Host-Pathogen Interactions During Campylobacter and Yersinia Infections

Host-pathogen interactions during Campylobacter and Yersinia infections

Nayyer Taheri

Department of Molecular Biology Umeå Centre for Microbial Research (UCMR) Umeå 2019 This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD Copyright © Nayyer Taheri ISBN: 978-91-7855-003-6 ISSN: 0346-6612; New Series Number 2005 Cover composition: Five years in one picture; (photo: EM OMV by Linda Sandblad, AFM Campylobacter by Monica Persson, rest by Nayyer Taheri, Umeå University, Sweden) Design: Nayyer Taheri Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU Print Service, Umeå University Umeå, Sweden 2019

This thesis is dedicated to my beloved family

“All wells, when you dig enough, share the same water source.”

Table of Contents

Table of Contents ...... i

Abstract ...... iii Sammanfattning på svenska ...... v

Papers included in this thesis ...... vii Introduction ...... 1

1. Gastrointestinal tract and its defense mechanisms ...... 1

1.1. Natural barrier defenses ...... 1

1.1.1. Bile ...... 2

1.2. Immunological defenses ...... 2

1.2.1. Innate immune response ...... 3

2. Enteropathogenic bacteria ...... 9

2.1. Bacterial secretion systems ...... 9

2.2. Outer membrane vesicles (OMVs) ...... 11

2.2.1. OMV biogenesis ...... 14

2.2.2. OMV-host interactions ...... 15

3. Yersinia pseudotuberculosis ...... 17

3.1. Biology and epidemiology ...... 17

3.2. Transmission and pathogenesis ...... 17

3.2.1. Post-infectious sequelae ...... 19

3.3. Virulence factors ...... 19

3.4. Infection models ...... 21

3.5. Y. pseudotuberculosis-host interactions ...... 21

4. Campylobacter jejuni ...... 23

4.1. Biology and epidemiology ...... 23

4.2. Transmission and pathogenesis ...... 24

4.2.1. Post-infectious sequelae ...... 25

4.3. Virulence factors ...... 25

4.4. Infection models ...... 28

4.5. C. jejuni-host interactions ...... 29

4.6. C. jejuni OMVs ...... 32 Aims of this thesis ...... 33 Results and discussion ...... 34

Paper I: Y. pseudotuberculosis blocks neutrophil degranulation ...... 34

Paper II: Exposure of C. jejuni to low concentration of bile influences OMVs protein content and bacterial interaction with epithelial cells ...... 37

Paper III: Accumulation of virulence-associated proteins in C. jejuni OMVs at human body temperature ...... 41

Paper IV: C. jejuni OMVs activate inflammasome without inducing cell death in immune cells ...... 43 Main findings and future prospects ...... 46 Acknowledgements ...... 48 References ...... 50

Abstract

The innate immune system is known for protecting the host against invading pathogens, for instance enteropathogens infecting the gastrointestinal tract. The production of e.g. antimicrobial peptides, cytokines, and chemokines by innate immune cells and intestinal epithelial cells contribute to bacterial clearance. Given the significance of this system in overall defense, pathogens affect and/or manipulate immune cells and responses in favor of their own survival. This thesis focuses on how the Gram-negative enteropathogenic bacteria Yersinia pseudotuberculosis and Campylobacter jejuni affect the host, either directly via type 3 secretion system (T3SS) effector proteins or via outer membrane vesicles (OMVs), and how host factors potentially affect their virulence.

Yersinia pseudotuberculosis uses its T3SS to translocate virulence factors that disable various immune responses and subvert phagocytosis. Neutrophils are main target cells during Yersinia infection. They release granules that contain proteins with antimicrobial properties to the cell's exterior upon activation through a process called degranulation. We found that extracellular Y. pseudotuberculosis could prevent neutrophil degranulation upon cell contact. Prevention of degranulation was shown to be mediated via co-operative actions of the two anti-phagocytic Yersinia outer proteins YopH and YopE. Bacterial contact with neutrophils resulted in a transient inhibition of degranulation and further prevented degranulation upon subsequent contact with avirulent Y. pseudotuberculosis (lacking YopE and YopH) as well as Escherichia coli. Thus, Y. pseudotuberculosis impairs several neutrophil defense mechanisms to remain in the extracellular environment and to increase its survival during infection.

Campylobacter jejuni lacks a T3SS and appears to use OMVs and flagella as its main secretion apparatus. During passage through the intestine C. jejuni is exposed to bile, an important physiological component and part of the natural barrier of the intestine, and ability to resist bile is advantageous for C. jejuni survival. We investigated how C. jejuni OMV production and protein content is affected by bile. The main invasion and colonization of C. jejuni occurs in the lower part of the intestine where the concentration of bile is low compared with the proximal intestine. The OMV proteomic profiles were radically altered when bacteria were grown in low concentration of bile corresponding to cecal concentrations. Twenty-five present of the detected proteins of OMVs showed an altered abundance in the presence of low concentration of bile. In contrast, the overall proteome of the bacteria was unaffected. Moreover, OMVs from bile- exposed bacteria could enhance adhesion as well as invasion of bacteria into intestinal epithelial cells, suggesting a role of OMVs to the virulence of C. jejuni in the gut. The body temperature differs between the asymptomatic avian carriers

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of C. jejuni and humans, which develop symptomatic disease. We investigated whether the bacterial growth temperature affects the OMV proteome and found that 59 proteins were differentially expressed at 37°C. Among the higher abundant proteins, significantly more proteins were predicted to be related to virulence. Thus, temperature has an impact on the property of the OMVs, and this might affect the outcome of infection by C. jejuni in different hosts.

C. jejuni OMV interactions with innate immune cells were studied by analyses of OMV-mediated inflammasome activation. OMVs were found to induce ASC- and caspase-1-dependent inflammasome activation in murine and human macrophages and dendritic cells as well as in human neutrophils. While C. jejuni infection induced a low level of inflammasome-dependent cell death, OMV- induced inflammasome activation did not result in cell death. Thus, OMVs disseminate into tissue without bacteria can be a vehicle for virulence factors without inducing inflammatory cell death.

Sammanfattning på svenska

Det medfödda immunsystemet skyddar värden mot invaderande patogena mikroorganismer, så som enteropatogena bakterier som koloniserar och infekterar mag-tarmkanalen. Produktion av t.ex. antimikrobiella peptider, cytokiner och kemokiner från immunceller och tarmepitelceller bidrar till det antibakteriella försvaret. De mest effektiva bakterierna påverkar immunceller till förmån för sin egen överlevnad. Denna avhandling fokuserar på hur de Gramnegativa enteropatogena bakterierna Yersinia pseudotuberculosis och Campylobacter jejuni påverkar värden via effektormolekyler translokerade via typ 3-sekretionssystem (T3SS) och yttre membranblåsor (OMV), men även hur värdfaktorer kan påverka bakteriell virulens.

Yersinia pseudotuberculosis använder T3SS för att translokera virulenseffektorer som inaktiverar olika immunfunktioner såsom fagocytos. Neutrofiler är huvudmålceller vid Yersinia-infektion och frisätter granula som innehåller proteiner med antimikrobiella egenskaper. Dessa frisätts till cellens yttre vid aktivering genom en process som kallas degranulering. Vi fann att extracellulär Y. pseudotuberculosis förhindrar neutrofildegranulering vid cellkontakt, och denna effekt medieras via de två anti-fagocytiska effektorerna YopH och YopE. Denna effekt håller i sig även vid en påföljande kontakt med avirulent Y. pseudotuberculosis (saknar YopE och YopH) liksom Escherichia coli. Denna upptäckt adderar ytterligare en mekanism som Y. pseudotuberculosis använder för att förbli i det extracellulära tillståndet under infektion.

Campylobacter jejuni saknar T3SS och använder istället OMV och flageller som huvudsekretionsapparater. Under passage i tarmen exponeras C. jejuni för galla och förmågan att motstå denna är fördelaktig för bakteriell överlevnad. Den huvudsakliga koloniseringen och invasionen av C. jejuni förekommer i den nedre delen av tarmen där gallkoncentrationen är låg jämfört med den proximala tunntarmen. Proteininnehållet i OMV förändrades radikalt när bakterier odlades i låg koncentration av galla, utan att galla påverkade det bakteriella proteomet. OMV från gallexponerade bakterier visades förbättra både vidhäftning till liksom invasion av bakterier i tarmepitelceller, vilket kan betyda att OMV påverkar C. jejuni virulens. Kroppstemperaturen skiljer sig mellan de fåglar/kyckling som är asymptomatiska bärarare av C. jejuni och människor som får symtomatisk sjukdom. Tillväxttemperaturen för C. jejuni visade sig ha effekt på proteininnehållet i producerade OMV, där 59 proteiner uttrycktes differentiellt vid 37°C (human temperatur) jämfört med 42°C (fåglars temperatur). Bland proteiner med högre förekomst vid 37°C fanns signifikant fler proteiner associerade med virulens, vilket tyder på att tillväxttemperaturen påverkar

egenskaperna hos OMV och därmed hypotetiskt påverka hur C. jejuni orsakar sjukdom i människa men inte i fåglar. C. jejuni OMV interaktion med immunceller viktige för det medfödda immunförsvaret studerades genom analys av OMV-medierad inflammasomaktivering. OMV inducerade ASC- och caspase-1-beroende inflammasomaktivering resulterande i aktivering och sekretion av IL-1 i både humana och murina immunceller. Till skillnad från inflammasomaktivering via levande C. jejuni resulterade OMV-inducerad inflammasomaktivering inte i celldöd. Detta kan hypotetiskt betyda att OMV som sprider sig i vävnad utan närvaro av bakterier används som ”tysta” spridare av virulensfaktorer utan att framkalla inflammatorisk död av immunceller.

Papers included in this thesis

I. Taheri N, Fahlgren A, Fällman M. (2016). Yersinia pseudotuberculosis blocks neutrophil degranulation. Infection and Immunity, 84(12): 3369-3378. doi: 10.1128/IAI.00760-16

II. Taheri N, Mahmud F, Sandblad L, Fällman M, Wai SN, Fahlgren A. (2018). Campylobacter jejuni bile exposure influences outer membrane vesicles protein content and bacterial interaction with epithelial cells. Scientific Reports, 8(1): 16996. doi: 10.1038/s41598-018-35409-0

III. Taheri N, Fällman M, Wai SN, Fahlgren A. (2019). Accumulation of virulence-associated proteins in Campylobacter jejuni outer membrane vesicles at human body temperature. Journal of Proteomics, in press. doi: 10.1016/j.jprot.2019.01.005

IV. Taheri N, Wai SN, Fällman M, Fahlgren A. Campylobacter jejuni outer membrane vesicles activate inflammasome without inducing cell death in immune cells. (Manuscript)

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Introduction

Host-microbe interactions can be studied on many levels; interactions, which are useful for the host and interactions, which cause damage to the host. The study of the harmful interactions is critical, as understanding of the involved mechanisms may lead to therapeutic improvements against infections. Host- pathogen interactions are investigated in vitro using different molecular approaches complemented by animal studies, which examine the infection in vivo. Studying host-pathogen interactions adds insights into the components of both the host and the pathogen that are required for a successful interaction.

1. Gastrointestinal tract and its defense mechanisms Gastrointestinal tract (GI tract) is one of the favorable niches for pathogens that have been extensively studied. It is an organ with diverse functions: it resists relatively strong hydrochloric acid without denaturing the stomach; it digests food but not itself; and it harbors more bacteria than the other parts of the body, yet does not allow them to take over. It is a highly complex organ, which forms the largest mucosal border between the external environment and the internal milieu. A single layer of epithelial cells are in direct contact with the external environment and provide a first line of defense against potentially harmful components such as microorganisms, toxins, enzymes, and intestinal breakdown products. These substances, which reside in the intestinal lumen, penetrate the mucosal epithelial barrier under pathologic conditions and gain access to the underlying tissues or enter the blood stream resulting in clinical disease manifestations such as infections, allergies, or autoimmune diseases (1). To control invasion across the mucosal barrier and to protect the internal milieu from harmful substances and pathogens, the host has created an elaborate system of defense mechanisms grouped as natural barrier defenses and immunological defenses.

1.1. Natural barrier defenses The first line of defense against invaders consists of the natural barriers including physical, chemical, and biological barriers. They are present in the lumen where pathogenic bacteria are degraded by digestive secretions from the stomach, pancreas, and liver including stomach acids, proteolytic enzymes, and bile salts. Commensal microbiota also contributes to the defense by competing for nutrients and by producing antimicrobial substances. The second line of defense is the mucus layer, which is rich in secreted Immunoglobulin A (IgA) and antimicrobial peptides preventing the access of bacteria to epithelial cells. Finally, a thin monolayer of epithelial cells forms the last barrier. Epithelial cells are joined to

each other by junctional complexes including tight junctions, adherens junctions, desmosomes, and gap junctions. However, natural barrier defenses are not perfect and breaking down of these barriers can result in invasion and colonization by pathogens and consequently development of different disease in the host.

1.1.1. Bile Bile is one of the well-studied and important natural barrier defense components of the intestine. Bile is a yellowish green aqueous solution that is synthesized from cholesterol in the liver and stored in the gall bladder. Bile emulsifies and dissolves ingested fats and denatures protein prior to proteolysis (2). It is secreted into the small intestine via bile duct by controlled mechanisms. In human bile, the most abundant bile acids are cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, which constitute approximately 95% of the total acids (3-5). Adult humans produce about 400-800 ml/day of bile, which comprises of bile salts (as the major organic constituents), cholesterols, phospholipids, biliverdin, carbohydrates, vitamins, mineral salts, and other trace elements (4-6). Bile salts are present in the duodenum, jejunum, and proximal ileum to solubilize, digest, and absorb lipids and lipid-soluble vitamins (6, 7). The concentration of bile salts in the small intestine ranges from 0.2 to 2% (w/v), depending on the individual, daily diet, and time of the day (8). Almost 95% of the bile salts are reabsorbed by passive diffusion in the length of the small intestine and do not enter the large intestine (9).

Bile also has antimicrobial effects (10). It affects bacterial dissemination by inhibiting of bacterial growth, disrupting bacterial membranes, inducting oxidative stress and DNA damage (11), misfolding and/or denaturing proteins, and by chelating iron and calcium (12). Enteropathogenic bacteria have developed mechanisms to adapt and respond to the antimicrobial activities of bile, including activation of efflux pumps, changing function of outer membrane proteins, and induction of stress response genes. Some bacteria have developed specific resistance mechanisms, e.g. the production of bile salt hydrolase by Listeria strains. Bile also affects the regulation of bacterial gene expression by acting as an environmental signal (13). The bile concentration in the intestine drops by a steep gradient from the small intestine to the large intestine. Responses of pathogens to bile are influenced by their intestinal localization, which might contribute to an altered expression of their virulence determinants.

1.2. Immunological defenses When the natural barriers are compromised, microbial invaders must be recognized and eliminated. The mechanisms permitting recognition of invaders

can be divided into two general categories: the nonspecific or innate immune response and the specific or adaptive immune response. The recognition molecules used by the innate immune system are expressed on a large number of cells; therefore, this system acts rapidly upon encountering an invading pathogen and thus establishes the initial host response. On the contrary, the adaptive immune system is consisting of a large repertoire of specific B and T lymphocytes (B- and T-cells) where a low number of cells are specific for a particular antigen that induces them. Hence, the responding cells must proliferate after encountering the antigen in order to achieve sufficient numbers and produce an effective response against the microbe. A key feature of the adaptive immune response is the ability to provide long-lasting protection by producing long-lived cells that stay in an inactive state, until further encounter with their specific antigen, even decades after the initial meeting. These two immune responses are connected by particular cell types that have functional characteristics of both systems (14) (Figure 1).

Figure 1. Cellular components of the innate and adaptive immunity. NK (Natural killer).

1.2.1. Innate immune response The innate immune response is the front line of host defense against invaders and plays a crucial role in host survival. It is characterized by a series of sensors on immune cells, called pattern-recognition receptors (PRRs). These receptors

recognize molecules on the pathogen surface called pathogen-associated molecular patterns (PAMPs) or on damaged cells called damage-associated molecular patterns (DAMPs) (15). Depending on the subcellular localization and biological characteristics, PRRs are divided into four main groups, the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the RIG-I like receptors (RLRs) and the NOD-like receptors (NLRs). PRRs involved in bacterial infection are mainly TLRs and NLRs. RLRs are intracellular receptors that recognize RNA viruses, and CLRs are receptors for endogenous ligands and recognize damaged cells. CLRs are also known to play a role in sensing fungi (16, 17).

TLRs involved in bacterial infections are TLR2/1/6 (detects lipoproteins), TLR4 [detects lipopolysaccharides (LPS)], TLR5 (detects flagellin), and TLR9 (detects DNA) (18). After activation of TLRs, a signal cascade involving the adapter protein MyD88 (all TLRs except TLR3) or TRIF (TLR3 and TLR4 only) results in the activation and translocation of nuclear transcription factors that in turn induce the transcription of cytokines and other immune genes (19, 20). NLRs that recognize intracellular bacterial infections are Nod1 and Nod2 (detect peptidoglycan from bacteria cell wall). Unlike TLRs, which are membrane-bound, Nod1 and Nod2 are located in the cytosol and can induce TLR-independent antibacterial responses (21). Other NLRs, such as NALP1, NALP3, IPAF, and NAIP5, are components of the inflammasome complex.

Multiple cell types can initiate innate immune responses. Three major ones, which are focus of this thesis, are neutrophils, macrophages, and dendritic cells. These cells employ several diverse mechanisms to destroy infectious agents, e.g. phagocytosis, degranulation, NET formation, reactive oxygen species (ROS) production, and inflammasome activation (Figure 2).

Phagocytosis: Internalization of particles by a phagocytic cell is termed phagocytosis. The innate immune system includes a number of phagocytic cells including monocytes, macrophages, neutrophils, and dendritic cells. Upon encountering invaders, phagocytic cells normally internalize pathogens into a compartment called phagosome. Phagosomes then fuse with lysosomes, which contain lytic enzymes and antimicrobial peptides, and internalized particles such as pathogens are degraded (22). During this recognition and response process, reactive oxygen species (ROS) and nitrogen oxide (NO) are produced. ROS has microbicidal capacity on invading pathogens, however it can also injure cells of the host (23).

Degranulation: Degranulation is triggered upon the activation of cells by microbial or inflammatory stimuli, and leads to the release of granule contents.

Figure 2. Roles of neutrophils, macrophages, and dendritic cells in innate immune responses.

Polymorphonuclear leukocytes (PMN) including neutrophils, basophils and eosinophils as well as mast cells contain granules that are released upon activation by stimuli. Neutrophils contain four types of granules, which are categorized as peroxidase-positive (azurophilic or primary) granules, peroxidase- negative (specific or secondary and gelatinase or tertiary) granules, and secretory vesicles (24, 25) (Figure 3). Secretory vesicles and tertiary granules are released during adhesion and transmigration of neutrophils through the endothelium, respectively. Primary and secondary granules hold the majority of the neutrophils antimicrobial activity, and are released at infectious and inflammatory sites (24- 26). Primary granules are rich in myeloperoxidase (MPO), defensins, elastase, heparin binding proteins, and proteinases. Secondary granules contain lactoferrin, collagenase and gelatinase, as well as the LL-37 precursor hCAP-18. However, these granules can be sub-divided further and granule heterogeneity depends on controlled synthesis of granule proteins during differentiation in the bone marrow (24, 27). Granule contents are released by exocytosis to the extracellular environment, or into the phagosome, in four separate steps: (i) translocation of granules from the cytoplasm to target membrane; (ii) granule binding resulting in contact between the outer surfaces of the granule membrane and the inner surface of the target membrane; (iii) development of a fusion pore between granules and target membranes; and (iv) granule fusion with target membranes resulting in the release of their content (28-30).

Figure 3. The granule subsets, summary of components, size, and tendency to exocytosis in neutrophils.

NET formation: Upon activation, neutrophils release granular proteins and chromatin that together form neutrophil extracellular traps (NETs), which help trapping and killing various bacterial, fungal, and protozoal pathogens by binding to them and provide a natural defense against inflammation (31-33). In vivo, NETs are released during a novel type of programmed cell death, named NETosis, which is distinct from other type of cell death e.g. apoptosis and necrosis (34). During NETosis, intracellular membranes are disintegrated and elastase enters the nucleus, followed by decondensation of chromatin and release of nuclear material from the cell. Then, extracellular DNA complexes together with histones and granular enzymes form a sticky network of NETs that can entrap endogenous (e.g. platelets) and exogenous (e.g. bacteria) particles and molecules (35). Release of NETs is not limited to neutrophils. Mast cells and eosinophils but not basophils, can release NETs (36, 37). While mast cells, similar to neutrophils, can release nuclear chromatin and may undergo NETosis, eosinophils release only mitochondrial chromatin, and its release does not result in active cell death.

Inflammasome activation: Inflammasome activation is an important component of the innate immune response in clearance of pathogenic microorganisms or damaged cells (38, 39). Inflammasomes are cytosolic multiprotein complexes formed in response to various stimuli, e.g. sensing of intruding pathogens or cell

damage. Inflammasome complexes consist of sensor proteins (members of the NLR or PYHIN protein family) and effector pro-caspases (mainly caspase-1), which are connected with the adaptor protein apoptosis-associated speck-like protein (ASC). After an initial signal and complex assembly, activation of caspase- 1 is induced through autocatalytic cleavage. Subsequently active caspase-1 cleaves the pro-forms of the cytokines interleukin 1-beta (IL-1β) and interleukin 18 (IL- 18) into active forms that are secreted to the exterior of the cell. In parallel active caspase-1 induces a programmed cell death known as pyroptosis (40-45). This is termed canonical inflammasome activation. The non-canonical pathway is an alternative mechanism of inflammasome activation, which is triggered by sensing cytosolic LPS. This pathway is independent of TLR4 and depends on caspase-11 (caspase-4/5 in human) (46). Caspase-11 is considered as the receptor for cytosolic LPS, and binding of caspase-11 to LPS results in its auto-activation, which together with NLRP3 and ASC triggers inflammasome activation. (47-49) (Figure 4).

Figure 4. Canonical and non-canonical inflammasomes. pro-Cas-11 (pro-caspase-11), pro-Cas-1 (pro-caspase-1).

The most studied inflammasomes are the NLRP3 and the NLRC4 inflammasomes (50, 51). Activation of NLRP3 inflammasome requires a two-signal process. The first signal (priming) leads to the transcription of NLRP3 and pro-IL-1β through activation of NF-ĸB via stimulation of pattern-recognition receptors e.g. TLRs. The second stimulus induces the formation of the NLRP3 inflammasome (52-54). NLRC4 inflammasome is formed after sensing cytosolic bacterial component such as flagellin or components of T3SS (55, 56).

2. Enteropathogenic bacteria Enteropathogens are organisms that cause disease of the GI tract. They include bacteria, viruses, protozoa, and fungi. The majority of enteropathogens studied in detail are bacteria, which contain both Gram-negative bacteria such as Helicobacter, enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), Shigella, Salmonella, Yersinia, Vibrio, and Campylobacter species and Gram-positive bacteria such as Clostridium difficile and Listeria monocytogenes. Infection with enteropathogenic bacteria occurs via the fecal-oral route, ingestion of contaminated food or water and/or through person-to-person transmission. The main infection site of enteropathogenic bacteria is intestine. A successful infection of the human intestine by enteropathogenic bacteria relies on the bacterial ability to adhere and colonize the intestinal epithelium. Ingested pathogens have developed abilities to resist non-specific host defenses, such as stomach acidity, peristaltic movements of the intestine, mucosal cell exfoliation, mucins, bile salts, proteolytic enzymes, and antimicrobial peptides (57), and to adhere and colonize the intestinal epithelial cells. In some cases, colonization involves host cell invasion, followed by either intracellular proliferation and dissemination to other tissues, or bacterial persistence (58).

To accomplish these tasks bacteria have developed an arsenal of virulence factors that interact with and manipulate normal functions of host cells. Virulence factors help pathogens to invade host tissue, protect them from host immune responses, and mediate their survival under harsh condition. They either act from the extracellular environment or are translocated into the cytosol of target cells where they interfere with host signal transduction pathways. In turn, the host defends itself against infection by initiating an inflammatory response to eliminate bacteria.

2.1. Bacterial secretion systems Gram‐negative enteropathogenic bacteria have developed highly specialized nano machines to secret a wide variety of materials such as proteins and nucleic acids across the bacterial membrane. Translocated materials have three fates; they remain associated with bacteria, they are released into the extracellular environment, or they are delivered into target cells. Translocated materials have key roles in the bacterial response to the environment, as well as in several physiological processes such as adhesion, adaptation and survival (59). In Gram- negative enteropathogenic bacteria, six types of secretion systems (T1SS-T6SS) with different compositions and mechanism of actions have been characterized (60) (Figure 5).

Figure 5. Schematic presentation of Gram-negative enteropathogenic bacterial secretion systems. IM: inner membrane, PG: peptidoglycan, OM: outer membrane, CM: cell membrane.

Based on the type of translocation, secretion systems are further subdivided into two groups: Sec‐dependent (two-step secretion) pathways, and Sec‐independent (one-step secretion) pathways. In the Sec‐dependent pathways (T2SS and T5SS), a protein to be secreted is first transported into the periplasmic space before translocated into the extracellular space. In the Sec-independent pathways (T1SS, T3SS, T4SS, and T6SS), the materials are translocated directly from the cytoplasm to the extracellular space or to the cytosol of a target cell (61, 62). Secretion systems are not constitutively active, but their activation can be triggered by for example contact with target cells (59).

T1SS mediates secretion of proteins with different sizes from the bacterial cytoplasm into the extracellular environment. Structurally, it is closely related to RND multidrug efflux pump (63). Secreted proteins by this system can be associated with nutrient acquisition and virulence. For example, E. coli metalloproteases and colicine V are secreted via T1SS (64).

T2SS secretes folded proteins from the periplasm into the extracellular milieu. Examples here are hemolysins, proteases, and lipases from different bacteria, thermolabile toxin of E. coli, and cholera toxin of Vibrio cholerae (65, 66).

T3SS is one of the best-characterized secretion systems utilized by Gram-negative enteropathogenic bacteria to deliver proteins directly from the bacterial cytoplasm into the cytosol of target cells (67-69). It is found in symbiotic bacteria such as Rhizobium and in pathogenic bacteria including Yersinia, Salmonella, Shigella, ETEC/EPEC/EHEC, and Vibrio species (67, 70, 71). Shigella uses T3SS to escape from the phagocytic vacuole and to disseminate to near cells (72), while Salmonella uses T3SS to reside and replicate within the Salmonella-containing vacuole (SCV) (73, 74). Yersinia targets the Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) by crossing the epithelial lining, where they use T3SS effector proteins to prevent phagocytosis to remain extracellular (69). EPEC and EHEC reside on the apical side of enterocytes. From there translocated T3SS effector proteins induce the formation of actin pedestals (75). Some Vibrio strains exhibit T3SS and are able to invade, survive, and replicate within epithelial cells using T3SS effectors (76). Despite their different lifestyles, the above-mentioned enteropathogenic bacteria can modulate host cell functions through their T3SS effectors.

T4SS is homologous to the pilus for bacterial conjugation and has a unique ability to mediate translocation of nucleic acids in addition to proteins directly from the cytoplasm of bacteria into the cytosol of target cell (77, 78).

T5SS is known as an autotransporter system that mainly secretes virulence proteins but is also associated with bacterial adhesion and biofilm formation, e.g. adhesin involved in diffuse adherence (AIDA)-I from E. coli (79, 80). Similar to T2SS, transportation of proteins from the bacterial cytoplasm by T5SS is subjected to a two-step secretion mechanism. Some cytotoxins and enterotoxins are secreted through T5SS (61).

T6SS translocates virulence proteins into target cells including both eukaryotic and prokaryotic cells and has an essential role in pathogenesis and bacterial competition (81, 82). It was first demonstrated in Vibrio cholerae (83), and is homologous to the bacteriophage tails (84-86).

2.2. Outer membrane vesicles (OMVs) OMVs are extracellular blebs released actively and deliberately by almost all Gram-negative bacteria, and in all cell growth phases both in vitro and in vivo (87), which are considered as a secretion system and proposed to be named type zero secretion system (88). The reason behind this suggestion is that OMVs can transport a variety of bacterial components in a protective way to the extracellular milieu or even into target cells (89-91). They were initially discovered in Vibrio cholerae during normal growth (92). They are nanoparticles diverse in the size (~10-300 nm in diameter) and in the content. OMV cargo mainly consists of outer

membrane components such as phospholipids, outer membrane proteins, and lipopolysaccharides (LPS), but also comprises periplasmic, cytoplasmic membrane or cytoplasmic components, such as toxins (93-96), DNA (97, 98), RNA (99-102), peptidoglycan, and enzymes including alkaline phosphatase, phospholipase C, proelastase, protease, and peptidoglycan (PG) hydrolase (87, 103-108) (Figure 6).

Figure 6. Composition of OMV released by Gram-negative bacteria. OMP (outer membrane protein).

OMVs have several advantages over other secretion systems: (i) transporting of lipids and insoluble proteins to the extracellular environment (109); (ii) diversity of secreted components e.g. proteins, lipids, DNA and RNA in one package (88, 110); (iii) carriage of bacterial components in a concentrated and therefore more effective form (91, 111); and (iv) protection of susceptible components from physical, chemical and enzymatic degradation until they reach their target safe and prolongation of their half-life (112, 113).

How bacteria pack the OMV cargo is not fully understood, but some bacteria seem to have a machinery for this purpose (114, 115). The OMV property enables them to carry bacterial components to the distal sites in the host, to mediate bacterial communication through OMV-associated signal molecules, to promote virulence, genetic transformation, elimination of unwanted components, and to modulate host immune responses (94, 103, 105, 110, 112, 116-129) (Figure 7). Proteomic

analyses of OMVs produced from different Gram-negative enteropathogenic bacteria such as Helicobacter pylori (130), Campylobacter jejuni (131, 132), Vibrio cholerae (133), and E. coli (134) have opened novel insights into the protein cargo carried by these OMVs.

Figure 7. Functions of OMV released by Gram-negative bacteria.

Examinations of animal and human biopsy specimens, tissues, and host fluids provide evidence that OMVs are produced during infection in vivo by several types of pathogenic bacteria, such as Helicobacter pylori, Neisseria meningitidis, Moraxella catarrhalis, and Borrelia burgdorferi and Salmonella (135). Inspection of H. pylori-infected biopsy samples by electromicroscopy (EM) showed presence of OMVs in contact with epithelial cells. Moreover, H. pylori OMVs were shown to absorb antibodies produced in the serum of infected patients (136, 137). OMVs from Neisseria meningitidis have been detected in blood, serum, or cerebrospinal fluid of infected patients (138-140). OMVs from a Salmonella strain were detected in the ileum of infected chicken (141). Moreover,

OMVs were proven in nasal sample from a patient with Moraxella catarrhalis infection (142). OMVs are not only produced in vivo by bacteria at sites of infection. They can disseminate to other tissues. For example, detection of OMV antigens in samples from infected animals and humans by B. burgdorferi indicate that OMVs disseminate to other tissues from the site of infection (143). It is still not clear whether they play any role in virulence in vivo.

2.2.1. OMV biogenesis OMV production by bacteria has been studied for decades but only recently, researchers have started to investigate the mechanisms behind OMV formation. Although several studies have tried to elucidate mechanisms behind the release of OMVs by different bacteria, a general and conserved molecular mechanism for the biogenesis of the OMVs is still missing. However, there are several models proposed for OMV biogenesis, which can be classified in three groups: (i) models where peptidoglycan plays a main role in OMV formation, e.g. reduction of cross‐ linking between the outer membrane and peptidoglycan layer as seen in Acinetobacter baumannii and E. coli (144, 145), modifications in the peptidoglycan structure (146), or accumulation of peptidoglycan fragments, LPS or misfolded proteins in the periplasmic layer (147) mediate OMV formation; (ii) models suggesting that changes in the outer membrane composition lead to OMV production, e.g. enrichment of the outer membrane with LPS and phospholipids, which increases membrane curvature or function of the Tol‐Pal system that is essential for maintaining bacterial membrane integrity as it is the case for Helicobacter pylori and Shigella boydii (111, 148, 149); and (iii) models proposing that environmental changes induce OMV formation. For example, a temperature change induces OMV production by Serratia marcescens and E. coli while Pseudomonas aeruginosa produces OMVs in the presence of antibiotics (119, 147, 150).

Recently, three novel mechanisms have been reported for OMV biogenesis. In the first study, an explosive cell lysis mediated by activity of a cryptic prophage endolysin was shown to result in OMV production by Pseudomonas aeruginosa through vesicularization of shattered membrane fragments (151). In another study performed on Salmonella Typhimurium, deacylation of lipid A resulted in OMV production (152). In the third study, a conserved phospholipid transporter (VacJ/Yrb ABC) was shown to be involved in OMV formation in Haemophilus influenza and Vibrio cholerae (153), which was discussed as a general mechanism for OMV biogenesis of Gram‐negative bacteria.

2.2.2. OMV-host interactions Bacteria can enter epithelial cells using different mechanisms, such as ligands specific for receptors on the cell surfaces. OMVs may also share such ligands and these properties may help bacteria to modulate host responses using OMVs as decoys (116, 154). OMVs can also transport bacterial toxins, LPS, proteases and virulence factors to the target host cells. Interactions between OMVs and host epithelial cells can modulate cellular mechanisms that control cell proliferation (155), apoptosis (156, 157), and immune responses. OMVs released by Cronobacter sakazakii are internalized by intestinal epithelial cells and induce cell proliferation and proinflammatory responses (IL-8 secretion) (158). OMVs produced by E. coli (100), Pseudomonas aeruginosa (101), Porphyromonas gingivalis (159), and Vibrio cholerae (102) have been shown to contain RNAs and have immunostimulatory capabilities in epithelial cells. OMVs produced by the highly virulent E. coli O104:H4 strain enters human intestinal epithelial cells via endocytosis, inducing caspase‐9‐mediated apoptosis, and IL‐8 production (156).

In addition, OMVs can enter host epithelial cells via lipid rafts and are subsequently detected by the cytoplasmic immune receptor NOD1, resulting in the production of innate immune responses in a NOD1‐dependent manner (160). Similarly, OMVs from Aggregatibacter actinomycetemcomitans and V. cholerae are internalized by human epithelial cells and activate NOD1- and NOD2- dependent inflammatory responses (88, 161). Once internalized by epithelial cells, OMVs are degraded intracellularly via autophagy in a NOD1‐dependent manner, resulting in an inflammatory response (162).

It has been shown that OMVs can also interact with TLRs expressed by both epithelial and immune cells to mediate inflammatory responses. It has been shown that pathogenic bacteria use OMVs as decoys to trigger or dampen the inflammatory responses. Initial studies identified that OMVs contain a number of PAMPs such as RNA, DNA, lipoproteins, LPS, and peptidoglycan that can interact with host PRRs, resulting in the induction of an innate immune response. For example, Salmonella Typhimurium, Yersinia pseudotuberculosis, and Yersinia pestis produce OMVs that mediate inflammatory responses via TLR4 on murine dendritic cells (163). Porphyromonas gingivalis OMVs abrogate TNF-α responses by monocytes in a TLR4‐dependent manner (164) or activate inflammasome in macrophages in vitro and in vivo (165). Bacteroides fragilis OMVs that contain capsular polysaccharides can interact with dendritic cells via TLR2 to induce the differentiation of IL‐10‐producing regulatory T cells and mediate protection from experimental colitis (166). More recently, this finding was expanded by identifying that Bacteroides fragilis OMVs use inflammatory bowel disease‐associated gene ATG16L1 within DCs to induce IL‐10 expression by regulatory T cells and to suppress intestinal inflammation in a colitis model

(167). Furthermore, OMVs can deliver LPS to the host cell cytosol to induce the activation of the caspase‐11 non‐canonical inflammasome pathway, leading to caspase‐11 activation and pyroptosis, which may be a critical mechanism against bacterial infections (168-173).

There are multiple studies suggesting that OMVs may play an important role in avoiding complement‐mediated killing of bacteria, thereby facilitating survival of the pathogen in the host. Vibrio cholerae OMVs contain the virulence factor OmpU that facilitates the survival of serum‐sensitive V. cholerae from complement‐mediated killing (174). Porphyromonas gingivalis OMVs contain the enzyme PPAD, which disables the functions of the complement factor C5a and facilitates immune evasion of P. gingivalis (175). OMVs from Neisseria meningitidis bind to long pentaxin 3, an acute phase inflammatory glycoprotein that has a role in regulating complement activation, pathogenesis of bacterial meningitis and sepsis (176). Moraxella catarrhalis OMVs have been shown to interact with the alternative pathway complement factor C3b and confine the complement activation (142, 177)

OMVs can modulate adaptive immune responses via multiple mechanisms. However, the differences in the ability of OMVs to activate or suppress immune responses may be dependent on their cargo and their bacterial origin. For example, Neisseria meningitidis OMVs have immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells (178, 179). Salmonella Typhimurium OMVs mediate Salmonella-specific immunity in mice (180). Helicobacter pylori OMVs modulate the host immune response by stimulating the release of both pro‐inflammatory (IL‐6) and anti‐inflammatory (IL‐10) cytokines from human mononuclear cells, thereby inhibiting CD4+ T-cell proliferation and inducing T‐cell apoptosis (181). OMVs from Borrelia burgdorferi and Haemophilus influenzae activate human B‐cells (182, 183). Moraxella catarrhalis avoids direct interaction with B-cells and instead redirect the adaptive immune response by using its OMV as decoys (184).

3. Yersinia pseudotuberculosis

3.1. Biology and epidemiology Yersinia, first isolated in 1894 by Alexander Yersin, is a zoonotic, Gram-negative bacterium distributed worldwide and constituting both pathogenic and nonpathogenic strains. The genus Yersinia consists of at least 17 species (185) and belongs to the family Yersiniaceae, in the class of Gammaproteobacteria. Among them, Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis are associated with human disease and have been extensively studied (186-188). Both Y. pseudotuberculosis and Y. enterocolitica are enteropathogenic bacteria that cause mild and self-limiting gastroenteritis (yersiniosis) presented as fever, diarrhea, vomiting, and abdominal pains. The gastrointestinal infections are usually cleared within 7-14 days and do not need antibiotic treatment, however septicemia can occur in immunocompromised patients. Y. pestis, which evolved from Y. pseudotuberculosis 1,500-20,000 years ago, is causative agent of plague. It is transmitted through the bite of an infected flea called Xenopsylla cheopsis into the bloodstream of human resulting in bubonic plague or pneumonic plague when Y. pestis reaches the lung. To date, three pandemics of plague have been reported (189, 190).

Yersinia pseudotuberculosis strain YPIII (serotype O:3) is characterized as a rod- shaped, extracellular, and facultative anaerobic bacterium. It is a foodborne and resilient pathogen, well adapted for survive in low-nutrient conditions and at temperatures ranging from 4 to 43°C (191), with an optimal growth temperature between 25–28°C (192).

3.2. Transmission and pathogenesis Y. pseudotuberculosis infects a variety of hosts, including both wild and domestic animals and birds (193-195). Humans are mainly infected via the oral route by consumption of contaminated food such as fresh vegetables, or water (196) (Figure 8). Y. pseudotuberculosis can survive the acidic condition of the stomach during the passage into the small intestine. Shortly after ingestion, Y. pseudotuberculosis leaves the lumen of the small intestine by crossing the intestinal barriers via passing through the microfold (M) cells into the Peyer’s patches (PPs) of the ileum (197, 198). Y. pseudotuberculosis replicates in PPs and can disseminate to the mesenteric lymph nodes (MLNs), spleen, and liver (199- 201). Once established in lymphoid tissues (PPs, and MLNs), Y. pseudotuberculosis replicates as aggregates of extracellular bacteria within necrotic lesions or abscesses. At this step of the infection, bacteria resist phagocytosis using Yop effectors.

Figure 8. Yersinia pseudotuberculosis transmission (A) and pathogenesis (B) in human.

Although it is clear that the bacterium replicates extracellularly by preventing phagocytosis, there is also indications that Y. pseudotuberculosis survives and multiplies in macrophages during the infection, which is more pronounced at early stages of colonization (202). The fact that macrophages are permissive cells for Y. pseudotuberculosis replication and survival in vivo is supported by in vitro experiments (203). There is also evidence showing that bacteria are able to subvert the functions of macrophages from the inside, by inhibiting phagosome acidification and the production of nitric oxide (202). All mentioned abilities are dependent on the expression of a functional T3SS and translocation of Yersinia outer proteins (Yops). In rare cases in which Y. pseudotuberculosis reaches the bloodstream, e.g. in immunocompromised hosts, they can resist the serum using YadA and the O-antigen of LPS, and thereby likely survive in bloodstream (204, 205).

3.2.1. Post-infectious sequelae Although the majority of infections with Y. pseudotuberculosis are self-limiting in humans, in some cases the infection can cause post-infectious sequela such as infectious ileocecitis (206, 207) that clinically mimics appendicitis (208, 209), reactive arthritis, erythema nodosum, or the Kawasaki autoimmune syndrome (187, 191, 210, 211). Y. pseudotuberculosis infections have also been discussed as players in the development of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and Crohn´s disease (212, 213).

3.3. Virulence factors Both chromosomal- and plasmid-derived virulence factors play roles in Y. pseudotuberculosis pathogenesis (214). Y. pseudotuberculosis carries a 70 kb plasmid (pYV), which is essential for virulence and encodes the type III secretion system (T3SS). The system was initially discovered in Yersinia in 1991 (215) and then has been extensively studied (200, 216, 217). T3SS plays a crucial role in both establishment and outcome of infection. It allows bacteria to escape host recognition and takes control of the host cell signaling system by translocating Yop effector proteins into the cytosol of target cells (216, 218). Delivery of Yops requires close contact between Y. pseudotuberculosis and its target cell, which is mediated by proteins called adhesins. Invasin (encoded by the chromosome), attachment-invasion locus protein (Ali; encoded by the chromosome), and Yersinia adhesin A protein (YadA; encoded by the virulence plasmid) belong to the most prominent and well-studied Yersinia adhesins. They work at different times and phases of infection and complementing each other by their ability to bind to various host molecules such as collagen, fibronectin, laminin, β1 integrins, and complement regulators. Adhesins are anchored to the bacterial surface (outer membrane), often forming fimbrial-like structures that protrude to the extracellular milieu (219-221).

Yops: Under in vitro conditions, secretion of Yops is induced at 37°C under depletion of calcium. During infection, direct interaction with target cells is required for secretion and translocation of Yops into host cells (222-224). According to their functions, Yops are divided into three groups: Translocators including YopB, YopD, and LcrV; effectors including YopH, YopE, YopM, YopK, YopJ, and YpkA; and regulators such as YopK, YopE, and YopN (200, 225, 226). YopB and YopD are transmembrane proteins, which by binding to each other form a pore for the translocation of effector proteins into the host cell (227). Besides YopB and YopD, LcrV (Low calcium response V or the V antigen) has been shown to be involved in translocation of effector proteins (228, 229). Yop effector proteins are secreted by T3SS and translocated into the cytosol of a host cell. They mimic activities of host cell enzymes such as kinases, phosphatases, proteases, acetylases, guanine nucleotide exchange factors, and GTPase-

activating proteins (GAPs). The activities of the Yop effector proteins lead to suppression of phagocytosis and proinflammatory cytokine production in the host cell (226).

 YopH is a protein tyrosine phosphatase (PTPase) (230, 231). Upon translocation, YopH localizes to peripheral focal adhesion complexes in the host cell and targets proteins involved in receptor signaling and adaptor proteins, such as FAK, p130Cas, Fyb (232-235). YopH inhibits calcium signaling in neutrophils and disrupts focal adhesion complexes resulting in inhibition of phagocytosis and the associated oxidative burst in macrophages, dendritic cells, and neutrophils (232, 233, 236-240).  YopE is a GTPase-activating protein that affects actin dynamics by inhibiting the small G proteins RhoA, Rac1, and Cdc42 (241-248). The GAP activity of YopE is required for the anti-phagocytic activity and virulence of Y. pseudotuberculosis (243). Beside its virulence factor activity inside the host cells, YopE has also a role as a regulator in Yop secretion and translocation (247).

Invasin: Y. pseudotuberculosis invasin is expressed at both 26°C (optimal expression) and 37°C under acidic condition (pH 5.5) and high sodium levels, suggesting that the small intestine condition may maintain invasin expression after ingestion of the bacterium (249). Invasin binds with high affinity to β1 integrins exposed on the luminal side of M-cells and facilitate entry of bacteria into the intestinal tissue (197, 250, 251). Y. pseudotuberculosis inv mutants are defective in colonization of PPs and the MLNs (252).

YadA: The adhesin YadA is encoded by the pYV plasmid, and its expression is upregulated at 37°C. YadA can adhere to both epithelial and immune cells by binding to different extracellular matrix (ECM) molecules such as collagen, fibronectin, laminin, and β-1 integrins. YadA contributes to adhesion and invasion independent uptake and serum resistance (220, 253-256). The Y. pseudotuberculosis strain lacking YadA, YPIII strain, does not show significant virulence defects. However, Paczosa et al. have recently shown a role of YadA and Ali in directing the translocation of T3SS effector proteins into the neutrophils (257, 258).

Ali: The expression of Ail is also upregulated at 37 ºC. Similar to YadA, Ail also mediates binding of bacteria to different ECM proteins on different cell types and is involved in serum resistance. Y. pseudotuberculosis Ail protein was found to be devoid of adhesion and invasion capacity (259-261).

LPS: Lipopolysaccharides (LPS) is the major outer membrane components of Y. pseudotuberculosis and is important for its virulence (262). LPS consists of lipid A, which is anchored in the outer membrane, an oligosaccharide core, and the O- antigenic polysaccharide (O-antigen). Most biological effects of LPS are due to the lipid A part, however, it has been shown that the O-antigen plays important roles in host tissue colonization, serum resistance, and the resistance to antimicrobial peptides (205). Mutations in genes involved in O-antigen biosynthesis result in attenuated bacteria (263).

Yersiniabactin: Yersiniabactin (Ybt) is a siderophore with high affinities for ferric iron and plays an important role in the pathogenicity of Y. pseudotuberculosis. This system is required for the initial colonization of Yersinia; as a mutant strain defective in Ybt is unable to colonize the host (264, 265).

OMVs: Y. pseudotuberculosis secretes OMVs that contain different proteins and virulence factors including Yops. Recently it has been shown that OMVs isolated from Y. pseudotuberculosis YPIII also harbor the cytotoxic necrotizing factor (CNFY), and stimulation of HeLa cells with OMVs causes multinucleation of these cells (266).

3.4. Infection models One main purpose to study Yersinia biology is the invention of new cures for plague caused by Y. pestis. To achieve this goal, studies of Yersinia virulence mechanisms are normally carried out with Y. pseudotuberculosis as a model organism for Y. pestis. The genetic similarities between Y. pestis and Y. pseudotuberculosis, and the fact that Y. pseudotuberculosis causes a systemic plague-like disease in rodents, makes this a convenient system to study more severe infections (267). Mice are commonly used in virulence infection models (268, 269). Rabbits are used to study Yersinia-mediated enteritis and the humoral immune response after oral infection. Rats are suitable models for studying Yersinia-induced aseptic arthritis, and the Caenorhabditis elegans model is often used to study biofilm formation by Yersinia (270, 271). In addition to in vivo models, multiple cell lines are used for in vitro studies of host-pathogen interaction and pathogenesis of Y. pseudotuberculosis including epithelial cells (ECs), fibroblasts, and primary cells such as macrophages, monocytes, dendritic cells, and neutrophils.

3.5. Y. pseudotuberculosis-host interactions After infection, Y. pseudotuberculosis encounters immune cells in PPs. PPs harbor resident macrophages and dendritic cells but also recruit neutrophils and inflammatory monocytes that participate in the bacterial clearance. However,

bacteria can resist the host defense and are not cleared efficiently from the site of infection leading to dissemination of bacteria to MLNs and the formation of bacterial aggregates. In the oral mouse infection model, bacteria can disseminate to the liver and spleen to cause systemic infection (226, 272). For this to happen, bacteria require active T3SS and secretion of Yops.

Y. pseudotuberculosis can effectively translocate Yops into different cell types including epithelial cells, macrophages, dendritic cells, neutrophils, B cells, and T cells grown in culture (240, 273, 274). During in vivo infection, Y. pseudotuberculosis appears to discriminate among cells it encounters and selectively translocates its effector proteins into professional phagocytes in PPs, MLNs and spleen (268). YopE, YopH, and YpkA alter signaling events that control cytoskeleton rearrangement (226). This inhibits the immediate bactericidal action of these cells (phagocytosis) and prevents initial killing of the bacteria. By translocation of YopJ, Yersinia can deactivate the NF-κB and mitogen-activated protein kinase (MAPK) signaling cascades of the host cell resulting in inhibition of the proinflammatory cytokine production and inducing apoptosis in macrophages (275-281). Apoptosis of macrophages in lymphoid organs of mice infected with Y. pseudotuberculosis has also been reported (281). YopM and YopK translocation into host cells result in inhibition of inflammasome activation in macrophages (282, 283).

It has been shown that during Y. pseudotuberculosis infection neutrophils are recruited in large numbers to the site of infection (284, 285). Two essential virulence effectors, YopH and YopE, impair the signaling machinery needed for phagocytosis by and degranulation of neutrophils, and mediate bacterial survival extracellularly in the lymph node (243, 245, 286, Paper I). Moreover, YopH blocks immediate early calcium signaling in neutrophils (236, 239). Y. pseudotuberculosis with defective YopD is impaired to mediate inhibition of ROS production in neutrophil-like cells and cell death in macrophages (287). YopJ, another important effector protein, which triggers apoptosis in macrophages and dendritic cells and inhibits inflammasome activation in macrophages, does not induce apoptosis in neutrophils but inhibits ROS production (288). The roles of YopH, YopK, and YopE have also been shown to be important for neutrophil targeting in the early stages of enteropathogenic Yersinia infections (285, 289). Moreover, Y. pseudotuberculosis blocks NET formation (290), while inducing survival of neutrophils (291).

4. Campylobacter jejuni

4.1. Biology and epidemiology Campylobacter species have been the focus of research for the past few decades. They can be isolated from human, animals, and environmental sources and are considered as an important human pathogen. The word Campylobacter is derived from two Latin words, where campylo means curved and bacter means rod, which reflects the microscopic appearance of Campylobacter as a Gram- negative spiral-, curved-, or rod-shaped bacterium (width of 0.2-0.8 μm, length of 0.5-5 μm) (292). Campylobacter could also appear as spherical or coccoid form when it is in the state of a viable but non-cultureable form. Transition to this form is the result of various stress conditions the bacteria might encounter. Since Theodor Escherisch first described it in 1886, the genus has grown to include multiple important human and animal pathogens (293). The genus Campylobacter contains at least 39 species and 16 subspecies (http://www.bacterio.net) and belongs to the family Campylobacteraceae, in the class of Epsilonproteobacteria. Except C. gracilis, which is a non-motile bacterium, Campylobacter species are motile with polar flagella at one or both ends (uni- or bi-polar flagella). C. showae is the only exception with multiple flagella. The motility of Campylobacter is a great advantage in colonization of the host intestine. Campylobacter can grow between 37°C to 42°C, with an optimal growth at 42°C for C. jejuni and C. coli. The majority of Campylobacter species are microaerophilic and capnophilic, with optimum atmosphere of 85% N2, 10%

CO2, and 5% O2 (294, 295).

Campylobacter jejuni is estimated to be responsible for 90% of the campylobacteriosis cases in humans, C. coli for the remaining 10% (296), and most of the available data are from C. jejuni infections. Campylobacteriosis is the most common infectious diseases in both developed and developing countries (297). While it is endemic in parts of Africa, Asia, and the Middle East, a significant increase in North America, Europe, and Australia has also been reported (298). In developed countries, contaminated animal products are assumed the main source of human infection while in the developing countries poor sanitation, contaminated water, and close contact of humans with animals are sources of infection.

C. jejuni strains 81–176 (serotype O:23/36) and 11168 (serotype O:2) are well characterized strains, and the most commonly used in pathogenesis studies. The genome size of C. jejuni is 1.6-1.7 Mbp with a GC ratio of ~30% (299). Almost 95% of the genome is encoding proteins. A distinct feature that might be involved in C. jejuni pathogenesis and immune evasion is the existence of hypervariable regions in the genome (299). Most of the hypervariable sequences are in regions

that encode proteins for biosynthesis or modification of capsule, lipooligosaccharides (LOS) and flagellum, which have key roles in C. jejuni interactions with hosts (300). Some C. jejuni strains harbor extra-chromosomal elements including plasmids and bacteriophages (301). C. jejuni 81-176 carries two plasmids, pVir and pTet (302). Plasmid pVir harbors ORFs encoding proteins orthologues to Helicobacter pylori T4SS (303). Plasmid pTet is a tetracycline resistance plasmid, which also encodes T4SS proteins (302).

4.2. Transmission and pathogenesis C. jejuni is considered as a commensal bacterium in avian hosts, e.g. chicken and poultries, where it rarely causes symptomatic disease (304, 305). It colonizes the chicken intestine in high numbers mainly in the mucosal layer and can be passed between chickens in a flock through the fecal-oral route. C. jejuni colonizes a wide variety of wild and domestic animals asymptomatically, and these animals may serve as reservoir (306). Humans are infected mainly via handling and consumption of contaminated poultry products, milk, and water, or contact with infected animals. Person to person infection is rare but cases are reported (298, 300) (Figure 9). In humans, C. jejuni can invade the intestinal epithelial layer to the mucosa by crossing the epithelial barrier, resulting in symptomatic infections with inflammation and diarrhea of different severities (307).

Figure 9. Campylobacter jejuni transmission and routes of infection in human.

An infective dose of 500-800 C. jejuni is sufficient to cause campylobacteriosis in humans (308-310). They can successfully compete with the human intestinal microbiota and colonize the human intestine. Symptoms of gastroenteritis such as abdominal pain, fever, vomiting, headache, and diarrhea develop one to three days after ingestion of bacteria. The severity of disease depends on both bacterial

invasiveness and the host immune response, and varies from mild self-resolving infection to inflammatory diarrhea. Occasionally an infection with C. jejuni can lead to extra intestinal manifestations such as septicemia, meningitis, cholecystitis, urinary tract infections, or septic abortions (300, 311, 312).

4.2.1. Post-infectious sequelae In rare cases, C. jejuni infection is associated with devastating post-infectious complications, including the acute inflammatory demyelinating polyneuropathy Guillain Barré syndrome (GBS) or Miller Fisher syndrome. It has also been suggested that C. jejuni infections have a role in the development of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn´s disease, unilateral facial paralysis Bell’s palsy, and reactive arthritis (300, 313-318).

To date, the post-infectious disease, which is most extensively studied, is GBS. It is an acute post-infectious ascending paralysis that can affect peripheral and cranial nerves and manifests as rapidly developing weakness and sensory disturbance in arms, legs, and sometimes in facial, bulbar, and respiratory muscles. GBS affects both children and adults and in most cases it is preceded by a bacterial or viral infection (319). C. jejuni infection is identified as the most common preceding illness in GBS patients. It has been estimated that almost 25– 40% of GBS patients world-wide have suffered from campylobacteriosis few weeks before the GBS onset (320). However, the risk of developing GBS after campylobacteriosis is only one to two cases per 100,000 persons (321). C. jejuni LOS contains a terminal tetra-saccharide identical to the one on the human GM1 gangliosides of neurons. It appears that, because of these molecular analogies, antibodies produced against C. jejuni LOS can also attack neuronal axons, leading to GBS (319, 321).

4.3. Virulence factors Although quite little is known about the virulence of C. jejuni, this microorganism possess different putative virulence factors related to motility, adhesion, invasion, toxin-activity, immune evasion, and iron-uptake.

Secretion systems: To date, several secretion systems have been identified in Campylobacter species including type 2, type 4, and type 6 secretion system. T2SS exists in almost all C. jejuni strains and is required for natural transformation. Some Campylobacter species contain T4SS. For example, C. jejuni strain 81-176 contains two plasmids (pVir and pTet), which are responsible for encoding the T4SS in this bacterium (303). T6SS in C. jejuni consists of both a needle-like structure that the bacterium utilizes to inject effector proteins into target cells, and a platform structure that connects the T6SS to the membrane of

the bacterium (322). The function of T6SS is not fully understood in Campylobacter, but has been suggested to contribute to the adhesion and invasion of human epithelial cells in vitro and mouse colonization in vivo, as well as to the adaptation to bile (323).

Flagella: To colonize the human intestine and establish infection, C. jejuni needs to be motile (309). The motility of C. jejuni is mediated by a combination of one or two polar flagella and the spiral shape of the bacterium that facilitates bacterial movement through the mucus layer (324, 325). The flagellum is 2.6-3.9 μm in length and ~21 nm in width (326) and considered as the primary adhesion factor (327). Flagellar motility is important for C. jejuni host cell invasion and colonization (328, 329). Non-motile C. jejuni mutants are attenuated for colonization of human and animal hosts (324, 325, 330, 331). Structurally, flagella are composed of FlaA (major flagellin), and FlaB (minor flagellin). They are highly homologous and approximately 59 kDa in size. Mutation of the flaA gene results in bacteria with a truncated flagella and severe reduction in motility but a flaB mutant has no significant disability in motility with a structurally normal Flagella (332). Although FlaA and FlaB are produced simultaneously, the expression level of flaB gene is lower than of flaA gene (333). C. jejuni lacks a type III secretion system (T3SS), commonly used by many Gram-negative enteropathogens. Instead, C. jejuni uses the flagellar apparatus to secrete virulence proteins to the surrounding milieu or into host cells (334). C. jejuni produces three sets of proteins that are secreted via the flagella and play roles in bacterial pathogenesis including Campylobacter invasion antigens (Cia), Flagella secreted protein A (FspA), and Flagellin C protein (FlaC). They induce host cell signaling, internalization of bacteria, apoptosis, and are critical for colonization of the ceca of chickens (334-336). C. jejuni flagella also help bacteria to evade the immune system by preventing activation of TLR5 signaling pathway (337).

Adhesins: Adhesion of C. jejuni to the host intestinal epithelium is a requirement for colonization. It is facilitated by multiple adhesins on the bacterial surface including Campylobacter adhesion to fibronectin protein (CadF), Campylobacter adhesion protein A (CapA), phospholipase A (pldA), Pei, Ellison and Blaser proteins 1-4 (PEB1-4), and many more (338). One of the most characterized adhesins in C. jejuni is CadF. It binds specifically to fibronectin on epithelial cells and triggers activation of the GTPases Rac1 and Cdc42 and internalization of bacteria (339-341). CadF is crucial for bacterial colonization and CadF mutants are shown to be unable to colonize chicken in vivo and in human intestinal epithelial cells in vitro (339, 342).

Capsule: Many pathogens express capsule polysaccharides (CPSs), which act as a protective shield on the bacterial surface and play roles in escaping phagocytosis, serum resistance, and in some cases antimicrobial peptide

resistance (343). C. jejuni is also surrounded by a capsule consisting of polysaccharides that is associated with survival and host colonization (344). Although the capsule might be crucial for successful adhesion to epithelial cells, it does not provide protection against initial host defense mechanisms such as potent antimicrobial action of human β- defensins 2 or 3 (345). Capsule genes are split in three different regions: the first and the last regions encode proteins involved in capsular assembly and transport whereas the central region is highly variable and is involved in polysaccharide synthesis. It has been shown that the C. jejuni capsule is essential for colonization of the host and mutations of the capsule synthesis genes significantly reduce the colonization ability of C. jejuni (338, 346).

LOS: LPS is the main component of outer membranes in Gram-negative bacteria. It comprises of a lipid A core, an oligosaccharides core and an O-polysaccharide chain or O-antigen (347, 348). Lipooligosaccharides (LOS) is analogous to LPS but lacks the O-antigen and comprises fewer saccharide units in the oligosaccharide core (349, 350). LOS of C. jejuni is highly variable and a target for modifications. In C. jejuni, LOS has roles in adhesion, invasion, and colonization of the host, protection of the bacterium from complement-mediated killing, and surviving in harsh environments (351, 352). Furthermore, Campylobacter with sialylated LOS have been suggested to have a higher potential for invasiveness and higher serum resistance ability (353, 354).

Glycosylation system: C. jejuni has two protein-glycosylation systems, the N- linked glycosylation system, which modifies serine or threonine residues, and the O-linked glycosylation system, which modifies asparagine residues (300). The N- linked glycosylation machinery is conserved in almost all C. jejuni strains and encoded by a single gene cluster named pgl cluster (355). The role of the N- glycosylation system is unclear in C. jejuni biology but it is demonstrated that it has a role in bacterial virulence. C. jejuni glycosylation mutants have reduced ability in adhesion and invasion of human intestinal epithelial cells in vitro and colonization of the chicken intestine (356-358). Involvement of this system in escape of bacteria from the host immune system is also suggested (359). In contrast, the genetic locus of the O-linked glycosylation system is more heterogeneous and genetically diverse (360). O-linked glycosylation is vital for successful flagellin assembly and motility, adhesion, invasion and virulence of bacteria (361)

CDT: A well-characterized toxin commonly produced by C. jejuni is the cytolethal distending toxin (CDT), which has also been found in several other Gram- negative bacteria (362). It is composed of three subunits. CDTA and CDTC are responsible for toxin binding to the target cell membrane and for the delivery of CDTB, which is the enzymatically active subunit (338, 363). CDTB enters the

nucleus of a target cell and by deoxyribonuclease activity induces DNA damage, leading to the activation of DNA repair responses and cell cycle arrest at the G2/M phase (338, 364). For CDT to be functionally active in C. jejuni, all three subunits must be biologically active (338). C. jejuni CDT has been shown to cause a rapid and specific cell cycle arrest in epithelial cells such as HeLa and Caco-2 cells (365), however its function on immune cells is not known.

Iron uptake system: The regulation and transport of iron is important for host colonization. An increase in transcription of hemin and hemoglobin receptor genes have been observed in both C. jejuni infected chicken and human (366, 367). Mutations in genes responsible for iron uptake in bacteria results in a lower ability of colonization in chickens (368, 369).

Multidrug and bile resistance: Resistance to bile salts, heavy metals and other antimicrobial agents is often mediated by the Campylobacter multidrug efflux pump (CME) encoded by the cmeABC operon, and expression of this operon is repressed by CmeR (338, 370). CME consists of three subunits including a periplasmic protein (CmeA), an inner membrane efflux transporter (CmeB) and an outer membrane protein (CmeC), and presence of all three subunits is required for CME to be functionally active (370).

4.4. Infection models Our knowledge about C. jejuni-human interaction is limited due to the lack of animal models that resemble the human disease (371). Chickens are the main host of Campylobacter and well suited to study colonization, while other models such as newborn pigs, weanling ferrets, gnotobiotic canine pups, and primates have numerous limitations including handling issues, and lack of reproducibility (371, 372). Mice are used but have limitations and show colonization resistance against C. jejuni mainly because of their microflora (373). Germ free mice has helped to overcome this problem to some extent but as they have an abnormal development of gut-associated lymphoid tissue and therefore may not be suitable because of lack of intact innate immune system (372, 374). Mice with defective MyD88 or IL-10 are used, but they do not have an intact immune system (375, 376). A mouse model with an intact immune system and complete human gut flora developed by Bereswill and colleagues may provide a better idea of the pathogenesis of Campylobacter (373). Three weeks old (weaned) mice show stable colonization, and significant clinical immunopathology mimicking human campylobacteriosis, and may provide the most useful murine C. jejuni infection model (377).

In vitro infection models with different cell lines are widely used to study adhesion, invasion, and induction of host cellular response. C. jejuni interacts

with different types of eukaryotic cells by inducing different cellular responses (378). For studies of Campylobacter adhesion to and invasion of the epithelial lining, intestinal epithelial cell lines such as HT-29, T84, and Caco-2, which can form tight and adherent junctions, are used (378, 379). Moreover, the polarized HT-29-MTX-E12 (E12) cell line that produces a mucus layer has been used for C. jejuni mucosal colonization studies in vitro (378, 380). In addition, immune cell lines, e.g. J774 and THP-1 and primary cells (monocytes, macrophages, dendritic cells, and neutrophils) are used to investigate C. jejuni-mediated immune responses in vitro. Interleukin 8 (IL-8) is a proinflammatory cytokine involved in recruitment of neutrophils and macrophages into the site of infection and is produced by both epithelial and immune cells. IL-8 levels are commonly used as an outcome of cellular responses to C. jejuni infection in vitro.

4.5. C. jejuni-host interactions The pathogenesis of Campylobacter is still largely unrevealed. Several studies have shown involvement of multiple putative factors in colonization and establishment of infection by this bacterium. The severity of disease caused by Campylobacter seems to be related to the genetics of this bacterium (381), infectious dose (309), and the host gut microbiota (382, 383).

C. jejuni mainly colonizes the distal ileum and colon in human (309, 384). The gastrointestinal tract contains surplus of nutrients, and the intestinal microbiota stimulates the expression of C. jejuni growth and colonization factors (306, 385, 386). To establish infection, C. jejuni has to survive in the acidic condition of the stomach during the passage into the intestine in humans. It has been reported that heat shock proteins may play a role in acid resistance of C. jejuni (387). Mucus is one of the defensive barriers of the gastrointestinal tract. C. jejuni crosses through the mucus layer using its corkscrew shape and darting motility (326). Once C. jejuni has passed through the mucus barrier, it will be in direct contact with the intestinal epithelial cells and has to overcome multiple host defense mechanisms to survive and establish a successful infection. Bacterial factors related to motility, glycosylation, and capsule are involved in C. jejuni adhesion to and invasion of epithelial cells (357, 388), and bacteria with mutations in these genes are defective in adherence and invasion of host cells (331, 389, 390).

One of the common adhesion mechanisms enteropathogenic bacteria use to adhere to intestinal epithelial cells are pili. Until now, C. jejuni has not been reported to contain any genes required for pilus synthesis. Instead, C. jejuni adhere to host cells mainly by using adhesin-receptor interaction. Multiple adhesins have been studied extensively in vitro and in vivo in C. jejuni and shown to play important roles in binding to host cells including CadF, JlpA and CapA

and PEB1-4. CadF binds specifically to Fibronectin (391). In vitro, CadF binds to fibronectin on the basolateral surface of cells (392). CadF is also crucial for C. jejuni to colonize the cecum of newly hatched chickens (342). The lipoprotein JlpA is required for binding onto epithelial cells and inducing activation of proinflammatory factors, nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinases (393, 394). CapA is an autotransporter and plays a role in host colonization by C. jejuni. It has been shown that a capA mutant is attenuated in chickens (395). C. jejuni major antigenic proteins PEB1 to PEB4 are important for adhesion of bacteria to host cells. PEB1 is an adhesin that plays an important role in the interaction with epithelial cells and in intestinal colonization in mice (396). PEB4 is involved in C. jejuni adhesion to epithelial cells, biofilm formation and colonization in a mouse model (397), and mutations in the peb4 gene result in reduced motility and host cells invasion by C. jejuni (398).

The translocation ability of C. jejuni has been studied in polarized Caco-2 colonic epithelial cells. It has been shown that clinical isolates of C. jejuni are able to translocate at a much higher frequency than non-clinical isolates (399). Flagella and capsule are two important factors involved in bacterial invasion (324, 388, 390). C. jejuni can transmigrate via both the trans-cellular and the para-cellular route (307, 400, 401). In addition, Microfold cells (M cells) seem to be implicated in translocation by C. jejuni in rabbits (402). In epithelial cells, C. jejuni can reside in a cytosolic compartment known as Campylobacter-containing vacuole (CCV) where it further prevents its delivery to lysosomes (390). Unlike in epithelial cells, C. jejuni appears to be unable to avoid delivery to lysosomes in professional phagocytes and is rapidly killed after engulfment (390, 403-407).

Different studies have demonstrated that C. jejuni has the potential to invade epithelial cells in vivo and in vitro. It has been shown that C. jejuni can invade the colonic mucosa and reside mainly in the submucosa without any major disruption of the mucosal barrier (408). Macaca mulatta monkeys orally challenged with high doses of C. jejuni showed clinical signs of campylobacteriosis, and C. jejuni were located both extracellularly in the mucosa and submucosa, and intracellularly, either in a membrane-bound vacuole, or free in the cytosol of epithelial cells (409). C. jejuni adhesion to intestinal epithelial cells induces the production of IL-8 resulting in recruitment of macrophages, dendritic cells, monocytes and neutrophils, which interact with C. jejuni. CDT and the surface-exposed heat-shock protein adhesion factor JlpA are responsible for the IL-8 production from the human epithelial cells (394, 410).

C. jejuni is equipped with mechanisms to avoid or delay detection by the immune system. Alteration in the flagellin recognition site and the heavy glycosylation of the flagella prevent flagellum-mediated activation of TLR5 (337, 355, 411). C. jejuni is also a poor TLR 9 stimulator due to its AT-rich genome that lacks the

unmethylated CpG dinucleotides responsible for TLR9 activation (412). However, the intracellular pattern recognition receptor NOD1 plays a critical role in the activation of the immune response by C. jejuni (413).

C. jejuni interaction with immune cells triggers a massive proinflammatory response through the activation of NF-κB, the cytokines IL-1ß, IL-6, IL-10, IL-12, and TNF-α (300, 414). Monocytes and macrophages are the key innate immune cells C. jejuni interacts with (415). Clinical isolates of C. jejuni survived for several days in murine peritoneal macrophages, human monocytes, and the macrophage cell line J774A.1 (403, 416, 417). In contrast, C. jejuni was killed immediately by macrophages derived from human monocytes (403). The possible reasons for the different observations might be the use of different C. jejuni strains and/or the different macrophage-like cell lines. Moreover, monocytic cells could be important contributors to the induction of gut inflammation because they express a range of cell receptors, e.g. CD14 and CD11a, which are involved in cell signaling in response to different PAMPs (418). The role of monocytes and macrophages in C. jejuni infection differs between cell lines and primary cells (300). Several studies have demonstrated that stimulation of the monocyte cell line THP-1 with C. jejuni activates NF-κB and induces IL-1β production (407, 419). However, a significant proportion of C. jejuni-infected monocytic cells also underwent apoptosis (407, 417). Dendritic cells produced IL-1β, IL-6, IL-8, IL-10, and a high level of IL-12 after internalization of live or heat-killed C. jejuni (414, Paper IV). The high levels of IL-12 indicate a Th1-polarized immune response in humans (414). Recently it has been shown that C. jejuni can induce inflammasome activation and IL-1β secretion in macrophages without cell death (420). However, the interaction of C. jejuni with innate immune cells in vivo is not well understood yet.

In addition, an adaptive immune response is observed during C. jejuni infection in humans, although multiple levels of variation in bacterial surface structure help the bacterium to evade antibody responses (300). Anti-Campylobacter specific antibodies can be detected in the serum of patients by the second week after infection (421). These antibodies are produced against several major C. jejuni antigens, including flagellin, LOS, major outer-membrane protein (MOMP), outer membrane proteins (OMP), and CDT (354, 422-426). Levels of specific immunoglobulin IgA, IgM and IgG can reach their peak within 8-14 days post-infection (427). IgA returns to a basal level within 20 days, and IgM gradually decreases within two months to a basal level. In contrast, IgG activity declines over the period of one year (428). Several studies suggest a protective role for these antibodies (309, 429, 430). The role of adaptive immunity in vivo in terms of T-cell subsets and their responses is not yet explored.

4.6. C. jejuni OMVs OMVs are constitutively released during the growth of C. jejuni (Figure 10) and consist of LOS, OMPs, lipids, and periplasmic proteins (131, 132, Paper II, Paper III). It has been shown that C. jejuni produces OMVs that contain biologically active CDT. The release of OMVs by C. jejuni is a route to deliver all CDT subunits to the surrounding environment, and to cause the typical cytolethal distending effects during in vivo and in vitro infections (95). Moreover, it has been demonstrated that apart from CDT, OMVs secreted by C. jejuni also contain serine proteases, e.g. HtrA which has proteolytic activity on epithelial cells (431- 433). OMVs from C. jejuni can trigger the production of a range of proinflammatory molecules, such as IL-8 in human epithelial cells resulting in modulation of the immune response (131, 132).

Figure 10. Electron micrograph of OMVs isolated from C. jejuni strain 81-176 (Linda Sandblad, Umeå University). The scale bar represents 200 nm.

Recently, it has been shown that OMVs from C. jejuni are highly immunogenic, and have a protective effect in chicken (434). Proteomic analyses of C. jejuni OMVs showed strain specific packaging of OMVs including periplasmic and outer membrane-associated proteins, but also many proteins known to be important for survival and pathogenesis (131, 132, Paper II, paper III).

Aims of this thesis

The over-all goal of this thesis was to gain further insights into host-pathogen interactions during enteropathogenic bacterial infections, using Campylobacter jejuni and Yersinia pseudotuberculosis as model organisms.

In order to address the over-all goal of the thesis, the main objective was subdivided into the following specific aims:

 To investigate the effect of Y. pseudotuberculosis on neutrophil degranulation

 To investigate OMV production and OMV protein content by C. jejuni in response to exposure to low concentration of bile

 To investigate the influence of growth temperature on C. jejuni OMV protein content

 To analyze the role of C. jejuni OMVs on inflammasome activation in immune cells

Results and discussion

Paper I: Y. pseudotuberculosis blocks neutrophil degranulation Here we investigated the effect of Y. pseudotuberculosis on neutrophil degranulation and showed that Y. pseudotuberculosis can block the release of both primary and secondary granules by neutrophils (Paper I, Figure 1A). This was determined by detection of myeloperoxidase and lactoferrin in the supernatants of infected neutrophils as markers of primary and secondary granules, respectively. A similar tendency was seen for the release of tertiary granules that was determined by detection of matrix metalloproteinase-9 (Paper I, Figure 1A). As a degranulation inducer, avirulent Y. pseudotuberculosis (virulence plasmid-cured) was used and inhibitory effects of virulent Y. pseudotuberculosis were compared to plasmid-cured bacteria. Together with the antiphagocytic trait of Y. pseudotuberculosis (237, 435), and its ability to block neutrophil NET formation (290), blocking of degranulation renders Y. pseudotuberculosis well equipped to disrupt multiple defensive strategies employed by neutrophils. Analyzing the effect of neutrophil-degranulated products on Y. pseudotuberculosis survival showed significant reduction of bacterial viability (Paper I, Figure 1C), which highlights the importance for Y. pseudotuberculosis to block neutrophil degranulation for its survival and for establishing a successful infection.

For degranulation, granules translocate to phagosomes or the plasma membrane. Analyses of neutrophils infected with Y. pseudotuberculosis for translocation of granules toward the plasma membrane revealed that Y. pseudotuberculosis wild- type strain could block translocation of secondary but not primary granules towards the plasma membrane (Paper I, Figure 1B, 3A). This is consistent with our finding that degranulation of secondary but not primary granules is a very rapid process. Already after 15 minutes post infection with plasmid-cured bacteria we could detect lactoferrin in cell supernatants. This was a consequence of a quick translocation and fusion of secondary granules with the plasma membrane. Accumulation of CD66b on the plasma membrane of neutrophils in response to avirulent bacteria but not virulent Y. pseudotuberculosis confirmed that CD66b-positive secondary granules fussed with the plasma membrane.

It has been shown that virulent wild-type Y. pseudotuberculosis adheres to cells less than the avirulent plasmid-cured bacteria (237). We artificially increased the attachment of wild-type bacteria to neutrophils by opsonization using anti- Yersinia antisera prior to infection (Paper I, Figure 3B). However, despite their increased adherence to neutrophils, no granule accumulation was detected in

neutrophils in contact with opsonized wild-type bacteria (Paper I, Figure 3C). Furthermore, the level of lactoferrin was still very low in supernatants from neutrophils infected with opsonized wild-type Y. pseudotuberculosis (Paper I, Figure 3D). We concluded that the wild-type strain efficiently modulates the degranulation machinery in order to prevent exocytosis of secondary granules.

It was clear that the presence of the virulence plasmid was required for the observed inhibitory effect, as plasmid-cured bacteria could still induce degranulation. The role of individual virulence effectors encoded by this virulence plasmid was investigated by infecting neutrophils using yopH, yopE, yopM, yopJ, yopK, and ypkA mutant strains. This revealed involvement of YopH and YopE in translocation of secondary granules towards the plasma membrane, as well as in the exocytosis (Paper I, Figure 4A, 4B). Moreover, the additive effect of YopE and YopH seen for both granule accumulation and release suggests their co-operation in blocking degranulation. Upon contact of the bacterium with the host cell, translocation and subsequent activities of YopH and YopE are instant (436). It is therefore likely that the translocated virulence effectors by the wild- type strain act on immediate early signals coming from receptors that the bacterium is bound to at the surface of neutrophils. This is an analogous situation previously shown for YopH-mediated blockage of macrophage and neutrophil internalization (230, 236, 239, 240). Both YopH and YopE are major effectors responsible for inhibition of phagocytic uptake (231, 236, 437). It is likely that these effectors target neutrophil signaling molecules that are important for the initiation of both phagocytosis and degranulation, ensuring that both of these antimicrobial functions are disabled.

To dissect the underlying mechanisms behind the inhibition of degranulation initiated by wild-type Y. pseudotuberculosis, we used inhibitors that target different signaling pathways. Inhibition of calcium signaling using the calcium chelator BAPTA-AM, to clamp the level of intracellular calcium showed significant decrease in release of secondary granule content (Paper I, Figure 5A). The same results were observed by using the general tyrosine kinase inhibitor genistein, cytochalasin D to impair actin dynamics and block phagocytosis, and PI3K-specific inhibitor LY294002 (Paper I, Figure 5C, 5E, 5G). Surprisingly, the accumulation of CD66b-positive granules at the bacterial contact site was unaffected by all those inhibitors (Paper I, Figure 5B, 5D, 5F, 5H). Taken together, inhibition of those pathways known to be targeted by effectors (calcium, tyrosine kinase signaling, and actin dynamics), together with a blockage of PI3K signaling, resulted in the prevention of secondary granules release, but not accumulation. Since wild-type Y. pseudotuberculosis blocked secondary granules accumulation via YopH and YopE, these data suggest that there might be other, yet unknown targets for these effector proteins, which are specifically involved in granule accumulation. In contrast to secondary granules accumulation, we noted an effect

of plasmid-cured bacteria on primary granule accumulation in the absence, but not in the presence of cytochalasin D. Thus, suggesting that recruitment of these granules is induced by phagocytosis. This was in contrast to secondary granules for which accumulation induced by the plasmid-cured strain was not blocked by pre-treatment with cytochalasin D, suggesting that the bacterial contact itself induces movement of these granules.

Neutrophils, at sites of infection, are expected to be primed due to pre- stimulation by low concentrations of bacterial products and inflammatory factors (438). Analyses of LPS-primed neutrophils prior to infection with plasmid-cured or wild-type bacteria showed that the priming per se increased the degranulation by both wild-type and plasmid-cured bacteria, but the level of released lactoferrin was still considerably lower for cells infected with the wild-type strain (Paper I, Figure 2A). The block of degranulation by Yersinia seemed to be very prominent, as even primed neutrophils are efficiently targeted. In the in vivo situation, neutrophils recruited to the site of infection are expected to arrive pre-activated and ready to attack invaders (438). Hence, the ability to block primed neutrophils shows a very essential aspect of Yersinia's ability to evade neutrophil defenses. Furthermore, we showed that neutrophils encountered wild-type Y. pseudotuberculosis was unresponsive to plasmid-cured Y. pseudotuberculosis and E. coli in term of degranulation (Paper I, Figure 2B, 2C).

Thus, Yersinia uses one strategy to immediately neutralize several mechanisms of neutrophil's antimicrobial function. We have previously shown that Y. pseudotuberculosis has an anti-apoptotic effect on neutrophils that is independent of Yops (291). The increased survival of neutrophils induced by the bacteria is assumed to benefit Yersinia pathogenicity, by preventing cell death, thereby promoting the retention of danger signals that would otherwise amplify immune responses. Our present finding, that Yersinia uses both YopH and YopE to inhibit degranulation, together with their previously shown inhibitory effects on phagocytosis, suggest that Yersinia uses these effector proteins to specifically target antimicrobial functions. Hence, Yersinia manipulates neutrophils in a highly sophisticated fashion, affecting neutrophil functions in both positive and negative manner. Such dual effects might allow both a direct avoidance of antimicrobial effects, while indirectly preventing any amplification of innate immune responses by suppressing the danger signals.

Paper II: Exposure of C. jejuni to low concentration of bile influences OMVs protein content and bacterial interaction with epithelial cells During passage through the GI tract, ingested enteropathogenic bacteria face multiple challenges, e.g. the acidic condition of the stomach, increased osmolarity, low oxygen, nutrient competition, the immune response, and exposure to several potentially harmful compounds such as bile, proteolytic enzymes, as well as antimicrobial peptides. In order to establish infection, enteropathogenic bacteria have to survive in these harsh conditions. Studying OMV content isolated from C. jejuni exposed to some of these stressors including low pH (4 and 5.5), bile, and polymyxin B showed that only bile alters the OMV protein profile (Figure 11, lane 4). C. jejuni resides in the large intestine, and its resistance to bile is a key factor for this enteropathogen to survive and establish infection. We analyzed how C. jejuni 81-176 responded to low bile concentrations in terms of OMV production and protein content, and whether that affected the virulence of the bacterium.

Figure 11. Protein profiles of OMVs isolated from untreated, 5 µg/ml Polymyxin B-treated, 0.05 % bile-treated, pH 5.5-treated, and pH 4.0-treated C. jejuni 81-176 were compared by 12% SDS-PAGE followed by visualization of proteins using silver staining. Lines to the left indicate the molecular masses of the protein standards in kDa. Lanes 1-4 represent whole cell lysate (1), supernatants after filtration (2), supernatants after ultracentrifugation (3), and OMVs (4).

First, we analyzed bacterial viability and OMV production after exposure to different concentrations of ox-bile (0.00625 to 0.5%) at 37°C for 20 hours (mid- exponential phase) under microaerobic conditions to resemble the human body

temperature and milieu. Although viability of bacteria and production of OMVs were altered by 0.5% of bile, no significant changes in viability (Paper II, Figure 1A) or OMV production (Paper II, Figure 1B) were seen after 20 hours incubation in 0.025% bile. OMVs isolated from 20 hours cultures with 0.025% ox-bile were chosen for further analyses. The concentration of bile salts is gradually declining along the intestine, estimated to be 20 mM in the duodenum, 4 mM in the ileum, and less than 1 mM in the cecum (3, 4, 439). The use of 0.025% ox-bile to study the effects on the OMV proteome is equal to approximately 0.5 mM bile salts. Thus, our experimental settings were reflecting a cecal milieu. Analyses of size and particle numbers of OMVs using Nanoparticle-tracking analysis (NTA) showed slight but not significant increase in the size of OMVs by bile (Paper II, Figure S1). Moreover, TEM analyses of OMVs showed similar morphology of OMVs from both conditions (Paper II, Figure 1C). Thus, growth of C. jejuni in low concentrations of bile does not affect production and size distribution of OMVs.

Next we measured the protein content of OMVs isolated from C. jejuni grown in the presence of bile (bile-treated OMV) or in the absebce of bile (untreated OMV). The same numbers of untreated and bile-treated OMVs were analyzed by SDS- PAGE and showed extensively different profiles (Paper II, Figure 2A). We therefore conducted a more detailed analysis by LC-ESI-MS/MS on OMV preparations from both conditions. 237 proteins were detected in bile-treated OMVs while untreated OMVs exhibited 229 proteins. Among them, 225 proteins were detected in both types of OMVs (Paper II, Table S1). The abundance of 56 out of 225 common proteins were altered by bile. Of these, 33 proteins showed increased, and 23 decreased abundance in bile-treated OMVs compared with untreated OMVs (Paper II, Figure 2D and Table S3). Proteomic analysis of bacteria grown with or without bile for 20 hours showed the same number (443) of identified proteins under both conditions (Paper II, Table S4) with no change in abundance of proteins using the same filtering parameters as used for OMVs. Thus, C. jejuni responds to low concentration of bile by changing the protein composition of OMVs only. OMVs are released by bacteria over the course of normal growth, and different stressors can affect this production. Here we show that low concentration of bile modulates the OMV protein content without affecting bacterial protein expression. The protein with highest increase in abundance in OMV after bile treatment was Cpp47, which is a 206 aa hypothetical protein encoded by the pTet plasmid. Further, CmeA and CmeC, important for bile resistance, were found in OMVs but only CmeA was up-regulated in bile- treated in response to bile.

OMVs released by bacteria upon exposure to low concentration of bile appear not only to contain certain proteins with altered abundance, but also to be packed with new proteins. In addition to 225 common proteins, we detected twelve proteins exclusively in bile-treated OMVs (Paper II, Table S7). The majority of the

twelve proteins were cytoplasmic proteins assigned to be involved in cell wall/membrane/envelope biogenesis. Among those, were GalU and GlmU, involved in LOS biosynthesis, and WaaD, involved in capsule biosynthesis. GalU has a role in resistance to antimicrobial peptides in C. jejuni (440) and in other enteropathogenic bacteria (441-443). The consequence of having these proteins selectively packed in OMVs upon bile exposure is not clear at this point and requires further investigations. Moreover, four proteins were found in untreated OMVs but not bile-treated OMVs. Three of them were cytoplasmic proteins involved in translation, ribosomal structure and biogenesis.

Analyses of the subcellular localization of differentially expressed proteins in OMVs showed different patterns among the proteins with increased and decreased abundance (Paper II, Figure 3A). The majority of proteins with increased abundance by bile were cytoplasmic proteins (76%). Although the mechanism for packing OMVs is still unclear, the increase in cytoplasmic proteins in OMVs upon exposure to bile suggests that bile exposure influences the packing of the vesicles. Bile is a detergent and can induce bacterial lysis. Live/dead staining of bacteria showed no differences in the amount of dead bacteria between different conditions (Paper II, Figure 3B, 3C) indicating that cytoplasmic proteins are sorted into OMVs and higher level of cytoplasmic proteins are not a consequence of bacterial lysis upon growth in low bile.

Of the 225 common proteins, we identified 23 known virulence-related proteins (Paper II, Table S6). Among them, 30% were decreased in abundance by bile, while 65% were unchanged. This indicates that the abundance of some known virulence proteins of C. jejuni is lower in OMVs from bacteria exposed to low concentration of bile. Since the total capacity of OMVs to load proteins appears not to be altered, it is possible that bacteria deliberately decrease the amount of proteins related to virulence upon exposure to bile. For example, CDT has been shown to be associated mainly with OMVs (95). We identified all three subunits of CDT in OMVs with decreased abundance by bile (Paper II, Table S3). We suggest that bile influences selective packaging of cellular components into OMVs of C. jejuni. This is in accordance with several other studies, which suggest selective packaging of OMVs (98, 110, 444-446).

Incubation of epithelial cells with bacteria grown with or without bile showed that bacteria grown in the presence of bile can adhere more efficiently to epithelial cells (Paper II, Figure 5A), an effect, which has also been reported for other bacteria. This can be a consequence of increased bacterial cell surface hydrophobicity by bile (Paper II, Figure 5B). Interestingly, co-incubation of C. jejuni unexposed to bile with bile-treated OMVs showed increased adhesion of bacteria to the same extent as bile-exposed bacteria (Paper II, Figure 5A). The underlying mechanism is not determined. The increased membrane

hydrophobicity of OMVs (Paper II, Figure 5B) might contribute to the enhanced adhesion of bacteria to epithelial cells. It is likely that this is caused by binding of OMVs to the bacteria facilitating interaction with the epithelial cells. This effect likely has implications for the infection situation where the bile concentration at the site of colonization is low. The increase in adhesiveness caused by bile exposure was not associated with increased cell invasion by bacteria, while addition of bile-treated OMVs elevated invasion by C. jejuni unexposed to bile (Paper II, Figure 5C). Thus, C. jejuni modifies the protein content of OMVs upon exposure to bile, which in turn enhances the adhesion to and invasion of the intestinal epithelium by C. jejuni.

Taken together, this study highlights how environmental stressors influence bacterial traits during infection. Here we show that environmental concentrations of bile affect C. jejuni adhesive characteristics, lead to changes in the OMV content, and that released OMVs have the capacity to enhance adhesion of bacteria not adapted to the environment.

Paper III: Accumulation of virulence-associated proteins in C. jejuni OMVs at human body temperature C. jejuni asymptomatically colonizes avian hosts but causes gastroenteritis in humans. The temperature is one of several parameters that differs between avian and human hosts and might have an impact on virulence of this bacterium. In this study, we analyzed whether different temperatures influence C. jejuni 81-176 OMVs production and content. C. jejuni 81-176 was grown under microaerobic condition for 20 hours at 42°C (avian body temperature), or at 37°C (human body temperature). The results showed a slight increase in growth (Paper III, Figure 1A) and OMV production (Paper III, Figure 1B) at 42°C compared to 37°C. Transmission electron microscopy of the OMVs showed no remarkable morphological difference between the two conditions (Paper III, Figure 1C).

Analyses of OMV protein profiles by SDS-PAGE showed comparable protein distribution patterns at 37°C and 42°C (Paper III, Figure 2A). By LC-ESI-MS/MS analysis of OMVs isolated from bacteria grown at 42°C and 37°C, we identified 181 proteins in both groups (Table S), one protein exclusively at 37°C and three proteins at 42°C (Paper III, Table 1). Comparison of the subcellular localizations and functional categorizations of two OMVs components revealed similar distribution pattern (Paper III, Figure 2B) and function (Paper III, Figure 2C) of the proteins. The majority of the detected proteins in both sample groups were predicted to be cytoplasmic proteins, with translation, ribosomal structure and biogenesis function. Thus, bioinformatic analysis of the whole proteomic data set showed that bacteria pack OMVs with proteins similar in function and cellular localization independent of the bacterial growth temperature.

Despite a similar protein distribution among the two growth temperatures of bacteria, a detailed comparative bioinformatic analysis of the 181 common proteins revealed that there were significant differences in the abundance of proteins between the OMV groups. Of these, 59 proteins were differentially regulated by temperature, with 30 proteins detected at higher amounts and 29 proteins at lower amounts at 37°C compared with 42°C (Paper III, Figure 3A, Table 2). Further analyses revealed that the differentially expressed proteins varied in subcellular distribution (Paper III, Figure 3B) and functional characteristics (Paper III, Figure 3C, 3D).

In silico analysis of the differentially regulated proteins revealed that more than 50% of the proteins with higher abundance at 37°C were predicted to be related to virulence (Paper III, Figure 4A, Table 3). This is particularly noteable as the presence of higher virulence-related proteins in OMVs produced in humans might be important for the outcome of the infection. Hence, we observed that OMVs from C. jejuni 81-176 induce IL-1β secretion in mouse macrophages and dendritic cells (Paper IV). Consistently, OMVs from bacteria grown at 37°C induced pronounced secretion of IL-1β in immune cells, while OMVs from

bacteria grown at 42°C did not (Paper III, Figure 4B) suggesting that OMVs from bacteria grown at 37°C, which contain more proteins related to virulence, may have a higher potential to induce inflammation during infection in humans.

Taken together, our study highlights the influence of environmental factors on OMV packaging by bacteria. We found that the host body temperature affects abundance of C. jejuni OMV proteins, and that OMVs isolated from bacteria grown at 37°C have higher amounts of proteins related to virulence. Moreover, the higher induction of IL-1β secretion in response to OMVs from bacteria grown at 37°C compared to 42°C agrees with the infection situation where the infection in humans but not avians is associated with inflammation.

Paper IV: C. jejuni OMVs activate inflammasome without inducing cell death in immune cells OMVs are produced in vivo at the site of infection by several pathogenic bacteria e.g. Helicobacter pylori, Neisseria meningitidis, Moraxella catarrhalis, Borrelia burgdorferi, and Salmonella (135). It has also been shown that B. burgdorferi OMVs in vivo can disseminate to other tissues from the site of infection (143). C. jejuni OMVs have been shown to disrupt epithelial tight junctions by proteolytic activities to promote bacterial migration through epithelial monolayers (431). Thus, in vivo, C. jejuni OMVs potentially due to their small size could easily get access to the underlying tissues to interact with and affect immune cells distant from the primary infection site. While the role of C. jejuni OMVs in the interaction with epithelial cells has been studied, the role of C. jejuni OMVs in the interaction with immune cells including macrophages, dendritic cells, and neutrophils has not been investigated. Here we show that C. jejuni OMVs interact with dendritic cells, macrophages, and neutrophils by means of activating inflammasomes to the same extent as live bacteria.

We investigated whether C. jejuni OMVs mediate the production of mature IL-1β in bone marrow-derived macrophages (BMMs) and dendritic cells (BMDCs) and found that stimulation of LPS-primed BMMs and BMDCs using OMVs isolated from C. jejuni 81-176 resulted in activation and secretion of IL-1β into cell supernatants (Paper IV, Figure 1A, 1B), as did live bacteria. Prolonged incubation resulted in increased activation of IL-1β (Paper IV, Figure S1A). Further analyses showed that both OMVs and bacteria could also induce rapid secretion of cleaved IL-1β in LPS-primed human monocytes, dendritic cells and neutrophils (Paper IV, Figure 1E, 1F, 1G). The induction of IL-1β release from immune cells upon exposure to C. jejuni or to OMVs was much more pronounced in BMDCs compared to BMMs. The levels of IL-1β released by BMDCs almost reached the levels seen with ATP, which was used as a positive control as it is a potent inflammasome activator. While no LPS-induced IL-1β secretion could be detected in BMMs, LPS priming itself induced low level of active IL-1β release by BMDCs, but OMVs and bacteria significantly increased this release.

In contrast to IL-1β activation by OMVs in LPS-primed cells, no secretion of active IL-1β was induced in unprimed cells after stimulation with neither OMVs nor bacteria (Paper IV, Figure 1C, 1D). However, addition of ATP to cells stimulated with bacteria or OMVs resulted in cleavage and release of mature IL- 1β, suggesting that both stimulants could efficiently prime and induce expression of pro-IL-1β in immune cells. While OMVs as well as bacteria induce pro-IL-1β transcription (priming) as efficient as LPS, induction of IL-1β maturation and secretion clearly depend on an initial priming event. This could mean that disseminated OMVs into the tissue do not induce inflammasome activation

without the contribution of bacteria. This could be of major importance during infection in which OMVs are used as vehicles for virulence factors without inducing prominent inflammasome activation. Together, our findings show that C. jejuni OMVs induce activation and secretion of IL-1β in LPS-primed mouse and human immune cells, but not in unprimed cells.

Inflammasome activation in dendritic cells and macrophages by OMVs did not lead to cell death, although the cells actively secreted IL-1β. Measurement of LDH release after 4h exposure of LPS-primed macrophages and dendritic cells confirmed that the presence of IL-1β in the supernatants was due to activated release by OMVs and bacteria and not by cell lysis (Paper IV, Figure 1H). Interestingly though, while prolonged stimulation of LPS-primed cells for 24 hours with bacteria resulted in significant increase in LDH release by BMMs and BMDCs, prolonged stimulation with OMVs did not have that effect, suggesting induction of cell death by bacteria but not OMVs upon prolonged stimulation (Paper IV, Figure S1B). The cytotoxicity we noted for primary immune cells exposed to live bacteria is in accordance with previous findings (414, 419). Hence, whether activation of inflammasome by C. jejuni OMVs without inducing cell death is beneficial for bacterial dissemination needs to be further elucidated.

ASC is the adaptor protein required for the assembly of almost all inflammasome components (447). By using ASC knockout (Asc-/-) immune cells, we showed that the mature IL-1β was totally abolished in Asc-/- cells compared to Asc+/+ cells, indicating that the OMV-induced IL-1β secretion is ASC-dependent (Paper IV, Figure 2A, 2B). Thus, inflammasomes are principally responsible for the rapid IL-1β secretion. It was previously shown that inflammasome activation by C. jejuni in macrophages is NLRP3 dependent in addition to ASC (420). Using the NLRP3 specific inhibitor MCC950 prior to stimulation with C. jejuni OMVs, we found complete abolishment of IL-1β secretion into the cell supernatants in macrophages but incomplete abolishment in dendritic cells (Paper IV, Figure 2C, 2D). Apparently, the NLRP3 inflammasome pathway is the main inflammasome activated by OMVs in macrophages as it was previously shown for live bacteria (420), while NLRP3 together with other component(s) are required for inflammasome activation in dendritic cells by both OMVs and bacteria. In addition, the OMV-mediated IL-1β activation and secretion was observed to be caspase-1 dependent (Paper IV, Figure 2C, 2D).

In epithelial cells, C. jejuni OMVs are internalized partially via lipid rafts and internalization of OMVs is required for cytokine release (131). In immune cells, we revealed that macrophages and dendritic cells internalized both bacteria and OMVs partly via lipid rafts (Paper IV, Figure 3A, 3B). This was not critical for inflammasome activation since blocking of internalization using the cholesterol-

depleting compound methyl-beta-cyclodextrin (MβCD) did not alter the IL-1β secretion level compared with non MβCD-treated cells (Paper IV, Figure 3C, 3D).

In previous studies, factor(s) involved in inflammasome activation by C. jejuni in dendritic cells and macrophages could not be defined, although an effect on IL- 1β secretion by purified LOS was seen (414, 419, 420). C. jejuni OMV membranes are enriched in LOS, membrane proteins and probably extracellular DNA. It appears that LOS of C. jejuni OMVs is not the responsible component for inflammasome activation as OMVs with nonfunctional LOS, i.e. C. jejuni LOS mutants with defects in LOS inner core oligosaccharide assembly (waaC::Cm) and (waaF::Cm) or neutralized lipid A moiety using polymyxin B, could induce pro- and mature IL-1β as efficient as OMVs from the wild-type strain (Paper IV, Figure 4A, 4B, S3A, S3B). Similarly, membrane proteins do not seem to be the activating components since proteinase K or heat treatment of OMVs to degrade surface-exposed proteins prior to stimulation did not reduce IL-1β secretion by primed cells, suggesting that membrane-associated proteins are not involved in activation either (Paper IV, Figure 4A, 4B). It has been shown that OMVs from a wide range of Gram-negative bacteria carry external and internal DNA, which could modulate host immune responses and activate the inflammasome (98, 448). The possible involvement of DNA as an inflammasome activator for LPS- primed cells was analyzed by DNase treatment of OMVs prior to addition to BMMs and BMDCs. No alteration in mature IL-1β production was detected by DNase treatment (Paper IV, Figure 4D, 4E), suggesting no role for OMV surface- exposed DNA in inflammasome activation.

In this study, we presented evidence that released OMVs by Campylobacter play a role in interaction with immune cells and might have effects on the bacterial pathogenesis. We showed that C. jejuni OMVs mediate inflammasome activation in pre-activated cells without inducing cell death, which has not previously been observed for OMVs from other pathogens. This adds new insights into how pathogens interact with host cells by manipulating inflammatory responses to thrive in the host.

Main findings and future prospects

The focus of the present thesis is to investigate host-pathogen interactions during C. jejuni and Y. pseudotuberculosis infections. C. jejuni-the main studied enteropathogen in this thesis-is the most prevalent food-borne bacterial pathogen and the leading cause of gastroenteritis worldwide. The disease is self- limiting but in rare cases, infections can cause extra intestinal manifestations and devastating secondary effects such as Miller Fisher or Guillain Barré syndromes. Y. pseudotuberculosis causes a self-limiting gastroenteritis in human with low prevalence. Nevertheless, further accumulation of knowledge about this bacterium, its virulence mechanisms, and host interactions is important and helps to increase the understanding about its close and more dangerous relative, Y. pestis, and other related pathogens.

Paper I

 Y. pseudotuberculosis blocks degranulation of human neutrophils, which might be important for its survival  YopH and YopE co-operate to block Yersinia-induced degranulation  Y. pseudotuberculosis inhibitory effect on neutrophil degranulation is prominent, and impairs the ability of neutrophils to respond to other bacteria

In this paper, we showed that Y. pseudotuberculosis efficiently blocks neutrophil degranulation, an important mechanism used by neutrophils to eliminate invaders. We also found that this effect is caused by the contribution of YopH and YopE. Analyses of host cell signaling pathways that are known to be targeted by these effector proteins indicated that there might be other, yet unknown targets important for neutrophil degranulation, which can be impaired by these effector proteins. Answer to questions such as what are the underlying mechanisms causing the inhibitory effect on degranulation and most importantly, is this effect also occurring during in vivo infection can increase our understanding of this bacterium and other related bacterial species.

Paper II

 C. jejuni alters protein content of OMVs upon exposure to low concentration of bile. They contain not only proteins with altered abundance but are also packed with exclusive proteins  OMV isolated from bacteria grown in bile are enriched in cytoplasmic proteins and reduced in known virulence protein abundance

 OMVs isolated from bacteria grown in bile enhance C. jejuni adhesion to and invasion of intestinal epithelial cells

The new findings described in this paper open up numerous research questions to study in future. Why and how are these proteins sorted into OMVs? Is the presence of bile driving an organized packaging of specific proteins into OMVs? If so, how is this coordinated? Is this a new mechanism by which C. jejuni resists bile? If so, does this also occur during in vivo infections? Further, mechanisms underlying the enhanced adhesion and invasion of C. jejuni in the presence of bile-treated OMVs are needed to be studied in the future.

Paper III

 OMVs isolated from C. jejuni grown at different temperatures differ in protein abundance  OMVs isolated from C. jejuni grown at 37°C are enriched in proteins associated with virulence  OMVs isolated from C. jejuni grown at 37°C induce stronger inflammasome activation compared to OMVs isolated from C. jejuni grown at 42°C

In this paper, we have shown that growth temperature can influence OMV packaging by C. jejuni and that OMVs isolated from bacteria grown at 37°C have higher amount of proteins associated with virulence. This is a new finding and underlying mechanisms should be addressed.

Paper IV

 C. jejuni OMVs induce inflammasome activation in primed immune cells, which is dependent on caspase-1, ASC and NLRP3 activation  OMV-mediated inflammasome activation does not induce cell death  Internalization is not necessary for inflammasome activation by OMVs

In this study, we demonstrate that OMVs isolated from C. jejuni can induce inflammasome activation in immune cells without inducing cell death. This is a new finding, which adds new insights into how pathogens interact with host cells and causes infections. The components of OMVs responsible for inflammasome activation are unknown and should be elucidated. Whether C. jejuni OMVs are produced and interact with immune cells in vivo should be further investigated. The role of C. jejuni OMVs on inflammasome activation should be addressed using in vivo infection models.

Acknowledgements

The work in this thesis has been carried out at Umeå University, Department of Molecular Biology. I would like to thank the institute and the department for providing me a motivating environment to work and write my thesis in. Without the help from many colleagues and friends, this thesis would not have been possible. I would like to express my greatest gratitude to all the co-authors, and donors for their invaluable contributions to this thesis. Also, an especial thanks to Dr. Tor Monsen at Department of Clinical Microbiology for letting me use incubator in his lab, it was great help during my PhD.

I feel very blessed to have so many magnificent people around me; you have all contributed to my work, thank you all!

Dr. Anna Fahlgren, my main supervisor, I am sincerely grateful for giving me the opportunity to do my PhD in your group. I especially appreciate for believing me and allowing me to explore my own ideas. I could not come to this point without your patience and encouragement. Thank you for everything!

Professor Maria Fällman, my co-supervisor, thank you for all your supports, scientifically and financially, for your invisible care and help in scientific thinking and writing. Professor Sun Nyunt Wai, my co-supervisor, I am so amazed that you always have an answer to any question I ask. Thank you for supporting me and providing me with your scientific knowledge, antibodies, and reagents during my projects. You both had time for me, no matter how busy you were. Without you, I could not finish my PhD. Thank you both!

Professor Åke Forsberg, my examiner, thank you for your trust and signatures. Dr. Nelson O Gekara and Dr. Christer Larson, thanks for your engorgements as members of my committee and providing me with valuable inputs regarding the direction of my projects and finishing my PhD. Professor Martin Gullberg, Thank you for nice discussions and providing me with reagents. Dr. Tomas Edgren, thank you for your help whenever I needed from ideas to materials for OMV preparations. All former and current members of Journal club & research meeting Groups, your invaluable suggestions, comments, and discussions helped me so much during my PhD. Thank you!

Dr. Saskia Erttmann, thank you for being my friend and sharing with me your extensive knowledge, thanks for all lunch and dinners, and especially thanks for reading my thesis.

Christian, Kristina, Joanna, Yang, Azhar, and Yngve, thanks for making teaching fun, interesting and even educational! Mikael, Marek, administrative people, media people, Marionella, and Johnny, thanks for all the great work you are doing to make our lives easier at MolBiol.

Kristina, I have so much to thank you for, from your helps in the lab with methods and reagents to your helps in the life with precious information, but the most especial thanks is for my cat. Kemal, Firoj, Roberto, and Rajdeep, thanks for being great colleagues and friends, for all the laughs and nice discussions, for being patient with me during my dark days, for helps whenever I needed and for all lunch and dinners.

My dear friends, Sabine, Ala, Alireza, Mehrnoush, Khalil, Hamedeh, Jalal, Ummehan, and Swarupa, your supports were more than I can thank you for. I appreciate all moments you spent with me, understood me, listened to me without judgment, for all lunch, dinner, and celebrations we had together. Sarp, Niko, Akbar, Lisa, Salah, Sveta, Henrik, Fu, and Jyoti, thanks for all talks and discussions we had during years in MolBiol, and for being my good colleagues and friends. Elda, Siama, and Leonie, thanks for helping me in my projects.

My family, the love, and supports you have given me throughout the years are priceless and I truly appreciate all you have done for me. I love you so much.

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