Download Cell-to-cell communication and virulence in Vibrio anguillarum

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
Jonna, Jonatan och Lovisa - Låt fantasin flöda
Till min familj
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
Virulence factors of V. anguillarum
4
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
Fish skin defense against bacteria
8
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
i
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 quorumsensing 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.
Relevance paper IV
32
32
Conclusions
34
Acknowledgements
35
References
36
ii
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.
iii
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
iv
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.
v
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 nonculturable 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
1
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.
2
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].
3
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
4
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 DNAbinding 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
5
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].
6
Figure
B
A
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 Opolysaccharide (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,
7
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
8
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 α1antiproteinase 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. Mucusassociated 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 Nacetylglucosamine components located in the peptidoglycan layer in the
bacterial membrane. Lysozymes are found in fish serum and mucus. The Creactive 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.
9
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 antitoxins, 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
10
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
II) Cytokines
- Anti-toxin
- Anti-invasin
- Interferon gamma
- Anti-adhesin
- Tumour necrosis factor
III) Activated macrophages
- Classical complement
- 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.
11
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 Sadenosylmethionine (SAM) and acylated acyl carrier protein (ACP) as
substrate for AHL synthesis [125-130].
12
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 414 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-(3oxo-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.
13
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
14
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 (3hydroxy-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
15
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
16
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 quorumsensing 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].
17
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 quorumsensing system, inducing bioluminescence and late colonization factors [129,
163, 166].
18
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
19
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 Nhexanoyl-L-homoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)-Lhomoserine 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].
20
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.
21
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
22
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. RR2 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 refolding 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
23
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 doublestrand DNA breaks. RecA is activated after binding to single-strand DNA
24
derived from damaged DNA [186]. The RecA/single-strand DNAnucleoprotein 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 growthrelated 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].
25
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 growtharrested cells and is produced as a response to a variety of different
environmental signals, such as nitrogen-, phosphate-, carbon- and aminoacid 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 DNAdamaging 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].
26
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].
27
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.
28
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 finetuned 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 quorumsensing regulation is unique and different from other vibrios. The fact that V.
anguillarum has multiple ways to regulate quorum sensing indicates the
29
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 quorumsensing 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 quorumsensing 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.
30
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 (C6HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6HSL). 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,
31
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 Oantigen, we suggest that the O-antigen masks mannose residues on the
bacteria surface not accessible for the phagocytic epithelial cells. In
32
conclusions, this study shows a new mechanism used by a pathogen to
colonize fish tissue.
33
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.
34
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!
35
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