Cell-to-cell communication and virulence in Vibrio anguillarum

Kristoffer Lindell

Department of Molecular Biology Umeå Center for Microbial Research UCMR Umeå University, Sweden Umeå 2012

Copyright © Kristoffer Lindell ISBN: 978-91-7459-427-0 Printed by: Print & media Umeå, Sweden 2012

"Logic will get you from A to B. Imagination will take you everywhere."

Albert Einstein

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Table of Contents

Table of Contents i Abstract iii Abbreviations iv Papers in this thesis v Introduction 1

Vibrios in the environment 1 Vibrios and Vibriosis 2 Vibriosis in humans 2 Vibriosis in corals 3 Vibriosis in fish and shellfish 4 Treatment and control of vibriosis due to V. anguillarum 4

Virulence factors of V. anguillarum 5 Iron sequestering system 5 Extracellular products 6 Chemotaxis and motility 6 The role of LPS in serum resistance 6 The role of exopolysaccharides in survival and virulence 7 Virulence factors required for colonization of the fish skin 7 Outer membrane porins and bile resistance 8

Fish immune defence mechanisms against bacteria 8 Fish skin defense against bacteria 8 The humoral non-specific defense 9 The humoral specific defense 10 The cell mediated non-specific and specific host defense 11

Quorum sensing in vibrios 12 The acyl homoserine lactone molecule 12 Paradigm of quorum-sensing systems in Gram-negative bacteria 13 Quorum sensing in Gram-positive bacteria 14 Hybrid two-component signalling systems 14 Quorum sensing in V. harveyi 15 Quorum sensing in V. fischeri 16 Quorum sensing in V. cholerae 19 Quorum sensing in V. anguillarum 20

Stress response mechanisms 23 Heat shock response 23 Cold shock response 24 Prokaryotic SOS response and DNA damage 24 Stress alarmone ppGpp and the stringent response 25 Universal stress protein A superfamily 26 Small RNA chaperone Hfq and small RNAs 26

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Aims of this thesis 28 Key findings and relevance 29

Paper I. The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.29 Relevance paper I 29 Paper II. The transcriptional regulator VanT activates expression of the signal synthase VanM, forming a regulatory loop in the Vibrio anguillarum quorum-sensing system. 30 Relevance paper II 30 Paper III. The universal stress protein UspA regulates the expression of the signal synthase VanM in Vibrio anguillarum 31 Relevance paper III 31 Paper IV. Lipopolysaccharide O-antigen blocks phagocytosis of Vibrio anguillarum by fish skin epithelial cells. 32 Relevance paper IV 32

Conclusions 34 Acknowledgements 35 References 36

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Abstract

Quorum sensing (QS) is a type of cell-to-cell communication that allows the bacteria to communicate via small molecules to coordinate activities such as growth, biofilm formation, virulence, and stress response as a population. QS depends on the accumulation of signal molecules as the bacterial population increases. After a critical threshold of the signal molecules are reached, the bacteria induce a cellular response allowing the bacteria to coordinate their activities as a population.

In Vibrio anguillarum, three parallel quorum-sensing phosphorelay systems channels information via three hybrid sensor kinases VanN, VanQ, and CqsS that function as receptors for signal molecules produced by the synthases VanM, VanS, and CqsA, respectively. The phosphorelay systems converge onto a single regulatory pathway via the phosphotransferase VanU, which phosphorylates the response regulator VanO. Together with the alternative sigma factor RpoN, VanO activates the expression of a small RNA, Qrr1 (Quorum regulatory RNA), which in conjunction with the small RNA chaperone Hfq, destabilizes vanT mRNA, which encode the major quorum-sensing regulator in V. anguillarum. This thesis furthers the knowledge on the quorum-sensing phosphorelay systems in V. anguillarum.

In this study, three additional qrr genes were identified, which were expressed during late logarithmic growth phase. The signal synthase VanM activated the expression of the Qrr1-4, which stands in contrast to Qrr regulation in other vibrios. Moreover, in addition to VanO, we predict the presence of a second response regulator which can be phosphorylated by VanU and repress Qrr1-4 expression. Thus, VanU functions as a branch point that can regulate the quorum-sensing regulon by activating or repressing VanT expression. Furthermore, VanT was shown to directly activate VanM expression and thus forming a negative regulatory loop, in which VanM represses VanT expression indirectly via Qrr1-4. In addition, VanM expression was negatively regulated post-transcriptionally by Hfq. Furthermore, a universal stress protein UspA repressed VanM expression via the repression of VanT expression. We showed that UspA binds Hfq, thus we suggest that UspA plays a role in sequestering Hfq and indirectly affect gene expression.

This thesis also investigated the mechanism by which V. anguillarum can attach to and colonize fish skin tissue. We show unequivocally that fish skin epithelial cells can internalize bacteria, thus keeping the skin clear from pathogens. In turn, V. anguillarum utilized the lipopolysaccharide O-antigen to evade internalization by the fish skin epithelial cells. This study provides new insights into the molecular mechanism by which pathogen interacts with marine animals to cause disease.

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Abbreviations

sRNA Small RNA QS Quorum Sensing Qrr Quorum regulatory RNA AHL N-acylated homoserine lactone

C-terminus Carboxy-terminus N-terminus Amino-terminus LPS Lipopolysaccharide HPt Histidine-containing phosphotransfer domain RNAP RNA polymerase UspA Universal stress protein A SAM S-adenosylmethionine TDH Thermostable direct haemolysin OMPs Outer membrane porins

ECP Extracellular products

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Papers in this thesis

This thesis is based on the following two publications and two manuscripts referred to by their roman numerals (I-IV)

I. Weber B*, Lindell K*, El Qaidi S, Hjerde E, Willassen NP, and Milton DL. 2011. The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum. Microbiology (2011), 157, 3324–3339. *Authors have contributed equally.

II. Lindell K, Weber B, Hjerde E, Willassen NP, and Milton DL. 2012. The transcriptional regulator VanT activates expression of the signal synthase VanM, forming a regulatory loop in the Vibrio anguillarum quorum-sensing system. Manuscript in preparation.

III. Lindell K and Milton DL. The universal stress protein UspA regulates the expression of the quorum-sensing signal synthase VanM in Vibrio anguillarum. Manuscript in preparation.

IV. Lindell K, Fahlgren A, Fällman M, Hjerde E, Willassen NP, and Milton DL. 2012. Lipopolysaccharide O-antigen blocks phagocytosis of Vibrio anguillarum by fish skin epithelial cells. PloS ONE. Minor revision before acceptance.

Paper not included this thesis

V. Gómez-Consarnau L, Akram N*, Lindell K*, Pedersen A, Neutze R, et al. (2010) Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation. PLoS Biol 8(4): e1000358. oi:10.1371/journal.pbio.1000358. *Authors have contributed equally.

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Introduction

Vibrios in the environment

Vibrios belong to the Gammaproteobacteria and are gram-negative rod shaped bacteria found in marine environments such as marine coastal waters, estuaries, sediments, and aquaculture facilities. The Vibrio genus consists of more than 50 species that may be associated with marine animals such as fish, corals, shrimps, sponges, mollusk, and zooplankton [1]. In pelagic waters, vibrios are present over the complete water column in a species and depth-specific manner [2]. Vibrios can also be found as commensal microflora on mucosal surfaces of marine animals, as symbionts associated with light organs of marine animals [3, 4] and as pathogens causing disease in humans, fish, and crustacean.

The coastal waters are a constantly changing environment and provide a wide ecological diversity. Attributes of the coastal waters such as temperature, pH, salinity, sunlight, and nutrient levels, often change rapidly both spatially and temporally depending on the season [1]. These environmental changes have a great impact on the occurrence and prevalence of vibrios in the marine environment [5, 6]. The ability to adjust to these environmental changes is manifested in the genetic diversity seen in vibrios [7-11].

Extracellular enzymes such as lipases, proteases, hydrolases, and chitinases produced by vibrios aid their ability to metabolize polymers in the aquatic environment. Vibrios are thought to be involved in nutrient cycling by taking up dissolved organic matter [12], in degradation of toxic aromatic compounds in polluted marine sediments [13], in producing essential unsaturated fatty acids for other aquatic organisms [14, 15], and in the degradation of chitin [16, 17].

Chitin, a homopolymer of N-acetyl-D-glucosamine, is an abundant amino sugar in the oceans and a component of the cell walls of mainly insects, fungi, and crustaceans. The ability to degrade and utilize chitin as an energy source is a vital nutrient source for vibrios [18]. The survival of vibrios in the environment is further promoted by their ability to undergo crucial physiological changes during stress or starvation, such as the viable but non-culturable state.

Vibrios are most commonly found as complex multispecies biofilms in or on marine animals or abiotic surfaces such as exoskeletons of crustaceans [1]. Life within a biofilm promotes survival of a bacterial community by genetic and metabolic exchange between species and protection against starvation, predation, and environmental stress [19-21]. Vibrios produce antimicrobials that are important in antagonistic interactions with other

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marine bacteria [22], which control the inter- and intraspecies composition in bacterial communities [23]. Thus, vibrios play an important role in the architecture and maintenance of bacterial communities in aquatic environments.

Vibrios and Vibriosis

Vibriosis is a complex disease defined as a haemorrhagic septicemia manifested by the interaction of a vibrio with its host. Vibriosis causes severe problems in the aquaculture industry around the world. Vibriosis is also a human disease, as twelve Vibrio species are described to cause disease in humans [24, 25]. Clinical signs of vibriosis in fish are septicemia, skin infections and diarrhea, and in humans the symptoms are primary septicemia, wound infections, and diarrhea [24, 26, 27]. Most vibrios are non-pathogenic or opportunistic pathogens causing disease only when the host is immuno-compromised.

Vibriosis in humans

Vibrios such as V. cholerae, V. parahaemolyticus, and V. vulnificus biotype 1 are known to cause disease in humans [28-30]. Vibrio cholerae, the causative agent of cholerae, causes a dehydrating diarrhea and vomiting of clear fluid in humans. The main route of infection is through contaminated food or water. Natural reservoirs of V. cholerae are copepods and chironomids. Moreover, the digestive tract of fish is a known reservoir for V. cholerae [31]. Since fish carrying V. cholerae swim from one location to another including sea and/or lakes to rivers, they act as vectors for V. cholerae on a minor scale. However, birds may consume infected fish and thus spread V. cholerae on a more global scale. Vibrio cholerae is known to cause severe problems in developing countries due to insufficient sanitation and water quality. After consumption of contaminated food or water, V. cholerae adheres to epithelial cells of the small intestines and secretes the cholerae toxin, an enterotoxin, which cause the severe diarrhea [32]. In addition to the cholerae toxin, V. cholerae has other important virulence factors required for disease such as the RTX toxin, motility and chemotaxis, outer membrane porins, lipopolysaccharide (O-antigen), haemolysins, proteases, and ToxR, a transcriptional regulator. [32-35]. ToxR is required for ToxT expression, which activates the transcription of multiple genes involved in virulence [36] including those that encode cholerae toxin and the toxin-coregulated pilus [37]. The toxin-coregulated pilus is essential for colonization of the small intestine epithelial cells.

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Vibrio parahemolyticus is recognized as the agent of seafood-borne gastroenteritis [38]. The virulence determinants include two haemolysins, a thermostable direct haemolysin (TDH) and a TDH-like haemolysin [39], and two type three secretion systems. TDH has been suggested to function as a membrane porin that modulates the cytoskeletal reorganization of ionic influx into the intestinal absorptive cells, enterocytes [40, 41]. The ionic influx caused by TDH affect the enterocyte osmotic balance, leading to death of the enterocyte.

Vibrio vulnificus is responsible for serious and often fatal infections in humans that are associated with biotype 1 and 3. These two biotypes cause a primary septicemia that is acquired from raw or under-cooked shellfish that is consumed [42], or from a wound that comes in contact with shellfish colonized with V. vulnificus or with aquatic environments with V. vulnificus present. Vibrio vulnificus is strongly associated with mollusks such as clams, mussels, and oysters. In oysters, V. vulnificus can reach up to 106 bacteria per gram [43]. The host susceptibility is important for the outcome of infection. Humans with chronic diseases of the immune system or the liver, or with an elevated serum iron level are more likely to be infected [44, 45]. The clinical signs of a primary septicemia include fever, chills, vomiting, and diarrhea normally followed by lesions on the extremities. The main virulence factor of V. vulnificus is the capsular polysaccharide (CPS), which covers the bacterial surface [46, 47]. In the host, the CPS resists phagocytosis of immune-cells and counteracts the effects of serum [43, 46, 48, 49]. The CPS also alters the level of the inflammation-associated cytokine tumor necrosis factor alpha, TNF-α, which contributes to septic shock [50]. Other factors modulating virulence are the lipopolysaccharide (LPS), pili, which are required for attachment to and colonization of human epithelial cells, and extracellular proteins such as the cytolysin VvhA and the metalloprotease VvpE.

Vibriosis in corals

Corals are built up of two parts, the coral host and the unicellular algae Zooxanthellae. Coral-Zooxanthellae symbiosis is considered mutualistic but this has been questioned [51]. Zooxanthellae provides the coral host with carbon and oxygen via photosynthesis; while, the coral host provides Zooxanthellae with carbon dioxide and ammonium [52, 53] and protection against predators. Coral bleaching appears either due to depletion of Zooxanthellae or to the degradation of the photosynthetic algae pigment. Vibrios involved in coral bleaching, V. shiloi and V. corallilyticus, are temperature dependent pathogens [54-61]. Therefore, global warming is proposed to affect the level of coral bleaching and to decrease the diversity of the corals on a global level [62].

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Vibriosis in fish and shellfish

Several vibrios cause disease in reared fish and shellfish. Vibrio salmonicida and V. anguillarum are known pathogens for many fish species reared in aquaculture [63-65]. Vibrio vulnificus biotype 2 is associated with vibriosis in eels [66]. In shrimps, V. harveyi is the main pathogen causing disease [67]. Clams with the brown ring disease are due to V. tapetis [68]. In wild and cultured salmonids, V. ordalii causes vibriosis, often with necrosis and hemorrhaging occurring at the site of infection [69].

Vibrio anguillarum is considered a major obstacle for many aquaculture settings due to the frequency of outbreaks, geographic distribution, and numbers of fish species affected [70]. Over 50 fish species in at least 17 countries have been reported with disease caused by V. anguillarum [71]. Vibrio anguillarum is found as normal and commensal flora on many fish species and is associated with planktonic rotifiers, which are the main food source for fish in aquaculture. Thus, rotifiers play an important role as vectors for vibriosis. Vibrio anguillarum consists of 23 serotypes (O1-O23). Almost all currently known serotypes are non-pathogenic except serotype O1-O3, which are considered opportunistic pathogens [72]. In fact, many disease outbreaks are due to over-crowded aquatic facilities, poor water quality, impaired fish health, and stress. Further, the presence of V. anguillarum in aquaculture settings is promoted due to high levels of nutrients resulting from excess of fish food remaining in the environment after feeding of the fish. The symptoms of vibriosis due to V. anguillarum are skin discoloration, ulcers, swollen intestines, dark lesions, and red necrotic lesions on the ventral and lateral side of the fish body. The gut and rectum are extended and contain viscous liquid [63, 73]. Late in infection, necrosis appears on the skin and internal tissues, such as gills, kidney, spleen, liver, and ulcers that release blood [74]. In acute epizootics, the course of infection is rapid and clinical signs are not detected prior to death of the fish. In a fish population, only a subset of animals may be susceptible to infection; however, transmission of the disease from infected to healthy fish occurs rapidly when the bacterial number has increased in the environment. Thus, a whole fish population in aquatic settings can be infected with V. anguillarum. The bacteria can sustain within a fish population for up to two years [75] making it hard to eradicate the bacteria, which leads to reoccurring infections and outbreaks.

Treatment and control of vibriosis due to V. anguillarum

Vibrio anguillarum infections are successfully controlled with antibiotics, vaccination, and probiotics. Antibiotics, which include oxytetracycline, erythromycin, carbencillin, chloramphenicol, and triomethoprim, are given

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via the water or food. High levels of antibiotics are required to treat and prevent an infection, which has lead to an increase in antibiotic resistance within V. anguillarum strains, an increase in the appearance of multi-drug resistance strains within the aquaculture settings [76], and an increase in the spread of resistance within the aquatic environment [77, 78]. A second means to control vibriosis within aquaculture settings is to vaccinate fish with inactivated V. anguillarum bacteria or membrane components. Vaccinations are mainly performed by intraperitonal injections or water immersion. The O-antigen structure of LPS and outer membrane porins are the most immunogenic components associated with the outer membrane [79, 80]. Probiotics, the co-growing of other bacteria to inhibit growth of pathogenic strains, is also an option for disease control. Probiotic bacteria such as Roseobacter 27-4 [81], Pseudomonas fluorescens AH2 [82], and Kocuria SM1 [83] can inhibit the growth of V. anguillarum and thus prevent vibriosis caused by V. anguillarum.

Virulence factors of V. anguillarum

Iron sequestering system

In vertebrates, iron is found bound to iron-binding proteins such as transferrin and lactoferrin. Consequently, iron is not freely accessible for the bacteria. To circumvent this problem, bacteria produce and secrete proteins with a high affinity for iron. These proteins sequester iron from the host proteins and transport the iron into the bacterial cell. In V. anguillarum, a 348-Da siderophore anguibactin is produced and is responsible for sequestering and transporting iron into the bacterial cell [84, 85]. The expression of anguibactin is negatively regulated by the transcriptional regulator Fur and the antisense RNA, RNAβ [86]. Two proteins, the DNA-binding regulator, AngR, and the transacting factor(s) (TAF) activate the expression of the iron sequestering system [86]. When iron levels are low in the cell, anguibactin is produced and sequesters iron. The outer membrane receptor FatA binds the iron bound anguibactin and transports the complex into the periplasm of the bacteria [87]. To internalize the anguibactin-iron complex, a periplasmic protein, FatB which is anchored in the inner membrane binds the complex and transports it to the cytoplasm [88, 89]. The iron is released and anguibactin is suggested to be recycled to sequester additional iron outside the bacteria [86]. An iron-independent system, which involves the uptake of heme and haemoglobin, may also be used by V. anguillarum [90, 91]. Genes essential for heme and haemoglobin uptake and utilization are huvAZBCD. These genes encode the heme receptor complex

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consisting of the receptor (HuvA) and the transport proteins (HuvZBCD) [92].

Extracellular products

Secreted extracellular products (ECP) cause severe tissue damage suggesting they play a role in virulence. The tissue damage observed in fish with vibriosis suggests that V. anguillarum produces proteases during infection. Both proteolysis and haemolysis are seen when fish are injected with ECPs. Vibrio anguillarum possesses five haemolysins (VAH1-5), which are described to lyse red blood cells of fish and to be involved in virulence [93, 94]. One of the most abundant secreted proteases is the 36-kDa zinc metalloprotease EmpA. In V. anguillarum, one role of EmpA is to function as a mucinase [95]. Mutations in empA or genes regulating EmpA result in decreased extracellular proteolytic activity and virulence [95, 96].

Chemotaxis and motility

Vibrio anguillarum must attach to and colonize host tissues for disease to occur [95]. To localize host tissues in the seawater, the bacteria utilize chemotaxis to find the host. Both intestinal and skin mucus are chemotactic attracts [97, 98]. The flagellar motility is crucial for V. anguillarum to enter the host to cause disease [99]. Chemotaxis requires a functional flagellum and the virulence of V. anguillarum is significantly decreased when mutations occur in structural flagellin genes, chemotaxis genes, or genes encoding regulators of flagellin motility or chemotaxis function [99-101].

The role of LPS in serum resistance

Lipopolysaccharides (LPS) are a major component of the bacterial outer surface. The LPS consists of three major parts: the lipid A, the core polysaccharide, and the O-antigen (figure 1a). LPS is an important structural component of the membrane integrity, induces a strong immune response in the host, and plays a role in serum resistance. In V. anguillarum, the LPS are localized within an outer sheath that covers the entire body including the flagellum (figure 1b) [99, 102]. The LPS levels and structure are important for virulence [99, 102, 103]. Furthermore, the LPS is required for a functional anguibactin-iron transport [104].

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Figure

Figure 1. The LPS molecule in Gram-negative bacteria. A) The LPS molecule consists of three major parts: the lipid A region, the core region, and the O-polysaccharide (O-antigen). The LPS molecule is anchored in the outer membrane. B) Electron-microscopy image with immunogold labeling to localize LPS on the V. anguillarum body.

The role of exopolysaccharides in survival and virulence

Exopolysaccharides are high molecular weight polymers composed of sugar residues and are secreted into the surrounding environment by microorganisms. In V. anguillarum, two operons, orf1-wbfD-wbfC-wbfB and wza-wzb-wzc are involved in exopolysaccharide biosynthesis and transport [95]. The exopolysaccharides of V. anguillarum are required for virulence and for colonization of the integument of the fish [95, 105]. Moreover, exopolysaccharides are required for lysozyme and antimicrobial peptide resistance [105].

Virulence factors required for colonization of the fish skin

Colonization of the fish skin is vital for V. anguillarum to cause disease [95, 105]. Many studies suggest that the fish skin is an important portal of entry into the fish [106, 107]. A recent study demonstrated a significant higher numbers of V. anguillarum on the fish skin compared to the intestinal tissues [108]. Thus indicating that colonization of the fish skin is vital for causing disease.

To colonize fish skin, V. anguillarum requires a functional exopolysaccharide transport and the small RNA chaperone Hfq, which repress the major transcriptional regulator in V. anguillarum [95, 105]. Interestingly, siderophore production is required for the skin colonization,

A B

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but not for the intestines. Thus suggesting that free iron is available in the intestines but not on the skin, or that V. anguillarum might possess a redundant iron-sequestering system.

Outer membrane porins and bile resistance

Outer membrane porins (OMPs) are abundant in the bacterial outer membrane and are suggested to play an important role in bile resistance. The ability to resist bile is important for survival in and colonization of the intestine. Bacteria regulate resistance to bile by altering their membrane permeability. In V. anguillarum, a 38-kDa major outer-membrane porin, OmpU, is required for bile resistance but not virulence [109]. Moreover, loss of OmpU in V. anguillarum results in LPS alterations that result in an increase in medium and high molecular mass O-antigen [109]. When OmpU is not present, a small subset of V. anguillarum cells express a 37-kDa OMP which may have redundant activities to those of OmpU. Thus, this may be one reason why OmpU is not required for virulence.

In vibrios such as V. cholerae, V. splendidus, V. alginolyticus, the outer membrane porin OmpU is involved in virulence [110-112]; whereas, in V. fischeri, OmpU plays a role in symbiosis with the squid [113]. In V. cholerae, the ToxR-regulated porin OmpU is important for bile resistance, antimicrobial peptide resistance, virulence factor expression and colonization of the intestines. Thus, OmpU plays a crucial role in bacterial survival in the human host [114-116].

Fish immune defence mechanisms against bacteria

Phagocytosis is a process involving the engulfment and ingestion of particles by a eukaryotic cell or a phagocyte (figure 2). Phagocytosis is a first line of defense for many hosts and a critical step in the host innate and adaptive immune response. Fish have both non-specific and specific humoral and cell-mediated mechanisms to prevent bacterial diseases (Table 1) [117].

Fish skin defense against bacteria

The main cell type of fish epidermis is the epithelial cell. Other names such as Malphighian cell, keratocyte, or keratinocyte are also common. Several roles for epithelial cells have been proposed such as protection against mechanical stress, primary wound closure, and keratinization which protects against pathogens. Moreover, the epithelial cell is crucial in wound repair. Immediately following wound damage; epithelial cells migrate as networks

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towards the wound. The network of epithelial cells quickly covers the wound, thus providing protection and a mechanical barrier against opportunistic pathogens. Phagocytic properties of the epithelial cells have been proposed [118], which could keep the fish skin clear of pathogens. Furthermore, mucus secreted by epithelial cells protects the fish from pathogens. The mucus is continuously sloughed from the fish, thus keeping the epidermis clear from bacteria.

The humoral non-specific defense

The humoral non-specific defense includes factors inhibiting the growth of the bacteria such as transferrin, antiproteases, and lectins. Transferrin binds free iron in the blood, limiting the availability of iron in the host deterring bacteria from establishing an infection. Antiproteases in fish such as α1-antiproteinase and α2-macroglobulin prevent bacteria from degrading host tissues as a nutrient source for amino acids [117]. Lectins in fish bind carbohydrates [117] and are present in the serum, ova, and mucus. Mucus-associated lectins have a high affinity for LPS on the surface of bacteria and can inhibit growth of the bacteria. Further, the lectins are thought to function as opsonins and thus, lectins target bacteria for phagocytosis and activate the complement system [117]. Several host proteins, such as lysozyme, C-reactive protein, complement, and antimicrobial peptides, function as lysins. Lysozyme hydrolyses the N-acetylmuramic acid and N-acetylglucosamine components located in the peptidoglycan layer in the bacterial membrane. Lysozymes are found in fish serum and mucus. The C-reactive protein interacts with the abundant bacterial surface molecule phosphorylcholine. The C-reactive protein activates complement and thus triggers a lytic and phagocytic defense. In rainbow trout, the C-reactive protein enhances phagocytosis of V. anguillarum [119]. Moreover, increased C-reactive protein levels are seen when rainbow trout are challenged with V. anguillarum [119]. The complement system is an important part of the fish immune defense. In teleost fish, two complement systems are present: the alternative complement system, which is antibody independent, and the classical complement system, which functions similar to that of mammals. The alternative complement system is found in high levels in fish serum.

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Figure 2. Principles of phagocytosis. The bacteria are recognized by surface receptors on a phagocyte. The phagocyte engulfs the bacteria and a phagosome is formed. Phagosome formation requires many antimicrobial factors during maturation. The phagosome then fuses with a lysosome, resulting in a phagolysosome in which the engulfed particles are degraded or digested. The LPS of bacteria directly activate the alternative complement system, which leads to lysis of the bacterial cell wall. To target bacteria for degradation, the C5a component from the alternative complement system acts as a chemotaxin for the fish immune cells. The C3b component from the alternative complement system acts as an opsonin, which binds to the bacteria and enhances phagocytosis by phagocytes [120, 121]. Antimicrobial peptides are small peptides, which can disrupt bacterial membranes, and are found on the fish skin within the mucus layer. The role of antimicrobial peptides in the fish defense to bacteria is still largely unknown.

The humoral specific defense The fish specific humoral defense involves antibodies and is an important mechanism to prevent bacterial disease. Antibodies in fish function as anti-toxins, anti-adhesins, and anti-invasins. Antibodies also activate the classical complement system [117]. Toxins produced and secreted by bacteria are efficiently neutralized when antibodies bind to them. To prevent bacterial adherence to fish epithelial cells, antibodies function as anti-adhesins by

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Table 1. Immune defense mechanisms in fish.

Humoral Non-specific

Cell-mediated Non-specific

I) Inhibitors I) Macrophages - Transferrin - Hydrolytic enzymes - Antiproteases - Respiratory burst - Lectins - Hydroxyl free radical II) Lysins II) Neutrophils - Lysozyme - Respiratory burst - C-reactive protein - Myeloperoxidase - Complement - Anti-microbial peptides

Humoral specific

Cell-mediated specific

I) Antibody I) T-lymphocytes and antigens - Anti-toxin II) Cytokines - Anti-invasin - Interferon gamma - Anti-adhesin - Tumour necrosis factor - Classical complement III) Activated macrophages - Increased bactericidal activity

binding to the adhesins on the bacterial surface. Anti-adhesin antibodies are found in the mucosal layers of the skin, gut, and gills [117]. Anti-invasins are used to prevent bacterial invasion of non-phagocytic cells. Bacteria invade non-phagocytic cells to evade the immune response of the host. Antibodies functioning as anti-invasins prevent bacterial invasion of host cells allowing phagocytic cells to remove the bacteria. Moreover, antibodies bound to bacterial surfaces can activate the classical complement system.

The cell mediated non-specific and specific host defense The main phagocytic cells involved in the immune defense are macrophages and neutrophils, which engulf and eliminate bacteria. The elimination is mainly due to the production of reactive oxygen species during the respiratory burst. In this process, hydrogen peroxide, superoxide anion, and hydroxyl free radicals, which have bactericidal functions, are produced. Moreover, macrophages and neutrophils contain hydrolytic enzymes, like lysozyme, and produce nitric oxide which is a precursor for bactericidal molecules.

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The cell mediated specific defense includes activated macrophages. The activation of macrophages occurs normally by interferon gamma, derived from antigen-stimulated T-cells. Activated fish macrophages have increased size, motility, lysosome and lysosomal enzyme levels, increased phagocytic activity, increased reactive oxygen species levels, and enhanced bactericidal properties.

Quorum sensing in vibrios

Quorum sensing (QS) is a type of cell-to-cell communication that allows the bacteria to communicate via small, diffusible molecules to coordinate activities such as growth, biofilm formation, virulence, metabolism, and stress response, as a population [122]. QS depends on the accumulation of signal molecules as the bacterial population increases. After a critical threshold of the signal molecules are reached, the bacteria induce a cellular response allowing the bacteria to coordinate their activities as a population. The QS signals are small chemical molecules or peptides called autoinducers as most signal molecules induce their own production. The best studied signal molecules are the acyl homoserine lactones (AHL) in Gram-negative bacteria. QS signals can cross bacterial species and kingdom barriers allowing interspecies communication [123, 124]. Some QS systems recognize human stress hormones and cytokines, which allow the bacteria to detect the physiological state of the host, and to coordinate an invasion when the host is most susceptible.

The acyl homoserine lactone molecule

AHLs consist of a common hydrophilic homoserine lactone ring moiety and a hydrophobic acyl side chain of variable length, allowing the water soluble AHL to freely pass cell membranes (figure 3). The acyl chain can also vary in substitutions at the β-position and the level of saturation. The specificity of an AHL towards its cognate receptor depends on the variability of the acyl side chain. Diffusion of the AHL through a membrane depends on the length and saturation of the acyl side chain. Shorter acyl side chains are less saturated and cross membranes easier; while, longer chain molecules may require a transport mechanism. In V. fischeri, two signal molecule synthases are present, LuxI and AinS, which require the substrates S-adenosylmethionine (SAM) and acylated acyl carrier protein (ACP) as substrate for AHL synthesis [125-130].

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Figure 3. Structure of N-acylated homoserine lactones (AHLs). The lactone ring is common for the AHLs but the acyl side chain (Cn) varies in length between 4-14 carbons and in substitutions at the β position (R = OH, O or no group).

S-adenosylmethionine is the source of the homoserine lactone ring and the acyl-ACP derives from fatty acid biosynthesis. LuxI-type signal synthases catalyze the amide bound formation the amino group of SAM and the acyl side chains of acyl-ACP [128, 131]. Synthesis is finalized with the lactonisation of the SAM-acyl resulting in an acyl homoserine lactone molecule.

Paradigm of quorum-sensing systems in Gram-negative bacteria

The first described example of QS was the regulation of bioluminescence in V. fischeri [132], a symbiont of the squid Euprymna scalopes [133, 134]. The V. fischeri QS system (figure 4) consists of the signal synthase LuxI and a transcriptional regulator LuxR. The LuxI/R quorum-sensing system has been described in over 40 bacterial species [135]. LuxI synthesizes the N-(3-oxo-hexanoyl)-homoserine lactone (3-oxo-C6-HSL). LuxR consists of two domains: the carboxy terminal domain and the amino terminal domain. The carboxy terminal domain contains a helix-turn-helix motif used for binding the promoter DNA of target genes. The amino terminal domain is regulatory and binds the AHL molecule [136-138]. At low cell densities in the absence of signal molecules, the three-dimensional structure of LuxR is such that the helix-turn-helix motif is masked. As cell densities increase, AHLs are abundant and bind the amino terminal domain of LuxR altering its conformation and exposing the helix-turn-helix motif.

At low cell density, LuxI synthesizes 3-oxo-C6-HSL at a low level, and LuxR is unstable and inactive. As the population increases, the AHL signal molecules accumulate and the equilibrium inside and outside increases above a threshold level. The AHL binds LuxR, which stabilizes and activates it. The active LuxR dimerizes and binds the conserved lux box in the promoter of the luxICDABE operon. LuxR induces expression of the light producing genes and LuxI. This autoinduction (positive feedback) induces the full expression of the LuxI/R system exponentially.

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Figure 4. The V. fisheri LuxI/LuxR quorum-sensing system. The mechanism of the LuxI/R quorum-sensing system is explained in the text.

Some bacteria lack the LuxI-type synthase and are not able to synthesize AHLs. However, the presence of a LuxR homologue in these bacteria allows them to utilize AHLs synthesized by other bacteria in the microenvironment to activate LuxR-dependent gene expression [139].

Quorum sensing in Gram-positive bacteria

The quorum sensing in Gram-positive bacteria is based on pheromone peptides as autoinducers. The autoinducers are synthesised within the cell and are transported by ATP-binding cassette transporters to the external environment [140]. In Gram-positive bacteria, the signal molecules are sensed by either two-component systems [141] or by direct sensing of the autoinducer by an intracellular receptor, which requires the autoinducer to be transported into the cell by an ATP-binding cassette permease [142].

In the two-component system, the autoinducer binds a cognate histidine sensor kinase located in the cell membrane. Binding of the autoinducer activates the signalling from the sensor kinase via autophosphorylation or dephosphorylation. The phosphate is transferred to an aspartate residue on a cognate response regulator leading to the activation of the response regulator. The activated response regulator then induces expression of target genes [143, 144].

Hybrid two-component signalling systems Bacteria utilize two-component systems to sense and to respond to external signals. The two-component system normally consists of a membrane

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localized hybrid histidine kinase, which senses an external signal, and a cognate response regulator which regulates gene expression in response to signals transmitted from the sensor kinase [141]. Two-component systems are important for bacteria to sense and respond to internal or external signals altering chemotaxis, metabolism, and gene expression accordingly [145]. Two-component signaling systems were previously thought to be present only in bacteria but have now been discovered in Archaea and eukaryotes such as Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, Dictyostelium discoideum, and Arabidopsis thaliana [146-148].

In vibrio quorum-sensing systems, a variant of the two-component system is used which is based on a phosphorelay cascade (figure 5). The hybrid histidine kinase autophosphorylates and transfers the phosphoryl group to an internal receiver domain. The phosphoryl group is transmitted to an uncoupled histidine phosphotransferase and subsequently to a response regulator. So far the phosphorelay quorum-sensing system has only been discovered in vibrios and is suggested to be a vibrio-specific quorum-sensing system [149].

Quorum sensing in V. harveyi

In V. harveyi, three parallel quorum-sensing systems regulate positively bioluminescence [150-152], siderophore production, EPS production [153], metalloproteases [154], and negatively regulate a type III secretion system [152] in a population-dependent manner. At least three quorum-sensing signal molecules are found in V. harveyi: 3-hydroxy-c4-HSL [131, 155], AI-2 (furanosyl borate diester) [131], and CAI-1 (cholerae autoinducer 1) [152, 156]. Figure 6 depicts the quorum sensing model in V. harveyi.

LuxM synthesizes N-(3-hydroxybutanoyl)-L-homoserine lactone (3-hydroxy-C4-HSL) [155, 157], which is sensed by the hybrid sensor kinase LuxN [155]. The AI-2 signal is produced by the synthase LuxS. The AI-2 signal binds the periplasmic protein LuxP. The LuxP-AI-2 complex is sensed by the hybrid sensor kinase LuxQ. CAI-1 is synthesized by CqsA (cholerae quorum sensing autoinducer) and sensed by the hybrid sensor kinase CqsS (cholerae quorum-sensing sensor). The three parallel quorum-sensing systems converge to a single regulatory system.

At low cell density and in the absence of signal molecules the hybrid sensor kinases LuxN, LuxQ, and CqsS autophosphorylate the H1 domain leading to phosphorylation of the internal D1 response regulator domain. LuxU, the phosphotransferase, accepts the phosphate [158, 159] and in turn phosphorylates the sigma-54-dependent transcriptional regulator LuxO [153, 160]. LuxO, together with sigma-54, activates the expression of five

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Figure 5. Mechanisms of the hybrid two-component signalling system. For details, refer to the text. H and D are conserved histidine and aspartate residues. Arrows indicate phosphorylation events.

small regulatory RNAs (sRNAs) Qrr1-5 [161, 162], which together with the small RNA chaperone Hfq, destabilize luxR mRNA [161].

At high cell density, the signal molecules accumulate and bind their cognate hybrid sensor kinase LuxN, LuxQ, and CqsS inhibiting kinase activity and allowing phosphatase activity of the hybrid sensor kinases to predominate. Dephosphorylation leads to inactivation of LuxO, loss of expression of the Qrr sRNAs, and induction of LuxR expression and the quorum-sensing regulon. LuxR positively regulate expression of bioluminescence, siderophore production, metalloproteases, and negatively regulates a type III secretion system.

In the vibrio phosphorelay quorum-sensing systems, V. harveyi is thought to be the paradigm. Within the vibrios the quorum-sensing systems are composed of similar components and the systems are believed to function the same (Table 2). However, despite the similarities of the phosphorelay quorum-sensing systems components within the vibrios, the cellular output is different. These differences are discussed below.

Quorum sensing in V. fischeri In V. fischeri, three quorum-sensing systems are utilized in a hierarchal regulatory cascade to sense the population density and to activate the induction of early and late colonization factors [129, 163] and genes for

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Figure 6. Model of V. harveyi quorum-sensing systems. For detailed information, refer to the text. Solid lines indicate gene regulation. Double arrowhead indicates phosphorelay, and a single arrowhead indicates gene activation. Lines with cross bar indicates gene inhibition. H1, H2, D1 and D2 are conserved histidine and aspartate residues in the hybrid two-component systems. bioluminescence, luxICDABE and luxR. Two V. harveyi-like quorum-sensing systems, LuxS and AinS work in parallel as well as a LuxI/R system, which is not found in all Vibrio species including V. harveyi. LuxS synthesizes an AI-2 signal, sensed by LuxP and LuxQ [129, 163]. AinS synthesizes the signal molecule N-octanoyl-L-homoserine lactone (C8-HSL), which is sensed by the V. harveyi LuxN homologue, AinR [164, 165]. Together these quorum-sensing systems induce a V. harveyi-like phosphorelay resulting in the expression of LitR, a V. harveyi LuxR homologue. LitR induces expression of a LuxI/R quorum-sensing system, thus linking LuxS/PQ and AinS/R to the LuxI/R quorum-sensing system [166].

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Table 2. Overview of quorum-sensing homologues in Vibrios.

V. harveyi V. cholerae V. fischeri V. anguillarum

LuxI VanI

LuxR VanR

LuxM AinS VanM

LuxN AinR VanN

CqsA CqsA CqsA

CqsS CqsS CqsS

LuxS LuxS LuxS VanS

LuxPQ LuxPQ LuxPQ VanPQ

LuxU LuxU LuxU VanU

LuxO LuxO LuxO VanO

LuxR HapR LitR VanT

One difference with V. fischeri compared to other vibrios is the presence of only one Qrr sRNA. Figure 7 depicts the quorum sensing model in V. fischeri in medium to high cell densities. When the bacterial population reaches moderate cell densities (108-109 cells/ml), C8-HSL and AI-2 accumulate and phosphatase activities of AinS and LuxN predominate leading to expression of litR and factors required for early colonization and inhibition of motility. At moderate cell density, 3-oxo-C6-HSL is limited; however, C8-HSL has a weak affinity to LuxR and induces a low level of bioluminescence and LuxI expression, which induces a strong induction of the lux genes. The LuxS/PQ system is required for bioluminescence; whereas, early colonization factors and motility are regulated by the AinS/R system [129, 163]. At high cell densities (>1010 cells/ml), 3-oxo-C6-HSL accumulates in the light organ of the squid, leading to full induction of the LuxI/R quorum-sensing system, inducing bioluminescence and late colonization factors [129, 163, 166].

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Figure 7. Model of V. fischeri quorum-sensing systems. For detailed information, refer to the text. Solid lines indicate gene regulation. Double arrowhead indicates phosphorelay, and a single arrowhead indicates gene activation. Lines with cross bar indicates gene inhibition. H1, H2, D1 and D2 are conserved histidine and aspartate residues in the hybrid two-component systems.

Quorum sensing in V. cholerae

In V. cholerae, the virulence gene expression is highly controlled by two V. harveyi-like quorum-sensing systems CqsA/S and LuxS/PQ. A V. harveyi LuxM/N system has not been detected nor a LuxI/R system. Figure 8 depicts the high cell density quorum-sensing model in V. cholerae. CqsA synthesizes CAI-1, which is sensed by the hybrid sensor kinase CqsS. LuxS synthesizes AI-2, which binds to the periplasmic protein LuxP. The LuxP-AI-2 complex is sensed by the hybrid sensor kinase LuxQ.

At high cell density, the signal molecules AI-2 and CAI-1 accumulate, and bind their cognate receptors LuxQ and CqsS inhibiting kinase activity and

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allowing phosphatase activity to predominate. Dephosphorylation leads to inactivation of LuxO, loss of Qrr1-4 sRNA expression, which induces expression of HapR, a V. harveyi LuxR homolog [161, 167].

HapR plays a crucial role in regulating virulence genes during the infectious cycle and a model is proposed [168]. In the environment, V. cholerae is mainly found in highly populated biofilms and not as free living bacteria. Therefore, it is thought that V. cholerae is ingested orally as biofilms and not as single bacteria. The biofilm protects the bacteria from acidic environments, allowing it to pass the gastric barrier to the intestines. Within the biofilm in the intestines, the expression of ctx, vps, and tcp are repressed, leading to detachment from the biofilm and colonization of the intestines. A low cell density promotes expression of cholerae toxin and toxin co-regulated pilus; however, as the population increases, the signal molecules accumulate; the virulence genes are repressed and the metalloprotease Hap is produced. The expression of the metalloprotease, which is a mucinase, leads to detachment of bacteria from the colonized intestinal epithelial cells and release back into the environment. Free in the environment again, the cell densities are low; HapR is repressed; and biofilm formation is promoted.

Quorum sensing in V. anguillarum

In contrast to V. cholerae where virulence is strictly regulated by quorum sensing, no direct correlation between virulence and quorum sensing has been described in V. anguillarum [169]. Both environmental and pathogenic strains of V. anguillarum produce AHLs suggesting a role in the physiology, ecology, as well as pathogenicity of this vibrio [170]. In V. anguillarum, three parallel phosphorelay quorum-sensing systems are found and one LuxI/R system. Figure 9 depicts the high cell density quorum-sensing model in V. anguillarum. The V. harveyi homologues LuxM/N and LuxS/PQ are named VanM/N and VanS/PQ [171, 172]. The VanM synthase produces both N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL), which are sensed by the hybrid sensor kinase VanN [171]. VanS produces AI-2 molecules that are sensed by VanP and the hybrid sensor kinase VanQ [172]. The CqsA synthase produces the CAI-1 molecule, which is sensed by the hybrid sensor kinase CqsS. The phosphorelay quorum-sensing systems regulate the expression of the transcriptional regulator VanT, a homologue of V. harveyi LuxR. VanT positively regulates metalloproteases, pigment production, serine, biofilm production, and negatively regulates type IV secretion system [173].

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Figure 8. Model of V. cholerae quorum-sensing systems. For detailed information, refer to the text. Solid lines indicate gene regulation. Double arrowhead indicates phosphorelay, and a single arrowhead indicates gene activation. Lines with cross bar indicates gene inhibition. H1, H2, D1 and D2 are conserved histidine and aspartate residues in the hybrid two-component systems.

At low cell densities, VanN, VanQ, and CqsS autophosphorylate and transmit a phosphoryl group to the phosphotransferase VanU, which phosphorylates and activates the Ϭ54-dependent response regulator VanO. Phosphorylated VanO, together with the alternative sigma factor RpoN (Ϭ54),

activates the expression of four small regulatory RNAs, Qrr1-4. The sRNAs Qrr1-4 together with the RNA chaperone Hfq, destabilize vanT mRNA. Thus, repressing VanT expression.

At high cell density, the threshold for the signal molecules are reached, and VanT expression is induced. Binding of the signal molecules to the cognate sensor kinases VanN, VanQ, and CqsS inhibits kinase activity, allowing phosphatase activity to predominate.

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Figure 9. Model of V. anguillarum quorum-sensing sytems. For detailed information, refer to the text. Solid lines indicate gene regulation. Double arrowhead indicates phosphorelay, and a single arrowhead indicates gene activation. Line with cross bar indicates gene inhibition. H1, H2, D1 and D2 are conserved histidine and aspartate residues in the hybrid two-component systems.

Thus, VanO is dephosphorylated and inactivated. Consequently, the sRNAs Qrr1-4 are not expressed, resulting in VanT expression.

In addition, the quorum-sensing systems in V. anguillarum are an integral part of stress response. The sigma factor RpoS indirectly induces VanT expression during late exponential growth by repressing expression of the RNA chaperone Hfq and thus stabilizing vanT mRNA [174].

In contrast to other vibrios, V. anguillarum vanT mRNA is stable at low cell densities [172]. Furthermore, an unusual feature of this quorum-sensing

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system is that the phosphotransferase VanU represses the expression of Qrr1-4 leading to activation of VanT expression, while VanO represses VanT expression via activation of Qrr1-4 [172, 175]. The quorum-sensing systems function the same as for other vibrios; however, the Qrr sRNA induction is different. The difference is due to a second response regulator, RR-2, which provides signal integration from quorum-sensing independent systems. RR-2 belongs to the NtrC protein family and may be phosphorylated by VanU. Active and phosphorylated RR-2 is predicted to inhibit Qrr1-4 expression (Milton, D.L unpublished data).

Moreover, VanM is directly regulated by VanT which binds vanM promoter and activates transcription [paper II]. In addition, the RNA chaperone Hfq repress VanM expression by destabilizing vanM mRNA [paper II].

A third system, VanI/R is homologous to V. fischeri LuxI/R. VanI produces N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL), which binds the transcriptional activator VanR [169]. VanR activates the expression of vanI and other putative target genes not yet described. Similar to V. fischeri, a link between the VanI/R system and VanM/N system is found since VanM regulates signal production via VanI [171]. Stress response mechanisms

In the environment, bacteria are constantly exposed to stressful conditions that require an immediate response by the bacteria to prevent death. The bacteria have evolved mechanisms to adapt and to respond to the stress conditions. Normally, a stress signal is sensed by the bacteria, which alter the gene expression profile, leading to phenotypic changes that are essential for survival. These changes occur quickly and must be reversible to adjust to a rapidly changing environment. Many signals are recognized as inducing factors such as temperature, DNA damage, oxidative stress, ultraviolet light, and pH.

Heat shock response

Heat shock is induced in response to denatured- or misfolded proteins due to a sharp increase in temperature. Bacteria respond to heat shock by producing a wide range of cytoplasmic heat shock proteins such as protein chaperones and ATP-dependent proteases. Both, of which, aid protein re-folding and protein degradation [176]. In E. coli, two global regulators modulate gene expression in response to heat shock: the alternative sigma factors RpoE (ϬE) and RpoH (ϬH). The heat-shock response initiates with the

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induction of RpoE expression in response to misfolded proteins in the periplasm or the outer membrane.

The rpoH gene has three promoters two of which require Ϭ70 and one requires ϬE. Consequently, RpoE regulates RpoH expression. RpoH expression is also determined by the secondary structure of rpoH mRNA [177]. RpoH stability is regulated by DnaJ and DnaK. During normal temperature, DnaJ and DnaK bind and inactivate RpoH, which also aids its degradation. However, when denatured proteins are present in the cell due to increased temperature, DnaJ and DnaK interact preferably with the misfolded proteins, extending the half-life of RpoH, which increases the amount of RpoH in the cell and induces the cytoplasmic heat shock response [178].

Cold shock response

In contrast to heat shock, no sigma factors have been identified to regulate the cold shock response for E. coli. During cold-shock, the secondary structures of DNA and RNA are stabilized, leading to rate limiting steps in the initiation of transcription and translation. Further, the membrane fluidity decreases. To increase the membrane fluidity, the levels of unsaturated fatty acids are increased in the membrane phospholipids [179]. The major cold-shock protein CspA is induced 200-fold upon a temperature shift from 37 to 10°C [179]. CspA prevents inhibitory RNA secondary structures at low temperatures, allowing translation to occur [180]. Moreover, CspA functions as a RNA chaperone which binds RNA nonspecifically with low affinity, resulting in increased translation or decreased RNA stability [181].

Prokaryotic SOS response and DNA damage

In bacteria, DNA damage occurs due to environmental factors and normal metabolic processes such as ultraviolet light, radiation and reactive oxygen species. In turn, the bacteria have evolved mechanisms to repair damaged DNA. A well studied defense mechanism is the inducible SOS response in E. coli, which controls DNA repair functions [182]. The SOS response is the result of the expression of approximately 30 genes involved in DNA repair such as recA, lexA, sulA, umuDC, and uvrAB. The SOS response requires RecA [183] and LexA [184] to be present. LexA represses the transcription of SOS response genes by binding the "SOS box" located in the promoter region, including those of lexA and recA [185]. RecA is responsible for the regulation of the SOS response as well as homologues recombination, and other DNA repair pathways such as SOS mutagenesis and repair of double-strand DNA breaks. RecA is activated after binding to single-strand DNA

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derived from damaged DNA [186]. The RecA/single-strand DNA-nucleoprotein complex then associates with LexA and activates LexA autoproteolysis [187]. Decreased LexA level results in the derepression of SOS genes such as sulA, umuDC, and uvrAB [188, 189]. SulA functions as an SOS checkpoint protein inhibiting cell division [190], allowing DNA repair prior to cell growth [191]. The SOS regulated umuDC operon is involved in a translesion DNA synthesis, which allows the bacteria to replicate over lesions that normally would block polymerization by DNA polymerase III [182]. Translesion DNA synthesis requires a post-translational form of UmuD called UmuD' [182]. This occurs via autodigestion after UmuD interacts with RecA/single-strand DNA-nucleoprotein complex. UmuC belongs to a superfamily of DNA polymerases that can replicate over lesions when in complex with UmuD' forming an error-prone DNA polymerase, allowing replication over unpaired abasic lesions [192-194] or thymine-thymine dimers. UvrA and UvrB are involved in the early stages of nucleotide excision repair. UvrA recruits UvrB to the damaged DNA site. A third protein UvrC binds UvrB creating UvrBC-DNA incision complex. This results in a DNA incision at the 3' and 5' side of the damage catalyzed by UvrC [195]. A fourth Uvr protein, UvrD, finally removes the damaged DNA strand, allowing DNA polymerase I to fill in the gap [195].

Stress alarmone ppGpp and the stringent response

The small nucleotide guanosine tetraphosphate, ppGpp, is the signal for the stringent response [196]. During the stringent response the ribosome production is down-regulated due to carbon- and amino acid starvation [197]. The production of ppGpp is a response to uncharged tRNA in the ribosomal A-site during amino acid starvation [197]. To produce ppGpp, E. coli activates the SpoT- and RelA-dependent pathways, where (p)ppGpp is produced from GTP and ATP and is subsequently converted to ppGpp. RelA is associated with the ribosome and produces ppGpp. The SpoT-pathway is mostly used for accumulation of ppGpp in response to other stress conditions and nutrient limitations [197]. The ppGpp molecule binds the β and β' subunits of the RNA polymerase (RNAP) core enzyme [198-200] and subsequently activates a wide array of physiological functions by transcribing target genes. After ppGpp has bound RNAP, the transcription of growth-related genes is down-regulated and genes involved in stress resistance and survival are induced. Besides the role in ribosomal down-regulation, ppGpp is also required for induction of many Ϭ70-dependent genes during starvation [201, 202].

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Universal stress protein A superfamily

The universal stress protein A (UspA) superfamily belongs to a conserved group of proteins found in many organisms including bacteria, Archaea, plants, fungi, and flies. In E. coli, UspA, is an abundant protein in growth-arrested cells and is produced as a response to a variety of different environmental signals, such as nitrogen-, phosphate-, carbon- and amino-acid starvation, and exposure to heat, metals, cycloserine, ethanol, and antibiotics [203, 204]. In E. coli, six usp genes are found, uspA, uspC, uspD, uspE, uspF, and uspG [205], that play various roles in resistance to DNA-damaging agents and to respiratory uncouplers [203]. The Usp proteins are divided into to two sub-families with UspA, UspC, and UspD belonging to one sub-family and UspF and UspG belonging to the second sub-family. Interestingly, UspE may belong to both since it contains both a UspACD and a UspFG domain. In E. coli, the expression of uspA is σ70-dependent and is regulated at the transcriptional level [206]. The requirement for σ70 is also predicted for uspC, uspD, and uspE. Furthermore, uspA, uspC, uspD, and uspE require the stress alarmone ppGpp for expression [202, 205]. This is exemplified during growth arrest due to cold-shock, which leads to reduced levels of ppGpp and repression of uspA expression [207]. uspA expression is also repressed by FadR, an activator of genes involved in fatty acid biosynthesis and a repressor of fatty acid degradation [208]. FtsK and RecA, involved in the SOS response, positively regulate uspA in a RecA-dependent manner further supporting that UspA plays a role in resistance to DNA damaging agents [205, 209].

Small RNA chaperone Hfq and small RNAs

The RNA chaperone Hfq is a global post-transcriptional regulator, which is found in both Gram-positive and Gram-negative bacteria (figure 10). In E. coli, Hfq is an abundant protein with approximately 50,000 to 60,000 copies per cell. Most Hfq molecules are associated with ribosomes [210]. Hfq has a high affinity for poly(A) tails and AU-rich RNA regions [211-213] at the base of a stem-loop structure [214-217]. However, Hfq can also bind DNA in the nucleoid [218] and RNAP in the presence of the ribosomal protein S1 [219], demonstrating that Hfq is also a transcriptional regulator [219]. Hfq plays an important role regulating cellular functions such as growth rate, sensitivity to ultraviolet light, and osmosensitivity [220]. In E. coli, Hfq regulates the expression of at least 50 proteins due to its role in the expression of the ϬS (RpoS) subunit of RNAP, which is important during stress conditions and stationary phase [221, 222]. Hfq regulates the expression of rpoS post-transcriptionally. Three sRNAs, OxyS, DsrA, and RprA regulate RpoS expression in response to specific stimuli [223, 224].

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Figure 10. The functions of the small RNA chaperone Hfq. In E. coli, Hfq functions as a modulator of protein activity, facilitating interactions between small RNA and a target mRNA, and to protect mRNA from RNasE cleavage. The sRNA OxyS, a regulator of oxidative stress, binds Hfq which facilitates the interaction of OxyS with rpoS mRNA, which inhibits rpoS translation [225, 226]. In contrast, DsrA and RprA activates translation of rpoS mRNA by preventing inhibitory secondary structures of rpoS mRNA [227-229]. This demonstrates the role of Hfq as an RNA chaperone, aiding the interaction of a sRNA with its target mRNA. This requires that Hfq alters the RNA secondary structure, allowing the sRNA to interact with the mRNA [230] and to regulate protein expression.

sRNAs are often between 40-400 base pairs in length, and allow the bacteria to respond quickly to environmental stimuli and to coordinate gene expression accordingly [231]. sRNA-gene regulation is beneficial for bacteria in terms of energy, filtering of noise from input signals, and response to a large input of signals [232]. sRNA can, together with Hfq, either repress or activate translation by alter the accessibility of the ribosomal binding site [232-235] or protect against RNase cleavage [236]. sRNAs are involved in a wide range of functions in the cell such as quorum sensing [237], virulence, and stress response [223, 238], glucose uptake [239], and modulation of outer membrane proteins [240]. The majority of sRNAs are incorporated in pathways responding to environmental signals, such as stress and nutrient limitation [232].

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Aims of this thesis

Quorum sensing is a part of the stress response in V. anguillarum. In this thesis, the characterization of the quorum-sensing phosphorelay systems in V. anguillarum was further analyzed with regard to stress response. Moreover, one stress response is the colonization of the fish skin. Therefore, a second aim of this thesis was to better understand mechanisms used by V. anguillarum to colonize fish tissue.

Specific aims

1. To further investigate the role of VanU and VanO in V. anguillarum quorum-sensing regulation.

2. To investigate the regulation of the signal synthase VanM and the role AHLs play in the quorum-sensing system of V. anguillarum. 3. To understand how V. anguillarum evades the fish innate immune system during the colonization of fish tissue to cause disease.

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Key findings and relevance

Paper I. The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.

Vibrio anguillarum uses three phosphorelay quorum-sensing systems to regulate stress response for survival in aquatic environments. At low cell densities, the quorum-sensing systems are relaying phosphates from the hybrid kinase receptors VanN, VanS, and CqsS to a single regulatory pathway involving the phosphotransferase VanU, which phosphorylates the response regulator VanO. Previously, phosphorylated and active VanO was shown to activate the expression of the sRNA Qrr1, which destabilizes and represses expression of VanT, a transcriptional regulator of V. anguillarum. In several vibrios, multiple qrr genes have been found in addition to the qrr1 gene [241]. In this paper, we investigated the possibilities of the presence of additional sRNAs, belonging to the Qrr family of RNAs in V. anguillarum. Using the qrr1 sequence, the draft genome of V. anguillarum was screened for additional qrr genes. We found three additional qrr genes and showed that all qrr genes were positively regulated by the sigma factor RpoN and the response regulator VanO. The Qrr1-4 sRNAs destabilized vanT mRNA, repressing VanT expression. Furthermore, we found that the expression profiles of the Qrr1-4 are different from Qrr sRNA expression profiles in other vibrios. The expression of Qrr1-4 was induced at high cell densities and repressed at low cell densities, which demonstrates a reverse expression of qrr genes to other vibrios [241-244]. This difference in expression suggests that signal molecules, which accumulate as the bacterial population increases, activate Qrr1-4 expression. Indeed, we were able to confirm that signal molecules activated rather than repressed Qrr1-4 expression. Therefore, we postulated in this study that the phosphotransferase VanU acts a branch point, which aids cross regulation between two independent phosphorelay systems that activate or repress Qrr1-4 expression. Consequently, the level of regulation on VanT expression can further be fine-tuned and controlled in response to stress.

Relevance paper I: This study demonstrates that bacteria, although having the same components of a regulatory system, may utilize the components very differently to respond and regulate gene expression. Moreover, this study strengthens the notion that V. anguillarum quorum-sensing regulation is unique and different from other vibrios. The fact that V. anguillarum has multiple ways to regulate quorum sensing indicates the

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ability to rapidly respond and adapt to a broad range of environmental signals. In addition to RpoS mediated regulation of VanT, a second potential quorum-sensing independent system is likely to affect expression of VanT.

Paper II. The transcriptional regulator VanT activates expression of the signal synthase VanM, forming a regulatory loop in the Vibrio anguillarum quorum-sensing system.

In V. anguillarum, quorum sensing plays a role in the physiology and stress response of the bacteria. Despite the similar components of the quorum-sensing systems within the vibrios, studies indicate that the cellular response of the V. anguillarum quorum-sensing phosphorelay systems is different to the same systems in other vibrios. In V. anguillarum, signal molecules produced by the AHL synthase VanM are suggested to repress the quorum-sensing regulon by activating the expression of the sRNAs Qrr1-4 [paper I]. Thus, we investigated the role of VanM in modulating the quorum-sensing regulon. In the present study, VanM was shown to activate the expression of Qrr1-4, which together with the RNA chaperone Hfq, destabilizes the mRNA of vanT, which encodes the main regulator of quorum sensing. Consequently, VanM represses the quorum-sensing regulon by activating the expression of Qrr1-4. This strengthens the observation that the phosphorelay quorum-sensing system in V. anguillarum responds differently to the same systems in other vibrios. Moreover, in this study regulation of vanM expression was also investigated. The transcriptional start site was identified 181-bp upstream and the 5'-untranslational region was characterized using bioinformatic analyses. Several putative Hfq-binding sites were found in the 5'-untranslational region and a putative VanT-binding site was found in the vanM promoter. This led us to investigate the role of VanT and Hfq in regulating VanM expression. VanT was shown to directly bind to and activate vanM expression, creating a negative regulatory loop between VanT and VanM. Consequently, VanT represses its own expression by activating VanM expression. Hfq was shown to destabilize vanM mRNA. Since Hfq also represses VanT expression, we suggest that Hfq plays a crucial role in regulating quorum sensing in V. anguillarum. Relevance paper II: In this study, VanM was shown in contrast to LuxM homologues in other vibrios to active the expression of Qrr1-4. Moreover, we give insight on how the signal synthase VanM is regulated. Since quorum-sensing is based on the signal molecules produced, a deeper understanding on how the signal synthases are regulated is crucial to fully understand how complex quorum-sensing systems are induced. Moreover, this study shows that Hfq has multiple roles in the V. anguillarum quorum-sensing systems.

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Paper III. The universal stress protein UspA regulates the expression of the signal synthase VanM in Vibrio anguillarum

In V. anguillarum, the signal synthase VanM is responsible for the production of the signal molecules N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). However, the studies on how this family of proteins is regulated are few. We have previously shown that VanM expression is directly activated by the master quorum-sensing regulator VanT and negatively regulated by the RNA chaperone Hfq post-transcriptionally [paper I]. In this study, we further investigated the regulation of VanM expression. In V. anguillarum, quorum sensing is tightly linked to stress response to aid survival of the bacterium. Located directly upstream of vanM is a uspA gene, which encodes a universal stress protein. In Escherichia coli, Usps are known to regulate stress response.

Thus, we asked if UspA plays a role in regulating VanM expression. UspA was shown to repress both VanM and VanT expression. VanM is suggested to repress the quorum-sensing regulon by activating the sRNAs Qrr1-4 [paper II], which together with Hfq destabilize vanT mRNA, thus repress VanT expression. Therefore, if UspA represses VanM expression, a decrease in Qrr1-4 expression is expected. Consequently, VanT expression should be activated. However, VanT was also repressed by UspA. This led us to propose a model where UspA repress VanM expression indirectly by repressing VanT expression.

Since UspA lacks a helix-turn-helix motif, we wondered how UspA may regulate gene expression. One possibility is that UspA interacts with another protein as the case for UspC in E. coli, which functions as a scaffolding protein. In V. anguillarum, VanM is negatively regulated by Hfq [paper II] and positively regulated by VanT [paper II]. However, E. coli does not contain a homolog of VanT but does contain Hfq. Thus, we investigated if UspA might bind Hfq. If so, UspA may derepress vanM expression by preventing Hfq from destabilizing vanM mRNA. Indeed, a strong interaction between UspA and Hfq was confirmed. As stated in this thesis, Hfq regulates the quorum-sensing system at multiple points. Therefore, an interaction with Hfq by UspA may alter Hfq activity and thus modulate quorum-sensing regulation in response to stress.

Relevance paper III: This study shows for the first time the involvement of a universal stress protein in regulating quorum sensing. Moreover, a novel regulatory role in the binding of Hfq suggests that UspA plays an important role in preventing Hfq mediated regulation in V. anguillarum. Furthermore,

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this study strengthens the fact that quorum sensing is a stress response in V. anguillarum.

Paper IV. Lipopolysaccharide O-antigen blocks phagocytosis of Vibrio anguillarum by fish skin epithelial cells.

Aquatic animals live in an environment that is rich in bacterial pathogens. The colonization of host tissues by bacteria is important during the initial stages of infection. However, even though bacteria have a great impact on bacterial disease in wild and farmed fish, very little is known about virulence factors required for colonization. Previous studies investigated factors utilized by V. anguillarum to colonize rainbow trout tissues. The RNA chaperone Hfq, siderophore production, and a functional exopolysaccharide transport were all shown to be essential for the bacteria to colonize skin tissues. However, the interaction between the host and V. anguillarum during colonization remains unknown. Thus, in this study we aimed to understand how V. anguillarum evades host immune defence associated with the skin tissues. The integument of the fish skin forms a mechanical barrier that protects the fish from bacteria in the marine environment. The outer most layer of the fish skin is mainly composed of highly motile epithelial cells, which plays a role in quick wound repair. We showed that the epithelial cells could efficiently phagocytize bacteria, thus giving the epithelial cells an antimicrobial role in the defence against bacterial colonization. Since V. anguillarum rapidly can colonize fish skin and thus cause disease, we proposed that a mechanism was used by V. anguillarum to evade the phagocytic epithelial cells. This study showed that V. anguillarum utilized the O-antigen of the lipopolysaccharide molecule to prevent internalization by the fish epithelial cells. The epithelial cells likely use a mannose receptor involved in the recognition of V. anguillarum since the phagocytic ability was blocked with mannose. Moreover, using in vivo bioluminescent imaging, we demonstrated that the O-antigen was required for skin colonization, but not for the intestines. In addition, a function for the O-antigen was shown in resistance to lysozyme and antimicrobial peptides.

Relevance paper IV: This study furthers the knowledge of bacteria-host interactions at initial stages of infection. Although suggested previously, we show unequivocally that fish skin epithelial cells play an important role in internalizing bacteria and keeping the fish clear from pathogens. Since V. anguillarum could evade internalization by using the lipopolysacchride O-antigen, we suggest that the O-antigen masks mannose residues on the bacteria surface not accessible for the phagocytic epithelial cells. In

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conclusions, this study shows a new mechanism used by a pathogen to colonize fish tissue.

34

Conclusions

Paper I. The phosphotransferase VanU represses expression of the four Qrr sRNAs and thus antagonizing VanO-mediated quorum-sensing regulation in V. anguillarum.

Paper II. The signal synthase VanM and the transcriptional regulator VanT form a regulatory loop in the phosphorelay quorum-sensing system in V. anguillarum. VanT directly activates vanM expression and VanM activates the expression of the Qrr1-4 sRNAs. Qrr1-4 destabilize vanT mRNA and repress VanT expression.

Paper III. The universal stress protein UspA regulates the signal synthase VanM in V. anguillarum.

Paper IV. Vibrio anguillarum lipopolysaccharide O-antigen blocks phagocytosis of V. anguillarum by fish skin epithelial cells. The phagocytosis by fish skin epithelial cells is likely receptor-mediated and involves a mannose-like receptor. The lipopolysaccharide O-antigen is required for fish skin colonization, but not for colonization of the intestines.

Figure 11. Quorum-sensing regulation of VanT and VanM. Solid lines with arrows and bars represent gene activation or repression, respectively. Solid lines with double arrowheads represent transfer of phosphoryl groups. RR2 is a second response regulator and HK2 is a histidine kinase.

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Acknowledgements

The time has now, after five amazing years, come to an end I would like to acknowledge the people who have made this possible.

Debbie, my supervisor, I'm so grateful for your all your help and support during my years in the lab. You have taught me so many things on every level when it comes to science. The present and previous lab members, you have made an impact on me for sure. Barbara, thanks for all the support. Roland and Kristina, keep it up. Also I would like to thank all the students that have been in the lab, especially: Sarp, Lisa, Makunda and Ali - my god you are funny... Thanks to all the members of GrpHWW, GrpMFR, GrpVSH, GrpÅF, GrpRR, and GrpMFÄ for the creative and interesting group meetings, journal clubs and other get-together meetings. I have learned a lot from you all.

Johnny, du gör mitt labbande enklare, men det är klart med Legatus så blir allt enkelt..eller?.. Labservice, tack för all service och roliga pratstunder vi har haft. Ni gör verkligen ett viktigt jobb! Tack! Några speciella personer som jag har lärt mig mycket från (inte så mycket viktigt dock..) Putte, Micke S, Tobbe, Stefan, Christian, Lelle, PA, och alla andra som jag glömt bort i skrivande stund. Dom flesta har sedan länge lämnat denna skuta... Putte vi får ta den där squaschen snart, är bara 10 år sen vi planerade att spela.. Tiden går fort lilla studiekamrat. Ett mycket stort tack till min underbara familj. Mina fantastiska barn Jonna, Jonatan, och Lovisa - ni är helt otroliga! Tack för all hjälp i labbet. Min älskade fru Anna, tack för att du står ut med mig och mina "kreativa" sidor. Min kära bror Tobias, du är också lite kreativ när det kommer till de "goda" sakerna i livet. Mina svärföräldrar Ola och Eva, ni har varit ett stort stöd och jag uppskattar er verkligen mycket! Ett speciellt tack till min mor och far, ni har/gör ett bra jobb och jag är stolt över er. Alla i Ume-familjen har alla uppskattat er hjälp ofantligt mycket!

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