UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series no. 1512, ISSN: 0346-6612, ISBN: 978-91-7459-451-5

Outer membrane vesicle-mediated export of virulence factors from Gram-negative bacteria

Pramod Kumar Rompikuntal

Department of Molecular Biology Umeå Centre for Microbial Research (UCMR)

Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University, SE-90187

Umeå, Sweden 2012

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) Copyright © Pramod Kumar Rompikuntal New series no. 1512 ISSN: 0346-6612 ISBN: 978-91-7459-451-5 Cover picture: Outer membrane vesicles from Vibrio cholerae (Photo: Mr. Akemi Takade) E-version available at http://umu.diva-portal.org/ Printed by: Print & media 901 87 Umeå, Sweden 2012

“The difference between what we do and what we are capable of doing would suffice to solve most of the world's problems” -Mahatma Gandhi

To my parents and family

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Table of Contents Abstract…………………………………………………………………………………………. vi

Papers included in this thesis…………………………………………………………………. vii

Introduction……………………………………………………………………………………... 1

1. Outer membrane vesicles (OMVs) from Gram-negative bacteria…………………………….. 1

1. 1. Vesicle biogenesis……………………………………………………………………….. 1

1. 2. Vesicle-mediated secretion of bacterial virulence factors……………………………….. 3

1. 3. In vivo production of OMVs during infection…………………………………………… 8

1. 4. Epithelial cells and OMV interaction……………………………………………………. 9

1. 5. Modulation of host immune response………………………………………………….. 11

1. 6. Use of OMVs as a potential vaccine…………………………………………………… 13

2. Membrane vesicle release (MV) from Gram-positive bacteria………………………………. 14

3. Membrane vesicles (MVs) from Archaea……………………………………………………. 15

4. Membranous extracellular organelles from eukaryotic cells…………………………………. 16

5. Vibrio cholerae……………………………………………………………………………….. 17

5. 1. Pathogenesis and virulence factors……………………………………………………... 18

5. 1. 1. Toxin co-regulated pili (TCP) and cholera toxin (CTX)…………………………….. 18

5. 1. 2. Vibrio cytolysin/hemolysin (VCC/HlyA)…………………………………………..... 19

5. 1. 3. Hemagglutinin/protease (HA/P)………………………………………….………….. 19

5. 1. 4. PrtV protease……………………………………………………….………………… 20

6. Protein secretion systems in Gram-negative bacteria……………………………………….. 21

7. Aggregatibacter actinomycetemcomitans…………………………………………………… 23

7. 1. Virulence factors and pathogenesis…………………………………………………….. 23

7. 1. 1. LPS…………………………………………………………………………………… 23

7. 1. 2. Fimbriae……………………………………………………………………………… 24

7. 1. 3. Leukotoxin (LtxA) ………………………………………………………………….. 24

7. 1. 4. Cytolethal distending toxin (CDT)…………………………………………………. 24

8. Campylobacter jejuni………………………………………………………………………… 25

8. 1. Virulence factors and pathogenesis………………………………………………………. 26

8. 1. 1. Flagella and chemotaxis……………………………………………………………... 26

8. 1. 2. CDT………………………………………………………………………………….. 26

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8. 1. 3. Lipooligosaccharide (LOS) …………………………………………………………. 27

Aims of the thesis………………………………………………………………………………. 28

Results and Discussion………………………………………………………………………… 29

Paper-I…………………………………………………………………………………………. 29

Paper-II………………………………………………………………………………………… 30

Paper-III……………………………………………………………………………………….. 32

Paper-IV………………………………………………………………………………………... 34

Conclusions…………………………………………………………………………………….. 36

Acknowledgements…………………………………………………………………………….. 37

Reference list…………………………………………………………………………………… 40

Articles I-IV……………………………………………………………………………………. 57

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Abstract The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue. The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.

The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.

In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

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Papers included in this thesis This thesis is based on the following three publications and one manuscript which will be referred to by roman numerical (I-IV). All the published papers in this thesis have been reprinted with permission from the original journal publishers. Paper I Lindmark B, Rompikuntal PK, Vaitkevicius K, Song T, Mizunoe Y, Uhlin BE, Guerry P, Wai SN. 2009. Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni. BMC Microbiol. 16;9:220. Paper II Bielig H, Rompikuntal PK, Dongre M, Zurek B, Lindmark B, Ramstedt M, Wai SN, Kufer TA. 2011. NOD-like receptor activation by outer membrane vesicles from Vibrio cholerae non-O1 non-O139 strains is modulated by the quorum-sensing regulator HapR. Infect Immun. 79(4):1418-27. Paper III Rompikuntal PK, Thay B, Khan MK, Alanko J, Penttinen AM, Asikainen S, Wai SN, Oscarsson J. 2012. Perinuclear localization of internalized Outer Membrane Vesicles carrying active Cytolethal Distending Toxin (CDT) from Aggregatibacter actinomycetemcomitans. Infect Immun. 80(1):31-42. Paper IV

Rompikuntal PK, Åhlund MK, Lindmark B, Johnson TL, Sandkvist M, Lundmark R, Uhlin BE, Wai SN. 2012. Outer membrane vesicle mediated export of PrtV protease from Vibrio cholerae (manuscript).

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Papers not included in this thesis The author has also contributed to the following articles that are not included in this thesis:

Vaitkevicius K, Rompikuntal PK, Lindmark B, Vaitkevicius R, Song T, Wai SN. 2008. The metalloprotease PrtV from Vibrio cholerae. FEBS J. 275(12):3167-77. Valeru SP, Rompikuntal PK, Ishikawa T, Vaitkevicius K, Sjöling A, Dolganov N, Zhu J, Schoolnik G, Wai SN. 2009. Role of melanin pigment in expression of Vibrio cholerae virulence factors. Infect Immun. 77(3):935-42. Ishikawa T, Rompikuntal PK, Lindmark B, Milton DL, Wai SN. 2009. Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains. PLoS One. 24;4(8):e6734. Ou G, Rompikuntal PK, Bitar A, Lindmark B, Vaitkevicius K, Wai SN, Hammarström ML. 2009. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease. PLoS One. 12;4(11):e7806. Syngkon A, Elluri S, Koley H, Rompikuntal PK, Saha DR, Chakrabarti MK, Bhadra RK, Wai SN, Pal A. 2010. Studies on a novel serine protease of a ΔhapAΔprtV Vibrio cholerae O1 strain and its role in hemorrhagic response in the rabbit ileal loop model. PLoS One. 30;5(9). pii: e13122.

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Introduction

1. Outer membrane vesicles (OMVs) from Gram-negative bacteria

Gram-negative bacteria naturally and constitutively release lipid bilayer vesicles from the outer

membrane during growth (Fig. 1). Outer membrane vesicles (OMVs) range in size from 20-200

nm in diameter. As the vesicles are extruded from the surface, they may entrap some of the

underlying periplasm so that they are actually small particles of Gram-negative cell wall

(Beveridge, 1999; Wai et al., 1995). OMVs possess outer membrane proteins, lipopolyaccharide

(LPS), phospholipids, and periplasmic constituents. They have diverse roles in bacterial

pathogenesis including involvement in bacterial communication through OMV-associated signal

molecules, as virulence, and in genetic transformation (Gankema et al., 1980; Hoekstra et al.,

1976; Horstman and Kuehn, 2000; Kadurugamuwa and Beveridge, 1995; Kolling and Matthews,

1999; Kulp and Kuehn; Mashburn-Warren and Whiteley, 2006; Wai et al., 2003; Wai et al.,

1995; Yaron et al., 2000).

Fig. 1. Electron micrograph showing OMV-release from Vibrio cholerae O1 El Tor Inaba strain

A1552. Scale bars represent 100 nm. The picture was taken by Mr. Akemi Takade, Kyushu

University, Fukuoka, Japan.

1. 1. Vesicle Biogenesis

The molecular mechanism behind OMV formation is not fully understood. OMVs are produced

in almost all Gram-negative bacteria and this phenomenon is not the result of cell lysis or death

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(Mug-Opstelten and Witholt, 1978). Vesicle formation was shown to be abundant at the site of

cell division in species such as Vibrio, Escherichia coli and Brucella melitensis (Chatterjee and

Das, 1967; Gamazo and Moriyon, 1987; Hoekstra et al., 1976). Hoekstra et al. reported that the

peptigoglycan binding lipoproteins play a considerable role in E. coli OMV production

(Hoekstra et al., 1976) (Fig. 2, Model. 1). They showed that OMV formation starts with an

outward bulging event towards the outer membrane, occuring mostly where there is less

peptigoglycan binding lipoprtotein, which can cause a weak linkage between the peptigoglycan

and the outer membrane of the bacterium. Another study demonstrated that the protrusions from

the outer membrane occur when periplasmic turgor pressure builds up due to accumulation of

peptigoglycan fragments in the periplasmic space, which eventually leads to the bulging of the

outer membrane (Zhou et al., 1998) (Fig. 2, Model. 2). In P. aeruginosa, it was reported that

ionic interactions between Pseudomonas Quorum Sensing (PQS) molecules and Mg2+ ion of LPS

destabilize the salt bridges in the outer membrane. This enhances anionic repulsion between LPS

molecules and consequently generates vesicles from the outer membrane (Mashburn-Warren and

Whiteley, 2006) (Fig. 2, Model. 3). Additional investigation is required to answer the intriguing

questions of whether a genetically regulated mechanism(s) exists for OMV formation.

In a recent study in our laboratory, we discovered a novel small RNA from V. cholerae (VrrA)

(Song et al., 2008). In characterizating the sRNA (VrrA) mutant V. cholerae, we found that the

ompA mutant released more OMVs. Therefore, we suggested that VrrA regulates the production

of OMVs from V. cholerae via regulating OmpA protein expression. OmpA is a β-barrel

membrane protein that is highly conserved among Gram-negative bacteria (Delcour, 2002). The

biological properties and functions of OmpA have been extensively studied in E. coli (Sugawara

and Nikaido, 1992). The mechanism underlying the greater OMV release in the V. cholerae

ompA mutant can possibly be explained by a role for V. cholerae OmpA analogous to that of E.

coli OmpA in stabilizing the cell envelope structure (Sonntag et al., 1978).

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Fig. 2. Proposed model for OMV formation (Mashburn-Warren and Whiteley, 2006)

1. 2. Vesicle-mediated secretion of bacterial virulence factors

Vesicle-mediated secretion of several bacterial proteins is considered a novel secretion system.

Biologically active toxins were found in association with OMVs from various Gram-negative

bacteria (Table 1). A major breakthrough in my research regarding the potential physiological

relevance of OMVs came from the earlier studies in our laboratory on a cytotoxic protein (ClyA)

in E. coli that was exported through outer membrane vesicles (Wai et al., 2003). The ClyA

protein seems to possess an intrinsic ability to translocate to the bacterial periplasm where it is

incorporated into OMVs during a process that includes redox-dependent oligomerization. The

activity of the ClyA protein on human target cells was much more potent when it was

incorporated into OMVs compared to isolated ClyA protein purified from the bacterial

periplasm. The explanation for this was that there are two cystein residues in ClyA that form an

intramolecular disulfide bond catalyzed by the Dsb oxidoreductase systems and this bond

seemingly prevents oligomerization when the protein is in the periplasmic compartment. Upon

localization to the OMV, the protein somehow adopts a reduced status and is able to form

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oligomers. These results provided evidence for a physiological role of the vesicle-mediated

transport mechanism. Earlier work by Wai et al. (1995) also showed that the cholera toxin-like

toxin LT (heat labile toxin) from enterotoxigenic Escherichia coli (ETEC) is excreted via OMVs

and later Kuehn and coworkers (Horstman and Kuehn, 2000, 2002; Kesty and Kuehn, 2004;

Kesty et al., 2004) made similar observations. A study by Balsalobre et al. (2005) found that the

α-hemolysin of E. coli, which is commonly expressed by extraintestinal isolates, to a large extent

is localized in OMVs. It was suggested that the α-hemolysin reaches the OMVs after it is

secreted through the TolC-dependent type I secretion route. The results of their studies, using

both natural and clinical isolates, confirmed that dissemination of α-haemolysin by OMVs is a

common feature among haemolytic E. coli strains (Balsalobre et al., 2006). These findings also

prompted investigations regarding the potential of other type-I-secreted proteins to localize to

OMVs and how components of the type I secretion machinery may influence the formation

and/or properties of OMVs.

Another cytotoxic protein commonly found in extraintestinal E. coli isolates is cytotoxic

necrotizing factor 1 (CNF1), a toxin that modifies small GTPases of the Rho family and is

internalized into the target cells. Earlier studies in our laboratory showed that the CNF1 protein

was released from the bacterial cells in association with OMVs (Kouokam et al., 2006). OMVs

play an important role in the release of Shiga toxin from periplasmic space of Shigella

dysenteriae and Enterohemorrhagic E. coli O157:H7 (Dutta et al., 2004; Kolling and Matthews,

1999; Yokoyama et al., 2000). It has been suggested that Shiga toxin residing within the vesicle

lumen is protected from hydrolysis of exogenous proteases. OMVs from Shigella flexneri have

been known to entrap the antibiotic gentamicin and deliver it to the target host cytoplasm

(Kadurugamuwa and Beveridge, 1998).

CagA, an effector oncoprotein, was found to associate with Helicobacter pylori OMVs

(Olofsson et al.). Upon internalization into host cells, CagA induced cell signaling and was

involved in cell proliferation, motility, differentiation and polarity (Hatakeyama, 2008).

Vacuolating cytotoxin, VacA from H. pylori is known as a potential virulence factor and is

expressed by 50-60% of clinical H. pylori isolates. However, vesicle-associated VacA exhibited

lower vacuolating activity than whole bacteria (Ricci et al., 2005). OMVs-associated VacA was

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Table 1. Vesicle-associated virulence factors from various Gram-negative bacteria Species Virulence-associated factors Functional role Reference Aggregatibacter actinomycetemcomitans Leukotoxin Cytotoxicity (Kato et al., 2002) Cytolethal discending toxin (CDT) Cell cycle arrest (Rompikuntal et al.) Bacteroides fragilis Haemagglutinin Hemagglutination (Patrick et al., 1996) Bordetella pertussis Adenylate cyclase-hemolysin Immune response (Hozbor et al., 1999) Borrelia burgdorferi Outer surface protein (Osp) A, B and D Adherence to the host cells (Shoberg and Thomas, 1993) Burkholderia cepacia Lipase, phospholipase-N, metalloprotease Enzymatic activities (Allan et al., 2003) Campylobacter jejuni CDT Cell cycle arrest (Lindmark et al., 2009) Escherichia coli Cytolysin A (ClyA) Cytotoxicity (Wai et al., 2003) Enterotoxigenic E.coli Heat labile enterotoxin (LT) Enterotoxicity (Horstman and Kuehn, 2000) Extraintestinal E. coli (ExPEC) α-hemolysin Cytotoxicity (Balsalobre et al., 2006) CDT Cell cycle arrest (Berlanda Scorza et al., 2008) Helicobacter pylori Vacuolating cytotoxin (VacA) Vacuolating activity (Keenan et al., 2000) Lewis antigen LPS Chronic immune stimulation (Hynes et al., 2005) Cytotoxicity-associated Immunodominant antigen (CagA) Cytotoxicity (Olofsson et al.) Blood group antigen-binding adhesin A (BabA) and sialic acid-binding adhesin (SabA), Adherence to epithelial cells (Olofsson et al.) Legionella pneumophila Metalloprotease ProA1 Hemolytic and Cytotxic activies (Galka et al., 2008) Macrophage infectivity Inhibit phagosome- lysosome fusion (Fernandez-Moreira et al., 2006) Hsp60, diphosphohydrolase Adherence to epithelial cells (Galka et al., 2008) Moraxella catarrhalis Ubiquitous surface protein A1 and 2 Interaction with C3 complements (UspA1/2) (Tan et al., 2007) Moraxella IgD-binding protein (MID) T cell indipendent B cell response (Vidakovics et al.) Myxococcus xantus TonB transporters Intercellular signaling (Kahnt et al.) Neisseria gonorrhoeae Peptidoglycan NOD1-dependent immune responses (Kaparakis et al.) Neisseria meningitides PorA, PorB Serum resistance (Vipond et al., 2006) Porphyromonas gingivalis Gingipain cysteine proteinases Cleavage of CD14 (Duncan et al., 2004) Heme-utilization protein (HmuY) Biofilm formation (Olczak et al.) Pseudomonas aeruginosa β-lactamase Resistant against β-lactam antibiotics (Ciofu et al., 2000) Hemolytic phospholipase C Cytotoxicity (Montes et al., 2007; Vasil et al., 1982) Alkaline phosphatase Biofilm formation (Huang et al., 1998; Xu et al., 1998) Cif CFTR-mediated Cl(-) secretion (Bomberger et al., 2009) PQS2-Heptyl-3-hydroxy-4-quinolone Quorum-sensing (Mashburn-Warren and Whiteley, 2006) Salmonella enterica PhoP-activated gene C (PagC) Accelerates vesiculation (Kitagawa et al.) Salmonella typhi ClyA Cytotoxicity (Wai et al., 2003) Shigella flexneri Invasion plasmid antigens Invasion IpaB, IpaC, IpaD (Kadurugamuwa and Beveridge, 1998) Shigella dysenteriae Shiga toxin 1 Cytotoxicity (Dutta et al., 2004) serotype 1 Shiga toxin-producing E. coli (STEC O157:H7) Shiga toxin Cytotoxicity (Yokoyama et al., 2000) Treponema denticola Proteases, dentilisin, adhesions Alteration of cellular tight junctions (Chi et al., 2003) Uropathogenic E. coli Cytotoxic necrotizing factor 1 (CNF1) Cytotoxicity (Kouokam et al., 2006) (UPEC) Vibrio anguillarum Metalloprotease, hemolysin, phospholipase Stimulation of cytokines (Hong et al., 2009) Vibrio cholerae LPS Stimulate antibody response (Bishop et al.) Peptidoglycan (Putative) NOD like receptor activation (Bielig et al.) RTX toxin Actin cross-linking (Boardman et al., 2007) PrtV protease Induce morphological effects Rompikuntal et al., 2012 (manuscript) Vibrio vulnificus Cytolysin–hemolysin (VvhA) Cytotoxicity (Kim et al.) Xenorhabdus nematophilus LPS, P pilin like protein, Larvicidal & chitinase activity Porins, Chitinase, Phage tail like protein (Khandelwal and Banerjee-Bhatnagar, 2003)

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biologically active and showed a significant role in pathogenesis (Keenan et al., 2000). In

addition, H. pylori OMVs containing either full-length VacA or a C-terminal truncated VacA

could induce cytochrome c-independent apoptosis in gastric adenocarcinoma (AGS) cells.

However, the truncated VacA is not secreted, therefore it was proposed that OMVs could

transport this truncated form of the toxin into target host cells (Ayala et al., 2006).

Several virulence factors have been proposed to association with P. aeruginosa vesicles,

including alkaline phosphatase, which promotes biofilm formation (Huang et al., 1998; Xu et al.,

1998); hemolytic phospholipase C, which induces a cytotoxc effect and enhances virulence of P.

aeruginosa (Montes et al., 2007; Vasil et al., 1982); and β-lactamase, which degrades β-lactam

molecules (Ciofu et al., 2000). It has also been shown that OMVs from P. aeruginosa are

important vehicles to transporting the antibiotic resistance protein β-lactamase from P.

aeruginosa to other bacteria. Therefore, antibiotic resistance can be acquired among bacterial

population via OMVs (Kadurugamuwa and Beveridge, 1995, 1996; Li et al., 1996). The Cif

protein, an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) protein

expression, is associated with P. aeruginosa OMVs (Bomberger et al., 2009; MacEachran et al.,

2007). P. aeruginosa vesicles loaded with the signaling molecule 2-heptyl-3-hydroxy-4-

quinolone (pseudomonas quinolone signal; (PQS)) have also been reported to play an important

role in bacterial cell-cell communication (Mashburn and Whiteley, 2005).

Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of

Campylobacter jejuni, but the mechanism by which CDT becomes surface exposed or is released

from the bacterium to the surrounding environment was not well understood. Our recent studies,

suggested that CDT is secreted to the bacterial culture supernatant via OMVs released from the

bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold

labeling and electron microscopy of OMVs. We hypothesized that a majority of the extracellular

biologically active CDT was tightly associated with the OMVs because OMVs from the wild

type C. jejuni could exert the cell distending effects typical of CDT on a human intestinal cell

line but there was no effect by the OMVs from the CDT mutant strain (Lindmark et al., 2009).

The oral pathogen Porphyromonas gingivalis is a causal agent of severe periodontal disease and

cardiovascular disease (Meurman et al., 2004). P. gingivalis vesicles contain several virulence

factors, such as, LPS, muramic acid, fimbriae, and protease like gingpains (Furuta et al., 2009;

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Grenier and Mayrand, 1987). It was reported that fimbriae-mediated bacterial adherence to the

periodontal tissue and gingipains provided proteolytic ability to destroy the tissue (Amano et al.,

2004; Kadowaki et al., 2007; Yilmaz et al., 2002). Therefore, vesicles with both adhesive and

proteolytic properties effectively contribute to periodontitis (Kadowaki et al., 2007).

Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis.

Leukotoxin, from this bacterium was found in association with OMVs. OMVs containing

leukotoxin showed four-to-five fold higher cytotoxic effects on human HL60 cells compared to

isolated outer membranes (Kato et al., 2002). Similarly to several other Gram-negative species,

A. actinomycetemcomitans produces and excretes a cytolethal distending toxin (CDT), a

genotoxin associated with cell distention, and induces G2 cell cycle arrest, and/or apoptosis in

many mammalian cell types (Guerra et al.; McSweeney and Dreyfus, 2004; Sugai et al., 1998).

In our recent studies, we have identified the OMVs from A. actinomycetemcomitans as a vehicle

for delivery of multiple proteins, including CDT, into human host cells. We showed that the

OMV-associated proteins were internalized into both the HeLa cells and the human gingival

fibroblasts (HGF) by fusion of OMVs with lipid rafts in the plasma membrane. The active toxin

unit, CdtB, was found to be localized inside the nucleus of the intoxicated cells and induced a

cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was

biologically active. Association of CDT with OMVs was also observed in A.

actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated

release of CDT may be conserved in A. actinomycetemcomitans. Our data suggested that OMVs

could deliver biologically active CDT and additional virulence factors into susceptible cells of

the periodontium (Rompikuntal et al.).

Vibrio cholerae produce large amounts of outer membrane vesicles (OMVs) during their growth.

We observed that an M6 family, zinc binding metalloprotease PrtV, was secreted from V.

cholerae wild type strain C6706 in association with OMVs. In addition, we observed that the

PKD-domain(s) of PrtV had a role in association of PrtV with OMVs. We also demonstrated that

OMV-associated PrtV was biologically active since the morphology of human colon carcinoma

(HCT8) cells was altered when the cells were treated with the OMVs isolated from the wild type

V. cholerae strain C6706, whereas cell morphology did not change after treatment with OMVs

from the prtV mutant. Moreover, OMV-associated PrtV might be released into the target

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eukaryotic cells by a vesicle fusion mechanism in association with lipid raft microdomains in the

plasma membrane (Rompikuntal et al. manuscript 2012).

In addition to their association with proteins, OMVs from E. coli, H. influenza, P. aeruginosa,

M. catarrhalis and N. gonorrhoeae reportedly contain DNase-resistance DNA, either surface

associated or inside the vesicle lumen (Deich and Hoyer, 1982; Dorward and Garon, 1989, 1990;

Kolling and Matthews, 1999; Renelli et al., 2004). In E.coli O157:H7, vesicle-associated DNA

could originate from various sources (Yaron et al., 2000), including both the chromosomal- and

plasmid-encoded DNA. The authors hypothesized that bacteria can utilize vesicle-mediated

DNA transfer as an alternative method of gene transfer. Trafficking of genetic material between

the donor and recipient cells reduce the necessity of pilus biosynthesis genes. Further studies

described that OMVs-associated DNA was not restricted to E.coli; it was shown in P.

aeruginosa that vesicles contained plasmid DNA, however, unlike E. coli, they were not capable

of mediating plasmid transformation into the recipient cells (Renelli et al., 2004). It would be

interesting to elucidate how cytoplasmic DNA enters these vesicles. Intriguingly, the study from

Renelli et al., (2004) demonstrated that DNA can enter into OMVs through the periplasmic route

or by re-association of extracellular DNA derived from the potentially lysed cells.

1. 3. In vivo production of OMVs during infection

Although several in vitro studies demonstrated how OMVs interact with different types of

epithelial cells immune cells, a few studies on in vivo production of OMVs have been performed.

These few studies have nevertheless shown a significant role for in vivo secreted OMVs in

bacterial pathogenesis. Examination of chronic H. pylori-infected biopsy samples revealed the

presence of vesicle binding to gastric epithelial cells (Fiocca et al., 1999; Heczko et al., 2000;

Keenan et al., 2000). Furthermore, serologically recognizable Lewis antigens were found on

OMV surface which might contributes to chronic immune stimulation of the host (Hynes et al.,

2005). In another study, OMVs from Neisseria meningitides were observed in cerebrospinal fluid

of an infant suffering from meningitis (Stephens et al., 1982). It was suggested that OMVs

significantly contributed to the severity of meningococcal endotoxemia. Electron microscopic

analysis from a nasal swap collected from a child suffering from sinusitis, showed the co

existence of OMVs producing bacteria with the host immune cells (Tan et al., 2007). In addition

to these findings, Tan et al., demonstrated, the presence of C3 complement binding factor(s)

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UspA1/UspA2 on OMVs from M. catarrhalis by using an immunogold labeling and EM analysis

of OMV samples (Tan et al., 2007).

1. 4. Epithelial cells and OMVs interaction

Entry of bacteria into host cell allows the pathogen to survive intracellularly, escaping various

conditions, such as immune surveillance and antibiotic pressure, and leading to intracellular

persistence, multiplication and dissemination to the adjacent cells. In vivo studies revealed that

invasive bacteria can enter non-phagocytic cells by utilizing various mechanisms, such as using

ligands that have the capability to adhere to host cell surface receptors. It was reported that

OMVs may share such adhesive and invasive ligands and that these properties ultimately may

help both invasive and non-invasive bacteria to modulate host responses (Furuta et al., 2009;

Kuehn and Kesty, 2005). OMVs can transport bacterial toxins, LPS, proteases and virulence

factors to the target host cells by various internalization mechanisms that are described below.

Enterotoxigenic E. coli (ETEC) is a prevalent cause of tranveler’s diarrhea and infant mortality

in developing counties. Heat-labile enterotoxin (LT) is one of the important virulence factor

expressed by ETEC. LPS-bound LT has been described as an adhesin responsible for the

interaction of OMVs from ETEC and the host cells (Horstman and Kuehn, 2000). LT is

translocated to the periplasm through the Sec machinery and is secreted through the outer

membrane via a terminal branch of the type II secretion system. The LT-B subunit forms a

pentamer and consists of both LPS and GM1 gnglioside binding sites (Horstman and Kuehn,

2002; Mudrak et al., 2009). Therefore, OMV-associated LT toxin can bind to the GM1 receptors

on the lipd raft of the plasma membrane and OMVs can become internalized through lipid raft-

mediated endocytosis. OMV endocytosis via cholesterol-rich lipid rafts was found to be caveolin

dependent. Once LT was internalized, it trafficked through golgi apparatus and the endoplasmic

reticulum by a retrograde pathway to the cytosol where LT-A subunit catalizes the ADP

ribosylation of G proteins which eventually led to the enterotoxicity. OMVs could efficiently

invade the host cells in a time-, temperature- and receptor-dependent manner (Kesty et al., 2004).

Porphyromonas gingivalis OMVs contains several virulence factors including, fimbriae, and

gingipain cysteine protenase. OMVs isolated from P. gingivalis could adhere to HeLa and

gingngival epithelial cells using one of its components and be efficiently internalized into the

epithelial cells via an actin-dependent, lipid rafts mediated pathway. OMV-associated gingipains

10

efficiently degrade cellular transferrin receptor, intgrin-related signaling molecules, paxillin and

focal adhesion kinase (FAK), and further inhibit cellular migration (Furuta et al., 2009). It was

suggested that OMV-associated virulence factors of P. gingivalis function as a powerful weapon

for destruction of periodontal tissue in patients with chronic periodotitis.

A recent study showed that using OMVs as a potential vehicle, P. aeruginosa delivered multiple

virulence factors directly into the host cell cytoplasm by fusion of OMV with lipid raft in the

host plasma membrane. Bomberger et al. described how the long-distance delivery of bacterial

virulence factors directly into the host cell excluded the necessity of host-pathogen direct

interaction (Bomberger et al., 2009). The internalized OMVs ultimately caused cellular

cytotoxicity and altered host cell biology to promote bacterial colonization. The actin

cytoskeleton, in particular N-WASP-initiated actin assembly, was critical for lipid raft-mediated

internalization of OMV cargo proteins to the host cytoplasm (Caron et al., 2006). Furthermore,

cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif), was used as

a marker protein to determine the intracellular localization. Cif co-imunoprecipitation with Rab5

GTPase, an early endosomal antigen (EEA)-1 suggested that OMVs had been encapsulated by

early endosomes and that OMV delivered Cif directly to the host cytoplasm (Bomberger et al.,

2009). However, in contrast to the entry of OMVs from P. aeruginoa by fusion with plasma

membrane, the OMVs of P. aeruginosa strains isolated from cystic fibrosis patients could be

internalized into human lung epithelial cells by an endocytic pathway. Endocytosis was mediated

by clathrin coated pits and surface bound vesicles were found co-localized with clathrin.

Internalized vesicle components were shown to be co-localized with endoplasmic reticulum (ER)

using the translocation associated protein α (TRAP α) as an ER marker. In the studies of

Bomberger et al., a zinc-dependent aminopeptidase, PaAP was also found to be association with

OMVs of P. aeruginosa. The OMV-associated PaAP promoted OMV association with lung

epithelial cells by proteolytically exposing OMV receptors on the epithelial cell, leading to

internalization of vesicles and contributing to the inflammatory response during infection

(Bauman and Kuehn, 2009). Salmonella enterica serovar Typhimurium is an intracellular

pathogen and one of the causal agents of food-borne illness. It was reported that a set of

virulence factors was translocated into the host cytoplasm via OMVs (Yoon et al.). OMVs

produced by intracellular Salmonella also contained lysosome-associated membrane protein 1

11

(LAMP1), suggesting the possibility of OMVs convergence with host cellular components

during intracellular trafficking (Yoon et al.).

1. 5. Modulation of host immune response

Many studies have shown that OMVs are deocys for the host immune system to trigger the

inflammatory responses. In addition to specific virulence factors, OMVs contain substances such

as the O-antigen of LPS that can be recognized by eukaryotic cells in the innate and acquired

immune response pathways. B-cells activatation upon co-incubation with OMVs from B.

burgdorferi has been described (Whitmire and Garon, 1993). N. meningitidis vesicles were

highly immunogenic when administered intranasally into mice, eliciting a mucosal and systemic

bactericidal antibody response (Saunders et al., 1999). In addition to a potent immune modulator

LPS, OMVs of N. meningitidis also consists several outer membrane porins and other immuno

modulatory factors.

OMVs from Salmonella typhimurium could stimulate professional antigen presenting cells

(APCs) in vitro. OMV-stimulated macrophages and dendritic cells displayed increased surface

expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators

nitric oxide (NO), TNF-alpha, and IL-12. In addition, OMV-vaccinated mice generated

Salmonella-specific antibodies and CD4 (+) T cell responses in vivo and were significantly

protected from infectious challenge with live Salmonella (Alaniz et al., 2007).

The respiratory pathogen Moraxella catarrhalis are endocytosed and killed by human tonsillar B

cells, although OMV can interact with and activate B cells, leading to rescue the bacterial cells.

Two components of the OMV, i.e., Moraxella IgD-binding protein (MID) and unmethylated

CpG-DNA motifs, were found to be important for B cell activation. The authors suggested that

Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune

response using its superantigen-bearing OMV as decoys (Vidakovics et al.). It was also

demonstrated that Moraxella OMVs containing virulence factors UspA1/A2 could interact with

the alternative pathway complement factor C3b and restrain the complement activation

(Nordstrom et al., 2005; Tan et al., 2007).

Recently, Kaparakis et al. reported that Gram-negative bacteria can deliver peptidoglycan to

cytosolic NOD1 in host cells via OMVs. The authors showed that OMVs from the Gram-

negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria

12

gonorrhoea contain peptidoglycan that induced innate and adaptive immune responses via a

NOD1-dependent but TLR-independent mechanism. They proposed that OMVs released by

bacteria in vivo may promote inflammation and pathology in infected hosts.

A trypsin like protease was secreted in association with P. gingivalis OMVs. The OMV-

associated protease could effectively degrade IgG, IgM and complement factor C3 in human

serum (Grenier, 1992). OMVs induce membrane biosynthesis of E-selectin and ICAM-1

adhesion molecules on human vascular endothelial cells, there by inhibiting interferon-γ-

mediated MHC class II synthesis (Srisatjaluk et al., 1999). P. gingivalis OMV-associated

gingipains degrade LPS receptor CD14 from the surface of U937 human macrophage-like cells,

leading to inactivation of the host immune system in periodontal disease (Duncan et al., 2004).

It was reported that OMVs isolated form a fish pathogen Vibrio anguillarum stimulated the

production of proinflammatory cytokines, such as TNF-α, IL-1β and IL-6 upon injection into a

flounder (Hong et al., 2009). In our laboratory, we analyzed the potential of outer membrane

vesicles (OMVs) derived from the non-O1 non-O139 V. cholerae (NOVC) strains to induce

inflammatory responses in human host cells. We observed that the OMVs were taken up by

human epithelial cells and induced an inflammatory response; the inflammatory potential of

NOVC OMVs was mediated by the nucleotide-binding domain-, leucine-rich repeat-containing

family member NOD1 and NOD2. Furthermore, we showed that a quorum-sensing regulator

HapR has a role in OMV-mediated activation of NOD1 and NOD2 (Bielig et al.).

The study by Kaparakis et al (2010) and our recent study on V. cholerae OMVs (Bielig et al.)

revealed an important role of OMVs as vehicles to deliver peptidoglycan (PGN) to host cells,

where it is subsequently sensed by members of the NOD-like receptor (NLR) family, most

importantly nucleotide-binding domain-containing protein 1 (NOD1). In addition, we suggested

that bacteria use the PGN content in OMVs, which is regulated by quorum sensing, to evade

NLR-mediated immune detection in the host. This showed that OMVs are critical to delivering

both membrane bound and soluble luminal (i.e. periplasmic) pathogen-associated molecular

patterns (PAMPs) to the host cell, resulting in activation of a variety of membrane associated and

intracellular pattern-recognition receptors (PRRs). Understanding how bacteria control the

PAMP composition of OMVs will give us the tools to manipulate OMV production and will

potentially encourage the use of OMVs as future vaccine candidates.

13

1. 6. Use of OMVs as a potential vaccine

Vaccines have highly reduced the morbidity and mortality of multiple infectious diseases and

effective cost-beneficial method to prevent the infectious disease spread

(http://www.cdc.gov/vaccines/events/niiw/index.htmlInvestigation). There are an increasing

number of licensed vaccines based on natural and engineered OMVs of Gram-negative bacteria

(Feiring et al., 2006; Jackson et al., 2009; O'Hallahan et al., 2004; Oster et al., 2005; Oster et al.,

2007). OMVs may be useful not only for understanding the pathogenesis of bacteria but also in

development of an effective vaccine against infections. OMVs harbour various immunogens and

appear to be safe when used as vaccines e. g. a protein-based OMV-vaccine against serogroup B

meningococcal disease for epidemic meningititis control in Cuba, Norway, Brazil, and New

Zealand (Danzig, 2004; Granoff; Rosenqvist et al., 1998). It has also been shown that OMVs can

be utilized as adjuvants (Danzig, 2004; Sanders and Feavers; Unal et al.). Previous studies

indicated that the main component of OMVs such as both outer membrane proteins and LPS

might play roles as PAMPs delivered to the host innate immune system, and could thus elicit

immune responses (Lee et al., 2007; Post et al., 2005). Since OMVs have antigenic properties,

such vesicles have been investigated as useful vaccine candidates against several Gram-negative

bacterial infections (Roberts et al., 2008; van de Waterbeemd et al.). For example, a Neisseria

meningitidis serogroup B vaccine was successfully developed using OMVs (Holst et al., 2009).

Recently, it was indicated that the OMVs from Edwardsiella tarda were able to induce synthesis

of several proinflammatory cytokines, and may stimulate the host innate immune system, thus

serving as PAMPs (Park et al.). The olive flounder injected with OMVs was protected to a

significantly higher extent than the control fish against bacterial challenge.

In an effort to devise a safer and effective pertussis acelullar vaccine, outer membrane vesicles

(OMVs) from Bordetella bronchiseptica were engineered. To decrease the endotoxic effect of

OMVs, the pagL gene which encodes a lipid A 3-deacylase, was expressed in B. pertussis strain

Tohama I. The resulting OMVs, designated OMVs (BpPagL), contained tetra- instead of penta-

acylated LOS, in addition to pertussis surface immunogens such as pertactin and pertussis toxin,

which are present in the wild type OMVs. The authors suggested that the OMVs (BpPagL)

obtained from B. pertussis may be an interesting candidates for development of a novel vaccine

against pertussis because the modified OMVs had lower endotoxic activity and retained

protective capacity in the mouse model in comparison with cellular vaccine (Asensio et al.).

14

Recently it was reported that vaccination with P. gingivalis OMVs elicited high levels of P.

gingivalis-specific IgA in nasal secretion and saliva, as well as serum IgG and IgA in a mouse

model. The authors suggested that the OMVs of P. gingivalis have an important role in mucosal

immunogenicity and proposed that P. gingivalis OMV is an intriguing immunogen for

development of a periodontal disease vaccine (Nakao et al.).

V. cholerae is the causative agent of cholera, a severe diarrheal disease that remains endemic in

many parts of the world and can cause outbreaks where sanitation and clean water systems are

not available. OMVs offer a new approach for an effective cholera vaccine. It was demonstrated

that immunization of female mice with OMVs induces a long-lasting immune response and

results in protection of their neonatal offspring from V. cholerae intestinal colonization. Transfer

of immunoblogulins to neonates via milk was sufficient for complete protection of the neonates

from colonization with V. cholerae. The authors reported that LPS was the major OMV

protective antigen (Bishop et al.; Bishop et al.; Roy et al.; Schild et al., 2009; Schild et al., 2008).

2. Membrane vesicle release (MV) from Gram-positive bacteria

The production of membranous spherical particles from microbial cell surfaces is conserved

among organisms from all three branches of the tree of life, spanning both prokaryotes and

eukaryotes (archaea, bacteria and eukaryotes). Although Gram-negative OMVs have been most

extensively studied, recently the release of Gram-positive membrane vesicles (MVs) was

demonstrated. MVs derived from Gram-positive bacteria, such as Bacillus spp., are

approximately 50-150 nm in diameter (Lee et al., 2009; Rivera et al.) and are rich in membrane

lipids as well as toxins including the anthrax toxin, the protective antigen (PA), lethal factor

(LF), edema factor (EF), and anthrolysin (ALO).

The recent study examined the production of MVs from Staphylococcus aureus and investigated

the delivery of MVs to host cells and subsequent cytotoxicity (Gurung et al.). The authors

showed that MVs were also produced during in vivo infection of a clinical S. aureus isolate in a

mouse pneumonia model. S. aureus MVs were shown to interact with the plasma membrane of

host cells via a cholesterol-rich membrane microdomain and then delivered their component to

host cells within 30 min. The authors suggested that S. aureus MVs play a role in delivery of

bacterial effector molecules to host cells. In addition, S. aureus-derived MVs contains penicillin-

15

binding proteins (PBP1/2/3) (Lee et al., 2009) which affects peptidoglycan-based cell wall

biogenesis and may contribute antibiotic resistance.

MVs release was also reported in Mycobacterium ulcerans (Marsollier et al., 2007). The authors

described that M. ulcerans derived-MVs significantly contribute to the formation of Buruli ulcer.

M. ulcerans MVs contains ulcerans polyketide toxin and mycolactone and are highly cytotoxic.

The MVs also confers bacterial resistance to antimicrobial agents and enhances the colonization

in the host. Therefore, knowledge of MVs may contribute to the development of a vaccine and

diagnostic tools for Mycobacterial infections.

3. Membrane vesicles (MVs) from Archaea

The production of vesicles from microbial cell surfaces is conserved among organisms and plays

an important role in cell physiology and pathogenesis of infection. In the archaea, little is known

about vesicle release. An electron microscopic investigation of Sulfolobus acidocaldarius and

Ignicoccus suggested that the release of vesicles might occur via an uncharacterized budding-off

process (Grimm et al., 1998; Nather and Rachel, 2004). Archaeal MVs range from form 90-230

nm in diameter and contain membrane lipids and S-layer proteins, which indicate that they are

indeed derived from the cell surface (Ellen et al., 2009). Interestingly, MVs isolated from the S.

islandicus were shown to contain a protein factor that inhibits the growth of other Sulfolobus spp.

(Prangishvili et al., 2000). Furthermore, proteomic analysis of the vesicles revealed the presence

of homologs of eukaryote endosomal protein sorting complex proteins (ESCRT-III) and vacuolar

sorting protein (Vps4) homologues, suggesting that MV release by archaea is controlled by a

regulated mechanism possibly similar to the endosomal vesicle sorting pathway in eukaryotes

(Makarova et al.). It was suggested that release of archaeal MVs may be related to the stress

response because the MVs contain the fork-head associated (FHA) protein along with the vWA-

containing proteins. In eukaryotes, vWA domains play an important role in the formation of

protein aggregates (Whittaker and Hynes, 2002) and protein-protein interactions (Smolka et al.,

2006) and are involved in various types of stress responses (Kolas et al., 2007). Future studies

are needed to address the mechanisms of MVs release from the cell envelope of arhaea and their

function(s) in the cellular physiology.

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4. Membranous extracellular organelles from eukaryotic cells

In addition to bacteria and archaea, eukaryotic cells also released membranous extracellular

organelles including exosomes, shedding microvesicles (SMVs) and apoptotic blebs (ABs), into

the microenvironment. Exosomes are 40- to 100-nm diameter membranous vesicles that are

released by different eukaryotic cells into the extracellular space (Simpson et al., 2008).

Exosomes originate from intracellular compartments such as the multivesicular bodies (MVBs).

Endosomal Sorting Complexes Required for Transport (ESCRTs) is involved in the

biogenesis/degradation of MVBs. The MVBs can either traffic to lysosomes where they are

subjected to proteosomal degradation or to the plasma membrane where, upon fusion with the

plasma membrane they release their contents into the extracellular space (Babst, 2005, 2006;

Hurley, 2008). Although the exosomal protein composition varies depending on the cell type of

origin, a conserved set of proteins were identified in exosomes, including clathrin and the Rabs

(the largest family of small GTPases). The Rabs regulate exosome docking and membrane fusion

by interacting with proteins involved in vesicular transport and fusion with membranes (Corbeel

and Freson, 2008). The increased levels of tumor-derived exosomes in plasma and malignant

effusions of patients with cancer suggest that exosomes can be a rich source for the discovery of

blood-based diagnostic biomarkers of disease (Andre et al., 2002).

Shedding microvesicles (SMVs) are large membranous vesicles (>100 nm diameter) shed from

the plasma membrane of a wide variety of cell types (Al-Nedawi et al., 2008; Heijnen et al.,

1999; Hess et al., 1999). The outward protrusion of the plasma membrane, the fission of the

membrane stalk, and the detachment of the protrusions result in the formation of SMVs (Cocucci

et al., 2009). Several enzymes such as calpain, flippase, floppase, scramblase and gelsolin are

involved in the formation of SMVs (Piccin et al., 2007).

Apoptotic cells also release 50–500 nm in diameter membrane vesicles into the extracellular

environment via protrusion of the plasma membrane, referred to as apoptotic blebs (ABs). The

extracellular microenvironment, such as ascites fluid and blood, contains a mixed population of

exosomes, SMVs and ABs (Piccin et al., 2007).

Recent studies reported the characterization of extracellular vesicles in pathogenic and non-

pathogenic species of fungi. C. neoformans, Histoplasma capsulatum, Candida albicans, and S.

cerevisiae were demonstrated to produce extracellular vesicles containing lipid, polysaccharide

and protein components (Albuquerque et al., 2008; Rodrigues et al., 2008; Rodrigues et al.,

17

2007). It was suggested that, as described for bacteria, extracellular vesicles in fungi may

represent an efficient mechanism of virulence factor delivery that may be crucial for the success

of the infection.

5. Vibrio cholerae

The Gram-negative, motile, comma-shaped facultative, anaerobic bacterium Vibrio cholerae is

the causative agent of cholera, a severe dehydrating diarrheal disease that occurs mostly in

developing nations and is transmitted via contaminated food or water (Faruque et al., 1998;

Kaper et al., 1995).

Two of the main virulence factors associated with the disease are cholera toxin (CT) and toxin

co-regulated pilus (TCP). Expression of CT and TCP is regulated via a complex cascade of

factors that respond to environmental signals (Weber and Klose). Out of more than 208

serogroups, based on the O-antigen in the LPS structure, only serogroups O1 and O139 have

caused pandemic cholera (Chatterjee and Chaudhuri, 2003; Faruque et al., 1998). The O1

serogroup is divided into two biotypes, classical and El Tor, depending on biochemical

properties, such as the Voges-Proskauer reaction, hemolysis, hemagglutination and sensitivity to

polymyxin B and phage infection (Sack et al., 2004). The first six cholera pandemics were

caused by V. cholerae O1 classical biotype. The seventh pandemic, which is still ongoing, started

in 1961 and is caused by the El Tor biotype of V. cholerae O1. In 1992, a new serogroup O139

(Bengal strain) of V. cholerae emerged as a causal agent of epidemic outbreak of cholera in India

and Bangladesh. The V. cholerae O139 strain arose from a strain closely related to the causative

agent of the present cholera pandemic, V. cholerae O1 El Tor, by acquisition of novel DNA

which was inserted into, and replaced part of, the O-antigen gene cluster of the recipient strain

(Bik et al., 1995). An insertion of ~35-kb DNA fragment encoding the O139-specific capsular

polysaccharide in place of 22-kb of the O1 El Tor rfb region resulted in the creation of new,

emerging O139 strain (Comstock et al., 1996). The serogroups other than O1 and O139 are

known collectively as the non-O1/non-O139 serogroups. Non-O1/non-O139 serogroups do not

produce cholera toxin (CT) and lack the filamentous bacteriophage CTXΦ and its receptor toxin

co-regulated pilus (TCP), as well as zonula occludens toxin (zot) and accessory cholera

enterotoxin (Ace) (Karaolis et al., 2001; Kurazono et al., 1995; Sharma et al., 1998). Although,

non-O1/non-O139 V. cholerae does not produce these major virulence factors, it was reported

18

that these strains could potentially cause septicemia, wound infection, bacteremia and liver

cirrhosis (Cover et al., 1989; El-Hiday et al., 2006; Gordon et al., 2001; Magnusson and Pegg,

1996).

5. 1. Pathogenesis and virulence factors

5. 1. 1. Toxin co-regulated pili (TCP) and Cholera toxin (CTX)

V. cholerae requires two coordinately regulated factors for full virulence: cholera toxin (CT); a

potent enterotoxin; and toxin co-regulated pili (TCP), surface organelles required for intestinal

colonization (Taylor et al., 1987; Waldor et al., 1997). The structural genes for CT are encoded

by a filamentous bacteriophage (designated CTXΦ). CTXΦ uses TCP as its receptor (Waldor

and Mekalanos, 1996). The TCP gene cluster is encoded by 41-kb region called TCP

Pahogenicity island or vibrio pathogenicity island (VPI) (Karaolis et al., 1999; Kovach et al.,

1996). Kovach et al., (1996), which suggested that V. cholerae epidemic strains obtained the tcp

gene cluster via inter-strain horizontal gene transfer. CT is the potent enterotoxin, encoded by the

ctxAB genes that reside in the genome of lysogenic CTXΦ. It is an A-B toxin consisting of one

enzymatic A-subunit and five receptor binding B-subunits (Finkelstein et al., 1974). The

holotoxin, is secreted from the bacteria via type II secretion sytem (Sandkvist et al., 1997). The

CT-B subunits bind to the ganglioside GM1 receptors localized mainly in lipid rafts on the

plasma membrane of the host cells (Chinnapen et al., 2007). The CT is endocytosed and

transported through the Golgi complex to the endoplasmic reticulum where the CT-A subunit

dissociates from the toxin complex. CT-A is transported to the cytoplasm of the target cell where

it ADP-ribosylates a GTP-binding protein that regulates adenyl cyclase activity, causing a

constitutively activated adenyl cyclase and a marked increase in the intracellular cAMP level.

The increased level of cAMP results in an imbalance in electrolyte movement in the epithelial

cell. The net increase in NaCl secretion leads to a large osmotic movement of water into the

intestinal lumen, resulting in the watery diarrheal disease cholera.

5. 1. 2. Vibrio cytolysin/haemolysin (VCC/HlyA)

Vibrio cytolysin (VCC), also known as hemolysin A (HlyA), is encoded by the hlyA gene, which

is a highly conserved gene in the V. cholerae species. It was suggested that VCC has a role in V.

cholerae pathogenesis, especially in stains that do not produce CTX (Pichel et al., 2003). VCC is

19

synthesized and secreted as a pro-VCC, a protein of about 80 kDa, which is proteolytically

activated by proteases (e.g., HA/P, trypsin, subtilisin, papain) to a 65-kDa mature VCC after

cleavage of the 15-kDa N-terminal region (Nagamune et al., 1996; Valeva et al., 2004). In the

presence of cholesterol- and ceramide-rich membranes, the mature VCC could form heptameric

transmembrane pores with an internal diameter of 1-2 nm (Harris et al., 2002; Olson and

Gouaux, 2005; Yuldasheva et al., 2001; Zitzer et al., 1997). It was also shown that VCC toxin

causes cytotoxic and vacuolizating activity in mammalian cells (Alm et al., 1991; Figueroa-

Arredondo et al., 2001). Recently, it was demonstrated that autophagy could act as a protective

cellular defence mechanism against VCC intoxication in human intestine Caco-2 cells (Gutierrez

et al., 2007). In our recent studies, we observed that VCC is capable of causing an inflammatory

response characterized by increased permeability and production of IL-8 and TNF-α in intestinal

epithelial cells (Ou et al., 2009).

5. 1. 3. Haemagglutinin/protease (HA/P)

HA/P was the first identified protease from V. cholerae and it belongs to the zinc metalloprotease

family M4 (Rawlings et al., 2006). The peptidase of this family contains a conserved HEXXH

zinc-binding motif in the catalytic site (Hooper, 1994). HA/P is secreted as 47-kDa protein and is

further processed to the 32-kDa active form (Hase and Finkelstein, 1991). The deduced amino

acid sequence of processed HA/P revealed 61.5% identity with the P. aeruginosa elastase. It was

suggested that HA/P might contributes to the detachment of V. cholerae from the gut epithelium

as it hydrolyzes fibronectin and ovomucin and cleaves lactoferrin (Finkelstein et al., 1983).

Moreover, it was described that HA/P could process and activate A-subunit of cholera toxin

(Booth et al., 1984) and 15-kDa N-terminal region of pro-VCC (Nagamune et al., 1996).

Recently, Jude et al., characterized a secreted attachment factor GlcNAc binding protein A

(GbpA), which functions in attaching to environmental chitin source as well as the intestinal

substrates. They showed that GbpA is degraded by HapA and PrtV. Consistent with this, ∆hapA

∆prtV strains attach to chitin beads more efficiently than either the WT or a ∆hapA ∆prtV ∆gbpA

strain. These results suggested a model in which GbpA levels fluctuate in concert with the

bacterial production of proteases in response to quorum-sensing signals (Jude et al., 2009).

20

5. 1. 4. PrtV protease

The PrtV protease of V. cholerae was identified in 1997 by Oigerman et al. as a 102-kDa

metalloprotease (Ogierman et al., 1997). The gene encoding PrtV is located on chromosome II

within a putative pathogenicity island. The members of the M6 family all display a

HEXXHXXGXXD motif. Full-length PrtV protein contains 918 amino acids and consists of: one

M6 peptidase domain, one zinc-binding domain and two PKD domains (Fig. 3). The two

histidines and the aspartate are zinc ligands and the glutamate is the catalytic residue. Further,

purified PrtV protease comes in two forms, as an 81- or 73-kDa protein, and is stabilized by

calcium ions. An assay using the pure PrtV protein showed a dose-dependent cytotoxic effect in

the human intestinal cell line HCT8 (Vaitkevicius et al., 2008).

The best characterized member of this protease family is immune inhibitor A (InA or InhA) from

Bacillus thuringiensis, a bacterium commonly used in agriculture for the control of insect pests.

InhA was first identified in B. thuringiensis as a secreted inhibitor of the humoral defense. In our

laboratory, using non-mammalian host models, we found that PrtV was required for the killing of

Caenorhabditis elegans, and was necessary for survival from grazing by the flagellate Cafeteria

roenbergensis and the ciliate Tetrahymena pyriformis. Moreover, supernatant from a V. cholerae

prtV deletion mutant caused increased interleukin-8 (IL-8) secretion in human intestinal

epithelial T84 cells and increased lysis of red blood cells compared with supernatant from the

parental strain (Ou et al., 2009; Vaitkevicius et al., 2006). The inflammatory response in T84

cells mediated by VCC was found to be modulated by the PrtV protease. The inflammatory

potential of VCC was almost completely abolished by PrtV-mediated degradation of VCC.

Therefore, PrtV might play a dual role: (i) protection from predator grazing in environmental

niche and (ii) maintaining low reactogenicity by modulating the activity of VCC.

The hemolytic and immunomodulating properties of the PrtV mutant were dependent on the

growth media (Ou et al., 2009). When grown in LB, the supernatant from the PrtV mutant was

more hemolytic and caused a stronger inflammatory response compared to wild type. On the

other hand, no differences between the PrtV mutant and wild type in hemolytic activity or

inflammatory response could be observed using supernatants from BHI broth. We suggested that

the content(s) of the BHI media might inhibit the activity or the expression of proteases from V.

cholerae. In addition, human serum proteins and extracellular matrix components fibrinogen,

fibronectin and plasminogen were potentially degraded by the PrtV protease (Vaitkevicius et al.,

21

2008). This suggests that in vivo, PrtV might contribute to the degradation of host tissue integrity

and dissemination of the infection.

In addition to the M6 family peptidase domain, the PrtV protein contains two C-terminal

Polycystic Kidney Disease (PKD) domains (Fig. 3) (Vaitkevicius et al., 2008). The PKD domain

is an 80–90 amino acid module originally found in the human Polycystic Kidney Disease

protein, polycystin-1 (PDK1), the cell surface glycoprotein involved in adhesive protein-protein

and protein-carbohydrate interactions (Bycroft et al., 1999). It is unclear, however, if the PKD

domain directly mediates any of these interactions. The PKD domain has also been found in

bacterial collagenases (Matsushita et al., 1999), proteases (Miyamoto et al., 2002; Oda et al.,

1996), cellulases (Ahsan et al., 1996) and chitinases (Perrakis et al., 1994). In our current studies,

we observed that the PKD-domain(s) of PrtV was essential for the association of PrtV with

OMVs (Rompikuntal et al. manuscript 2012). The functional significance of the two PKD

domains in V. cholerae PrtV is not yet completely understood. Since V. cholerae resides in the

marine niche, elucidating the function and the mechanism of action of the PKD domains in PrtV

will further increase our general understanding about the bacterial degradation mechanisms of

marine biopolymers.

Fig. 3. Schematic representation of V. cholerae PrtV protein

The M6 peptidase domain, its predicted catalytic Zn2+-binding site and PKD domains are

indicated with respect to the 918 amino acid, full-length PrtV protein.

6. Protein secretion systems in Gram-negative bacteria

Both Gram-negative and Gram-positive bacteria secrete a variety of proteins into the

extracellular space, which requires secretion systems specifically equipped for the transport of

macromolecules across two bacterial cell membranes as well as the host plasma membrane,

directly interfering with and altering host processes. These molecules, which have been referred

to as effectors or toxins, have a wide range of biological functions, including cytotoxicity,

hemolysis, proteolysis, protein phosphorylation, dephosphorylation and inter-bacterial virulence.

22

Bacteria coordinate the expression of secretion system genes to maintain/control physiological

responses to environmental cues. Several distinct types of secretion pathways for proteins that

are important for the bacterial interaction with host environments have been identified in Gram-

negative bacteria. The type I secretion system (T1SS) uses a one-step mechanism by which the

secreted proteins cross directly from the cytoplasm to the extracellular space. For example, the

cytolytic HlyA and V. cholerae repeat-in-toxin (RTX) toxins utilize the T1SS (Boardman and

Satchell, 2004; Koronakis et al., 1991). In the type II secretion system (T2SS), also known as the

general secretion pathway, proteins are first transported through the inner membrane to the

periplasm and then are transported across the outer membrane by either of several distinct

“terminal branches” of the process (Pugsley, 1993). For example, ETEC E. coli heat-labile

enterotoxin, V. cholerae hemagglutinin protease, cholera toxin and chitinase are transported by

the T2SS (Connell et al., 1998; Davis et al., 2000; Sandkvist, 2001; Sandkvist et al., 1997; Scott

et al., 2001). The type III system (T3SS) functions both as a pathway for secretion across the

bacterial membranes (both inner and outer membranes) and for translocation of the secreted

bacterial proteins across the plasma membrane of eukaryotic host/target cells (Galan and

Collmer, 1999; Hueck, 1998). Although the T3SS is not present in V. cholerae O1 strains, the

Vibrio outer protein F (VopF) of non-O1 non-O139 V. cholerae strains is secreted through the

T3SS (Tam et al., 2007). The type IV system also usually translocates proteins in a single step

from the cytoplasm to the intracellular compartment of a target cell (Christie and Vogel, 2000).

The type V pathway is utilized by so-called autotransporter proteins (Henderson et al., 2000).

Such proteins possess an N-terminal signal sequence and utilize the Sec system for translocation

across the inner membrane, whereas they mediate their own translocation across the outer

membrane by forming a pore structure. Recently, the type VI secretion system (T6SS) has

emerged as a protein secretion system important to several Gram-negative bacterial species. The

gene cluster-encoded T6SS mediates the extracellular secretion of four distinct proteins. The

functional significance of these genes were characterized in V. cholerae, where the ability to

secrete hemolysin co-regulated protein (Hcp) and valine-glycine repeat (VgrG) proteins into

supernatant fluids was shown to be important as a defense against predation by D. discoideum

and interbacterial virulence (Ishikawa et al., 2009; Ishikawa et al.; Mougous et al., 2006;

Pukatzki et al., 2006).

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7. Aggregatibacter actinomycetemcomitans

A. actinomycetemcomitans, formerly Actinobacillus actinomycetemcomitans, is a small Gram-

negative, non motile, facultative anaerobic, coccobacillus. The oral cavity is its natural habitat,

and A. actinomycetemcomitans was implicated in aggressive form of periodontitis, which is

characterized by chronic inflammation and severe loss of alveolar bone and connective tissue

that support the teeth (Henderson et al., 2003; Meng et al., 2007; Slots and Genco, 1984;

Socransky and Haffajee, 1992). However, the organism can spread form the oral cavity into the

blood circulation, thereby disseminating to other regions of the body and causing several non-

oral infections, including endocarditis, bacteremia, pericarditis, septicemia, pneumonia,

infectious arthritis, osteomyelitis, synovitis, skin infections, urinary tract infections, and

abscesses (van Winkelhoff and Slots, 1999).

7. 1. Virulence factors and pathogenesis

Periodontitis is a common chronic infectious inflammatory disease in man, characterized by the

inflammatory destruction periodontal tissues. A. actinomycetemcomitans is present frequently

and in high numbers in biofilms at sites with localized, aggressive periodontitis. A.

actinomycetemcomitans produces several putative virulence factors. The most studied A.

actinomycetemcomitans virulence factors were lipopolyaccharide (LPS), fimbriae, leukotoxin

(LtxA) and cytolethal distending toxin (CDT).

7. 1. 1. LPS

LPS, as a major component of the outer membrane of Gram-negative bacteria, is considered to

be a major factor in the pathogenesis of many Gram-negative bacterial species, including A.

actinomycetemcomitans. LPS acts as a microbe-associated molecular pattern recognized through

pattern-recognition receptors on resident immune and non-immune cells within the periodontium

(Akira and Hemmi, 2003). The LPS molecule from A. actinomycetemcomitans is very active in

biological systems and capable of eliciting the inflammatory and destructive manifestations of

this disease. In earlier studies it was reported that A. actinomycetemcomitans LPS induces the

production of IL-1β, IL-6, IL-8 and TNF-α in various host cells (Agarwal et al., 1995;

Lindemann et al., 1988; Patil et al., 2006). An in vitro experiment by (Ito et al., 1996) suggested

that the activity of LPS from A. actinomycetemcomitans to augment osteoclast-like cell

24

formation in bone marrow cell cultures was derived from a common structure of the lipid A

portion. This is consistent with the finding of A. actinomycetemcomitans LPS inducing bone

resorption as well as local IL-1α and IL-1β synthesis observed in the periodontal tissue of mice

(Nishida et al., 2001).

7. 1. 2. Fimbriae

The adhesion of A. actinomycetemcomitans to oral surfaces is essential for its colonization and

persistence within the oral cavity. Adhesion and subsequent biofilm formation are strategies that

A. actinomycetemcomitans has evolved to overcome the constant pressure of the mechanical

clearance mechanisms in the oral cavity. Initial adhesion of A. actinomycetemcomitans to an

abiotic surface is mediated by type-IVb-like, bundle-forming fimbriae encoded by flp-1, also

known as the tight adherence [tad] locus (Inoue et al., 2003; Schreiner et al., 2003; Wang et al.,

2005). Recently, (Wang et al., 2005) described that mutations at the promoter region of the flp

operon might explain the rough-to-smooth colony conversion of A. actinomycetemcomitans.

7. 1. 3. Leukotoxin (LtxA)

Leukotoxin (LtxA), a member of RTX family of pore-forming toxins, can selectively kill human

leukocytes by triggering apoptosis or lysis (Kachlany; Kelk et al., 2003; Korostoff et al., 2000;

Lally et al., 1999). The lysis of human monocytes/macrophages by leukotoxin is mediated by

activation of the cysteine proteinase Caspase-1, which is also associated with the secretion of

bio-active IL-1β (Kelk et al., 2003). In addition, the strong association between expression of the

highly leukotoxic LtxA of A. actinomycetemcomitans and disease progression indicates an

important pathogenic role for the leukotoxin.

7. 1. 4. Cytolethal Distending Toxin (CDT)

Bacterial cytolethal distending toxins (CDTs) are a family of heat-labile proteins with the

unusual property of blocking the mammalian cell cycle. In certain eukaryotic cell lines, CDTs

cause cell cycle arrest and progressive cellular distension, resulting in enlargement of both the

cell and nucleus, and leading to cellular death (Cortes-Bratti et al., 2001; Frisan et al., 2002;

Lara-Tejero and Galan, 2002). The CDTs are produced by a number of Gram-negative bacteria,

including diarrheic enteropathogens such as Salmonella typhi, Escherichia coli, Shigella

25

dysenteriae, Campylobacter species, and A. actinomycetemcomitans (Cortes-Bratti et al., 2001;

Haghjoo and Galan, 2004; Smith and Bayles, 2006; Sugai et al., 1998). CDT cause DNA damage

and cell cycle arrest at the G2/M stage in eukaryotic cells (Cortes-Bratti et al., 2001; Frisan et al.,

2002; Lara-Tejero and Galan, 2000). CDT is a tripartite A-B2 toxin comprised of CdtA, CdtB

and CdtC subunits, which each contributes to CDT-mediated cytotoxicity (Lara-Tejero and

Galan, 2001). The molecular masses of the cdtABC encoded proteins were 24.5, 31.5, and 20.7

kDa, respectively. It was demonstrated that CdtB alone is sufficient to induce CDT associated

cytotoxicity in cultured cells (Lara-Tejero and Galan, 2000) and CdtA and CdtC were required

for binding of the holotoxin to the plasma membrane of the target cells. Moreover, CdtB exhibit

type I deoxyribonulease activity (D NaseI) (Elwell and Dreyfus, 2000; Lara-Tejero and Galan,

2000), which involved in CDT mediated DNA damage and cell cycle arrest through blocking of

CDC2 kinase involved in the entry into mitosis (Pickett and Whitehouse, 1999). A.

actinomycetemcomitans CdtB showed 97% amoniacid sequence identity with the CdtB of H.

ducreyi and 45-48% identity with the CdtB of E.coli and C. jejuni (Mayer et al., 1999; Sugai et

al., 1998). In addition, the effect of CDT was dependent on cholesterol on plasma membrane

lipid microdomains (Boesze-Battaglia et al., 2009). The toxin is reported to be internalized via

Golgi complex to the endoplasmic reticulum and, from there, it reaches the nucleus by a

retrograde pathway (Guerra et al., 2005; Heywood et al., 2005). A. actinomycetemcomitans is the

only oral bacteria that produces CDT (Yamano et al., 2003), and it was reported that A.

actinomycetemcomitans might contribute to the pathogenesis of the aggressive form of

periodontitis (Tan et al., 2002). In our recent studies, we have identified A.

actinomycetemcomitans OMVs as a vehicle for the delivery of CDT into human cells. The

OMV-associated CDT proteins were internalized in both HeLa cells and human gingival

fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The

active toxin unit, CdtB, was found to be localized inside the nucleus and induced a cytolethal

distending effect on both HeLa and HGF cells.

8. Campylobacter jejuni

Campylobacter species have been associated with a wide range of gastrointestinal diseases,

particularly gastroenteritis, irritable bowel disease and periodontitis. The most well-known

species is C. jejuni, a leading cause of bacterial gastroenteritis in humans worldwide. Acute

26

campylobacteriosis, which is characterized by fever, severe abdominal cramps, and diarrhea

containing blood and leukocytes, is associated with C. jejuni invasion of intestinal cells. C. jejuni

pathogenesis involves different strategies compared with other well-characterized enteric

organisms. In vitro cell culture models and in vivo animal models unraveled a number of

pathogenic mechanisms of C. jejuni. It was shown that C. jejuni can attach to and invade

intestinal epithelial cells and generate toxins that kill host cells and impair host functions

(Everest et al., 1992; Hickey et al., 2000; Hu et al., 2006). C. jejuni also strategically deliver

effector proteins into the host cell, via specialized secretion systems. In addition, these organisms

possess surface structures that promote evasion of host immune defenses (Man).

8. 1. Virulence factors and pathogenesis

8. 1. 1. Flagella and Chemotaxis

During infection of the host, C. jejuni colonizes primarily within the mucous layer of the

intestine (Beery et al., 1988). Flagellar motility is an important colonization determinant for this

organism, enabling migration to and movement within the mucus to reach microenvironments

suitable for bacterial growth. Non-motile C. jejuni mutants are attenuated for colonization of

human or animal hosts (Ottemann and Miller, 1997). Like other motile bacteria, motility of

Campylobacter is regulated by a chemotactic signaling system that allows the organisms to

follow favorable environments. A non-chemotactic C. jejuni mutant was reduced for

colonization, indicating that the chemosensory system might have a role for in vivo growth

(Hartley-Tassell et al.).

8. 1. 2. CDT

CDT is a tripartite protein toxin composed of three subunits, CdtA, CdtB, and CdtC, encoded by

an operon comprising cdtA, cdtB, and cdtC as described in A. actinomycetemcomitans. CDT

holotoxin functions as an AB2 toxin in which CdtA and CdtC form a binding unit and CdtB is an

active unit. In addition, the nuclear-translocated CdtB subunit exhibits type I DNase activity,

which causes DNA damage resulting in cell cycle arrest at the G2/M phase (Lara-Tejero and

Galan, 2000). CDT appears to be common in C. jejuni strains. In a study of 117 isolates, there

was positive evidence for CDT in 114 of the isolates in Vero cell assays (Bang et al., 2004). The

earlier studies by (Fox et al., 2004) suggested that the mechanism of clearance of C. jejuni from

27

the intestine was NF-κB-dependent and that CDT may have proinflammatory activity in vivo, as

well as a potential role in the ability of C. jejuni to escape immune surveillance. CDT has been

identified as one of the virulence factors required for induction of interleukin-8 (IL-8), a

chemokine associated with local acute inflammatory responses in C. jejuni (Zheng et al., 2008).

In our recent studies, we demonstrated that all CDT subunits from C. jejuni were released from

the bacterial cells in association with OMVs. We suggested that the release of OMV-associated

CDT is as a mechanism by which C. jejuni delivers all the subunits of CDT toxin (CdtA, CdtB,

and CdtC) to the surrounding environment, including infected host tissue (Lindmark et al.,

2009).

8. 1. 3. Lipooligosaccharide (LOS)

The lipooligosaccharide (LOS) of C. jejuni is highly variable. Some C. jejuni LOS structures

resemble human neuronal gangliosides. This molecular mimicry can lead to autoimmune

disorders, including Guillain-Barré syndrome (GBS), an ascending neuritis neuropathy that

occurs following C. jejuni infection in approximately 1 in every 1,000 cases (Hughes et al.,

2004). C. jejuni mutants impaired in LOS biosynthesis are impaired in serum resistance, as well

as adherence to and invasion of epithelial cells (Fry et al., 2000).

28

Aims of the thesis

The aim of this thesis was to investigate the outer membrane vesicle-mediated virulence factors

from various Gram-negative bacteria.

Specific Aims

To investigate the OMV-mediated release of CDT from C. jejuni.

To analyze the potential role of non-O1/non-O139 Vibrio cholerae OMVs in modulating

the host innate immune response.

To examine the biological activity and host cell interaction of OMV-associated CDT

from A. actinomycetemcomitans.

To investigate the vesicle-mediated export of PrtV protease from Vibrio cholerae.

29

Results and Discussion

Paper-I

Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from

Campylobacter jejuni

Cytolethal distending toxin (CDT) is the best characterized virulence factor from C. jejuni.

Similar to many other Gram-negative bacteria, C. jejuni also produces OMVs (Fig. 1A & 1B,

paper I). It has been previously reported that several bacterial protein toxins are released in

association with OMVs of Gram-negative bacteria (Balsalobre et al., 2006; Kouokam et al.,

2006; Kuehn and Kesty, 2005; Wai et al., 2003). However, it was not known how CDT from C.

jejuni becomes surface-exposed and released to the surrounding environment. Our data suggest

that CDT is secreted to the bacterial culture supernatant via OMVs. Immunoblot analysis of

OMVs isolated from C. jejuni wild type strain 81-176 revealed that all the subunits of CDT

(CdtA, CdtB, and CdtC) were found in association with OMVs (Fig. 3, paper I). Furthermore,

immune-gold labeling of OMVs isolated from wild type strain 81-176 (using anti-CdtA, anti-

CdtB, and anti-CdtC antibodies) and electron microscopy strongly supported the suggestion that

the CDT subunits were indeed associated with C. jejuni OMVs and that the proteins appeared to

be internal or integral to the vesicle membrane. The presence of CDT in OMVs would imply that

the proteins should be localized, at least transiently, in the periplasmic compartments of the

bacterial cells. We analyzed the localization of the CDT subunits in different subcellular

(cytosolic, inner membrane, periplasm, outer membrane) fractions of the bacterium. All CDT

proteins were clearly found in the periplasmic fraction (Fig. 6B, Paper I). From this data, we

suggest that substantial amounts of the CDT subunits are translocated into the periplasm of the

bacterial cells, and from the periplasmic space CDT may be included in the OMVs that are being

released from the bacterial cell surface. Furthermore, data from the dissociation assay support the

idea that the majority of CDT toxin is tightly associated with OMVs (Fig. 7A, paper I). It is

known that CDT, a genotoxin has the ability to induce DNA damage and cell cycle arrest at the

G2/M stage in eukaryotic cells (Lara-Tejero and Galan, 2000). In our study, OMV-associated

CDT toxin from C. jejuni exhibited a cytolethal distention effect i.e., distinct enlargement of

human ileocecum carcinoma cells (HCT8) (Fig. 8A, paper I). In addition, a radioactive

30

nucleoside ([3H]-labeled thymidine) incorporation assay clearly indicated that the OMV-

associated CDT from C. jejuni wild type strain 81-176 induced a significant cell cycle arrest and

reduced DNA proliferation, as assessed by lower thymidine uptake in HCT8 cells (Fig. 8B, paper

I). Taken together, the results in this study demonstrate that biologically active CDT of C. jejuni

is secreted from the bacterium in tight association with OMVs. We suggest that OMV-mediated

release could be a mechanism for delivery of CDT to the surrounding environment and this

process may be involved in the pathogenesis of Campylobacter infections.

Paper-II

NOD-like receptor activation by outer membrane vesicles from Vibrio cholerae non-O1

non-O139 strains is modulated by the quorum-sensing regulator HapR

V. cholerae belonging to the O1 and O139 serogroups cause cholera that appears frequently as

epidemics in many developing countries. Strains belonging to serogroups other than O1 and

O139 are collectively known as non-O1 non-O139 V. cholerae (NOVC). The majority of NOVC

isolates lack the virulence gene encoding cholera toxin (CT) and toxin co-regulated pili (Tcp).

The NOVC may produce other extracellular virulence factors, such as the V. cholerae cytolysin

(VCC), encoded by the hlyA gene; the repeat-in-toxin (RTX) toxin, hemagglutinin protease

(HA/P); type III secretion effector protein (VopF); and type VI secreted proteins (e.g., VgrG-1)

that may be responsible for the pathogenesis of such strains in humans (Ghosh et al., 2006; Nair

et al., 1988; Tam et al., 2007). In our recent studies, we observed that the culture supernatant

from a NOVC strain V:5/04 induced a cytotoxic effect in HeLa cells, suggesting that either a

soluble protein or OMVs in the culture supernatant might be involved in the cytotoxicity.

We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain

V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by

human epithelial cells and appeared to accumulate in the vicinity of the nucleus, as assessed by

indirect immunofluorescence analysis with an antibody specific for the outer membrane protein

OmpU (Fig. 2, Paper II). An siRNA-mediated gene knock-down in THP-1 cells revealed that the

inflammatory potential of NOVC OMVs was partially mediated by NOD1 and NOD2, members

of a family of proteins containing nucleotide-binding domains, leucine-rich repeats.

In HeLa cells and human myeloid-like THP1 cells, V:5/04 OMVs induced significant release of

the proinflammatory cytokine IL-8 (Fig. 3A and 3B, Paper II). Interestingly, OMVs from the

31

ΔhapR mutant of V:5/04 strain induced a significantly lower IL-8 response (Fig. 3A, Paper II).

Similarly, V:5/04 OMVs also induced NF-κB activation in the THP1 cell line derivative THP1-

Blue, which expresses secreted embryonic alkaline phosphatase (SEAP) under the control of an

NF-κB-responsive promoter (Fig. 3C, Paper II).

OMVs contain a mixture of different pathogen-associated molecular patterns (PAMPs). A

ubiquitous bacterial PAMP is peptidoglcan (PGN). NOD1 and NOD2 are well-characterized

PGN sensors that modulate inflammatory responses mediated by activation of NF-κB and

mitogen-activated protein kinase (MAPK) signaling cascades in the host. To investigate if

NOVC OMVs contain PGN, we assayed activation of NOD1 and NOD2 by OMVs isolated from

the wild type V. cholerae strain V:5/04 and the ΔhapR mutant. When OMVs were applied to

human embryonic kidney (HEK293T) cells with a NOD1 or NOD2 expression plasmid which do

not express TLR4 and TLR5, OMVs significantly induced NOD1- and NOD2-dependent NF-κB

activation (Fig. 4A and 4B, Paper II). The Deletion of hapR significantly decreased the potential

of the OMVs to activate NOD1 and NOD2 (Fig. 4A and B, Paper II). Depletion of endogenous

NOD1 and NOD2 by specific siRNA in THP1-Blue cells led to the specific reduction of nod1

and nod2 mRNA levels, respectively (Fig. 5B, Paper II). Knock-down of NOD1 led to a

significant reduction of the OMV-induced NF-κB response (Fig. 5A, Paper II). Surprisingly,

depletion of NOD2 had no significant effect, suggesting that NOD1 might be the physiologically

relevant sensor of OMV-delivered peptidoglycan under these experimental conditions (Fig. 5A,

Paper II). In summary, these results suggest that V5:/04 OMVs contain PGN that could activate

NOD1 and NOD2.

To obtain additional evidence for the presence of PGN in the V:5/04 OMVs and to analyze

whether HapR deficiency might affect the PGN content of the OMVs, we analyzed the chemical

composition of the V. cholerae OMVs by X-ray photoelectron spectroscopy (XPS). This

revealed a significant difference in the chemical surface compositions of OMVs from the wild

type V. cholerae strain V5:/04 and the ΔhapR mutant (Fig. 5C, Paper II). In conclusion, we

demonstrate that NOVC OMVs contain PGN capable of activating both NOD1- and NOD2-

mediated inflammatory responses. Interestingly, whole bacterial lysate from the ΔhapR mutant

showed more NF-κB activity in both THP1-Blue cells and HEK293T cells compared to cell

lysate from the wild type strain (Fig. 6, paper II).

32

Understanding how bacteria control the PAMP composition of OMVs will give us the tools to

control the immunogenic properties of OMVs and potentially lead to the development of OMVs

as vaccine candidates.

Paper-III

Perinuclear localization of internalized Outer Membrane Vesicles carrying active

Cytolethal Distending Toxin (CDT) from Aggregatibacter actinomycetemcomitans

A. actinomycetemcomitans is the only oral bacteria that produce CDT (Yamano et al., 2003), and

several studies have suggested that CDT from A. actinomycetemcomitans might contribute to the

pathogenesis of periodontitis (Belibasakis et al., 2005; Tan et al., 2002). Previously, it was

reported that in E.coli and C. jejuni all subunits of CDT are secreted into the culture media

(Berlanda Scorza et al., 2008; Lindmark et al., 2009). In this study, we also observed that the

biologically active CDT subunit proteins are secreted from the bacterial cells in association with

OMVs from A. actinomycetemcomitans. OMVs were obtained from both plate- and broth-grown

cultures from the wild type strain D7SS and its derivateive, the cdtABC triple mutant. Western

immunoblot analysis suggested that all CDT subunits were present in OMV preparations of the

wild type strain D7SS, whereas they were absent in the OMVs of the cdtABC mutant (Fig. 1,

paper III). This phenomena was also observed in serotype a, as well as in serotype b and c,

suggesting that OMV-associated CDT is not strain specific. In addition, a dissociation assy also

confirmed that the active toxin unit CdtB was tightly associated with A. actinomycetemcomitans

OMVs (Fig. 2, paper III).

OMV-associated CDT from A. actinomycetemcomitans induced CDT-dependent cytolethal

distending activity in HeLa and HGF cells. OMVs of the wild type strain D7SS from both plate-

and broth-grown cultures induced a distinct cell enlargement, which was not observed in cells

treated with OMVs from the cdtABC triple mutant (Fig. 3, Paper III). Confocal microscopy

anlyses suggested that the OMVs might facilitate the simultaneous delivery of multiple proteins

into the human cells in vitro. Fluorescent labeling of whole bacterial cells and of A.

actinomycetemcomitans OMVs with antibodies against outer membrane protein A (OmpA)

clearly indicated that the OMVs were distributed in the cytosol, preferentially in the perinuclear

region of intoxicated HeLa and HGF cells (Fig. 4, Paper III). These findings suggeting that both

the A. actinomycetemcomitans OMV cargo internalized into the HeLa and HGF cells and the

33

OMV internalization process are independent of CDT because OMVs from the cdtABC mutant

could also be internalized into host cells. Previously, it was reported that OMV were internalized

through the retrograde pathway via the Golgi complex and endoplasmic reticulum (Bauman and

Kuehn, 2009; Kesty et al., 2004). Therefore, we investigated the possibility that A.

actinomycetemcomitans OMV internalization occurs through the retrograde pathway. However,

the retrograde pathway blocker, brefeldin A, could not block vesicle internalization (Fig. 5, paper

III), suggesting that internalization of A. actinomycetemcomitans OMV does not require an intact

Golgi apparatus. Furthermore, confocal microscopy indicated the presence of CdtB in nuclei of

intoxicated HeLa cells. OMVs from the wild type strain D7SS revealed a clear nuclear

distribution of fluorescent signal in intoxicated cells (Fig. 6, paper III). Previous studies, using H.

ducreyi recombinant CdtB reported that CdtB passes directly from the endoplasmic reticulum to

the nucleus of the host cells (Guerra et al., 2009). To assess if CdtB entered into the nuclei of the

intoxicated HeLa cells, we performed confocal microscopy using a CdtB-specific antiserum and

a TRITC-conjugated secondary antibody. The experiment revealed apparent nuclear distribution

of fluorescence in HeLa cells treated with OMVs from strain D7SS (Fig. 6). Taken together,

these results indicated that A. actinomycetemcomitans OMVs were internalized into host cells

and that the active toxin subunit CdtB was localized into the nucleus of the intoxicated cells in a

retrograde-independent manner.

We further investigated how the OMVs from A. actinomycetemcomitans were internalized into

the host cells. The OMVs were labeled with the rhodamine isothocyanate B-R18, which is

dequenched upon fusion with the host cell plasma membrane, resulting in red fluorescence

(Bomberger et al., 2009; Hoekstra et al., 1984). Confocal microscopy suggested that rhodamine-

B-R18-labeled OMVs (red) co-localized with the FITC-conjugated lipid raft marker CtxB

(green) in HeLa and HGF cells (Fig. 7A and B, paper III). This effect was abolished upon

treatment with the cholesterol-sequestering agent Filipin III (Fig. 7C and D, paper III). These

findings suggested that A. actinomycetemcomitans OMV-associated proteins including,

biologically active CDT, were delivered to the susceptible host cell cytoplasm via fusion with

lipid raft microdomains in the plasma membrane.

Taken together, our results provide a molecular basis for the OMV-mediated delivery of CDT

from A. actinomycetemcomitans into the host cells. Further studies are needed to fully

34

understand how OMV-mediated transport of virulence factors in A. actinomycetemcomitans may

contribute to human periodontal disease.

Paper-IV

Outer membrane vesicle mediated export of PrtV protease from Vibrio cholerae

Several observations have suggested that Gram-negative bacteria secrete various virulence

factors in association with OMVs, to the extracellular environment and into target host cells

(Aldick et al., 2009; Balsalobre et al., 2006; Kouokam et al., 2006; Lindmark et al., 2009;

Olofsson et al.; Rompikuntal et al.; Wai et al., 2003; Wai et al., 1995). In previous studies from

our laboratory, we discovered a novel M6 family Zn2+-binding metalloprotease, PrtV, which was

identified as an essential secreted virulence factor for V. cholerae survival against predator

grazing under natural environmental conditions (Vaitkevicius et al., 2006). Furthermore, pure PrtV

protein effectively degraded human blood plasma components and also showed a dose-

dependent cytotoxic effect in the human intestinal HCT8 cell line (Vaitkevicius et al., 2008).

PrtV protease was secreted efficiently from the various V. cholerae O1 El Tor strains to the culture

supernatant (Fig. 1A, paper IV). Similar to other Gram-negative bacteria, V. cholerae also

produces a large amount of OMVs during growth (Fig. 1B, paper IV). Therefore, we tested

whether the PrtV protein could be detected in association with OMVs. The crude OMVs samples

from the wild type V. cholerae strain C6706 and the prtV mutant were purified by Optiprep

gradient centrifugation (Fig. 2, paper IV), and the OMV-containing fractions were analyzed by

SDS-PAGE and immunoblotting using anti-PrtV polyclonal antiserum. Our data showed that the

PrtV protein was released from the bacterial cells in association with OMVs from the V. cholerae

wild type strain C6706 and no PrtV protein was found in association with OMVs from the

isogenic prtV mutant (Fig. 3B, paper IV). The relative amounts of vesicles released from the two

strains were quantified by immunoblot using anti-OmpU antiserum and found to be similar (Fig.

3B, paper IV). Moreover, the OMV-associated PrtV secretion was observed in different V.

cholerae isolates, indicating that the OMV-associated release of PrtV is not a strain specific (Fig.

S1, paper IV). Furthermore, the data from immunogold labelling and electron microscopy

revealed that PrtV protein was indeed strongly associated with vesicles of V. cholerae strain

C6706 but not with OMVs of the prtV mutant (Fig. 3C c and d, paper IV).

35

The PrtV protein consists of two PKD-domains at the C-terminus (Vaitkevicius et al., 2008). The

PKD-domain(s) might function as a ligand-binding site for protein-protein or protein-

carbohydrate interactions. Therefore, we tested whether the PKD-domains are essential for the

association of PrtV with OMVs. Interestingly, a reduced level of PrtV was found in both the

bacterial cells and culture supernatant of the PKD-domain-deficient strain compared with the

wild type. Moreover, PrtV association with OMVs was completely abolished in the PKD-

domain-deficient strain (Fig. 4A, paper IV). This data suggested that the PKD-domains are

necessary for the association of PrtV with OMVs. In order to test the biological activity of

OMV-associated PrtV, we treated the HCT8 cells with OMVs from the wild type strain C6706

and the prtV mutant. Cells incubated with the wild type OMVs showed significant morphological

changes, whereas no morphological changes were observed in human colon carcinoma (HCT8)

cells incubated with OMVs from the prtV mutant (Fig. 5, paper IV). However, there was no

increase in lactate dehydrogenase (LDH) released from the cells incubated with OMVs,

suggesting that PrtV-associated OMVs did not induce cytotoxicity in host cells. The mechanism

of the effect of OMV-associated PrtV on host cells remains to be elucidated. Furthermore, an

OMV-internalization assay and confocal microscopy suggested that FITC-conjugated lipid raft

microdomains co-localized in the plasma membrane with rhodamine-isothiocyanate B-R18 labeled

OMVs. This co-localization was blocked upon treatment with a lipid-raft-disrupting compound

Filipin III (Fig. 6, paper IV). Therefore, our data suggests that the OMV-associated, biologically

active PrtV from V. cholerae might enter into the target eukaryotic cells by a mechanism

independent of OMV fusion with the plasma membrane.

36

Conclusions

All three subunits of CDT toxin from C. jejuni were released to the surrounding

environment in association with OMVs and vesicle-associated CDT caused the cytolethal

distending effects on mammalian cells.

NOVC OMV-containing peptidoglycan elicits an immune response in host cells and is

mediated by NOD1 and NOD2. Furthermore, V. cholerae quorum-sensing machinery

might play an essential regulatory role in OMV-mediated immunogenicity.

A. actinomycetemcomitans could deliver biologically active CDT into the susceptible

host cells in association with OMVs. Vesicle-associated CDT is internalized via

membrane fusion, and is dependent on lipid raft microdomains in the plasma membrane.

The V. cholerae protease PrtV is secreted into the culture supernatant in association with

OMVs. The PKD domain(s) is essential for PrtV association with vesicles and OMV-

associated PrtV protease is biologically active.

37

Acknowledgements First of all, I would like to thank all the funding resources: Swedish Research Council,

the Faculty of Medicine, Umeå University, J. C. Kempe foundation, and this work was in

part performed within the Umeå Centre for Microbial Research (UCMR).

I express my deepest gratitude to my PhD thesis supervisor, Prof. Sun Nyunt Wai, for

giving me the opportunity to do my PhD studies in her lab. Thank you so much for believing in

me and surely, I would not have stayed here without your constant encouragement, support,

scientific supervision and kindness. I am also very much thankful to my co-supervisor, Prof.

Bernt Eric Uhlin, for the thoughtful suggestions, organizing scientific meetings, workshops and

also many gathering parties. I would like to express my special thanks to my master thesis

supervisor and my PhD studies examiner Prof. Maria Fällman. I am very much thankful to all

people from the Department of Molecular Biology for all the help.

I am deeply grateful to all the present and past members of the group, thanks for making

things easier: Takahiko - such a polite person I never seen in my life! It is always nice to discuss

with you science and many other things. Karolis - for the initiation of PrtV project. Barbro -

without you the lab is quite calm and boring! :-( Tianyan – for your impressive organization

skills. Mitesh - all-rounder, you have the solution for every problem. Sridhar – it is so nice that

you are here; always happy to share my feelings with you in my mother tongue. Amit pal –

thanks for your critics and joyful discussions. Annika – you are like, the coolest person I’ve ever

met. Marylise – for your frank opinion on several things. Dharmesh – impressing wise answers

and like your focus on specific things. Svitlana – the next OMV-generation :-) very enthusiastic

person and thanks for interesting discussions on your travel experiences. Tetsuya & Ryoma –

You were unforgettable colleagues. Sony – it was a nice time with you. Kent – nice companion

and I enjoyed the time working with you. Aurelia & Soma – hope you enjoyed here! Kristina,

Jalal, Alberto, Vincent, Erik, Lin Lin, Fatima and Suha thanks for maintaining nice

environment in the lab.

Monika, Hyun-Sook, Connie, Stina, Patricia, Junfa, Bhupender (thanks for your help

regarding the reprinting permission from the original publisher), Ting and Nikola thanks for

sharing lab chemicals, equipments and many more things. I am also thankful to the people

Teresa, Jonas, Sakura, Christopher, Berit, Jörgen and the rest of JC team members:

BEU/SNW/JO/JJ/EC, I appreciate all your suggestions during my presentations.

38

My sincere thanks to all the collaborators, Dr. Jan Oscarsson, Bernard & Khanzeb

Khan; Dr. Thomas A. Kufer; Dr. Richard Lundmark & Monika; Prf. Elisabeth Sauer-

Eriksson & Aaron; Dr. Andreas Wieser; Prof. Marie-Louise Hammarström, Gangwei &

Aziz.

I am also thankful to all administrative people: Maria, Ethal, and Ingela for your well

organizing skills. My special thanks to Johnny, for all the ordering stuff and Media people for

marvelous help on cleaning & autoclaving. Marek, for solving computer related problems,

Gunilla, for maintaining the hazardous chemicals properly, Marie, for teaching confocal

microscopy, Kristoffer, for your help with Legatus and Live-cell microscopy.

My teaching team: Sofia, Damini, Edvin, Christopher, Jonas, Hanna, Linus, chaz,

Connie, Stefanie, Maria, and Lena - it was a great pleasure to share the teaching responsibility

with all of you.

I am grateful to Mikael Wikström, for spending time and quick update of my completed

graduate courses in Ladok system. Åke Forsberg - one of the best course leaders for the course

which I taught and I was very much enjoyed with the discussions on the planning of laboratory

part, in addition, I am very much thankful to your kind generosity in finding space to write my

thesis.

My sincere thanks to Dr. M. F. Rahman (IICT), for inspiring me towards the field of

research. I am very much grateful to my best and childhood friend Dr. Siva Ram Garimella,

you are the best role model.

Kiran, Sireesha, Anish & Sourish – we miss you people :-( time goes very fast while

talking with you. Whenever, I met you, always I felt “I am not away from my home-town”.

Thanks for the homely atmosphere and all the initial help & guidance when I came to Umeå.

Hopefully soon we will meet again. Suresh & Vani – very nice couple. Suresh, you are my best

batch-mate in Umeå. Even though we couldn’t able to meet personally, it’s always nice to share

many things with you on phone. Vijay & Kamla – You are the good neighbors. It is a nice time

together with variety of Indian food. Always we have a lot to discuss. We really enjoyed your

company in Umeå. Mitesh, Sushma & Aarav – Thanks for arranging good gathering parties and

nice dinners especially for a huge number of delicious food. Mitesh you could have opened a

restaurant in Umeå :-). Always it happens, never ending non-stop discussions, I am quite sure; if

no one stops us we continued till the next morning :-). Takahiko, Kumi & Masahari – Thank

39

you for the nice dinners. I enjoyed your traditional Japanese food. Mohan, Madhavi & Krishiv

– One another nice telugu couple. We have a very nice time with you. It is always nice to have

with you a good Andhra food. Philge, Nisha & Jessica - We enjoyed your company in Umeå.

Mohan Pabba & Anupama – Mohan, it was a nice time with you in Umeå. However, we

enjoyed talking with you on phone.

I am also thankful to all other Indian friends – Ramesh, Azar, Karunakar, Kishan,

Jyotshna, Bindu, Dinesh, Jannek Hauser, Deepak, Sreenivas, Krishna, Pradeep, Kishore,

Shyam, Bhaskar, Venkat, Munendar, Sharvani, Aaron, Shani, Balasubramanian, Uma,

Ravi, Vivek, Shankar, Geetanjali, Mrudula, Ganesh, Kamraj and the list goes on…

I thankful to my beloved grand-parents (Late: Manikya Rao & Sowbhagyamma; Late:

Prasada Rao & Tulasamma) - for such a lovely memorable childhood. I am indebted to my

parents (Venkateswarlu & Sree Lakshmi) without your love and support I wouldn’t be here.

Thank you so much for everything. I am also thankful to my father-in-law and mother-in-law

(Venkatappa Rao & Padmaja) for your faith in me and for the precious gift to the rest of my

life. At this time, I would like to thank all my close family relatives: Vasudeva Rao (Late) &

Seetamma; Rama Mohan Rao & Lakshmi; Muralidhar Rao & Jaya Sri; Raghava, Vasu,

Raghu ram, Swaroopa & Madhu (I promise, surely, I learn this time f**h spelling:-)), Krishna

(I can’t remember how many nights we played “Chess” without a sleep!), Jyothi, Rahul &

Rohini; Ram narayana (one other unforgettable childhood friend). My deepest thanks to

Kamalamma & Damayanti - for your wholehearted blessings. Hari & Gayatri; Kishore &

Souji - it’s a wonderful time together. Chinnu & Samyu - amazing kids, I have the most

auspicious moments with you.

Last but not least; I can’t simply express my thanks in a few words to the wonderful god’s

gift, my soul mate and lovely wife Deepthi. However, still I will try to mention a few words; a

very big thanks for your unforgettable love, affection, everlasting patience, encouragement for

concentrating on thesis project(s), and caring. I am sure, without you my life wouldn’t be this

much easier.

Thank you all

Pramod

40

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