Studies of Pore-Forming Bacterial Protein Toxins in Escherichia Coli

Home , Bacteriolysin, Defensin, Exotoxin, Lipopolysaccharide, RpoF

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No 1677, ISSN 0346-6612, ISBN 978-91-7601-131-7

Studies of Pore-forming Bacterial Protein Toxins in Escherichia coli

Constance Enow

Department of Molecular Biology 901 87 Umeå Umeå 2014

This thesis is dedicated to my late sister Veronica Tabotorock and Ferdinand Ashu Etta, who did not live long enough to see my success. May your souls, and the souls of all other departed ones in the family continue to Rest In Peace.

"It is God's privilege to conceal things, but the kings' pride is to research them.'' (Proverbs 25:2)

TABLE OF CONTENTS

ABSTRACT……………………….……..….……….………………………..…….……....4

ABBREVIATIONS………………..………………………….……………..……….……..5

PAPERS IN THIS THESIS…….…….……………………………..……………………8

1. INTRODUCTION..….…………………………..…………………………..….….9

1.1. Bacterial virulence and evolution...... …………………….……………….…...... 9

1.2. Escherichia coli (E. coli)...... …………………..……………..………………..….10

1.2.1. Commensal versus pathogenic E. coli variants...……….…………………………..…11

1.2.2. Intestinal and extra-intestinal pathogenic E. coli…….….…….……………………...13

Pathogenicity island (PAI)…….……………………………………….………………….…..15

1.3. Vibrio cholerae (V. cholerae)…………………………………..………….………………….17

2. FACTORS THAT CONTRIBUTE TO BACTERIAL COLONIZATION....…..18

2.1. Adhesin……………………………….……..……………………………………….……..……….18

2.2. Invasion factors…………………………..………………..…………………..………….….….19

3. BACTERIAL PROTEIN TOXINS……………………….……………..………..19

3.1. Pore-forming toxins (cytolytic toxins)…….…….……..……….……………………...…20

3.1.2. Alpha pore-forming toxins (Cytolysin A; ClyA)……………………………………....…22

3.1.3. Beta pore-forming toxin (alpha-hemolysin; HlyA)……….………………………….…24

4. GENE EXPRESSION AND REGULATION.…....……………….……….……25

4.1. Transcriptional regulation……………………………………………..…...... ….27

4.2. DNA binding protein….………………………………………………….……………………….27

4.3. Nucleoid-associated proteins………………………………………….…………………..….28

4.3.1. H-NS (Histone-like Nucleoid Structuring Protein)………………………………….…..28

4.3.2. Anti-silencing of H-NS ……………………………………………………….…………..……..30

4.4. FIS (Factor for Inversion Stimulation)…….……………………………………….……...31

4.4.1. IHF (Integration host factor)………………………………………………………………….31

4.5. Transcriptional activators…………………………………………………..……………….…31

4.5.1. SlyA (Salmolysin A)...…………………………………………………….………………………32

4.5.2. Cyclic adenosine monophosphate (cAMP) receptor protein (CRP)..…………....32

4.5.3. SfaX …………………...... ………33

4.5.4. C-di-GMP (Cyclic diguanylate guanosine monophosphate )...... …………………34

4.5.5. Stationary phase sigma factor (RpoS)..…………………………………………………..34

4.5.6. ppGpp (Stress alarmone)………………. ..………….……….…………….……..…………35

5. PROTEIN SECRETION IN BACTERIA....….…………………………....…..35

5.1. Type I and type III secretion mechanisms……………………………………………….36

5.2. Type II and IV secretion system mechanisms…….……………………………...... 36

5.3. Outer membrane vesicles (Type Zero secretion system).....……………………..36

6. OTHER GENES OR PROTEINS OF INTEREST………...……………………38

6.1. Alkaline phosphatase (phoA) gene fusions……………….………………………………38

6.2. Dsb system in E. coli ……..…………………………………………………………………….38

7. AIMS OF THIS THESIS.…….…………………………….…………….………39

8. RESULTS AND DISCUSSION.....……..…….…………….……………….....40

8.1. Paper I…………………………………………………………………………………………………40

Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain pathoadaptive mutations in a subset of extraintestinal E. coli strains…………………………………………………………………..41

8.2. Paper II …..……………………………………..……….…………………..…………………….45

Localization and structure of the ClyA protein in Escherichia coli before secretion and pore-formation…………………….…….………………………………..…..45

8.3. Paper III……………………………………………………………….………………………...... 50

Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains....……………………..………….50

9. CONCLUDING REMARKS…………..…………………..…….………………..55

ACKNOWLEDGEMENTS…………………………………………..…………….………56

REFERENCES.…………………….…………………………………………..….……….59

ABSTRACT

Escherichia coli, a Gram-negative bacterium, which can be classified into three groups: the commensal, intestinal pathogenic (IPEC) and extra-intestinal pathogenic (ExPEC) E. coli. The cytolysin A (ClyA) protein, a 34-kDa pore-forming toxin, encoded by a gene found in both non-pathogenic and pathogenic E. coli and in Salmonella enterica serovars Typhi and Paratyphi. It mediates a cytotoxic effect on various mammalian cells. ClyA is released by E. coli via outer membrane vesicles (OMVs) after reaching the periplasm via an unknown mechanism through the inner membrane. The gene is silenced by mutations in some of the most studied ExPEC strains suggesting that the locus would be subject to patho-adaptive alterations.

To study if the mutations of the clyA gene in E. coli strains was particular to certain strains, the sequences of the clyA gene locus of a set of ExPEC isolates and of the E. coli collection of reference strains (ECOR) were compared. The ExPEC strains - uropathogenic and neonatal meningitis E. coli (UPEC and NMEC) strains contained various ΔclyA alleles. Next, a functional clyA gene locus was restored and tagged with luxAB in the chromosome of the UPEC strain 536. Luciferase activity of the bacteria carrying the restored gene showed that the clyA gene expression is highly increased at the late logarithmic growth phase when compared to the non-pathogenic E. coli K-12 strain. A higher transcriptional level of the clyA+ gene was observed when the SfaX regulatory protein was heterologously overproduced. It was concluded that the clyA+ gene is expressed at elevated levels in the UPEC strain and this is at least in part due to the SfaX/PapX transcriptional regulators.

Studies of clyA::phoA fusions obtained by transposon TnphoA insertion mutagenesis showed that the first 12 amino acid residues of ClyA was sufficient for translocation of the protein chimera into the periplasm and to the OMVs. The role of the two cysteine residues in ClyA for protein translocation was tested by introducing substitution mutations. The results indicated that the C-terminal Cys (ClyAC 285S) is important for localization and/or stability of the protein in the periplasm. Structural analysis of ClyAwt purified from the periplasm revealed that the protein forms dimeric complexes. Upon treatment with the reducing agent DTT the ClyA protein readily assembled into typical pore complexes as revealed by electron miscroscopic analysis. In conclusion, the ClyA protein is present in the periplasm in a conformation that prevents it from forming pores in the bacterial membranes.

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin which induces lysis of mammalian cells by forming transmembrane channels. Although the biophysical activities of VCC were well studied, there was no detailed analysis of VCC secretion. Our study demonstrated that a fraction of the VCC was secreted in association with OMVs. OMV- associated VCC from the wild type V. cholerae strain V:5/04 is biologically active as shown by toxic effects on mammalian cells, interestingly, OMV-associated VCC was more active than purified VCC. Both environmental and clinical V. cholerae isolates transport VCC via OMVs. In addition, when the vcc gene is heterologously expressed in E. coli, OMV-associated secretion of VCC was also observed. We suggest that OMV-mediated release of VCC is a feature shared with ClyA.

ABBREVIATIONS

Aa Amino acid; cAMP Cyclic adenosine monophosphate; c-di-GMP Cyclic diguanylate guanosine monophosphate;

ClyA Cytolysin A protein;

Dps DNA-binding proteins;

DR Direct Repeat;

DTT Dithiotreitol;

E RNAP core enzyme;

ExPEC Extra-intestinal pathogenic Escherichia coli;

Eσ70 σ70-programmed RNAP;

FIS Factor for inversion stimulation;

FPLC Fast protein liquid chromatography; foc Gene cluster coding for F1C fimbriae; GIT Gastrointestinal tract; HapR Haemagglutinin protease regulator;

HGT Horizontal gene transfer;

H-NS DNA-binding, histone-like nucleoid structuring protein;

HU Heat unstable protein;

IHF Integration host factor;

IM Inner membrane; int Integrase gene;

LPS Lipopolysaccharide;

MIC Minimal Inhibitory Concerntration; mob Mobility gene;

1mob Pseudomobility gene;

OM Outer membrane;

OMV Outer membrane vesicle;

OmpU Outer membrane protein U; pap Pyelonephritis - associated pili gene cluster; PapX Regulatory DNA-binding protein from the MarR family, homologous to SfaX; PDE Phosphodiesterase;

PG Peptidoglycan; ppGpp Guanosine tetraphosphate;

RelA ppGpp synthetase;

RNAP E. coli RNA Polymerase holoenzyme;

Rsd Regulator of sigma D, anti sigma factor for σ70; sfaX S-fimbrial X gene (coding for MarR-like type transcriptional regulator); sfaY S-fimbrial Y gene (codes for a putative EAL - phosphodiesterase);

SpoT ppGpp synthetase and hydrolase;

SlyA Salmolysin A;

StpA DNA-binding protein, Suppressor of td phenotype A;

SheA Silent hemolysin A;

Tat Twin-arginine translocation system;

UPEC Uropathogenic E. coli; UTI Urinary tract infection; vir Virulence-associated gene; VCC Vibrio cholerae cytolysin; YOPs Yersinia outer membrane proteins;

σ28 Sigma F ( RpoF) - the flagellar alternate sigma factor;

σ32 Sigma H (RpoH) - the heat-shock alternate sigma factor in E. coli;

σ38 Sigma S (RpoS) - the stationary phase alternate sigma factor in E. coli;

σ54 Sigma N (RpoN) - the nitrogen deprivation alternate sigma factor in E. coli; σ70 Sigma 70 (RpoD) - the general house-keeping gene sigma factor in E. coli.

PAPERS IN THIS THESIS

The papers included in this thesis will be referred to in their roman numerals as I – III.

I. Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain pathoadaptive mutations in a subset of extraintestinal E. coli strains. Enow C, Oscarsson J, Zlatkov N, Westermark M, Duperthuy M, Wai S, Uhlin B. BMC Microbiol. 2014 Sep 2;14(1):216

II. Localization and structure of the ClyA protein in Escherichia coli before secretion and pore-formation. Constance Enow, Jan Oscarsson, Yoshimitsu Mizunoe, Shenghua Huang, Elke Meier, Roland Benz, A. Elisabeth Sauer-Eriksson, Sun Nyunt Wai, Bernt Eric Uhlin. Manuscript.

III. Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains Elluri S, Enow C, Vdovikova S, Rompikuntal PK, Dongre M, Carlsson S, Pal A, Uhlin BE, Wai SN. PLoS One. 2014 Sep 4;9(9)

1. INTRODUCTION

1.1. Bacterial virulence and evolution

Many bacteria that inhabit the human body are harmless commensals, such as the residents of the gastrointestinal tract (GIT) with a unique ecological niche and with its own physicochemical properties. The intestinal microflora can reach a density of 1011 bacterial cells per gram of the intestinal contents in the colon and rectum (1). The gut bacteria co-exist with their host by having a non-harmful and beneficial interactions. The microflora is mainly composed of anaerobic Gram-negative and Gram-positive bacteria such as Bacteroides spp., Prevotella spp., Fusobacterium spp., Clostridium spp. Peptostreptococcus spp. and Bifidobacterium spp. Facultative anaerobic bacteria such as Escherichia coli are also present in the gut microflora. Some of these bacteria can cause severe diseases outside the intestine such as urinary tract infection (caused by uropathogenic E. coli) or within the intestine such as peptic ulcer (caused by Helicobacter pylori, a Gram-negative bacterium which normally inhabits the upper part of the GIT). The opportunistic pathogens are another group of bacteria which lacks obvious virulence traits such as toxin production but they can still cause diseases in the host when the normal human defense is weakened (2). A pathogen is different from a non-pathogenic microorganism because of the additional virulence genes present (3). These few additional genes can be introduced by horizontal gene transfer (HGT or lateral gene transfer, explained in Figure 1). In short, E. coli strains can become virulent due to a large number of virulence determinants found on acquired mobile genetic elements (called pathogenicity islands, PAIs) which can be also transferred from the pathogenic strains via conjugation, transformation, and transduction (Figure 1). E. coli is an example of a bacterial species that shows great diversity and has been able to establish many different interactions with its host (4, 5).

DNA acquisition Genome by HGT (transformation, optimization transduction and (rearrangements conjugation) and mutations))

Genome reduction (deletions)) Genome optimization (rearrangements and mutations))

Figure 1: Genome optimization and patho-adaptation of E. coli. Modified from (3)

1.2. Escherichia coli (E. coli)

E. coli is a Gram-negative bacterium that groups together with Salmonella spp. and Shigella spp. in the Enterobacteriaceae family. E. coli belongs to the commensal flora of the GIT of humans and various other warm-blooded animals which it colonizes just few hours after birth (6, 7). It is a rod shaped-like (bacillus-type), non- sporulating, motile bacterium, with facultative metabolism and peritrichous flagellar organization (8, 9). E. coli is a model organism with well annotated genomes from several strains. Past works on E. coli generated tremendous amount of data that explain many molecular mechanisms that exist in E. coli but also in other species such as human.

In my thesis, I used E. coli as a model organism to study cytolysin A protein (ClyA), which is a pore-forming toxin produced by E. coli and many other Gram-negative bacteria.

1.2.1. Commensal versus pathogenic E. coli variants

Most intestinal E. coli isolates and also variants of commonly adapted laboratory strains such as E. coli K-12 BL-21 (DE3) and DH5α are regarded as non-pathogenic, also called “the harmless’’ E. coli. But some strains of E. coli are pathogenic to mammals and birds, and they can cause both intestinal and extra-intestinal infections (6). The pathogenic strains of E. coli evolved from the non-pathogenic variants via HGT (Figure 1), i.e. they have either acquired PAIs in their chromosomes, or host a plasmid carrying virulence determinants (Figure 2) (3). The different variants of E. coli are listed in Table 1

Commensal E. coli

They are harmless E. coli strains

Intestinal pathogenic E. coli (IPEC)

EPEC Enteropathogenic E. coli EIEC Enteroinvasive E. coli ETEC Enterotoxigenic E. coli EHEC Enterohaemorrhagic E. coli Diarrheal diseases EAEC Enteroaggregative E. coli DAEC Diffusely adherent E. coli

Extraintestinal pathogenic E. coli (ExPEC) NMEC Newborn meningitis E. coli causing meningitis UPEC Uropathogenic E. coli causing UTI and pyelonephritis APEC Avian pathogenic E. coli causing colibacillosis SEPEC Septicemic E. coli causing sepsis ABU Asymptomatic bacteriuria

Table 1: Summarized from (10). Different E. coli variants. The commonly used abbreviations are shown on the left column.

The difference between disease-causing and non-disease-causing strains lies in their ability to express and produce various molecules, called virulence factors, which enable them to subvert or elude the host defenses and establish infection. Adhesins, toxins, siderophores and invasion factors are examples of well-described virulence factors that counteract host immune defenses. With few exceptions, genes encoding such factors are absent in non-pathogenic strains including E. coli K-12. The commensal enterobacteria gain or lose virulence-associated genes by HGT, when they come in contact with different HGT agents which happens when they occupy different niches outside the intestinal tract, such as the blood stream, the urinary tract and also crossing the blood-brain barrier. The genome of the commensal E. coli is about 4 Mb while the pathogenic strains of E. coli have a genome that range from 5 to 6 Mb (Figure 2). Such difference is due to acquisition of PAIs by HGT (10).

E. coli K-12, MG1655 E. coli UPEC 536 NC_000913 NC_008253 4,641,652 bP 4,938,920 bp ClyA

Figure 2: Differences between the genome of E. coli K-12 (non-pathogenic strain) and UPEC 536 (pathogenic strain). E. coli K-12 MG1655 with an accession number: NC_000913 has a genome size of 4,641,652 bp while E. coli UPEC 536 with an accession number NC_008253 has a bit bigger genomic size of 4,938,920bp shown in the maps. The arrow shows the position of ClyA in K-12. The figure is generated with the “Snapgene” software program.

1.2.2. Intestinal and Extra-intestinal pathogenic Escherichia coli strains (IPEC and ExPEC)

Pathogenic E. coli strains can be divided into two groups according to their abilities to cause infections either inside or outside of the GIT. Although mammals carry E. coli in their intestine, some strains can cause a variety of symptoms such as abdominal cramps, diarrhea, dysentery and in some severe cases hematuria (presence of blood in the urine). Intestinal pathogenic E. coli strains are transmitted via fecal contamination due to unhygienic conditions.

In cases where E. coli bacteria escape the intestinal tract through a gut perforation, and enter the abdominal cavity, they can cause abscesses or peritonitis which can be fatal. Virulent E. coli can cause a variety of extraintestinal diseases. Some examples of ExPEC variants are the uropathogenic E. coli (UPEC) strain 536 and the new born meningitis strain IHE3034 (Table 1). UPEC strains are the causative agent of the urinary tract infections (UTIs) and it accounts for 90% of the UTIs in anatomically- normal, unobstructed urinary tract (11). UTIs are one of the most common group of bacterial infections encountered in clinical practice in Europe and North America. UPEC strains are the causative agent of about 90% community acquired UTIs and 50% of all cases of nosocomial infections. E. coli UTI is the most frequently diagnosed kidney and urologic disease in women (11, 12) Recurrent infections (three or more episodes during a 12-month period) occur frequently in healthy girls and women who have anatomically normal urinary tracts (13).

Bladder infections are 14 times more common in females than in males by the virtue of the shorter urethra in females. The typical patient with uncomplicated cystitis is a sexually-active female who was first colonized in the intestine by a UPEC strain. The microorganisms migrate into the bladder from the peri-urethral region during sexual intercourse. With the aid of specific fimbriae-associated adhesins (such as: FimH adhesin that recognises mannosylated receptors, PrsG and PapG adhesins (which recognise digalactosylated receptors)) they are able to colonize the bladder (14-16). Most of the virulence factors associated with the UPEC strains are:

Adhesion factors: They allow the bacteria to attach and invade the host. Fimbriae (such as type 1, P- and S-fimbriae), are straight, proteinaceous, hair-like structures on the surface of prokaryotic cells (16, 17). Fimbria range from 3-10 nanometers in diameter and can be up to several micrometers long. They carry adhesins located on their tips with which they attach differentially to different sugar-protein conjugates of the host tissue, so that the bacteria can withstand shear forces and obtain nutrients from their host.

The iron uptake systems: Bacteria use siderophores, i.e. iron chelator compounds to take up iron (Fe3+) from their host’s environment (18).

The capsule (The K antigen): A polysaccharide structure that covers and protects the bacterium from its host immune system. Some examples are K1 antigen from E. coli IHE3034 and K15 antigen from UPEC 536 strain.

Endotoxin or Lipopolysaccharides (LPS; The O antigen) has been shown to activate host response, induces nitric oxide and cytokine production (19)

Exotoxins are a group of bacterial toxins that can be secreted extracellularly. Such as the alpha-hemolysin, a pore-forming toxin; TIR domain-containing protein C (TCPC), a newly identified virulence factor in UPEC strains, common among the patients with cystitis and pyelonephritis thus contributes to the severity of the UTI (20, 21).

IHE3034 is another example of an ExPEC strain, it produces the S-fimbriae, K1 capsule and Ibe10 as its virulence factors (22). IHE3034 strain causes neonatal meningitis which is a serious medical condition in infants. The mortality rate in developing countries is about 50% while in developed countries it ranges from 8%- 12.5% (23, 24). Bacteremia is the most common cause of neonatal meningitis and it occurs when the bacteria enter the blood and cross the blood-brain barrier. Complications such as cerebral palsy, blindness, deafness, and learning deficiencies can occur due to delayed treatment of neonatal meningitis (23-25).

Both the non-pathogenic E. coli K-12 strain (MG1655) and the ExPEC strain IHE3034 (O18:K1:H7) were used to study the regulation of the expression of their virulence factors. In addition, the 536 (O6:K15:H31) strain isolated from a patient with acute pyelonephritis was used because it carries the P- , Prs- and also the S-fimbriae. The UPEC 536 strain encodes several virulence genes which are scattered among seven pathogenicity islands and the virulence of the strain is attenuated by the deletion of individual pathogenicity island (26).

Pathogenicity islands (PAIs)

PAIs are mobile genetic elements in the chromosome of bacteria which carry virulence genes. They range in size from 10 to 200Kb and are more susceptible to incursion by foreign DNA than the pangenome (or supra-genome). The insertion of PAIs to a strain is not a permanent event. PAIs are present only in the pathogenic variants of E. coli and rarely or not in the non-pathogenic variants. PAIs are also present in animals and plants genomes (27). The presence of PAIs in pathogenic strains of E. coli makes the difference between commensal K-12 strains and the pathogenic variants ( Figure 2 and Figure 3), (28).

Pathogenicity island

tRNA int virA virB mobA mobB

Pathogenic

DR DR

tRNA

Non-Pathogenic

Figure 3: Model of a bacterial pathogenicity island. Adapted from (27)

The thin bold line represents regions of the core genome. The light green boxes represent PAI-specific genes. The arrows indicate the presence of direct repeats (DR) at the ends of the pathogenicity island. int , intergrase gene and mob genes, encode proteins involved in mobility of the prokaryotic genome.

There are four different PAIs in the E. coli ExPEC strain (UPEC 536 (O6:K15:H31)) and they encode a variety of different virulence factors: adhesins, toxins, invasins, protein secretion systems, iron uptake systems, and others (27, 29). PAI I and PAI II are inserted into two genes that encode for Leu and Sec (Selenocysteine) tRNA s, (30, 31). PAIs are not unique just to E. coli species but they are also found in a variety of both Gram-negative bacteria (such as Yersinia species, Vibrio cholerae, Salmonella enterica Typhimurium) and also Gram-positive bacteria (such as Listeria monocytogenes, Staphylococcus aureus etc). The PAIs of Gram-positive bacteria are more stable and do not carry mobility genes as compared to the PAIs of Gram- negative bacteria.

Some pathogenicity islands are more stable than others in terms of deletion frequencies due to environmental stimuli such as temperature, nutrient availability and cell density (32). The recombination processes that lead to the integration of PAIs are not specific, since both UPEC and EPEC code for different genes but are located at identical base pairs in selC tRNA genes of the PAIs.

Point mutations, genome rearrangement and the acquisition of new genes by horizontal gene transfer is the current basis of understanding the evolution of microbial pathogens from related non-pathogenic bacteria, as well as for the generation of new variants of pathogens. Following the acquisition of new genetic information, the stabilization and optimization of the expression of the new genetic elements becomes important (10). Loss-of-gene function by point mutation in the genome may enhance bacterial virulence without horizontal gene transfer of a DNA fragment, carrying a specific virulence factor (33).

Virulence factors Examples

Adherence factors Uropathogenic E. coli Intestinal pathogenic E. coli Vibrio cholerae

Toxins Uropathogenic E. coli Vibrio cholerae

Iron uptake system Uropathogenic E. coli

Invasions, Intestinal pathogenic E. coli modulins,effectors

Table 2: Some major virulence factors encoded in pathogenicity islands. Summarized from (27)

1.3. Vibrio cholerae

Vibrio cholerae are Gram-negative comma-shaped facultative anaerobic bacteria (from the family Vibrionaceae), with a single polar flagellum for movement. The natural habitat for V. cholerae is estuarine and aquatic environments particularly the surface of water. The pathogenic strain of V. cholerae serotype O1 El Tor causes the current pandemic disease cholera and does not have a polysaccharide capsule, while the other pathogenic strain of V. cholerae serogroup O139 does form a polysaccaride capsule (34). The O139 serogroup comprises different clones of epidemic and non-epidemic strains that were derived from many V. cholerae strains with O1 or non-O1 progenitors. When V. cholerae gets into the human through contaminated food or water, it passes through the acid barrier of the stomach. It causes cholera using two major virulence factors: toxin co-regulated pilus (TCP) important for colonisation of the intestine and cholera toxin (CT) which is responsible for typical symptoms; the rice water stool and vomiting which result in extreme dehydration and if not treated, death (35). V. cholerae produces a pore forming toxin

called Vibrio cholerae cytolysin (VCC) or HlyA. VCC is a beta (β)-barrel pore-forming exotoxin, distinct from staphyloccal–haemolysin both functionally and structurally. It is a unique class of pore-forming toxins, produced by all strains of V. cholerae. VCC facilitates V. cholerae to colonise the intestine by permeabilising the human intestinal and immune cells. VCC has also been suggested to be the major diarrheagenic factor for the non-cholera producing strains (36). The VCC protein is regulated by haemagglutinin protease regulator (HapR) of V. cholerae.

2. FACTORS THAT CONTRIBUTE TO BACTERIAL COLONIZATION

Pathogenic bacteria have acquired genes whose products improve their strategies to gain entry to a preferred host, find a unique niche within the host, evade the host´s innate defense mechanism, multiply and exit the host in a manner designed to maximize the likelihood of a transmission to a new susceptible host (1). Many bacteria have co-evolved with the organism that they infect and they often use the host cellular components for their entry survival and growth (28, 37-39). Virulence factors can be divided into two categories: (i) those that promote bacterial colonization and invasion of the host and (ii) those that cause damage to the host. Some of the virulence factors that promote bacterial colonization and invasion are discussed below:

2.1. Adhesins

Adhesins are cell surface components of bacteria that facilitate the bacterial adherence to specific receptor molecules on the surface of host cells and enable colonization. Not all adhesins are essential virulence factors but the gene encoding adhesins are found in the genome of most bacteria (40).

2.2. Invasion factors

Invasins are proteins produced by bacterial cells that promote bacterial penetration into mammalian cells. Bacteria have evolved mechanisms for entering host cells by attaching to the host cell membrane and causing changes in the host cytoskeleton (38, 41). Invasins force non-phagocytic cells to engulf bacteria. For UPEC strains, a newly recognized virulence strategy is the invasion of bladder epithelial cells. The EPEC and EHEC strains, cause diarrheal diseases by disrupting the intestinal environment through the intimate attachment of the bacteria to the intestinal epithelium. This process is mediated by intimin, an outer membrane protein that is homologous to the invasins of pathogenic Yersinia (42). In order for the pathogen to evade the host defense system, it produces capsule to side step non-specific host defense and LPS that protects the bacterial membrane from certain kinds of chemical attack (43-45). The bacteria also use intracellular strategies to avoid being phagocytosed by professional phagocytic cells and rather escape the hostile environment (28).

3. BACTERIAL PROTEIN TOXINS

Actively dividing bacteria (in their exponential phase of growth) usually secrete bacterial toxins and this exotoxin production is species specific. Only the virulent bacterial strains produce toxins, although it was discovered by (46), that the non- virulent strain of E. coli K-12 (an hns mutant E. coli strain) produces a toxin called cytolysin A toxin (ClyA) (46). Toxins are a major determinant of virulence. Bacterial protein toxins (such as enterolysin, hemolysin, streptolysin, bacteriolysin, botulinum toxin (BTX) and tetanus toxin) are major determinants of virulence. The lethality of the most potent bacterial exotoxins (botulinum toxin) is higher than the lethality of the most poisonous snake venom (28). These bacterial toxins often play a central role in pathogenesis and they are biologically active at very low concentrations (47).

Bacterial toxins can be grouped by their modes of action and by the variety of their mechanisms.

Cytolytic toxins are one group of toxins produced by many Gram-negative bacteria (48, 49). Cytolysins of Gram-negative bacteria are usually synthesized as precursor proteins which are converted to active toxins by post translational modifications. Such requirement for activation is not common for cytolysins produced by Gram- positive bacteria. Nowadays many toxins such as cytolytic toxins, AB toxins, E. coli subtilase cytotoxin, cyclomodulins are well characterized (50). This study has been mainly focused on cytolysin A (ClyA), which is a pore-forming toxin that lysis erythrocytes and other cell types by forming oligomeric pores on the target host membrane but its role in virulence in E. coli is not yet known.

3.1. Pore-forming toxins (Cytolytic toxins)

Some exotoxins are typically produce but not exclusively, to wage a battle against rival bacteria and also to attack host cells (51). Most pathogenic Gram-negative bacteria synthesize cytolytic toxins as virulence factors. The toxins generate pores in eukaryotic (host) cell membranes and the cytoplasmic content get released and lyse the host cell membrane (52). Pore-forming toxins are not limited only to Gram- negative bacteria, since it was shown that Gram-positive bacteria also produce pore- forming cytolysins. Cytolysins of Gram-negative bacteria usually are first produced as precursor proteins which are later converted to an active toxin by proteolytic processing (49). Gram-positive bacteria do not imply this process for toxin conversion. Depending on the secondary structure of the membrane-spanning region, these pore-forming toxins are categorized into two classes: alpha-PFTs and beta-PFTs. Alpha-PFTs insert into the membrane with alpha helices while the beta-PFTs form transmembrane beta-barrel (53). Pore-forming toxins remain the most potent and versatile weapons which an invading microbe uses to damage host cells. The pore- forming toxins disrupt the selective influx and efflux of ions across the plasma

membrane by inserting a transmembrane pore. PFTs are a class of potent virulence factors that are converted from a soluble form to a membrane-integrated oligomeric form within the host membrane (Figure 4) (53). They exert their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores (49). Some of the pore forming toxins used for this study are listed below on Table 3.

Toxin type Examples

Alpha-pore forming toxin Cytolysin A of E. coli

Beta-pore forming toxin 1. Alpha-hemolysin 2. Panton-Valentine leukocidin

Table 3: Subcategories of pore-forming toxins. Summarized from (54)

Figure 4: Mechanism of ClyA pore assembly and the insertion into the target membrane. The monomeric (soluble) ClyA has the disulphide (S-S) bond and is non-active, does not form pores but when the S-S bond is reduced they form prepore and when the prepore gets in contact with a target membrane, inserts into the membrane with the amphipathic alpha- helix and lysis the membrane (53).

3.1.2. Alpha pore-forming toxins (Cytolysin A; ClyA)

Some enteric bacteria synthesize a pore-forming toxin, called cytolysin A (ClyA) also called hemolysin E (HlyE) or Silent Hemolysin A (SheA), which is cytolytic and cytotoxic to host cells (49, 54). Hemolysin E (HlyE) was first identified in a non- pathogenic hns mutant Escherichia coli K-12 (46). It is present in most of the intestinal pathogenic E. coli strains tested so far but absent in some but not all strains of the extra-intestinal pathogenic E. coli tested so far (55). The clyA gene is present in the highly pathogenic enteric pathogens Salmonella enterica serovars Typhi and Paratyphi but not Typhimurium, and also in Shigella flexneri. The clyA gene has also been found to be present in the avian pathogenic E. coli (56, 57). It is a secreted hemolysin which encodes a 34-kDa protein, with 302 amino acid residues (58). The gene is “cryptic” in E. coli K-12 (no phenotype). It has been shown that the protein has several forms: monomeric, hexameric, octameric and 13-mers (59- 62). The monomeric form has a disulphide bond between its two cystein residues (at position 87 and 285). When the disulphide bond is reduced by a reducing agent for example, DTT, the protein can form oligomers.

OMV ClyA oligomers

S SH ClyA ClyA S SH

ClyA monomers from Pore-forming the periplasm assemblies

Figure 5: Adapted from (59). Monomeric (folded) form of ClyA from the periplasm and and oligomeric (the disulphide bonds are reduced: unfolded) form of ClyA from the OMVs.

The three dimensional (3D) structure of ClyA has been shown to have both the head and tail domains (Figure 6). The tail domain contains both the N- and C-terminal ends of the protein while the head domain contains the beta tongue. The beta- tongue is hydrophobic, and has a unique hairpin formed by residues at position 177 to 203. This hairpin interacts with the lipid bilayer of host cell membranes (57). Studies with avian pathogenic strain JM4660 (which produces ClyA), that has an Arg at position 188 revealed that when the Arg 188 was substituted with Gly the effect of beta-tongue was abolished in E. coli K-12. This further support the functional role of beta-tongue in the ClyA protein (57, 63). The N-terminal amphipathic alpha-helix of ClyA is located at the tail domain. It is crucial for the formation and the properties of the transmembrane channel, and for hemolytic activity (61). The ClyA protein can lyse erythrocytes and mammalian cells in a Ca2+-independent manner, by forming transmembrane pores with a minimum internal diameter of 25 Å (64, 65).

β Head domain

C87 C285

Tail domain

Figure 6: The 3D structure of ClyA. It was generated by protein Workshop, a molecular viewer from RCSB PDB. The structure shows the head domain that has the beta-tongue. The tail domain that has the N- and C-terminal and the disulphide bond formed between C87 and C285 is also shown (60). 23

The expression of clyA gene is positively regulated by many transcription factors such as FNR and CRP by binding at the same site as the H-NS protein (H-NS is the repressor of clyA). But in vivo transcription studies revealed that FNR protein occupies the clyA promoter more frequently than CRP, providing a mechanism for the moderate upregulation of clyA expression in response to two distinct environmental signals (oxygen and glucose starvation) (66). ClyA is also regulated positively by SlyA. When SlyA is upregulated, it induces the synthesis of ClyA which confers a haemolytic phenotype (67, 68).

ClyA serves as a platform for vaccine delivery, by increasing the immunogenicity of an antigen that has low immunogenicity when no adjuvants are added (69). The ClyA protein is translocated to the periplasm via an unknown mechanism. The N- terminal part of the protein is not subject to processing and ClyA has not been connected to any of the genetically defined secretion systems in E. coli (Type I-VI, see below). However, it is known to be exported from the bacteria via outer membrane vesicle-mediated transport (70).

3.1.3. Beta pore-forming toxin (hemolysin A, HlyA)

E. coli alpha hemolysin (HlyA) belongs to the family of Repeats-in-Toxin (RTX). The RTX family of proteins functions as toxins or adhesins and consists classically of cytolysins and hemolysins (71). The RTX protein family consists of two sub-families: (i) proteins involved in adhesions and biofilm formation and (ii) the multifunctional- autoprocessing RTX toxins subfamily which is associated with cytotoxicity and pathogenesis. The RTX family of proteins has glycine-aspartate (GD)-rich nonapeptide repeats and the consensus sequence is G-G-X-G-(N/D)-D-x-(L/I/F)-X near the C-terminus of the proteins (72). RTX is an exotoxin whose expression is frequently associated with IPEC and ExPEC strains, such as EHEC and UPEC respectively (73, 74). The HlyA (alpha-hemolysin) protein present in UPEC is a labile pore-forming toxin (75). Its secretion is mediated

by type I secretion system, and it is directly secreted to the extracellular space through a pore consisting of HlyB, HlyD and TolC without any periplasmic intermediates (73). It can also associates with OMVs and thus be exported. Alpha (α-) hemolysin present in OMVs is active (cytolytic) and cytotoxic, it causes lysis of red blood cells and other cell types, such as granulocytes. HlyA is calcium-dependent and in E. coli it has a molecular size of 107-kDa (70, 76). The activity of alpha- hemolysin is growth-phase dependent. Indeed, HlyA activity increases during the exponential phase and is stabilized by Ca2+ ions during the stationary phase (71, 77, 78). It has been demonstrated that E. coli cytotoxin has a cytocidal activity against some epithelioid cultured cell lines (such as Vero, HeLa and Hep-2 cells) but was almost inactive for avian-fibroblast cells.

In V. cholerae, Vibrio cholerae cytolysin (VCC) or HlyA is a beta (β) barrel pore- forming exotoxin, distinct from the staphyloccal hemolysin both functionally and structurally. VCC is a unique class of pore-forming toxins, produced by all strains of V. cholerae. It assembles into pentameric channels, lyse erythrocytes and HeLa cells. The VCC protein is secreted to the periplasm by type 2 secretion system (T2SS). In V. cholerae, the molecular weight of HlyA is 79 kDa and it is a water-soluble inactive pro-cytolysin or pro-hemolysin that undergoes post-translational N-terminal cleavage mediated by haemagglutinin protease A (HapA) of V. cholerae to form an active 65- kDa toxin (79-81).

4. GENE EXPRESSION AND REGULATION IN BACTERIA

Enteric pathogenic bacteria can survive in different ecosystems - in soil, water, and air, but when the bacteria suddenly find their way to the gut, these bacteria adjust their lifestyle to the elevated temperature (37ºC) in the human body, and high osmolarity. All these adaptations take place within a couple of seconds to minutes. In order for bacteria to adapt to the new conditions, they have evolved a number of regulatory mechanisms at every step to handle these environmental changes by regulating the expression levels of their housekeeping and virulence genes. Some of

the common environmental signals which cause these changes are: iron, temperature, osmolarity, Ca2+, carbon source, and many others. It has been shown that adaptation to these signals can occur at different levels such as phase variation, antigenic variation and transcriptional initiation. The role of phase variation and antigenic variation is to help the bacterium circumvent the host immune system.

Phase variation The random switching of bacterial phenotype due to environmental changes occurs at a frequency that is much higher (sometimes >1%) than classical mutation rates. Bacteria can acquire new genetic material from surrounding bacteria by HGT or they can duplicate the same gene on the chromosome. Phase variation contributes to virulence by generating heterogeneity, certain environmental or host pressures select those bacteria that express the best adapted phenotype (82).

Antigenic variation It is another type of gene rearrangement where a bacterium can alter its surface proteins in order to evade a host immune response. They change the gene arrangement so that the gene product can no longer be recognized by the host immune defense. This is an approach used by Streptoccocus pyogenes where the N-terminal region of the M protein is hyper-variable (83). This can also be observed with the immune mimicry of Campylobacter flagella and Campylobacter jejuni where they induce the anti-ganglioside antibody and the components on the host cell-surface apart from lipooligosaccharide (LOS) do not appear to induce cross-reactive antibodies (84, 85).

Transcription initiation Transcription initiation is controlled by the sequence of the promoter and by activating or repressing proteins that affect binding of the RNA polymerase. Transcription regulation is the most complex and most intensively studied area of gene regulation and this regulation can be very diverse. When a set of 26

genes is under the regulation of the same promoter, they form one transcriptional unit (an operon) and they can be co-expressed as polycistronic mRNA. Gene regulation can also be global, i.e. operons can be further arranged into regulons. A regulon is a group of operons that is controlled by the same regulator. The different regulatory mechanisms relevant for this study are described below.

4.1. Transcriptional regulation

Transcriptional regulation is the change in gene expression levels by altering the rates of transcription. In E. coli, RNAP consists of two α- subunits, one β-subunit, one β’-subunit and Ω-subunit, that builds up a core enzyme, and one sigma (σ) subunit which together with the core enzyme builds up the holoenzyme (86). The initiation starts with the sigma factor which recognizes the core promoter region composed of -35 (TTGACA) and -10 (TATAAT) elements and also some promoters which have upstream elements are recognized by the C-terminal domain (CTD) of the α-subunits. All these events are required for transcription to occur. There are multiple alternate sigma factors; each of them recognizes a distinct set of promoters (87).

4.2. DNA-binding proteins

These proteins are characterized by DNA-binding domains (such as HTH-motifs) that bind to either a single or double stranded DNA. DNA-binding proteins include transcriptional factors that modulate the transcription process such as various repressors, activators and nucleases that cleave DNA molecules.

4.3. Nucleoid-associated proteins

The nucleoid is a protein-DNA complex, which represents the bacterial chromosome. It is attached to the inner-leaflet of the bacterial membrane. The nucleoid-associated proteins are small in average molecular weight. These proteins play a critical role in the maintenance of the bacterial genome architecture by altering the DNA supercoiling levels upon binding. This is achieved by constraint supercoiled loops and random coiling (88). The major and most abundant nucleoid-associated proteins in E. coli are: FIS, (factor for inversion stimulation), HU (heat unstable protein), IHF (integration host factor) and H-NS (histone-like nucleoid structuring) protein. The order of abundance of several major chromosomal proteins in the exponential phase of growth is FIS > HU > H-NS > IHF, whereas on entry into the stationary phase this order changes to Dps > IHF > HU > H-NS > FIS. FIS is the major DNA - binding protein during the exponential phase of growth.

4.3.1. The H-NS (Histone-Like Nucleoid Structuring) Protein

H-NS influences the expression of many genes, such as the clyA gene of E. coli, the rtxACHBDE gene cluster of Vibrio anguillarium. It is a neutral protein with a molecular weight of 15.6 kDa (89). H-NS binds preferentially to A/T-rich curved DNA region (ATATATAT) and the initial binding region can function as a site of nucleation from which the protein can polymerize along the DNA (Figure 7) (90, 91). H-NS can form heteromeric protein–protein complexes with full-length or partial paralogues such as StpA, Sfh, Hha, YdgT, YmoA or H-NST (92-95).

Figure 7: Adapted from (96). Transcriptional regulation by H-NS molecules. A): H-NS recognizes and binds to curve DNA element. B) Polymerization of H-NS along the DNA resulted to the repression of transcription.

The deletion of H-NS protein has a pleotropic effect due to the implication of this protein in the regulation of several pathways that are important for the bacterial physiology (92, 96-98). H-NS possesses two domains spaced by a linker sequence (Figure 8). The N-terminal domain (residues 1-64) is responsible for dimerization and higher order homo- or hetero- oligomerization while the C-terminal domain (residues 91 - 137) mediates binding to DNA (99, 100).

Figure 8: Modified from (95). Domain organization of H-NS protein.

H-NS has about 52% amino acid sequence identity to E. coli StpA (99). StpA has 133 amino acids and has been identified as a multicopy suppressor that can complement the effect on H-NS-dependent genes and operons such as hns, proU, bgl, pap. H-NS plays a crucial role in the global gene regulation of enteric bacteria (98, 101-103). In an hns background, the expression of about 200 proteins is either repressed or activated (91, 96, 104-107).

4.3.2. Anti-silencing of H-NS

It has been shown that H-NS is involved in the processes of anti-silencing, where it represses the transcription of repressor genes (108, 109). Silencer sequences are located several base pairs downstream from the gene and upon binding to a transcription factor the transcription is repressed. While a repressor is a protein that prevents the synthesis of mRNA by binding to DNA at a specific site near the start of a gene or set of genes.

For transcription to occur, it is necessary to relieve gene repression which allows free access to anti-repressors, and this is achieved by modulating the binding of H-NS and other nucleoid-associated proteins. H-NS has two binding sites distant from each other. As studied in the virF promoter region of S. flexneri, it was shown that H-NS binds with low affinity and it can be displaced easily without the help of other proteins (110). Transcription cannot occur at lower temperature, since the DNA is bent and the H-NS can keep the DNA in a closed loop. However at elevated temperatures the bending of DNA decreases which increases the distance between the H-NS binding sites allowing other factors to bind to the DNA and initiate transcription (110). In E. coli H-NS binds to the upstream end of the promoter site of clyA and inhibits the transcription of the clyA gene.

4.4. FIS (Factor for Inversion Stimulation)

It is a homodimeric protein thought to constitute the major protein component of bacterial chromatin during the exponential growth phase (111). FIS binds to DNA in a sequence-specific manner but the consensus binding site is very degenerated (AA/T/CATA/TTTA). FIS has a preference for sites flanked by A/T regions and it induces strong compaction of DNA when it is at its maximal expression (112, 113).

4.4.1. IHF (Integration Host Factor)

IHF is a heterodimeric nucleoid protein encoded by the himA and himD genes. It shows a strong homology to the HU protein and binds with high affinity to specific DNA at specific sequences, then induces strong DNA bending. It has a consensus binding sequence but it can also bind DNA non-specifically and can be substituted by HU. The HU protein usually binds preferentially to supercoiled DNA and it shows little sequence specificity although it recognizes nicks and gaps in DNA (114-116).

4.5. Transcriptional activators

A transcriptional activator is a protein that binds and stimulates gene expression. Transcriptional activators are DNA-binding proteins that bind to specific site of the DNA sequences and create protein-protein interaction with the general transcription machinery (RNA polymerase and general transcription factors). The activator binds upstream of the promoter and interacts directly with the RNA polymerase through a contact with the CTD of the α- subunits (Figure 10), (117, 118).

4.5.1. SlyA (Salmolysin A)

SlyA (a hemolytic protein) belongs to the MarR family which is a growing family of transcriptional regulators. This protein consists of two domains, an N-terminal domain involved in dimerization and a C-terminal domain indispensable for DNA recognition and binding (119). SlyA was initially identified in Salmonella, and it has been it demonstrated that SlyA recognizes a short palindromic DNA sequence of 12 base pairs TTAGCAAGCTAA. The binding of SlyA to this sequence increases the expression of hemolysin E (or ClyA) by antagonizing the negative effects of H-NS (66, 120, 121). SlyA regulates a wide variety of biological processes such as antibiotic resistance and environmental sensing of aromatic compounds. The slyA gene of E. coli and S. enterica serovar Typhimurium can activate the expression of cytolysin A in E. coli K-12 strain (67, 68). Even though RovA of Yersinia, SlyA of Salmonella and PecS of Dickea dadantii all belong to the MarR/SlyA family, some differences were identified in their regulation mechanisms. Indeed SlyA negatively regulates its own expression by interfering with the binding of RNA polymerase, whereas RovA appears to interfere with the progression of RNA polymerase and competes with H-NS binding.

4.5.2. Cyclic adenosine monophosphate (cAMP) Receptor Protein: (CRP)

The cyclic AMP receptor protein (CRP) is a prokaryotic global transcriptional regulator; which regulates the expression of nearly 200 genes (122). In Escherichia coli, the CRP exists as a homo-dimeric transcriptional activator that is triggered by cAMP. One of the most well-studied transcriptional regulators in E. coli is the CRP also known as the catabolite gene activator protein (CAP) (123-125). CAP exists in three forms: apo-CRP, CRP-(cAMP) 2, (for transcription activation), and CRP-(cAMP), dominates in solutions (124). CAP functions through contact with RNAP and the CRP only functions with cAMP bound (CRP-cAMP). Under glucose limitation, its level rises and then cAMP binds to homo-dimeric CRP protein which in turn binds to the

activator site (126). The CRP-cAMP complex bends the DNA and makes contact with the alpha-CTD of the RNA polymerase (Figure 10) (127, 128). CRP consists of two domains the N-terminal cAMP-binding domain (residues 1–137) and the C-terminal DNA-binding domain cAMP-CRP (129). CRP acts as a sole regulator for three different promoters, the class I (Plac), class II (Pgal), and class III promoters.

The cAMP-CRP consensus binding site is: TGTGA -N6-TCACA. CRP activates the transcription of clyA through the class I promoter of clyA and this can be transcriptionally activated by CRP and/or FNR. The clyA promoter region has a high intrinsic curvature which is a regulatory region to silence H-NS (130). For the class I promoter (Plac), the CRP protein makes contact with the alpha-CTD of the RNA polymerase and thus stabilizes its binding to the promoter. This allows transcription to occur (131, 132). Figure 9, shows the model for activation of transcription by CRP and the role of RNA polymerase holoenzyme (RNAP) α-subunit in promoter recognition and transcriptional activation.

Activator

DNA Activator -35 -10 site

Figure 9: Modified from (132). A promoter that contains an activator site makes contact with α-CTD of RNA polymerase ( the lac promoter). And the α-CTD makes specific protein protein interaction (the black circle) with the activator.

4.5.3. SfaX

The SfaX protein is a 17-kDa transcriptional regulator from the MarR family found in some ExPEC strains (some UPEC and NMEC strains). Its gene, sfaX, belongs to a gene family whose members (papX, focX, sfaX) are located downstream (300- 3000bp) of different fimbrial operons (pap, foc and sfa, respectively), acquired via 33

HGT. The sfaX transcriptional analysis showed that the gene is part of the main fimbrial operon and it is transcribed together with the rest of the fimbrial gene (133, 134). In addition, the sfaX gene can be expressed from a more proximal promoter and is found to be a subject for a strong down-regulation by the nucleoid structuring protein H-NS (135). A new gene, the sfaY, that codes for a putative c-di-GMP EAL - phosphodiesterase is located between the sfaX gene and the fimbrial biogenesis genes array. sfaY is located upstream of the sfaX gene in the fimbrial operon and its product can influence the cyclic di-GMP turnover in the bacteria (136).

4.5.4. c-di-GMP (Cyclic diguanylic acid)

Cyclic di-GMP [bis-(3′-5′)-cyclic di-GMP] is a novel second messenger used in signal transduction in a wide variety of bacteria. This nucleotide is produced by diguanylate cyclases (DGCs which have the GGDEF domain) and it is degraded by specific phosphodiesterases (two types of PDEs, carrying either EAL or HD-GYP domains). This second messenger is unique for bacteria but not present in all bacterial species. It regulates a wide range of functions including, adhesion, motility, biofilm formation, and the virulence of animal and plant pathogens (137). The levels of cyclic di-GMP in bacterial cells are influenced by both synthesis and degradation. (138). In E. coli bacteria there are ORFs coding for 12 GGDEF-, 7 GGDEF + EAL- and 10 EAL- proteins, hence the levels of c-di-GMP in E. coli are tightly regulated.

4.5.5. Stationary phase sigma factor (RpoS)

RpoS also known as (SigS, OtsX, AbrD, AppR, Csi2, DpeB, KatF, Nur, sigma 38 factor, sigma S factor, σ38, σS) is a key (master) response regulator to stress conditions in Escherichia coli and many other proteobacteria (139, 140). When RpoS binds to RNAP, the complex recognizes -10 and -35 boxes that are upstream of the genes coding for proteins needed for the transition from vegetative-to-stationary phase or for other stress conditions (141). RpoS controls the expression of approximately 100 genes involved in different physiological functions that help the 34

cells to adapt with starvation and stressful conditions (140, 142, 143). In contrast to its conserved well-understood role in stress response, effects of RpoS on pathogenesis are highly variable and dependent on species (144-146).

4.5.6. (p)ppGpp – stress alarmone

The small molecule (p)ppGpp also called guanosine pentaphosphate, or tetraphosphate (ppGpp) or magic spot acts as a global regulator of gene expression in bacteria. (p)ppGpp is a modified nucleotide which acts as a stress signal in bacteria. It is involved in stringent response of bacteria that causes inhibition of RNA synthesis during shortage of amino acids. ppGpp/DksA activates RpoS which can directly or indirectly upregulates ClyA expression in E. coli. ppGpp is an effector molecule of the stringent control unit, it is a nucleotide produced not only in response to amino acid limitation but also in response to many different kinds of nutrient limitations and circumstances that can cause growth arrest (147-149). (p)ppGpp is synthesized from GTP (or GDP) and ATP by RelA and/or SpoT in response to any stress condition that will result in growth arrest. The (p)ppGpp is not restricted only to Gram-negative bacteria, it is also found in Gram- positive bacteria and also in the chloroplasts where it also functions in stress-related processes (148, 150).

5. PROTEIN SECRETION IN BACTERIA

Secretion is a process that Gram-negative bacteria use for transporting proteins and other cellular constituents to the outer membrane or to the extracellular space. Before a protein is transported to the outside of Gram-negative bacterial cells, it must be transferred through different compartments: the inner membrane, the periplasmic space, and the outer membrane. To be transported, proteins need a

signal peptide, (also called signal sequence, leader sequence or leader peptide) which is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins to enable the translocation of the protein from the bacterial cell to the outside of the cell. Many types of secretion systems have been identified in Gram-negative bacteria for the export of protein and virulence factors (151). These secretion mechanisms present in bacteria so far are: type I, type II, type III, type IV, type V, type VI and secretion via outer membrane vesicles (or type Zero secretion). E. coli ClyA protein uses outer membrane vesicles as its secretion mechanism while E. coli HlyA uses type I secretion system for delivery but can also be exported out of the bacterial membrane within OMVs. In V. cholerae, VCC is secreted via type II secretion mechanism.

5.1. Type I and type III secretion mechanisms

These secretion mechanisms are referred to as the sec-independent pathways: and the proteins are secreted in a stepwise manner through a trans-membrane channel (28, 152).

5.2. Type II and type IV secretion mechanisms

Type II secretion is the major secretory pathway for Gram-negative bacteria (153). Type II secretion system allows secretion of fully folded proteins and even oligomers with S-S bridges. Proteins secreted through the type II system depend on the Sec or Tat systems for initial transport into the periplasm. In Pseudomonas and Erwinia some T2SS substrates are not exported in the periplasm per se but by the Tat pathway. In Gram-negative bacteria, type IV pili and type II system are phylogenetically linked.

5.3. Outer membrane vesicles (Type Zero secretion system)

The secretion of extracellular products is the major mechanism by which Gram- negative pathogens communicate with and intoxicate host cells. Vesicles released

from the envelope of growing bacteria serve as a secretory vehicles for proteins and lipids of Gram-negative bacteria (154). Outer membrane vesicles are small bubble- like nano-compartments budded from the bacterial outer membrane. They range in size from 20-200nm and are produced during normal growth conditions (155, 156). As being released from the surface of the outer membrane, they ensnare some of the underlying periplasmic constituents. OMVs contain outer membrane proteins, lipopolysaccharides (LPS), phospholipids and periplasmic components. They have different roles in the pathogenesis of bacteria such as virulence, bacterial communication through OMV-associated signal molecules and genetic transformation (157-159). Vesicles are another method used by almost all Gram-negative bacteria to release material, and the phenomenon is not due to cell lysis or death but the molecular mechanism behind is not fully understood (158, 160, 161). Vesicle formation is abundant at the site of cell division in species such as E. coli, V. cholerae, and Brucella melitensis (162). The peptidoglycan lipoproteins play a role in the production of outer membrane vesicles in E. coli. Where there is less peptidoglycan lipoproteins, there is a weak linkage between the peptidoglycan and the outer membrane of the bacterium and thus the OMV formation starts with outward bulging event towards the outer membrane (158). It has been demonstrated that the release of vesicles is a result from a general response to stress conditions, where the process of loading cargo proteins seems to be selective (163). The protrusion from the outer membrane occurs when periplasmic turgor pressure increases due to accumulation of peptidoglycan fragments in the periplasmic space which eventually leads to the bulging of the periplasmic space (163).

6. OTHER GENES OR PROTEINS OF INTEREST

6.1. Alkaline phosphatase (PhoA)

Alkaline phosphatase is encoded by the phoA gene in E. coli. The wild type PhoA protein has a signal-sequence that allows the export of alkaline phosphatase into the periplasm where it is located and active (164-166).

6.2. E. coli Disulphide bond system (Dsb)

Disulphide bonds are covalent, single, non-polar, σ-bonds that are formed between two sulfur atoms of the thiol groups of cysteines, and they are important features for the three-dimensional structure of many proteins. The disulfide-bond (Dsb) system is responsible for the formation of disulfide bonds in bacteria during the process of protein folding (167). In Escherichia coli K-12 the DsbA and DsbB proteins are involved in the oxidative pathway where they introduce disulfide bonds into newly synthesized proteins translocated into the periplasm. The other pathway is the isomerase pathway where the DsbC, DsbG and DsbD catalyze the disulphide bond reshuffling/isomerisation, when incorrect disulphide bonds are introduced either by DsbA or under the conditions of copper oxidative stress (168, 169).

7. AIMS OF THIS THESIS

To examine possible clues to the apparent patho-adaptive selection of the ΔclyA alleles among different ExPEC strains. By restoring an intact clyA gene locus in variants that have truncated clyA genes we aimed to monitor the expression and activity of the clyA gene in the ExPEC genetic background.

To study ClyA as a model protein for the export mechanism involving outer membrane vesicle-mediated release of proteins from the periplasmic space in Gram-negative bacteria. We asked if some particular domain could be identified as responsible for subcellular localization of the ClyA protein and also analyzed the different forms of the protein in the periplasm.

To investigate the secretion mechanism of a pore-forming cytolysin (VCC) in V. cholerae and to analyse how VCC is secreted when the vcc gene is heterologously expressed in E. coli.

8. RESULTS AND DISCUSSION

During my Ph.D, I performed studies on pore forming protein toxins present in E. coli and Vibrio cholerae.

8.1. Paper I

Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain patho-adaptive mutations in a subset of extra-intestinal E. coli strains

The ClyA protein is a pore-forming protein toxin, whose expression is activated by CRP and SlyA but repressed by H-NS. The ClyA protein was discovered in an hns mutant of E. coli K-12. The gene is present in the IPEC and APEC strains, but absent in some of the well-studied E. coli ExPEC strains. In this study we analyzed the DNA sequence from different ECOR strains obtained from different hosts and geographical location. We identified four different variants which have ΔclyA alleles (denoted I to IV) Figure 1, paper I. Variant III which was identified in UPEC strain 536, which belongs to the B2 phylogenetic group (E. coli strains involved in extraintestinal infections), have two deletions: one deletion was at the promoter sequence of the clyA locus and the other deletion was identified in the clyA coding sequence. Escherichia coli O6:K15:H31 (UPEC strain 536), and the UPEC strain J96 O4:K6 (from Variant I) were used in this study. Both strains were originally isolated from patients with pyelonephritis.

To examine why the clyA gene was absent in the UPEC strains, we performed an experiment to analyze whether the absence of the gene was advantageous or disadvantageous to these UPEC strains. The intact allele of the gene was introduced into the chromosome of J96 and 536, using a suicide plasmid pJON176 containing

clyA wild type allele and a kanamycin cassette as a selectable marker located 350bp downstream at the clyA stop codon. The resulting strains 536 clyA+ (JON53) (Figure 10) and J96 clyA+ (JON47) were generated.

umuD clyA+

E. coli K-12

UPEC 536 (493 bp) (217 bp)

Km JON53

(UPEC 536 clyA+)

Km COE2

+ (JON53 clyA , clyA::luxAB)

Figure 10: Schematic representation of the clyA loci in E. coli K-12 and in derivatives of UPEC strain 536. The strain JON53 (536 clyA+) was created using the plasmid pJON176 and the strain COE2 is a derivative of JON53 with a chimeric clyA::luxAB fusion.

By PCR analysis, we confirmed the presence of the clyA gene in the chromosome of 536 and J96. The expression of alpha-hemolysin by UPEC strains is calcium- dependent. Production of alpha-hemolysin can be detected using blood agar plate on which bacteria can lyse the red blood cells forming a dark green transparent hemolytic zone because of the release of HlyA protein from the bacteria. The hemolytic activity of ClyA was Ca2+-independent. In my study, the clyA gene was introduced into the chromosome of the UPEC strains to construct JON53 and JON47 strains. We undertook a phenotypic characterization of these strains using Ca2+- depleted blood agar plates in order to block the hemolytic activity of alpha- hemolysin protein. No hemolysis was observed on the blood agar plate demonstrating that the ClyA protein was not released by the cells. However, when mitomycin C was added to these plates to induce pore formation in the bacterial membrane via phage production, beta hemolytic phenotypes were observed on 41

blood agar plates. Hemolysis was due to the release of the ClyA protein from the bacteria through the pore in the cell envelop. These data indicated that the ClyA protein was produced and is active in the UPEC strain JON53 (Paper I, Figure 2b).

Proteins from sub-cellular fractionation experiments were separated by a SDS-PAGE and transferred onto a nitrocellulose membrane for immunodetection with anti-ClyA antibodies. ClyA was detected in the periplasm of the JON53 strain suggesting that ClyA was efficiently transported through the inner membrane of the bacterial cells (Paper I, Figure 2c).

In order to monitor the transcriptional expression level of clyA, a clyA::luxAB operon was integrated in tandem, in the chromosome of JON53 by a single recombination event, using the suicide plasmid pMWK4 generating COE2 (JON53 clyA::luxAB). The strain COE2 was generated to determine the expression profile of clyA using the luciferase activity measurement. Luciferase activity measurements were performed with COE2 and JON53 strains at different phases of growth. Our data clearly showed that clyA expression was elevated at the late logarithmic phase of growth. A 5-fold increase of clyA expression was observed in COE2 compared to the positive control strain E. coli K-12 clyA::luxAB derivative JON33. (Paper I, Figure 4A). With this analysis we demonstrated that expression of the clyA gene is growth phase dependent and elevated level of expression was observed at the late logarithmic phase of growth. SfaX/PapX are encoded in the fimbrial gene clusters that are found typically in UPEC and NMEC isolates and are absent in E. coli K-12. The SfaX/PapX proteins show resemblance to the SlyA protein since these proteins belong to the MarR family transcriptional regulator. We then analyzed whether SfaX would affect the expression of clyA and included tests with another regulator of fimbrial genes, PapI, for comparison. The following strains were used: COE2, COE3 (COE3/pAES1; a sfaX+ clone), COE4 (COE2/pBR322; a vector control) and COE6 (COE2/pHMG94; a papI+ clone). A luciferase activity measurement to monitor the transcriptional levels of the clyA gene was performed. The clyA expression was greatly enhanced at the late

log/early stationary phase of growth in the strain COE3 (carrying the sfaX+ plasmid) when compared to the papI+ strain COE6 (Figure 6, paper I). When the sfaX gene was ectopically expressed, there was an increased level of ClyA which indicated that the increase of the ClyA protein was influenced by the expression of SfaX protein. Furthermore, we found that the level of SfaY, a phosphodiesterase that decreases the c-di-GMP levels, also might be involved in the upregulated level of ClyA production in the UPEC strains (Figure 11). In an sfaY deficient IHE3034 strain the level of ClyA was reduced. From these studies, we suggest that both SfaX and SfaY can cause higher level expression of ClyA. The elevated levels of the clyA gene might also be due to the other genes acquired by PAIs that are present in the ExPEC strains but not in the strain E. coli K-12.

Model of clyA regulation in E. coli K-12 and in ExPEC (536 UPEC strain)

A) B) FIS FIS CRP/FNR H-NS +? +? + - CRP/FNR clyA H-NS clyA + - ? ? + + + + SfaX RpoS ppGpp (+) + (+) RpoS ppGpp SlyA + SfaY SlyA

Figure 11: Schematic representation of the clyA gene regulation in (A) E. coli K-12 and (B) in ExPEC (UPEC, 536 clyA+).

We hypothesized that bacteria with high expression of pore-forming toxin might be more sensitive against membrane targeting antimicrobial agents. To prove this hypothesis, we have performed antimicrobial peptide sensitivity tests using the UPEC strain JON53 (536 clyA+) and the K-12 strain expressing ClyA protein. Briefly, the bacterial strains were cultured in an artificial urine medium supplemented with

serially diluted antimicrobial peptides (Polymyxin B, β-defensin and LL-37), and also with different concentrations of urea and creatinine. The UPEC construct JON53 (536 clyA+) shows a growth inhibition in presence of polymyxin B when compared to the parental 536 (ΔclyA) strain (Paper I, Figure 7). High concentrations of urea affected the growth of clyA + derivative JON53 more than the parental ΔclyA UPEC 536 isolate. JON53 showed a delay in growth with a long lag phase as compared to the wild type 536 strain. Also the E. coli K-12 clyA+ strain MWK11 showed a reduced growth when 0,48 µg/ml of polymyxin B was added as compared to the wild type MC4100 (that has a low chromosomal expression of clyA). Taken together, these results indicated that the restored clyA locus made the UPEC strain more susceptible to the antimicrobial peptide polymyxin B. Presumably, such a phenotype would be more of a disadvantage in the intestinal locations where commensal bacteria can produce antimicrobial peptides. It will also be worthwhile to test with other antimicrobial peptides like cathelicidin that plays a role in mammalian innate immune defense against bacteria that invade the host.

We can hypothesize that the presence of intact clyA + allele in UPEC strains might be unfavourable for the bacteria in the urinary tract where the bacteria can face antimicrobial peptide stress or other bacterial membrane targeting stresses. The mutations in the clyA gene may adapt the bacteria to these stress conditions and thereby contribute to the bacterial survival as pathoadaptive events.

8.2. Paper II

Localization and structure of the ClyA protein of Escherichia coli before secretion and pore-formation

We performed a study to determine if ClyA has a “Membrane Translocation Domain”. Since no classical signal peptide has been identified (for Sec- or Tat- dependent translocation) within the ClyA sequence, therefore the mechanism through which the ClyA protein is translocated from the cytoplasm to the periplasm is not yet known.

In order to determine if there would be a particular domain that promotes translocation of ClyA from the cytoplasm to the periplasm via inner membrane, we generated hybrid proteins between truncated variants of ClyA and the alkaline phosphatase (PhoA). PhoA was used as a reporter of periplasmic export since PhoA is active only in the periplasm where the disulfide bonds necessary for the protein activity are formed. The TnphoA transposon was used for this purpose and a collection of plasmids with the transposon inserted in the cloned clyA gene was obtained. The plasmids encoding the different fusions were introduced into the phoA deletion mutant strain CC118. Enzymatic activity of PhoA was detected by streaking the strains on plates containing chromogenic 5-Bromo-4-Chloro-3-Indolyl phosphate (XP) which turns blue when hydrolyzed by PhoA. For further analysis we selected three constructs that correspond to the fusion of the first 12, 83 and 284 residues, respectively, of ClyA to the N-terminus of PhoA. Dark blue colonies were observed with the strain that carried only 1-12 ClyA amino-acids, while the bacteria with the longest truncated variant of ClyA produced slightly blue colonies. The presence of blue colonies showed an active alkaline phosphatase in the periplasm indicating that the 12 first residues of ClyA constitute the peptide of ClyA that can promote translocation into the periplasm (Figure 12). Previous data revealed that 2 to 15 N-

terminal amino-acids of the ClyA protein are essential for translocation and phenotypic expression (61).

ClyA a.a residues

PhoA phenotypes + + + + + +

Figure 12: Alkaline phosphatase (PhoA) fusion sites in ClyA. The arrows indicate the different fusion point amino-acid. +++ = Dark blue colonies, ++ = blue colonies and + = faint blue colonies, on XP- plate.

The results from sub-cellular fractionation experiments performed with transformants carrying different lengths of the ClyA::PhoA constructs (ClyA 1- 12::PhoA, ClyA1-83::PhoA and ClyA1-284::PhoA) revealed that the fusion construct with the shortest ClyA amino-acids (1-12) at the N-terminal was sufficient for translocation of the PhoA to the periplasm. However, ClyA (1-12) was not detected in outer membrane vesicles after western blot analysis.

The monomeric ClyA has two Cystein residues, at position 87 in the N-terminal and at position 285 in the C-terminal. When ClyA protein is not active (not able to form pores), it means it has the intra-peptide disulphide bond present between the two Cys, making the protein folded, monomeric and soluble. When the bond is reduced by some reducing agent like DTT, the protein turns to its active form and can form pores on the target membrane.

This study was performed to determine the role of the two Cys residues in dimer and oligomer formation, and also the translocation and stability of the ClyA protein.

Earlier results indicated that the ClyA C-terminal region (a.a 281-303) is important for translocation and/or stability of ClyA protein in the periplasm (61, 170).

We created single and double mutants by substitution of the Cys with Ser giving the following mutants: ClyAC87S, ClyAC285S, ClyAC87S,C285S. Plasmid carrying the substituted mutant genes were introduced into the DH5α strain and the transformants were cross-linked with 2.5mM gluteraldehyde at different time points (Paper II). ClyA C87S, cross-linked for 10 minutes, showed both monomeric and multimeric forms of the protein while the ClyA C285S and ClyAC285,C87S, demonstrated only the monomeric form of ClyA after 10 minutes of cross-linking with gluteraldehyde (Figure 3, paper II). These results suggest that the Cys residues are important for dimer and/or oligomer formation by ClyA.

Results from phenotypic tests on blood agar plate and contact hemolytic assay in 96- well plate format revealed that the Cys residues are important for the activity of the protein. Subcellular fractionation experiments showed a decrease of ClyA C285S and

ClyAC285S,C87S export into periplasm. On the other hand, ClyAC87S export level was similar to the wild type pYMZ80. It was concluded that the CysC285 is important for translocation. Figure 13 illustrates the model proposed for the role of the Cys residues of ClyA in E. coli.

Figure 13: The role of Cys residues and the fate of ClyA protein in E. coli.

ClyA protein was purified using FPLC (paper II) and the purity of the protein was analyzed by SDS-PAGE gel electrophoresis. ClyA is able to form giant cation-selective channels in lipid bilayers and it has a high channel-forming activity when small amounts of detergents are added to the protein. The purified protein was treated with a reducing agent and both the reduced protein (detergent treated) and the non-treated protein were applied on a black lipid bilayer membrane to determine the pore sizes and channel forming activity of the two forms of the protein. It was observed that the non-treated protein (monomer) form small pores with a conductance that ranges between 9 to 11 nS, while the treated form (multimeric form) of ClyA form larger pores which are homogenous in sizes. This data suggested that the channel-forming activity of the oxidized form of ClyA was indeed much lower than that of the reduced form.

The purified periplasmic protein was treated with DTT and stained with the negative stain then visualized under the electron microscope for the pore-forming ability at different time points. The pure protein treated for longer than 30 minutes showed a ring like pore formation, Figure 14. Both the electron microscopic analysis and lipid bilayer assay showed that the reduced form of ClyA can form pores which if in contact with a target membrane will disrupt the influx and eflux of ions across the membrane. The purified periplasmic fraction of ClyA also showed an arrangement of Head-to-Tail dimers arranged in pairs of three beside each other which showed the hexameric form of the ClyA protein (Figure 5, paper II). This head to tail organization prevents premature folding of the protein in the periplasm.

Figure 14: Purified ClyA from periplasm treated with DTT (50 mM) in vitro: ring like pore formation with an octameric assembly. EM analysis using negative staining (Paper II).

We have shown from this study, that the first 12 ClyA amino acids from the N- terminal are involved in translocation of ClyA to periplasm. Cys is important for translocation of the ClyA protein. The reduced form of ClyA protein can form homogenous pores and treatment of ClyA with DTT at longer time points (30 minutes) shows the ring-like pore formation.

8.3. Paper III

Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains

The V. cholerae non-O1 non-O139 strain V:5/04 was used, for the studies to determine VCC secretion, export and activity. The sub-cellular fractionation experiment was performed using the cultured NOVC V:5/04 strains grown to an optical density of 2.0. Results from immunoblot assay revealed that the VCC protein was present in the whole cell (WC), inner membrane (IM) periplasmic and supernatant fractions. OMVs were isolated, purified and examined under a transmission electron microscope and the results revealed that the wild type strain V:5/04 showed the release of OMVs from the surface of the bacterial cell (Figure 2A Paper III). Immunoblot experiments performed using both OmpU (outer membrane protein U as a vesicle marker) and VCC anti-serum, further proved that the VCC protein was associated with OMVs from the wild type V. cholerae strain V:5/04. These data clearly show that VCC is released from the bacterial cells in associated with OMVs. To analyse how VCC was associated with OMVs, dissociation assay was performed.

Purified OMV samples were treated with urea, NaCl, Na2CO3 and 1% SDS. When the VCC was treated with 1% SDS that can rupture the vesicle membrane, the VCC protein was not detectable in the pellet associated together with OMVs, but the VCC was released to the supernatant fraction, (Figure 3, lane 10, paper III). Resistance to urea and release of VCC from the OMVs after SDS solubilization of vesicle membrane, as well as presence of VCC in the periplasmic space showed that VCC most likely located inside the OMVs during the formation and release of OMVs from the bacterial cells. Proteinase K experiment was also performed to assess if the VCC protein was protected by the vesicle architecture. When the VCC associated OMVs was treated with proteinase K in the presence or absence of 1% SDS, there was a clear proteolytic digestion of VCC when treated with 1% SDS and this showed that

the VCC was actually protected by the OMVs (Figure 3 B, paper III). Further evidence to prove that VCC is associated with OMVs from the NOVC V:5/04 strain was verified by the immunogold labeling experiments (Figure 4B, paper III). The immunogold labeling test was performed between the wild type and vcc mutant NOVC V:5/04 strain and the results showed gold particles associated with only ruptured vesicles from the wild type NOVC strain. This indicates that the vesicles from the wild type were ruptured and the gold particles were able to attach to the

VCC protein further confirming that VCC was internal to the vesicle membrane. Re-association assay was performed to determine if VCC can be re-associated on the surface of OMVs but this proves that the presence of VCC in the OMVs from the wild type V. cholerae strain V:5/04 occurred as a result of internalization or encapsulation during formation of OMVs and not due to binding of VCC on the surface of OMVs.

Biological activity of the VCC associated with OMVs was detected by using 5% rabbit red blood and HeLa cells. Contact hemolytic assay was performed with OMV- associated VCC and purified VCC. The purified VCC and OMV-associated VCC were co-incubated with 5% rabbit red blood cells and the rate of hemolysis was monitored by measuring the release of hemoglobin. The release of hemoglobin showed that both purified VCC and OMV-associated VCC provoked a dose-dependent hemolysis of the red blood cells when compared to the OMVs isolated from the vcc mutant. Moreover, at each of the tested samples with different dilutions, the hemolytic activity of OMV-associated VCC was higher than that of the corresponding amount of purified VCC. To analyze the cytotoxicity effects of OMV-associated VCC, cultured HeLa cells were treated with OMVs for 6 h. Microscopic observations showed that when the HeLa cells was treated with OMVs from the wild type V. cholerae, the HeLa cells showed a cytotoxic phenotype due to detachment and lysis of the cells, but this was not the case with OMVs isolated from the vcc mutant (Figure 6B, paper III).

In order to identify a putative link between autophagy induction and bacterial pore- forming toxins, HEK293 cells expressing GFP-LC3, were incubated with different concentrations of OMVs isolated from the wild type V. cholerae, purified VCC and

OMVs from the isogenic vcc mutant V. cholerae for 6 h. Epithelial cells treated with 0.3 and 0.15 nM VCC associated OMVs from the wild type strain V:5/04 showed active autophagy response visualized as bright GFP-LC3 particles (Figure 7A, panel a and b respectively) and showed clear LC3-II form (Figure 7B, lanes a and b respectively) in a dose dependent manner. To obtain the same level of autophagy response, 2.4 and 1.2 nM of purified VCC was required (Figure 7A, panels e and f; Figure 7B, lanes e and f respectively) indicating that OMV-associated VCC is more active than the purified VCC in different biological activities.

All the V. cholerae strains analyzed by western blot have a band that shows the presence of VCC in the vesicles. Also we use E. coli strain MC1061, to detect if OMV- associated VCC secretion occurred when the vcc gene is heterologously expressed in E. coli. The vcc gene was cloned into a plasmid and introduced to an E. coli strain. Sub-cellular fraction followed by immunoblot assay showed that the OMV-mediated release of VCC is a general mechanism, since it was also secreted from the E. coli cells in association with OMVs.

The study showed that OMVs from the NOVC V:5/04 strain are biologically active and can also be toxic to mammalian cells and they can induce autophagy.

DISCUSSION

From these studies, we have demonstrated that ClyA is highly expressed in the late logarithmic growth phase of the UPEC strain COE2 that carries the clyA::luxAB fusion gene and its level was super induced by the SfaX/PapX regulatory proteins. Also the restored ClyA + strain showed increased susceptibility to an antimicrobial peptide.

Although the clyA gene was discovered in an ∆hns mutant E. coli K-12 strain which is a non pathogenic variant, studies with the clyA gene on other enteric bacteria such as Salmonella enterica serovars Typhi and Paratyphi have shown that the gene has a role in virulence. It yet remains to be verified if there is any role as a virulence factor of the clyA gene in E. coli, e.g. in the case of some IPEC strains.

Why is the clyA gene present in the non-harmful K-12 strains and IPEC but absent in some of the ExPEC strains? The sequence analysis from K-12, IPEC and some ExPEC strains with non-functional clyA showed about 98% sequence similarity of clyA in the promoter and the coding region. Does this gene have a beneficial role for the K-12 bacteria and also for IPEC? The patho-adaptation events might be due to a tight selective pressure on the part of the bacteria to keep the clyA gene inactive.

The SfaX/PapX regulation on ClyA should be further investigated - why the clyA gene expression is highly elevated when SfaX is overexpressed? What is the role of H-NS in these restored strains (COE2 and COE3)? It will be of interest to further investigate the effects of other regulators like Hfp, StpA, H-NS, FNR and RpoS in the ExPEC (UPEC and NMEC) strains with restored clyA locus.

Studies performed on the secretion and localization of the ClyA protein have revealed that the protein is exported to the periplasm and secreted from the bacteria through the type Zero secretion system as illustrated below in Figure 15, (59).

Figure 15: Suggested ClyA translocation mechanism in E. coli. The ClyA protein from cytoplasm is translocated to the periplasm where the proteins are folded with the DsbA and DsbB subunits. The protein attached to the outer membrane of the bacteria and when the vesicle in the outer membrane blebs they will also be exported.

ClyA protein isolated from the periplasmic fraction revealed that the protein is active and have different forms. Also the Cys residues are important for translocation, what will happen if the Cys mutant plasmid are introduced into a ClyA overproducing strain or ClyA negative strain?

Vibrio cholerae was used for this study because it also has a pore forming toxin, VCC. Both VCC and ClyA can form pores on target host membrane and also lyse erythrocytes. Studies of VCC expression in E. coli have revealed that the VCC protein might also act as a reporter for the transport of proteins (without a signal sequence) to the periplasm. It remains yet to be investigated if the OMVs-associated VCC can provide a virulent trait in an E. coli background.

Although VCC and ClyA are both pore-forming toxins produced by Gram-negative bacteria, the VCC protein and ClyA also have some differences which are discussed below.

The VCC protein is a beta-barrel pore-forming toxin that contributes to the pathogenesis of V. cholerae after VCC is proteolytically activated by exogenous

protease HapR. VCC exhibits specificity for membranes enriched in cholesterol and ceramides. It contains a prodomain that is required for its folding and it must be cleaved to produce the active and mature 65-kDa form. The mature VCC protein assembly within the target membrane to form pentameric channels.

While ClyA from E. coli belongs to the alpha-pore forming toxin, it uses the amphipathic alpha helices to insert into the target membrane. Its role in virulence of E. coli is not known. It has two forms the non-active form that has a S-S bond between its two Cys (at position 87 and 285) residues and the active form of ClyA has its disulphide bond reduced. The ClyA protein can be arranged into dimeric, octameric and 13-meric forms. They can form pre-pores in solution without any biological or target host membrane and when they get attached to a target host membrane then they exert their functions (i.e. disrupt the influx and efflux of ions).

9. CONCLUDING REMARKS

1) The absence of clyA might be due to a selective pressure, and it might be disadvantageous for these UPEC variants to keep the clyA gene. This might be the suggested reason for their deletion in the ExPEC strains.

2) The ClyA protein has an N-terminal motif that can act as its signal sequence during the transport of the protein to the periplasm.

3) The VCC protein can be released within the OMVs of Vibrio cholerae and E. coli.

ACKNOWLEDGEMENTS

The long and rough track towards obtaining my Ph.D. degree have finally come to an end. I will like to start by thanking The Almighty LORD GOD and my Ancestors for all my achievements in life. My years as a Ph.D. student in Umeå university, Department of Molecular Biology, have finally come to an end. The time I spent as a Ph.D. student in Umeå university has been enjoyable, challenging and filled with surprises. During this period, I had the opportunity to reinforce my knowledge of Molecular Biology. I learn a lot of new concepts in Microbiology, especially in Gram-negative bacteria. I also had the privilege of experiencing the harsh winter in Umeå, 22 hours of sunshine, and the northern lights (Aurora borealis). I will like to thank all the co-authors and collaborators who relentlessly contributed to the publications and manuscripts in this thesis.

There are some people who deserved special thanks.

First I will like to thank my supervisor Bernt Eric Uhlin for giving me the opportunity to work in your lab, I learn a lot of new concepts and I develop a lot of independence. Thank you Sun Nyunt Wai my co-supervisor for our scientific discussions and for all the nice gatherings at the BEU-SNW house. A big thanks to Olle Heby my master´s thesis supervisor, in short your guidance during my time in your lab makes me to develop interest to continue in science. Lennart Johansson my study coordinator you are such a nice and international person, thanks for the guidance and orientation.

Let me also extend my appreciation to Monica for your support and help in the lab. A very special thanks to my small brother Nikola Zlatkov you are the best indeed when it comes to scientific discussions, keep up with the good work and I like your enthusiasm towards science (smile), thank you for carefully reading my thesis. Nabil for your encouragement. And to some former members in the group of BEU- Bhupender I enjoy your little crazy jokes. Patricia Paracucs (Buenos días) my good friend, I like your hospitality and to your dearest one Tiago for your politeness and sense of dressing, thanks for the parties in your apartment and in INVITO. Annika for your seriousness and likeness in sport. Stina, for your help. Hyun Sook my able friend for your kindness. Mariette for our driving trips and for all the fika in Holmsund, thanks to your parents for the fika in their house. Junfa for always accepting to assist. Kyaw for always smiling. Ting for being a colleague. Ryoma for nice Japanese cookies. Jurate it was great to work with you during the early years of my Ph.D.

And to all the present and past members of group SNW – Marylise and Shridhar for scientific collaboration, Svitlana for your sense of beauty, Mitesh for your jokes, Dharmesh for your braveness, Tianyan for being so ambitious, Barbro for your hospitality and kindness, Pramod, Sony, for the nice time we share. Jan Oscarsson for collaborating and Bernard for our lunch discussion. Jörgen Johansson for your scientific discussions and Berit for treating everyone equally. Group JJ team members Teresa for your positive

discussion, Edmund for your chocolates. Isabelle, Sakura, Carolina, Jessica, Christopher, Sabine, Stephanie, Karolis for the morning coffee and some scientific discussion.

Åsa Gylfe, for your unending support and help, in times of sorrow and in times of joy. Åsa you are like a sister and not a friend and to your love one Matti for updating me about new computer games. A big thanks to your parents and parent in-law for all the gatherings at home and in the summer house. Fredéric and wife Hande Login the most holy Fred what a nice hearted brother you are, always ready to help thanks so much for carefully reading my thesis. And to your wife Hande, aka sunshine, thanks for all the unending support, gatherings in your apartments and also at “INVITO” pub. I wish you people the best in your marriage life, enjoy Denmark. Ola and wife, thanks for giving me tips on how to survive in Sweden. And to my friend Teresa Del Peso and boy friend Martin thanks for all the gatherings in your apartment. Ikenna and wife Ego for all the birthday parties and nice discussion. Ala thanks for being such a nice and sociable girl. Anjana for your nice smiles. Ava for your encouragement. Nelson for your smart ideas. Ulli for your toughness, Olena, Anna Kauppi, for being nice friends. Geetanjali, Anne Laure, Khan, Roshani, Steffi, Laura, Akbar, for the Wednesday night movies. Kemal, and wife Ummehan, Saskia, Edwin, Tomas and wife Helen for our lunch discussion. My friend Salah thanks for your encouraging words.

Thanks to the media ladies. Thanks to all the secretaries in Molecular Biology and to Jonny and Mareck. Thanks to Marinella for the best cleaner ever. To the Catholic community in Umeå for all the times we worship together. And to all Cameroonians in Umeå for the good times we spent together.

Thanks to my Dearest Mats for the great care, support and encouragement during this harsh period I encountered towards the last year(s) of my Ph.D. studies. A big thanks to your mom Birgitta, for her very knowledgeable and encouraging discussion.

Lastly my very special thanks goes to my kids Bryde, Ayamba, Etengeneng and Nkongho for their patience, you people are my encouragements/inspiration and the reason why I continuously work hard. Bryde, I wish you one day to become the best Doctor in the world. Ayamba, I hope that you will not ask me anymore if I am still a student. Etengeneng I hope that you will follow my example and go for science. Nkongho I hope that one day you will write a better thesis than your mommy. Thanks to my mother Ma- Nchong Margaret, you are the best mom and grand mom in the world. To my senior sister Achare, her husband Mr. Atong and the kids, for the great care and unending support you gave to my kids during my Ph.D studies. Sister Achare you are such a nice and kind hearted big sister, who else can be better than you? My brother Pascal aka PK, for your encouragements and uncountable support. A very big thanks PK for everything and thanks to your wife Mafua Miriam and kids. Thanks to my brother Ayuketang D-in fact I like your strictness, seriousness and readiness to always help. My junior sister Stella and family for always preparing my food stuffs, you are the best. Derrick, Manyi, Ma-Nchong and 57

Baby, I wish you people all the best in whatever you do, auntie Connie love you people so much. And thanks to all the other members of my family and in-laws for their continuous support to me and the kids.

This work was supported by the Swedish Research Council project grants 2010–3031 (VR- MH) and 2012–4638 (VR-NT). The Faculty of Medicine Umeå university. The Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University and the Swedish Research Council (353-2010-7074). This work was performed as part of the Umeå Centre for Microbial Research (UCMR) Linnaeus Program supported by Umeå University and the Swedish Research Council (349-2007-8673). The work was also supported by the Swedish Research Council project grants 2006–4702 (VR-NT), 2013–2392 (VR-MH), and the Swedish Foundation for International Cooperation in Research and Higher Education IG2008–2049 (STINT). The work was performed in the framework of Umeå Center for Microbial Research (UCMR) Linnaeus Program. The work was carried out in the framework of European Virtual institute for functional Genomics of Bacterial pathogens (CEE LSHB-2005-512061) and the ERA-NET project "Deciphering the intersection of commensal and extraintestinal pathogenic E. coli

“If you can’t fly then run, if you can’t run then walk, if you can’t walk then crawl, but whatever you do you have to keep moving forward.” - Martin Luther King Jr

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