has been bitten by

a tick or been exposed to areas where infected ticks are present (12). To detect

B. burgdorferi in skin biopsies or cerebrospinal fluid, PCR can be used. A

drawback of this method is that it does not distinguish between live or dead

bacteria (17). Serological tests and analysis of the patient’s antibody response

towards known antigens in Borrelia by Western blot or Enzyme-linked

immunosorbent assay (ELISA) are other, commonly used methods that are

constantly being improved (13, 18). Early in an infection the patient has not

yet produced antibodies and therefore PCR can be more useful at this stage of

infection than serological methods. Even in the absence of an ongoing

infection, the patient can still have antibodies reacting to the spirochete which

can be a drawback of serological tests.

1.1.5. Treatment

Often penicillin, ceftriaxone, cefotaxime and doxycycline are used as

treatment but differences in choice of antibiotics may exist depending on the

patients age, clinical manifestations and location of the patient (12, 19). The

tissue damage and symptoms arising in Lyme disease are thought to be caused

by the inflammatory response provoked by the bacteria in the human host,

rather than by the bacteria themselves. This because the genome of B.

burgdorferi is deficient in genes encoding for secreted toxins that could cause

such damage (20-22). Furthermore, it has not been possible to demonstrate

that post-treatment symptoms of Lyme disease originate from persistent

infection, instead these symptoms are most likely caused by the tissue damage

the bacterial infection initially caused (23). Extended treatment with

antibiotics to treat post-Lyme disease symptoms is for this reason not

effective, since there are no viable B. burgdorferi bacteria present to

eliminate.

4

1.1.6. Prevention

The best way to avoid a tick bite and subsequent Borrelia infection is to avoid

areas where the tick vector is present. If exposure to the tick vector cannot be

avoided, protective clothing and tick repellents can be useful (12). Checking

your body for ticks every day after spending time in areas were ticks are

present reduces the risk of Borrelia infection. The transmission of the

infectious agent does not always occur instantly after a tick bite and removal

of a tick within 24 hours of attachment reduces the risk of infection (24).

5

1.2. General characteristics of Borrelia

B. burgdorferi s.l., the bacteria causing Lyme disease, is a spiral-shaped, thin

bacterium belonging to the phylum Spirochetes (Fig. 1) (25). They can be up

to 30 μm in length and less than 1 μm thick (26). Unlike most other bacteria,

spirochetes have their flagella localized to the periplasmic space, between the

outer and the cytoplasmic membrane. The flat-wave morphology and

spinning/wave-like motility is generated by rotating the flagella in a clockwise

or counter-clockwise direction. Assembly of the flagella has been studied in

detail and motor rotation of the flagella has been seen to not only confer shape

and motility of the bacteria but is also important for infectivity, persistence

and transmission within hosts (27-29). The B. burgdorferi s.s. genome has a

low guanosine-cytosine content and is composed of a linear chromosome and

21 additional plasmids, 9 circular and 12 linear (20, 30). Borrelia spp. are

considered to be Gram-negative due to their Gram stain properties and

because they have both a cytoplasmic and an outer membrane, so called

diderm. However, compared to ordinary Gram-negatives, the outer

membrane of Borrelia is more fluid and consists of 45-62% protein, 23-50%

lipid and 3-4% carbohydrates (25). Another striking difference compared to

typical Gram-negative bacteria is the lack of lipopolysaccharides covering the

surface of the outer membrane; instead, Borrelia species have large amount

of lipoproteins (20, 25).

Fig. 1. Differential interference contrast (DIC) microscopy picture of

the B. burgdorferi strain B31-A. Photo: Mari Bonde

6

1.2.1. Life cycle

Lyme disease Borrelia are obligate parasites, maintained in a complex life-

cycle within the host. The hosts can be ticks of the species Ixodes or different

mammals such as small rodents or humans. Usually, rodents serve as

reservoirs and are a source of infection during feeding of ticks, the vector by

which the pathogen is transmitted. Also, migratory birds have been found to

be important reservoirs of Lyme disease and also as carriers over large

distances (31-35). Ticks have three developmental stages: larvae, nymph and

adult. The larvae are uninfected when they hatch since B. burgdorferi cannot

be transmitted transovarially from adult to eggs. After the first blood meal,

where B. burgdorferi might be acquired if the larvae are feeding on an infected

reservoir host, the larvae molt into nymphs. When the infected nymph feeds,

it will transfer the pathogen to the animal that supplies the next blood meal.

Every developmental stage of the tick requires one blood meal in which the

spirochetes can be ingested and thereafter transmitted to a new mammalian

host by the next blood meal (33, 35). A schematic picture of the B. burgdorferi

transmission cycle can be seen in Fig. 2 (36). In Europe and Asia, ticks of the

species I. ricinus and Ixodes persulcatus, respectively, transmit the

spirochetes, Ixodes scapularis and Ixodes pacificus in North America (35, 37).

Fig. 2. Transmission cycle of B. burgdorferi. This figure is reprinted with

permission from Elsevier, Little, S. E., et al. 2010, Trends in Parasitology, 26(4)

213-218.

7

1.2.2. Genome organization

The B. burgdorferi B31 type strain was the first strain to be sequenced. The

genome comprises a linear chromosome of 910 kbp and 21 additional

plasmids, 9 circular and 12 linear, which together contain more than 600 kbp

of DNA (20, 30). It is believed that the B31 type strain lost two plasmids

between the time it was isolated and its genome was determined and hence

originally contained 23 plasmids (30, 38-40). The gene content of the

chromosome in Lyme disease and the closely related relapsing fever Borrelia

strains is fairly similar whereas the plasmids are more variable in size, number

and content (41-45).

The B. burgdorferi chromosome contains 815, tightly packed, predicted

genes that encode for proteins mostly involved in bacterial housekeeping

functions. Plasmid genes generally encode for lipoproteins and virulence

factors, gene products involved in Borrelia host interactions (20, 30, 44). Only

one plasmid, cp26, carries genes for housekeeping functions important for

nutrient uptake and enzymes for nucleotide metabolism (46-48). This plasmid

also carries the important major outer surface protein antigen, OspC,

expressed by B. burgdorferi in the mammalian host (49-51). resT is another

central gene present on cp26 that encodes for the telomere resolvase, aiding

in replicating the ends of the linear DNA molecules (52, 53). Genes located on

cp26 hence encode for many vital Borrelial proteins and the plasmid is the

only one that cannot be lost without affecting bacterial growth in culture (50,

54, 55). The importance of plasmids in Borrelia is hence inconsistent with the

classical definition of plasmids as nonessential DNA elements.

1.2.3. Paralogous genes and sequence similarities on

plasmids

In contrast to the tightly packed chromosome, the gene density of the B.

burgdorferi linear plasmids is low and contains many paralogous genes as

well as several pseudogenes (20, 30). In fact, most B. burgdorferi B31 plasmid

genes have plasmid-encoded paralogs (44). The porin P13 belongs to gene

family 48, which has 8 additional plasmid-encoded paralogs or pseudogenes

that have been considered non-functional or thought to only be used in the

absence of a functional P13 protein (56, 57). In this thesis (Paper V), we

demonstrate that at least two p13 paralogs are up-regulated at the

transcriptional level in bacteria cultured under osmotic stress applied by

glycerol, even in the presence of a functional P13 protein. This indicates that

8

paralogs of P13 have unique functions, which might also be the case for other

paralogs. Repeated and extended sequence similarities can also be found among the

B. burgdorferi plasmids. Of the 9 circular plasmids present, practically only

three types exists, cp9, cp26 and, interestingly, seven distinct types of cp32

plasmids (30). The cp32 plasmids are homologous nearly throughout their

length, are present in all isolates studied and are thought to be prophages (30,

39, 58-64). Cp32 plasmids encode for surface-exposed proteins of which

some, the erp genes, are known to bind the host complement regulatory

protein, factor H (65). This family of genes, also called ospEF, bind

plasminogen and laminin in addition to factor H (66, 67). Because the

plasmids encode for surface-exposed proteins and extended sequence

similarities exist throughout and between plasmids, it has been speculated

that antigenic variation could be a plausible cause of this plasmid

arrangement. This idea has not been experimentally confirmed, hence the

reason for this genome organization remains a mystery, although it probably

provides an important function in bacterial survival that promotes persistence

of several cp32 plasmids. Plasmids homologous to Lyme disease Borrelia cp32

plasmids have also been found in relapsing fever Borrelia (68).

1.2.4. Importance of plasmids

Extended in vitro cultivation of B. burgdorferi s.l. leads to plasmid loss (69).

This is associated with changes in infectivity of the pathogen, which has been

predominantly analyzed in B. burgdorferi s.s. strains (41, 69-72). It is believed

that plasmid loss occurs because genes important for virulence and

pathogenicity on B. burgdorferi plasmids are not required in the artificial and

safe world of the laboratory milieu. Often, lp25 and lp28-1 are among the first

plasmids that are lost during in vitro cultivation and these plasmids are also

highly correlated with an infectious phenotype of the bacteria (73-75). B.

burgdorferi isolated from different tissues can contain different plasmid

profiles indicating that some sort of tissue tropism exists (73). Lp28-1 contains

the vlsE gene that encodes for a surface-exposed lipoprotein undergoing

antigenic variation, used by the bacteria to escape recognition and elimination

by our immune system (76). Interestingly, lp28-1 and the vlsE gene appear to

be required most often during a B. burgdorferi infection in mice, except when

the bacteria are present in joint tissues (73). Lp25 seems to be the only plasmid

required at all times and in all tissues for an infectious phenotype of B.

burgdorferi due to the gene bbe22/pncA. bbe22 encodes for a protein with

9

nicotinamidase activity, involved in the production of NAD. This gene is

capable of restoring infectivity of a B. burgdorferi clone lacking lp25 (77). Genetic manipulation of B. burgdorferi is difficult because of its low

transformation capacity. Because genetic manipulation also involves harsh

treatment of the bacteria (78) and several passages in culture, often plasmid

loss and loss of infectivity occurs. Bacterial strains that are easier to transform,

maintain their plasmids and hence keep their infectious phenotype have been

created to simplify the laboratory work (79). These strains have an inactivated

restriction-modification gene, bbe02, located on lp25. The gene was first

identified when researchers observed that bacteria lacking lp25 were easier to

transform and its function was later supported by the observation that first

methylating DNA to be introduced enhanced the transformation of B.

burgdorferi (80, 81). Elimination of this gene product, normally involved in

protecting the bacteria from foreign DNA by degrading DNA without correct

modifications, leads to a higher transformation capacity of the bacteria. The

gene is inactivated through insertion of a kanamycin resistance-cassette and

selection with kanamycin for transformants. These transformants

consequently have higher transformation capacity and maintenance of the

lp25 plasmid, which is important for infectivity. A positive effect is that

maintaining lp25 also leads to maintenance of other plasmids for a longer time

(70). A problem with these strains has been that one of the few selective

markers that function in Borrelia is already utilized for interruption of bbe02.

In a recent study B. burgdorferi was complemented with a suicide vector that

inserted the complementing gene into the bbe02 locus (82). This method

overcomes problems with shuttle vector copy number and in vivo stability of

a shuttle vector in the absence of selection (83). In addition, maintenance of

lp25 is ensured and the selective marker kanamycin is available for utilization

in genetic modifications of Borrelia. Multiplex PCR methods have also been

developed in order to quickly and easily keep track of the plasmid content in

bacterial clones and hence the infectious phenotype. Methods both for

screening the plasmid content of a large number of clones (84) and more

suitable for creating single mutants for infectivity studies have been developed

(70).

10

1.3. Laboratory mimicry of the B. burgdorferi hosts

In vitro cultivation of Borrelia is associated with plasmid loss that is believed

to occur because plasmid-encoded genes, important for virulence and

pathogenicity, are not required in the laboratory milieu (41, 69-72). Hence,

gene transcription and protein expression of Borrelia in vitro is not similar to

that within a host, since not all host-associated conditions can be replicated in

the laboratory. These limitations need to be kept in mind when studying

Borrelia and analysing the results of in vitro experiments. Gene transcription

associated with respective hosts can still be triggered to some extent by, for

example, alternating the pH of the growth media and the culturing

temperature in vitro (85).

Cultivation of B. burgdorferi in chambers implanted subcutaneously in mice

or intraperitoneally into rats keeps the bacteria in a more natural

environment, exposed to host factors that affect expression of many B.

burgdorferi proteins (86, 87). Dialysis membrane chambers (DMC)

implanted intraperitoneally into rats have been utilized in many recent studies

and is a cultivation method that allows host factor exposure while the bacteria

are still maintained in rich Barbour-Stoenner-Kelly (BSK) II medium (88),

used for culturing B. burgdorferi in vitro (86). A p66-deficient mutant

behaved much like a wild-type strain within a DMC and the results were

discussed in the context that the porin activity of P66 is not essential for the

bacteria in this protected environment (89). From the nutrient availability

point of view, the DMC surroundings are similar to that of bacteria cultured

in the lab and the results are therefore not so surprising. B. burgdorferi

mutants deficient in porins, usually show a phenotype similar to wild type

when cultured in BSK II in vitro (90). This is thought to be due to the excess

nutrients that surround the bacteria, therefore abrogating the need for the

bacteria to sequester nutrients. This might also occur when B. burgdorferi

porin mutants are cultured in DMC. Results from an amplification-microarray

approach study were recently published analyzing B. burgdorferi gene

transcriptomes in fed larvae, fed nymphs and bacteria cultured in DMC (91).

Interestingly, none of the transcriptomes resembled that of bacteria cultured

in BSK II medium. The bacteria also had unique expression patterns during

each tick stage and in the DMC. Genes encoding lipoproteins, proteins

involved in different nutrient uptake mechanisms, lipid synthesis and carbon

utilization had profound differences in transcription levels (91). It is known

that Borrelia can utilize different carbon sources and that different

carbohydrates serve as the primary carbon source within mammalian and tick

hosts (92, 93). The availability of building blocks hence varies within

11

mammals and ticks and consequently activates different synthetic pathways

within Borrelia. By substituting the main carbon source glucose with glycerol

under tick host conditions in vitro, an even more natural environment would

be established for the bacteria. In addition to the temperature and pH changes

usually made, this carbon substitution would trigger bacterial transcriptional

changes even closer to that naturally occurring within ticks. Although

culturing Borrelia in vitro is artificial, it is essential for studying the bacteria.

For this reason, all components of the media that mimic the bacterial natural

environment are of importance. Components that trigger bacterial gene

transcription and protein expression towards that which naturally occur

within different hosts aid in the research of this important human pathogen.

12

1.4. B. burgdorferi metabolism and carbon

utilization.

Borrelia spp. have reduced metabolic capabilities due to their small genome

and have adapted to a parasitic lifestyle within their different hosts, including

mammals, birds and ticks. The genome of B. burgdorferi lack, for instance,

genes encoding enzymes for fatty acid, nucleotide and amino acid biosynthesis

as well as genes for the TCA cycle (20). By utilizing amino acids, nucleotides

and fatty acids sequestered from the host, the bacteria can utilize energy that

is required to synthesize these building blocks for synthesis of membranes,

proteins, nucleic acids, motility and cell division instead. B. burgdorferi lack

known systems for sequestering iron and interestingly does not seem to

require iron, which is fundamental for many synthetic pathways in other

bacteria (94). Borrelia degrades sugars through the Embden-Myerhof

pathway to lactate (20). Glucose is the carbohydrate utilized in BSK II medium

and it is also the primary carbohydrate in mammalian blood that B.

burgdorferi can use to support growth (88, 93). Glycerol is the major carbon

source for B. burgdorferi within ticks (92). B. burgdorferi can support growth

with, except glucose and glycerol, chitobiose, mannose, N-acetylglucosamine

(GlcNAc) and maltose (93). B. burgdorferi use a phosphotransferase system

(PTS), a family of sugar transporters, for uptake of sugars across the

cytoplasmic membrane. Six PTS systems have been predicted in B.

burgdorferi for glucose uptake, GlcNAc, mannose/galactose and chitobiose

(20, 93, 95). Glycerol uptake is believed to be managed through a predicted

operon consisting of four genes (bb0240-bb0243) with glpF (bb0240) being

a glycerol transporter (20, 93, 95).

13

1.5. Gene regulation in B. burgdorferi

In the complex parasitic life cycle, with alterations between arthropod and

vertebrate hosts with high variability in pH, temperature, carbon source and

osmotic pressure, expression of various Borrelia proteins changes. Borrelia

spirochetes have only two two-component signal transduction systems in

addition to the CheA and CheY orthologs which are associated with

chemotaxis (20, 96). The two-component signal transduction system, Hk2-

Rrp2, along with the Rrp2/RpoN/RpoS regulon promotes mammalian

infection, tick to mammal transmission and is involved in differential

expression of membrane lipoproteins like OspA, OspC and DbpA (97-100). B.

burgdorferi RpoS has been shown to have a different role in gene regulation

when associated with pathogenesis rather than with environmental stress

adaptation, as for example, Escherichia coli (101-103). The other two-

component system, Hk1-Rrp1, has been suggested to be involved in bacterial

feeding within ticks (92). Rrp1 controls, expression of genes involved in

uptake and dissemination of glycerol which has been shown to be a carbon

source that Borrelia can utilize for energy and also the major carbon source

for B. burgdorferi within ticks (92, 93, 104).

14

1.6. Borrelia membrane composition

The membrane is a selective and protective barrier between the bacterial cell

and the outside environment. It must allow passage of nutrients and

substances important for the bacteria while at the same time function as a

selective barrier, excluding harmful substances. The membrane also lie at the

borrelial host-pathogen interface and constantly needs to be optimized for

interaction with the changing environment within different hosts. Borrelia

spp. are often described as Gram-negative bacteria because of the presence of

both a cytoplasmic and an outer membrane with a peptidoglycan layer in

between (25). Profound differences exist and partly explain why Spirochetes

are a phylogenetic group, distantly related to other bacteria. In Fig. 3, a

schematic picture of the B. burgdorferi membrane composition compared to

that of a general Gram-negative bacterium, E. coli is shown. The most striking

difference between the membrane compositions in these bacteria is probably

the lack of lipopolysaccharides (LPS). Instead there is the presence of large,

bulky, immunogenic lipoproteins on the surface of the B. burgdorferi

membrane (105, 106). Lipoproteins are the most abundant proteins in

Borrelia and are often involved in host-pathogen interactions. Several

genome analyses have predicted about 105 to 140 lipoproteins in the genome

of B. burgdorferi. Around 7.8% of the B. burgdorferi open reading frames

were predicted to be lipoproteins by Setuba et al. (107) This is a significantly

higher proportion of lipoproteins than in other bacterial genomes such as

Treponema pallidum (2.1%) and Helicobacter pylori (1.3%) (20, 30, 91, 107,

108). Antigenic variation, evasion of complement killing and adhesion

mechanisms are proposed functions that lipoproteins, anchored to the outer

leaflet of the outer membrane, have in B. burgdorferi pathogenesis (65, 76,

109-117). Other special features of the borrelial membrane are the relatively

low density of transmembrane-spanning proteins and localization of the

flagella to the periplasmic space, in between the outer and the cytoplasmic

membrane (25, 27, 118, 119).

The outer membrane of B. burgdorferi consists of 45-62% protein, 23-50%

lipid and 3-4% carbohydrate and is more fluid than that of typical Gram-

negative bacteria since it contains fewer carbohydrates (25). The phospholipid

content of borrelial membranes differs even from more closely related

bacteria, such as the spirochete T. pallidum. The major lipids in B.

burgdorferi were identified as α-monogalactosyl diacylglycerol (α-MGalDAG

36.1%) and the phospholipids phosphatidylcholine (PC, 11.3%) and

phosphatidylglycerol (PG, 10.5%). The presence of diglyceride-based

glycolipids like MGalDAG in the membrane is most widespread among Gram-

15

positive bacteria and is hence yet another feature separating Borrelia spp.

from typical Gram-negatives. The predominant fatty acid is palmitate (120,

121).

Fig. 3. Outer membrane composition of a common Gram-negative

bacterium, E. coli, and the spirochete B. burgdorferi. The membranes are

composed of both an outer membrane (OM) and a cytoplasmic membrane (CM).

Borrelia spp. have their flagella located in between the outer and cytoplasmic

membrane and, unlike E. coli, have large lipoproteins covering the surface instead of

lipopolysaccharides (LPS).

1.6.1. Transport systems in Borrelia

Porins are responsible for passage of substances across the outer membrane

into the periplasmic space, through diffusion of solutes down a concentration

gradient (122-124). Inner membrane translocators take care of transportation

of substances over the cytoplasmic membrane. Uniporters, symporters and

antiporters are electrochemical transport systems that couple electrochemical

proton gradients to move solutes across the cytoplasmic membrane, against a

concentration gradient. In B. burgdorferi, fatty acids are transported into the

cytoplasm in this manner (20, 95, 125). Resistance-nodulation-division

(RND) transporters also belong to electrochemical transport systems and can

mediate bacterial efflux of antibiotics (125, 126). The best characterized RND-

transporter is the AcrAB-TolC complex in E. coli. ArcAB forms a complex in

the inner membrane that binds to TolC, a membrane fusion protein spanning

the outer membrane and the periplasmic space (127, 128). The complete

complex makes it possible to export substances across both the outer and the

16

cytoplasmic membrane (129). In B. burgdorferi, an RND-transporter,

BesABC, has been discovered that confers B. burgdorferi resistance to various

antibiotics (130). BesC has the same role as TolC in the efflux pump but

differences exist between the proteins. BesC forms larger channels of 300 pS

in 1 M KCl than TolC, which has a channel size of 80 pS under the same

conditions (131). Structural differences between the TolC and BesC channels,

especially in the residues lining the periplasmic entrance, could explain why

BesC has a larger SCC and is non-selective compared to TolC (130). PTS

systems transport carbohydrates across the cytoplasmic membrane (20, 93,

95). Transport can also require energy and by use of ATP hydrolysis, i.e.,

hydrolysis of ATP, the ABC transporters transport substances against a

concentration gradient into the cell. ABC transporters can also function as

secretion systems, exporting, for instance, protein toxins out of the cell (132).

The best characterized oligopeptide transport systems in B. burgdorferi are

encoded on the oligopeptide permease (Opp) operon (20, 95).

1.6.2. Outer membrane proteins

Outer membrane proteins (OMP) are located at the cell surface and facilitate

many interactions between the pathogen and its host. OMPs can act as

adhesins or receptors for different molecules aiding in attachment,

transmission and escape of the pathogen. It is well known that Borrelia spp.

express different types of OMPs depending on whether it is within a

mammalian or tick host. The plasmid-encoded outer membrane protein OspC

has been shown to be important for infection in mammals, while OspA is

abundantly expressed by bacteria within the tick host (114, 133). OMPs can be

targets for bactericidal antibodies since the proteins are exposed on the

surface, towards the host, and are consequently potential vaccine candidates.

OMPs are also responsible for uptake of nutrients that can occur through

porins, water-filled channels that allow diffusion of compounds across the

membrane (134). Passage of solutes across the membrane can also be more

specific and receptor-mediated (135).

17

1.7. Lipoproteins involved in infection and

pathogenicity

1.7.1. Osps

Borrelia spp. have their surface covered with large, bulky lipoproteins called

outer surface proteins (Osps). There are several different types of Osps, OspA,

-B, -C, –D, -E and, -F that Borrelia can alternate expression of depending on

which host they inhabit and their stage in the life cycle. The coding sequence

of OspA was the first one determined and OspA and OspC are the two most

studied Osps (136, 137). OspC expression is coupled to B. burgdorferi

transmission to and presence within the mammalian host. OspA/B expression

is associated with the arthropod host (51, 138). Both OspA and OspC have

functions as adhesins, which aid in spirochete transmission from tick to

mammal. Transmission does not take place before 24 hours of tick attachment

(24) and this delay is thought to be due to alteration in the surface protein

expression of the reciprocally regulated proteins OspA and OspC. The

transition to OspC expression allows the spirochete to detach from the midgut

epithelium of the tick because of down-regulation of OspA. Using the adhesin

OspC, the spirochetes can move from the midgut to the salivary glands of the

tick and thereafter be transmitted into the mammalian host (51, 114, 115).

Antibodies against OspA in the blood of a vertebrate host kill Borrelia in the

midgut of the tick, before the spirochetes have a chance to migrate to the

salivary glands, and hence block transmission (138, 139). Antibodies targeting

OspC are also efficient and probably block transmission by preventing the

spirochete from migrating to the salivary gland (140). Immunization with

either OspA or OspC confers protective immunity in animal studies (141, 142).

A human vaccine, based on immunization with recombinant OspA (rOspA),

was developed that protected against subsequent B. burgdorferi infections

but was withdrawn from the market because of supposed side effects (143,

144). However, a canine vaccine based on rOspA is still available in the USA.

The structure of OspA has been solved and revealed a protein structure of

four β-sheets formed by 21-antiparallel β-strands and a C-terminal α-helix

(145). The three-dimensional structure of OspC is different from that of OspA

and is predominantly helical, containing a dimer of two molecules each

comprising five parallel α-helixes and two short β-strands (146, 147).

Since the Osps are large and bulky, they cover and hide other proteins

present in the membrane. Because of this and the fact that OspA is abundantly

expressed and also relatively resistant to proteases, it has been proposed that

OspA may function in shielding and protecting other proteins in the

18

membrane (148, 149). When studying membrane proteins other than Osps,

the abundant expression, of for example, OspA can be troublesome. Surface

labeling of, for instance, P66 is difficult because antibodies do not reach the

protein in the presence of Osps. Purification of proteins or pull-down

experiments often result in co-purification of OspA. To circumvent this and

other similar issues, a B. burgdorferi strain, B313, has often been utilized

because it lacks OspA, OspB, OspC and OspD expression (148, 150, 151).

1.7.2. The vls locus

VlsE is a 35-kDa lipoprotein in Borrelia that undergoes antigenic variation

and is used by the bacteria to escape recognition and elimination by the host

immune system. The expression site vlsE together with a continuous site of 15

vls silent cassettes comprise the vls locus (76, 117). The plasmid carrying VlsE,

lp28-1, is highly correlated with an infectious phenotype of B. burgdorferi,

which is believed to be mainly due to the vls locus (73, 74). VlsE has been

structurally characterized and contains eleven α-helixes and four short β-

strands (152).

1.7.3. OspE/F-like proteins

A functionally diverse group of B. burgdorferi proteins are encoded on

identical loci on both the circular plasmids cp32 and cp18 (153-155). OspE and

OspF are prototypes of the OspE/F-related proteins (Erp) and the OspE/F-

like leader peptide proteins (Elp), which are members of this family. The

proteins are differentially expressed within the mammalian and tick hosts and

during transmission. Host-specific signals alter the expression pattern (154-

157). Gene rearrangements within this group of proteins are believed to have

generated sequence diversity that has helped Lyme disease Borrelia in its

strategic parasitic lifestyle and with its mechanisms for evasion of the immune

system (153, 158, 159). Some members of this diverse group of proteins, the

complement regulatory acquiring surface proteins (CRASPs), bind

complement inhibiting factor H (CFH), factor H-like protein 1 (CFHL-1) and

several other factor H-related proteins (CFHRs). By binding to these

complement-inhibiting proteins, the pathogen can evade host-mediated

complement killing (116, 160-163). Five CRASPs have been identified in B.

burgdorferi, i.e., CspA, CspZ, ErpA, ErpC and ErpP. The crystal structure has

recently been solved for OspE, ErpC and ErpE (164, 165). To elucidate why

19

ErpA and ErpP can bind CFH while ErpC cannot, the authors compared the

binding sites of OspE with ErpC and ErpP. An extended loop region was found

in the potential binding site for CFH in ErpC. This loop was not present in

ErpE and ErpP and can potentially explain why these two proteins can bind

CFH while ErpC cannot (165).

1.7.4. Mlp

One of the many paralogue protein families in B. burgdorferi is the Mlp

(multicopy lipoproteins) family. Up to ten mlp genes are encoded on the

circular plasmids cp32 and cp18 (158, 166). The Mlp proteins are lipoproteins

expressed on the surface of B. burgdorferi and elicit an immunological

response in the host, thus these proteins are believed to be involved in host-

pathogen interactions (167, 168). The exact function of the protein family is

not known but besides eliciting an immunological response, expression of Mlp

proteins has been seen up-regulated by, for example, elevated temperature

(23°C to 37°C) and mammalian host signals (86, 169).

1.7.5. DbpA/B

DbpA and B are two B. burgdorferi surface-exposed adhesins, binding the

proteoglycan decorin (112, 170, 171). Decorin is present in most tissues and is

present on collagen fibers. Expression of both DbpA and DbpB is up-regulated

at 35°C versus 23°C and antibodies against DbpA can prevent a B. burgdorferi

infection (85, 171, 172). These data indicate that adhesion to facilitate

colonization in the mammalian host via these proteins occurs during the

pathogenesis of B. burgdorferi.

1.7.6. BBK32

BBK32 is a fibronectin-binding protein that promotes B. burgdorferi

attachment to glycosaminoglycans (173). Expression of BBK32 is up-regulated

at 35°C versus 23°C, and inactivation of BBK32 leads to a significantly

attenuated infectious phenotype of B. burgdorferi (85, 174). The fibronectin-

BBK32 interaction hence appears to be important for B. burgdorferi

pathogenesis.

20

1.8. Integral outer membrane proteins, porins

Freeze-fracture electron-microscopy (EM) studies have shown that Borrelia

spp. have a relatively low abundance of integral outer membrane proteins

(118, 119). These bacteria are limited in their metabolic capacity (20, 30) and

sequester nutrients from their host surroundings. Since they are highly

dependent on their surroundings to obtain nutrients, the integral outer

membrane proteins present in the membrane are believed to be porins. Porins

are water-filled channels that allow diffusion of substances into the bacterial

cell and serve as an efficient route of nutrient uptake that is vital to Borrelia

(134). The molecular sieving properties of the outer membrane as a selective

and protective barrier between the bacterial cell and the outside environment

are taken care of by these proteins. Hence, porins allow passage of nutrients

and substances important for the bacteria while at the same time functioning

as a selective barrier, excluding harmful substances.

1.8.1. General characteristics of porins

Porins can be general diffusion porins that sort mainly according to the

molecular mass and size of the solutes or specific porins that have a binding

site for a particular solute inside the channel, such as carbohydrates or

nucleosides (122). In addition to their role in nutrient acquisition, porins can

also function in adaptation to new environments with variations in osmotic

pressure (175). Environments with variations in osmotic pressure might be

encountered by B. burgdorferi when alternating between mammalian and

arthropod hosts as well as when transmigrating through various types of

tissues and body fluids. Porins can also be involved in interactions with other

cells and function as adhesins, binding extracellular matrix proteins (176). The

B. burgdorferi integral outer membrane protein P66 has, for example, dual

function as an adhesin, binding to integrins, and as an active porin with an

extremely high single channel conductance (SCC) (110, 177-179). Msp, the

major surface protein in T. denticola, also forms large pores on the bacterial

surface and, similar to P66, has both a porin and an adhesin activity (180, 181).

By modulating expression of porins, bacteria can survive in the presence of

antibiotics and use the potential to alter porin expression as a survival

strategy. Bacteria can also alter the charge of channel domains, which

consequently disorders diffusion through the pore or modulates the pore size

(176, 182).

21

The E. coli OmpF porin (Fig. 4) is a well-studied, typical porin that form β-

barrels and assembles as a trimer in the outer membrane (124, 183).

Generally, porin monomers consist of 16- or 18- stranded β-barrels and often

have an inward folded loop, attached to the inner side of the barrel that

stabilizes and constricts the internal diameter of the channel. Porin properties

such as diameter, substrate affinity and selectivity are determined by this loop

(124, 184). In addition, α-helical structures are usually not found in porins,

while inner membrane proteins mostly have α-helical structures.

Fig. 4. Three-dimensional structure of the Escherichia coli porin, OmpF.

Internal and surface loops are shown in dark grey, beta sheet in light grey. A side view

of the porin trimer can be seen to the left and top view to the right. This figure is

reprinted with permission from Bentham Science Publishers, Galdiero, S., et al. 2012,

Curr. Proten Pept. Sc., 13, 843-854.

22

1.9. Porins in Borrelia

1.9.1. P13 and its paralog, BBA01

The integral outer membrane protein P13 is encoded by the chromosomal

gene bb0034 and belongs to paralogous gene family 48, which has eight

additional plasmid-encoded genes or pseudo-genes (20, 56). P13 was first

discovered in the B. burgdorferi B313 strain, deficient in OspA-D and DbpA/B

(151). As mentioned, outer membrane proteins without surface structures and

epitopes exposed far from the membrane can be shielded and hidden from

antibody accessibility by Osps (148). Using the B313 strain, it was possible to

generate monoclonal antibodies (Mab) detecting P13, thus confirming the

outer membrane localization of P13. The Mab inhibited growth of the Osp-less

strain but not of a wild-type strain, which implicated lipoprotein coverage of

the P13 epitope (151). Furthermore, immunoelectron microscopy and protease

sensitivity assays confirmed surface-exposed parts of P13. Computer analysis

of the P13 sequence revealed an N-terminal signal peptide, a signal peptidase

I cleavage site and, unusual for porins, three transmembrane α-helices. In

addition, P13 was both N- and C-terminally processed (56). This unusual C-

terminal processing of P13 is performed by carboxyl-terminal protease A

(CtpA). Cleavage of P13 by CtpA is believed to be important for translocation

of P13 to the outer membrane. However, P13 was also present in outer

membrane preparations from ctpA knock-out mutants (185), indicating that

the C-terminal processing has another function. CtpA in photosynthetic

organisms is known to cleave proteins at the C terminus to allow correct

assembly of the target protein (186, 187). Whether this is the function of CtpA

in B. burgdorferi remains to be elucidated.

The pore-forming ability of P13 was first described in 2002. Purified from

outer membrane protein fractions and utilized in the Black lipid bilayer (BLB)

assay, P13 showed an average SCC of 3.5 nS (57). The pore formed by P13 was

voltage independent and slightly selective for cations over anions, although

anions also penetrated the P13 porin in the formed bilayer. The p13 gene was

inactivated by allelic-exchange mutagenesis. Planar lipid bilayer experiments

revealed that the channel-forming activity of P13, at 3.5 nS, was deficient in

outer membrane protein preparations extracted from the mutant (57). In this

thesis, new planar lipid bilayer experiments utilizing P13 are described (Paper

II). Surprisingly, the SCC of P13 was much smaller than previously thought.

Thus, P13 has, according to the new BLB study, a SCC of 0.6 nS. Analysis of

the P13 protein complex by Blue Native Polyacrylamide gel electrophoresis

(BN-PAGE) revealed a molecular mass of about 300 kDa, solely composed of

23

P13 monomers. The P13 channel had an apparent diameter of about 1.4 nm

(188). These results will be discussed in more detail in the Results and

Discussion section, Paper I and II.

BBA01 is the closest paralog to P13, sharing 40.9% identity and 54.1%

similarity at the amino acid level and also showing pore-forming activity.

BBA01 has a SCC comparable to that of the 3.5 nS activity seen for P13

previously. BBA01 is up-regulated in p13-deficient mutants and is present in

B. burgdorferi strains but not preferentially found in outer membrane

preparations unless P13 is absent (56, 57, 189, 190). In Paper V, the response

of B. burgdorferi cultured under osmotic stress was studied. An interesting

finding was that two p13 paralogs were up-regulated at the transcription level

in bacteria under osmotic stress, even in the presence of a functional P13

protein. These results indicate that at least these P13 paralogs might have a

function of their own separate from the function of P13. These results will be

discussed in more detail in the Results and Discussion section, Paper V.

1.9.2. P66, with dual function

P66 is an integral outer membrane protein identified in Borrelia spp. with a

remarkably high SCC of 11 nS in 1 M KCl (177, 191, 192). It is the most-studied

porin in Borrelia and has, interestingly, besides porin activity also integrin-

binding activity (110, 178, 179, 193, 194). The surface-exposed P66 antigen was

initially recognized in many studies focused on serological analysis of sera

from patients with Lyme disease. Patient sera contained antibodies

recognizing a 66-kDa protein (195-198). In 1995, the chromosomal

localization of the 66-kDa protein gene (bb0603) was identified and surface

exposure of the P66 protein could be confirmed by proteinase K treatment of

B. burgdorferi bacteria as well as with computer predictions of a surface-

exposed epitope (192, 199). Many studies have subsequently been performed

on P66 and the surface-exposed, immunogenic domain. The protein has been

found to be present in both Lyme disease and Relapsing fever Borrelia

spirochetes (177, 199). The surface-exposed domain is variable in both size and

sequence and confirmed to be immunogenic by monoclonal antibodies and

sera from Lyme disease patients as well as from infected mice (200-202).

These properties suggest selective pressure on the surface-exposed region and

involvement in antigenic variation and evasion of the host immune system.

The first porin activity of native P66 was demonstrated by using liposomes in

1997 and the integrin-binding activity of P66 was discovered by Coburn et al.

using a phage display library (110, 194). The p66 gene was later inactivated in

24

B. burgdorferi, and the channel-forming activity was eliminated, thereby

confirming the porin activity of the protein. Loss of P66 porin activity also

abolished the integrin-binding activity and the tertiary structure and surface-

exposed domains of P66 were revealed as being important for receptor

recognition and hence integrin binding (178, 179, 193). P66 mediates the

interaction between B. burgdorferi and β3-chain integrins found on different

immune cells, blood platelets and endothelial cells. The integrin-binding

activity of P66 is thought to aid in Borrelia escape from the site of the

inoculum and in dissemination of the bacteria into the tissues, throughout the

mammalian host (89, 203). In paper IV, we further show that deletion of

surface-exposed regions in P66 reduced integrin-binding activity but did not

instigate loss of P66 porin activity or surface localization (203).

Recent studies on the pore-forming activity of P66 have focused on

determining the structure, actual pore size and oligomeric constitution of this

unusually large porin. Kenedy et al. (204) used the P66 sequence in

computational algorithms to study the structure of P66. The predictions

showed that P66 forms a β-barrel with 22-24 membrane strands, has a

surface-exposed loop, and that recombinant P66 is made up of 48% β-sheets.

They also observed that P66 co-immunoprecipitates with OspA and OspB but

the porin activity does not require this interaction. This association between

P66 and OspA has been previously reported by (148). Structural analysis of

the P66 complex has also been studied using BN-PAGE and the BLB assay in

combination with nonelectrolytes. The results revealed that the lumen of P66

is less than 1.9 nm and that the P66 complex consists of 7-8 subunits (205).

These results will be discussed in more detail in the Results and Discussion

section, paper I.

1.9.3. DipA

Dicarboxylate specific porin A (DipA) is the first and only porin in Borrelia

that has been found to be selective for specific compounds. DipA isolated from

B. burgdorferi preferentially allows diffusion of dicarboxylates, such as

oxaloacetate, succinate and maleate (206). The SCC of the protein is 50 pS in

1 M KCl. DipA was first found and characterized in Relapsing fever Borrelia

and the protein was named Oms38 (207). Later studies found the protein to

be present in the Lyme disease species B. burgdorferi, B. garinii and B. afzelii.

Polyclonal antibodies raised against DipA detected DipA in the Relapsing

fever strains B. crocidurae, B. duttonii, B. hermsii, B. hispanica and B.

recurrentis (206).

25

2. Methods

General molecular biology and biochemistry methods have been utilized, for

instance SDS-PAGE, PCR, anion-exchange chromatography. In this section, I

want to describe more specific methods utilized for obtaining data for this

thesis.

2.1. Black lipid bilayer assay

The method for Black lipid bilayer experiments has been described previously

and used for channel size determinations of both eukaryotic and bacterial

proteins (208, 209). The bilayer equipment is illustrated in Fig. 5. and consists

of a Teflon chamber with two compartments, separated by a thin wall with a

small circular opening (0.4 mm2) that connects the two compartments. Over

the small hole, an artificial bilayer membrane can be painted where proteins

with channel-forming capabilities can insert. Insertion of pore-forming

proteins causes a change in the membrane current that can be measured and

used to calculate the size of the channel formed. Since the conductance

determines the size of the pore and the conductance is measured in Siemens,

the size of the pore is specified in Siemens units by this method.

Fig. 5. Schematic picture of the Black lipid bilayer assay equipment. Figure

reprinted with permission from R. Benz.

The membrane solution utilized for BLB assay studies in this thesis was

formed from 1% (w/v) diphytanoyl phosphatidylcholine (DPhPC) dissolved in

n-decane. The protein-containing solution was diluted 1:1 in 1% Genapol and

26

added to the electrolyte on both sides of the Teflon chamber when the

membrane turns black. The two compartments contain in standard

measurements a 1 M KCl salt solution and the voltage applied is 20 mV. The

membrane current is measured with a pair of Ag/AgCl electrodes with salt

bridges switched in series with a voltage source and a current amplifier

(Keithley 427). The experiments were performed at room temperature.

The pore diameter of P13 and P66 were studied in the BLB assay in

combination with nonelectrolytes (NEs) with known hydrodynamic radii, as a

function of the spherical size of the NE (Fig. 6) (210). Polyethylene glycols

(PEGs) were utilized because in aqueous solutions these molecules have a

spherical shape. The method is based on the facts that small NEs that can flow

through a channel-forming protein will decrease the SCC of the channel

proportionally to that of the bulk solution conductivity. The NE will cause an

increase in viscosity of the solution in the channel that will cause changes in

the ion flux (211). Hence, the closer the NEs are to filling the channel

completely, the smaller the SCC will be of the protein studied. When NEs too

large to flow through the channel are used, the SCC will remain unchanged

since the NE is too large to disrupt the bulk solution conductivity through the

pore. The filling of the channels by nonelectrolytes, seen by a decreased SCC,

can be used to calculate the channel size.

The constriction zone inside channels can also be analyzed as a function of

the spherical shape of the NEs (211). In this concept the radius of a channel

constriction zone should be close to the radius of the smallest NE that does

not fill the channel completely and therefore does not pass freely through the

channel.

Fig. 6. Schematic illustration of how NEs with known hydrodynamic radii

can be used to determine channel size of pore forming proteins.

27

2.2. Nanodiscs

Nanodiscs are small, artificial membranes in a solution in which proteins of

interest can be inserted in an active form without the requirement for

detergents that often cause inactivation of the proteins (212-214). The discs

hold integral membrane proteins in a soluble form in a native-like lipid

environment. The naturally folded and assembled proteins can by studied by

EM and structural analysis can be done. High-resolution single-particle cryo-

electron microscopy (cryo-EM) has, for example, enabled subnanometer-

resolution structural determinations of membrane protein complexes in these

lipid bilayer environments (215). We used in this study Nanodiscs to reveal

structural information of the P13 porin in a biologically relevant environment,

using negative staining and EM as well as single particle cryo-EM.

The Nanodiscs are composed of a phospholipid bilayer that is encircled and

limited in space by two α-helical membrane scaffold proteins (MSPs) (Fig. 7)

(213).

Fig. 7. Schematic picture of an assembled Nanodisc. Two membrane scaffold

proteins, shown in blue and green, encircle a lipid bilayer and determine the size of the

Nanodisc. The lipid headgroups are shown in orange and the tail in gray. This figure is

reprinted with permission from Shih, A. Y., et al. 2007, Nano Lett., vol. 7, No 6. 1692-

1696. © 2015 American Chemical Society.

MSPs can be found naturally and are called high-density lipoproteins (HDL).

The natural function of HDL is to flow through blood, transporting cholesterol

to the liver for degradation (216). The human apolipoprotein A-1 (apoA-1)

sequence has hence been used as a template to further design MSPs with

different sizes and modifications (213, 214). Because of the large size of both

the P66 (around 420 kDa) and the P13 (around 300 kDa) complexes, the

28

commercial available MSP1E3D1 (217) and MSP2N2 (218) were used in this

study. MSP1E3D1 and MSP2N2 provide Nanodiscs of about 12 nm and 17 nm,

respectively. The MSPs were expressed in E. coli BL21 Gold cells and a

His/Trap column was used to purify the Histidine-tagged MSPs. POPC, 1-

palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, was utilized as lipid. MSP,

lipid and protein of interest are mixed together with the detergent cholate.

Upon removal of cholate, Nanodisc assembles spontaneously (219).

29

3. Aims of the thesis

The general aim of this thesis was to study characteristics of the two B.

burgdorferi pore-forming proteins P13 and P66. This was performed by

analysing complex formation and molecular organization of the protein

complexes. Possible functions of the porins were studied in vitro and in the

case of P66, also in vivo. The specific goals were:

To study channel size and constitution of the P66 outer membrane protein

complex by use of Black lipid bilayer analysis in combination with

nonelectrolytes and Blue Native PAGE.

To analyse the channel size of the P13 porin and the molecular organization

of the P13 complex by use of the Black lipid bilayer assay in combination

with nonelectrolytes and Blue Native PAGE.

To evaluate and study the structure of the P13 protein complex in artificial

bilayer membranes called Nanodiscs using electron microscopy.

To determine the importance and function of P66 in a Borrelia infection in

mice.

To investigate the possible role of P13 and P66 by transcriptional analysis

under osmotic/membrane stress applied by glycerol

30

4. Results and discussion

4.1. Paper I and II

4.1.1. Characterizations of the actual pore sizes and sizes

of the protein complexes formed by the P13 and

P66 porins.

The pore sizes formed by P13 and P66 have been assessed with biophysical

measurements, i.e., the BLB assay. The SCC of P13 was earlier determined to

be 3.5 nS in 1 M KCl (57) but was in Paper II re-evaluated and instead shown

to have pore-forming activity at 0.6 nS. The P13 protein is unusual considering

that this protein forms pores in the outer membrane despite its small

molecular mass (13 kDa) and predicted α-helical structures (56, 57). The porin

P66 can form extremely large pores with a SCC of 11 nS in 1 M KCl. P66 pores

are nonselective for anions or cations and exhibit voltage dependent closure

(177, 194). Porins from other spirochetes such as T. denticola and Spirocheta

aurantia also display large SCCs (180, 220). This is a feature otherwise rare

to porins in Gram-negative bacteria. An approximate channel diameter of 2.6

nm has also been calculated for P66 (194). This calculation was based on the

assumption that the conductance of P66 is equal to the conductivity of a

straight cylinder in an aqueous salt solution. The length of P66 was assumed

to be equal to the thickness of the membrane and factors such as form of the

channel were not taken into account.

4.1.2. Pore-forming activity of P13

In Paper II, the pore-forming activity of the P13 complex was studied using

the BLB assay after elution of the P13 complex from BN-PAGE gels.

Surprisingly, the 3.5 nS activity previously seen for P13 could not be detected

(57). The pore-forming activity of P13 was now 0.6 nS. In previous studies of

the pore-forming activity of P13, the protein was purified with anion exchange

chromatography and thereafter studied in the BLB assay (57). Here, the P13

complex was eluted from BN-PAGE gels. Variations in purification of the P13

complex could therefore be the reason for the different results obtained in the

two studies measuring the pore-forming activity of P13. To test the integrity

of the eluted P13 protein complex, additional BN-PAGE separations were

31

made and the P13 complex retains its native state. This indicates that the

smaller P13 activity seen in these experiments was not caused by degradation

of the complex or P13 protein. Activity at 0.6 nS has been observed in a

previous BLB assay analysis of outer membrane vesicles from B. burgdorferi

(221). The protein thought to be responsible for the activity was Oms28

(BBA74) (222). Oms28 was first believed to be a porin but has subsequently

been re-evaluated because of its predicted α-helical structures and apparent

periplasmic localization, as determined by insensitivity to proteinase K

treatment and is now designated BBA74 (223).

The pore-forming activity of P66 was similarly investigated after eluting the

460-kDa complex from BN-PAGE gels and P66 retained its pore-forming

activity (Paper I). In addition to the controls performed in Paper II, retention

of the P66 pore-forming activity after elution from BN-PAGE gels shows that

the measurements of the P13 pore-forming activity of 0.6 nS are reliable. In

Paper II, the P13 pore was also shown to be a general diffusion channel

without preference for a specific compound and voltage-independent up to ±

100 mV.

4.1.3. Size of the pores formed by P13 and P66

In Paper I and II, the actual pore size of P13 and P66 were determined with

the BLB assay using NEs. After reconstitution into BLBs, the conductance of

the P13 and P66 pores were studied as a function of the spherical size of NEs

with known hydrodynamic radii (210). The method used is described in detail

in the Methods section.

NEs with hydrodynamic radii larger than 0.94 nm did not enter the P66 pore

and had no influence on the conductance through the pore. NEs with

hydrodynamic radii up to 0.6 nm caused a decreased conductance through

P66, proportional to the conductivity of the salt solution. From these

experiments, the entrance radius of P66 was calculated to be 0.94 nm. A

possible restriction zone diameter of the P66 channel was estimated to be 0.78

nm based on Sorbitol (r=0.39 nm) being the smallest NE that could not pass

freely through the P66 channel and did not fill the channel completely.

Interestingly, PEG400 and PEG600, with hydrodynamic radii of 0.7 and 0.8

nm, respectively, caused a drastically reduced SCC that was not proportional

to the bulk aqueous solution. The conductance decline after addition of these

NE compounds was very slow and the level of noise very different from that of

titration experiments with substrate-binding porins and channels (224, 225).

A nonchemical interaction between the polymer and the P66 channel interior

32

therefore appeared to be the cause of the reduced SCC, a result not reported

previously for any other channel.

NEs with a hydrodynamic radii of 0.7 nm or larger did not change the SCC

of P13 and were therefore not able to enter the P13 channel. NEs with a

hydrodynamic radius of 0.6 nm and smaller could enter the channel and

caused a decrease in P13 SCC. From these experiments, the diameter of the

P13 pore was calculated to be 1.4 nm. We could not identify NEs that only

partly filled the P13 channel. Therefore, the P13 channel is unlikely to contain

a constriction inside the channel although this possibility cannot be excluded.

4.1.4. Oligomeric constitution of the P13 and P66

complexes

P66 forms exceptionally large channels in the outer membrane of B.

burgdorferi. Knowledge about the molecular organization of the complex is

therefore of high interest and could explain the high conductivity of the porin.

To study the molecular organization of the complex, NEs was used to induce

blockage of the P66 pore. A single 11 nS P66 protein complex unit was

reconstituted into the BLB assay membrane and PEG-400 was added. PEG-

400 was shown to induce a step-wise closure of the P66 pore in 7-8

subconductance steps, with a conductance of about 1.5 nS in 1 M KCl each. To

study the oligomeric constitution and content of both the P13 and P66 protein

complexes, BN-PAGE was also utilized. BN-PAGE analysis of native P66

revealed protein complex of approximately 460 kDa that in a second

dimension SDS-PAGE was shown to only contain P66. Thus, the obtained

results fitted well with the suggested hepta- or octameric complex of P66 seen

in the BLB assay with PEG-400.

The P13 pore-forming activity obtained from eluted 300-kDa protein

complexes showed intrinsic noise after reconstitution of a few insertions. This

made it difficult to further analyze the exact conductance of new insertions

and to analyze the oligomeric constitution of the complex by addition of NEs

as we did for P66. This requires stable insertion of the protein complexes. The

300-kDa protein complex formed by P13 was further studied by second

dimension SDS-PAGE instead and was seen to be homo-oligomeric complexes

composed of only P13 protein. The actual size of protein complexes seen by

BN-PAGE might vary by ± 15% normally. The reason for this is because

proteins are separate in their native, folded state. Protein complexes formed

of proteins with a high pI value, like P13, can show a molecular mass up to

30% higher than its actual molecular mass (226). In Western blots of second

33

dimension SDS-PAGE gels, spots of around 200 kDa were shown to contain

P13. These spots could correspond to P13 complexes retained in their native

state and thus reflect its actual oligomeric size. The results still show that the

small P13 protein of only 13 kDa, forms a quite large complex composed of

only P13 monomers in the outer membrane of B. burgdorferi. The P13 paralog

BBA01 was not detected in the complex.

The SCC of BBA01 has been studied previously in complete outer membrane

protein fractions isolated from a p13-defcient B. burgdorferi strain with bba01

introduced on a plasmid. The activity of BBAo1 was similar to that previously

seen for P13, 3.5 nS (189). SCCs of recombinant forms of P13 and BBA01 were

analyzed in the same study and activity at 3.5 nS activity was observed for both

proteins. In studies of the pore-forming activities of both P13 and BBA01, the

proteins have been purified or analyzed in different ways than eluted from BN-

PAGE gels. Analyzing the complex formation of BBA01 compared to that of

P13 on BN-PAGE gels would therefore be of interest, as well as the pore-

forming activity of BBA01 eluted from gels.

34

4.2. Paper III

4.2.1. Structural analysis of P13 in Nanodisc

To further investigate the protein complexes formed by P13 and P66,

structural analysis in form of 3D-crystallization or use of artificial membranes

called Nanodiscs were considered. Since membrane proteins are difficult to

express and purify in large amounts, especially in Borrelia, we investigated B.

burgdorferi porin structures using Nanodiscs. By this method, less purified

protein is needed and in Nanodiscs membrane proteins can be inserted and

assembled in their natural confirmation in a lipid environment without the

presence of detergents (212, 213), which could otherwise influence protein

folding and the activity of the protein.

Nanodiscs are described in detail in the Methods section. Briefly, they

consist of a membrane scaffold protein (MSP) that encircles the artificial lipid

membrane and inserted protein of interest. The MSP consequently

determines how large the membrane can be (213). P66 forms complexes of

460 kDa (Paper I). The MSP for the largest artificial membranes available to

date is MSP2N2 (218), creating a membrane size of 17 nm. These membranes

could be too small to fit an entire P66 complex. In addition, it has been seen

that protein complexes that theoretically would fit into an MSP only form

complexes in an MSP forming a larger membrane. This as a consequence of

the fact that a number of lipid molecules need to be associated with the

Nanodisc-protein complex and therefore also fit into the MSP to allow native-

like structures to form (227, 228). P13 assembles into smaller protein

complexes as seen by BN-PAGE (Paper II) and therefore P13 was chosen to

evaluate Nanodiscs as a method to study membrane proteins in Borrelia.

Negative staining and cryo-EM was further used to visualize the Nanodiscs in

these early studies of the P13 structure.

4.2.2. P13 is a unique channel-forming protein

Structural predictions using the P13 sequence has shown that P13 is composed

of α-helical structures and not β-sheets like typical porins (56, 124, 190).

Membrane topology modeling and predictions of tertiary structure have been

performed on P66 utilizing the RED-TMBB

(http//biophysics.biol.uoa.gr/PRED-TMBB/) and TMBpro

(http://tmbpro.ics.uci.edu/) servers, respectively (204, 229). Utilizing

TMBpro, β-sheet structures were also predicted for P13. Since porins and

35

outer membrane proteins usually contain β-sheets and form β-barrels, it is not

surprising that software designed to predict membrane proteins also predict

β-sheets in the sequence of P13. I-TASSER

(http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (230-232) is another

method by which structures of proteins can be predicted and this server

predicts structures of various types of proteins, not only membrane proteins.

By I-TASSER, P13 was predicted to contain α-helical structures. This further

enhanced our desire to gain actual structural information on this atypical

porin, P13, in its native state, reconstituted into a lipid membrane Nanodisc.

Proteins inserted into Nanodiscs visualized by high-resolution single-particle

cryo-EM have enabled subnanometer-resolution structural determinations of

membrane protein complexes (215). Hence, this method could be a valuable

tool for our goal to gain more information on the P13 protein structure.

4.2.3. P13 in Nanodiscs

In this study, two MSPs were utilized, MSP1E3D1 (217) and MSP2N2 (218)

which create membranes with sizes of 12 nm and 17 nm, respectively. In the

previous BLB assay, we determined that P13 and P66 porins insert into

diphytanoyl phosphatidylcholine (DPhPC) environments and therefore a

phosphatidylcholine lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

(POPC) was further utilized for assembly of Nanodiscs. In initial attempts to

insert P13 into MSP1E3D1 and MSP2N2, quite homologous populations of

Nanodiscs could be seen but appeared to contain various amounts of P13

monomers. The correct ratio between lipid, MSP and protein of interest is

important to create optimal Nanodiscs and this needs to be further optimized

and could explain the varying results. A limitation of MSPs is that they restrict

the size of the membrane formed. Amphipols are amphiphatic polymers that

could be utilized to further study the P13 complex with EM but without

limiting the space required. Amphipols have been developed as stabilizing

surfactants for handling membrane proteins in their native, functional state

in an aqueous solution (233-235). This is done without the addition of

detergents and without limiting the space availability for the protein complex

as MSPs do. Since the size of the P66 protein complex is thought to be even

larger than the P13 complex, Amphipols should be more useful in those

studies as well.

36

4.3. Paper IV

4.3.1. Integrin binding and porin function of P66 during

B. burgdorferi infection.

P66 has dual function. Besides the porin function, P66 also binds integrins

located on the surface of immune cells and epithelial cells, working as an

adhesin (110, 179). During dissemination within the host, it is known that B.

burgdorferi attaches and interacts with different cell types, for instance

epithelial cells in the microvasculature. Three forms of interactions with the

vasculature—tethering, dragging and stationary interactions—have been

identified (236, 237). BBK32 is a B. burgdorferi surface adhesin involved in

two of these interactions. BBK32 binding to fibronectin is required for

tethering and BBK32 binding to glycosaminoglycans is required for dragging

interactions (236). Proteins necessary for stationary interactions of B.

burgdorferi and transmigration from the vasculature to the surrounding

tissue have yet to be identified.

P66 can bind both β3 and some β1 integrins, located on, for instance, blood

platelets, epithelial, endothelial and smooth muscle cells (236, 238-240). A

portion of P66, amino acid (a.a.) 142-384, contains all the sequences required

for the integrin-binding activity of P66. A synthetic peptide of the a.a. 203-

209 can compete with intact B. burgdorferi for binding to αIIbβ3 (179).

∆p66 bacteria inoculated subcutaneously or intradermally are cleared from

the site of inoculum within 48 h. Clearance of the bacteria is thought to be

mediated through host defences already present at the site of inoculum and

not by defence cells migrating to the site (89). Up-regulation of the vascular

endothelial growth factor/vascular permeability factor pathway has been

noted in cells incubated with wild-type bacteria compared to cells incubated

with ∆p66 bacteria (241). A function of P66 might therefore be to facilitate

bacterial migration into the vasculature across endothelial barriers.

Because of the integrin-binding activity of P66 and the protein’s possible role

in bacterial migration, we wanted to study if P66 integrin-binding activity

contributes to B. burgdorferi infection in mice. Recombinant MBP-P66 fusion

proteins (110) were constructed with mutations in the suggested sequence

implicated in integrin binding of P66. Aspartic acid residues 205 and 207 were

changed to alanine in D205A and D207A mutants. Another mutant, Del202-

208, containing a seven-a.a. deletion, was also constructed.

37

4.3.2. Pore formation and integrin binding analysis of

P66 mutants

The D205A, D207A and Del202-208 mutants were created in an infectious

B31-A3 strain and in a high-passage, non-infectious HB19 (WT) strain. The

BLB assay was used to study the pore-forming activity of P66 isolated from

P66 mutants and from the WT strain. The 11 nS SCC characteristic for P66

could be seen for the WT and the mutated forms of P66, indicating that

assembly of the P66 porin complex was not interrupted by mutations in the

surface-exposed parts of P66. In addition, a band of 460 kDa co-migrated with

the WT protein when mutant P66 protein complexes were analyzed by BN-

PAGE. This further confirmed that the mutated P66 proteins were able to

assemble into protein complexes of the same size as WT P66.

The integrin-binding abilities of the P66 variants to integrins from both

humans and mice were tested. Greatly reduced integrin-binding activity could

be seen for the Del202-208 mutant while D205A, D207A had a binding

affinity closer to that of WT P66. These results indicated that the integrin-

binding affinity of P66 is dependent on several a.a. in the region and not only

205 and 207. Together, the results from the pore-forming activity and

integrin-binding activity analysis also showed that the integrin-binding

function of P66 can be separated from its porin function. Alterations in

regions important for integrin-binding of P66 did not affect the ability of P66

to form 11 nS pores, or to form the complete oligomeric P66 complex.

4.3.3. Importance of the integrin-binding region of P66

in the establishment of a B. burgdorferi infection

and endothelial transmigration

C3H/HeN mice were infected with the WT B31-A3 strain, and the mutants

D205A, D207A and Del202-208. Four weeks after inoculation, heart,

tibiotarsus, skin (at the inoculation site) and ear tissues were harvested for

culture and qPCR analysis. From the results, it was clear that WT level of β3

chain integrin binding is not required for B. burgdorferi to establish a

disseminated infection. All strains that had mutations in p66 had ID50 values

comparable to the WT strain. A more substantial colonization defect was seen

for bacteria with the Del202-208 mutation of P66.

In further in vitro experiments, the ability of P66 mutants to cross

endothelial cell monolayers were tested. By 48 h, a significant defect in the

ability of Del202-208 bacteria to cross the monolayer was seen compared to

38

WT bacteria, while there was no significant difference between WT and

D205A, D207A mutant bacteria. The ability of the P66 mutants to cross

endothelial cell layers and exit the vasculature in vivo was also tested.

Intravenously inoculated Del202-208 mutants could not be recovered in

tissues two weeks after inoculation. When the infection was allowed to

progress for 4 weeks, the Del202-208 and D205A, D207A mutants were,

surprisingly, as infectious as WT bacteria. Variations in bacterial burden in

tissues isolated could be seen between mice inoculated with WT and mutants,

which could be explained by this delay in colonization. Spirochetes with an

altered P66 protein might have to establish a stable bacterial burden first, to

be able to maintain the infection and later disseminate to various tissues. As

seen in Paper V, elimination of P66 leads to up-regulation of other

extracellular matrix binding proteins. This might also be of importance for the

P66 mutants studied here and could be the reason for the observed delay in

colonization. Possibly, the bacteria already made changes to adapt to the

mutated P66 protein in culture. These alterations might, however, not be

sufficient when the bacteria are exposed to host factors and situations where

integrin binding is needed and further changes therefore need to be made.

The results of this study showed that the integrin-binding function of P66

seems to be involved in bacterial endothelial transmigration in vitro and in

vivo. In addition, the integrin-binding affinity of P66 seems to be dependent

on several a.a. in the region 202-208, not only a.a. 205 and 207. The integrin-

binding activity of P66 was, however, not shown to be required for B.

burgdorferi to establish an infection in mice. These results, in combination

with the complete loss of infectivity seen for ∆p66 in previous studies (89),

indicates that an additional or several additional functions of P66 are

important for establishing a B. burgdorferi infection. Possibly, the porin

function, P66 induction of the vascular endothelial growth factor/vascular

permeability factor pathway, or yet another unknown function of P66 is

involved in the early phase of Lyme borreliosis.

39

4.4. Paper V

4.4.1. Function of P13 and P66 in B. burgdorferi during

osmotic stress adaptation induced by glycerol.

In Paper I, II and III, structural determinations of the P13 pore and pore

complexes were studied and in Paper IV, the integrin-binding function of P66

was studied. The function of P13 and P66 as porins is, however, still

uncharacterized and has not been studied extensively. It is known that porins

have an important function in the sieving mechanism of the outer membrane,

are involved in nutrient uptake, can function as adhesins and can also be

involved in osmotic adaptation of bacteria (122, 175, 176). B. burgdorferi

might encounter changes in osmotic pressure during its natural life cycle, i.e.,

when alternating between mammalian and arthropod hosts as well as when

transmigrating through various types of tissues. Porin mutants deficient in

p13, p66 or double mutants have previously been created in the B. burgdorferi

strain B31-A (57, 193). Herein, these mutants were utilized to analyze the

function of P13 and P66 in B. burgdorferi adaptation to a form of membrane

stress, i.e., osmotic stress induced by glycerol.

4.4.2. Porin transcription and expression in B.

burgdorferi bacteria cultured under osmotic

stress

Osmotic stress was induced by 7% glycerol because at this percentage the B31-

A strain showed a significantly reduced growth rate compared to that in BSK

II medium. The porin mutants, ∆p13, ∆p66 and ∆p13∆p66, also had a reduced

growth effect in glycerol compared to that in BSK II medium, where the

mutants had similar growth as the B31-A strain. When analyzing expression

of P13 and P66 in bacteria exposed to osmotic stress, it was obvious that both

porins were down-regulated. This indicated that P13 and P66 are not actively

involved in regulation of osmotic stress induced by glycerol. The bacteria

adapt to the variation in osmotic pressure by down-regulating P13 and P66. A

full-genome transcription analysis was performed and from these results it

was clear that the genes encoding for P13 and P66 are not actively transcribed

in bacteria subjected to glycerol stress. Instead, two p13 paralogs had

increased transcription in bacteria cultured under osmotic stress. This was

surprising since one paralog, BBA01, was previously thought to only be

utilized in the absence of a functional P13 protein (189). The results here

40

indicate that paralogs of P13 can have a function of their own even in the

presence of a functional P13 protein. Whether the P13 paralogs up-regulated

here have functions in regulating other types of stress conditions has not been

investigated.

4.4.3. Increased transcription of an extracellular matrix

binding protein and membrane localization of a

heat shock protein in response to p66 deficiency.

When analyzing transcription of genes highly up-regulated in osmotically

stressed B. burgdorferi strains, bacteria deficient in p66 had, interestingly,

up-regulated transcription of another extracellular matrix binding protein.

This up-regulation could be seen in both ∆p66 and ∆p13∆p66 but not in the

B31-A strain, nor in ∆p13. These results indicate that up-regulation of the

adhesin was more a consequence of p66 deficiency and not caused by osmotic

stress. Interestingly, in separated outer membrane protein fractions from B31-

A, ∆p13, ∆p66 and ∆p13∆p66 cultured in BSK II medium, a heat shock protein

was present in membrane protein fractions from p66-deficient mutants.

Increased expression of the heat shock protein could, however, not be seen in

total protein samples from p66-deficient mutants compared to those from

B31-A. The normally expressed heat shock protein in p66-deficient mutants

was therefore present in a different location from the cytoplasm, i.e., in the

membrane. Surface localization of a heat shock protein, GroEL, has been

found on mucosal pathogens, like H. pylori, Lactobacillus johnsonii and E.

coli, and has been implicated in cell attachment and immune modulation in

these bacteria (242-244). Interestingly, surface-localized heat shock proteins

and the extracellular binding protein up-regulated in this study have

implications in binding extracellular matrix proteins, which is the function of

P66 as an integrin-binding protein (110, 178, 179). Because of this, we believe

that the effects seen in response to p66 deficiency are a consequence of the

lost integrin-binding function of P66. Thus, the bacteria up-regulate other

extracellular matrix binding proteins in the absence of P66.

B. burgdorferi interact with the microvasculature during dissemination

within the mammalian host (236, 237). To be able to do this, many different

outer membrane proteins involved in binding various extracellular

components are encoded in the genome of the pathogen. Because of lost

plasmids in the B31-A strain utilized in this study, transcriptional differences

in response to p66-deficiency of, for example, bbk32 could not be seen. BBK32

is another extracellular matrix binding protein, binding fibronectin and

41

glycosaminoglycans (173, 174). Transcriptional changes in response to p66

deficiency need to be further assessed in a B. burgdorferi strain possessing

the full genome content.

4.4.4. Similarities in transcriptional regulation of

putative transporters and stress factors in B31-A

and ∆p13∆p66 in response to osmotic stress.

The B31-A and ∆p13∆p66 B. burgdorferi strains could adapt best to glycerol

stress conditions, as assessed by analyzing bacterial growth. These strains had

the highest number of live bacteria at day 7. When analyzing genes highly

differentially regulated in all strains exposed to osmotic stress, it could be seen

that B31-A and ∆p13∆p66 bacteria also shared similarities in gene

transcription. This was especially clear for transcription of genes encoding

putative transporters in the inner membrane and also several stress factors.

We therefore decided to analyze how gene transcription of several

transporters and stress factors were regulated in B31-A and ∆p13∆p66

compared to ∆p13 and ∆p66. Generally, many transporters and stress factors

were down-regulated in bacteria exposed to osmotic stress, indicating shut-

down of many cellular processes. Overall, when studying the changes in gene

transcription of stressed bacteria to the BSK II control, genes on the

chromosome were down-regulated. These genes are often involved in borrelial

housekeeping functions (20) and therefore this result also points to the fact

that the bacteria focus on something else than housekeeping functions under

osmotic stress. An apparent pattern was also that transporter and stress factor

genes up-regulated in B31-A and ∆p13∆p66 were down-regulated in ∆p13 and

∆p66 and vice versa. Although not all genes in the groups were regulated

similarly by B31-A and ∆p13∆p66, these similarities possibly explain why

these strains coped best under osmotic stress conditions.

42

5. Conclusions

P66 form large membrane complexes of about 460 kDa, with an oligomeric

constitution of about 7-8 subunits. P66 is the only protein present in the

complex.

P13 pore-forming activity was found to be 0.6 nS in 1 M KCl.

The membrane complex formed by P13 is approximately 300 kDa, and

solely composed of P13 monomers.

Initial evaluation of Nanodiscs as a method to study structure of membrane

proteins in Borrelia looks very promising.

The integrin-binding function of P66 is not important for establishment of

a B. burgdorferi infection but is instead important for bacterial endothelial

transmigration and dissemination.

P13 and P66 are down-regulated and therefore not actively utilized in B.

burgdorferi adaptation to osmotic stress induced by glycerol.

Two P13 paralogs seem to be the porins preferentially utilized in B.

burgdorferi under osmotic stress.

p66 deficiency caused increased transcription of an extracellular matrix

binding protein and membrane localization of a heat shock protein as a

possible response to the loss of integrin-binding function of P66.

43

6. Acknowledgements First of all I would like to thank Sven, the best boss I could have ever had! You are

inspiring with your positive and laid back attitude despite a stressful environment. I

appreciate that I have been allowed to work under my own responsibility. It has been

a challenge and I like that. Also, that you have allowed me to create my own education

although it has meant that I have been away from the lab sometimes. I have learnt a lot

about research and it has been a time for great personal development. Thank you for a

wonderful time!

Ingela, or was it Thelma or Louise :) my travelling companion for so many enjoyable

and exciting trips. Thank you for all the fun times we have had both in- and outside of

the lab and of course for your excellent lab work! What would Grp. SvB be without

you?! No offense, Sven! Maria N., my big-sister ;) for a great conference-trip and for

all discussions we have had about life in general during the years and especially the last

months. I am glad to have had somebody to ventilate my thesis-issues with and that

goes for the rest of the DA-group also, Hanna and Lena. Annelie for always being

positive and cheering me up. It has been fun working with you and I hope the rest will

be piece of cake. Thanks to both you and Linda S. for working in high speed with the

ND-manuscript. Maria F., Yngve and Betty for evaluating this thesis and for

helping me with the stress manuscript. Elisabeth, my jungle-friend, for great

company during our Rwanda adventure. I know we were struggling sometimes but still,

I can only remember how much fun we had. It was a work-trip to remember! Thank

you, Johan and Sven, for allowing me to go! Johan, for being inspiring with all your

ideas and multitasking and for the work you have done with the stress-manuscripts!

Ignas, my Baloo, for teaching me, if not all that you can, at least many valuable things

I have had great value of in the lab and for showing me that this world is not always an

easy world. Elin, Lisette, Christer, Pär, Marie and Lelle for the great spirit in the

SvB lab when I first started and for fun times. Patrik for being a great fellow PhD

student. When I hear Broder Daniel songs, all I think of are our long days in the lab

with our hands stuck in the sterile bench, singing along. To Ivan, Marcus and

members of prof. Roland Benz’s lab for always taking care of me in my second lab-

home on various places in Germany. Jenifer Coburn and Laura Ristow for superb

collaboration. Thank you to all the people at pysselkväll and in the bunker for

discussions about everything and nothing during the years. Helena, Jenny, Reine,

Lisa, Christian, Tobias, Jim (not for throwing blue balls in my face), Sveta, Maria

L., Micke S. Linda W for all the fun times and for introducing me to the Lindy hop

environment. To former and present members of the SvB lab. Haitham and Wael for

looking after me and making sure that I am OK. Mukunda for our nice discussions.

Thank you to Grp UvPR, CU, LS for interesting and educational journal clubs and

WIPs. To Johnny for saying what needs to be said, for your wonderful humor and of

course for ordering stuff. You, the media ladies and secretaries have made my work

at the Department so much easier. I am grateful and we are lucky that you exist and

keep the department running. To all present and former members of the Department

44

of Molecular Biology that has made it a perfect environment and a place I have enjoyed.

Cecilia Elofsson and Lars Åberg for good, short and quick answers and for all

valuable help in moments when I needed it the most!

To all my friends during the years that have been lightening up my life outside work.

You know who you are!

Tack till mamma, pappa, Anna, Nicke, Vilmer och Amanda för alla fina tider när

ni hälsat på eller jag varit hemma och laddat batterierna och bara fått komma bort från

allt. Mamma för att du alltid fixar, ställer upp och pysslar om mig och för våra fina

”frissa”-stunder tillsammans. Pappa för alla våra underbara ”stjären” turer, talko-

jobb och andra äventyr. Då laddar jag batterierna som bäst! Anna, för alla roliga

samtal som lättat upp min vardag genom alla år. Vilmer och Amanda för att ni är de

ni är och jag uppskattar att jag kan komma hem och vara lite som ett barn själv när jag

är runt er. Ni är alla som en frisk fläkt och jag är tacksam för att ni stått bakom mig i

detta och låtit mig vara jag! Det är tråkigt att du morfar var tvungen att lämna oss nu

men jag är tacksam för att du alltid brytt dig. Du har det nog bra där du är nu, kanske

tillsammans med mina andra favoriter.

Tack, bästaste Kennet för att du är precis som du är! Att du finns i mitt liv, allt ditt

stöd, uppmuntran och kärlek! Vad hade jag gjort utan dig under den här tiden?! Jag är

tacksam och uppskattar verkligen att du alltid ställer upp för mig när jag behöver det

och det har varit rätt ofta nu. Hoppas det blir min tur nästa gång!

Du gör mig glad och lycklig! Älskar dig!

This work has been financially supported by the Swedish Research Council, the J.

C Kempe Memorial Foundation and Knut och Alice Wallenbergs stiftelse.

45

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