Báo cáo khoa học: The secretome of Campylobacter concisus pot

The secretome of Campylobacter concisus
Nadeem O. Kaakoush
1
, Si Ming Man
1
, Sarah Lamb
1
, Mark J. Raftery
2
, Marc R. Wilkins
1
, Zsuzsanna
Kovach
1
and Hazel Mitchell
1
1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
2 Biological Mass Spectrometry Facility, University of New South Wales, Sydney, Australia
Introduction
Since the discovery of Campylobacter jejuni, which is
recognized as the leading cause of bacterial gastroenteri-
tis in both the developing and developed world [1], a
considerable body of research has focused on this patho-
genic bacterial species. The virulence factors integral to
the pathogenesis of C. jejuni include motility, adhesion,
expression of toxins, and invasion into host cells [2],
making it well suited to the conditions of the gastroin-
testinal tract. Following colonization of the intestinal
mucosa, C. jejuni adheres to epithelial cells via surface-
associated adhesins [2]. The bacterium then employs its
flagellar apparatus as a secretion organelle, through

which it secretes invasion antigens that promote cellular
invasion [3]. Evidence also supports a paracellular
invasion mechanism by which C. jejuni disrupts tight
junctions of epithelial cells [4,5]. Additionally, C. jejuni
can induce apoptotic cell death through the expression
and secretion of a cytolethal distending toxin within the
cells [6].
Although research into the pathogenesis of C. jejuni
has expanded over the past decades, little is known
about other members of the Campylobacter genus.
Over recent years, evidence has emerged suggesting
that a number of non-jejuni Campylobacter species
may also be potential pathogens of the human intesti-
nal tract. For example, Van Etterijck et al. [7],
Vandamme et al. [8] and Johnson and Finegold [9]
have reported the isolation of Campylobacter concisus
from fecal samples of patients with gastrointestinal
disorders. As a result of these and other studies,
Keywords
Campylobacter concisus; Crohn’s disease;
secretome; virulence; zonula occludens
Correspondence
H. Mitchell, School of Biotechnology and
Biomolecular Sciences, The University of
New South Wales, Sydney, NSW 2052,
Australia
Fax: +61293851483
Tel: +61293852040
E-mail:
(Received 3 November 2009, revised

18 January 2010, accepted 20 January
2010)
doi:10.1111/j.1742-4658.2010.07587.x
A higher prevalence of Campylobacter concisus and higher levels of IgG
antibodies specific to C. concisus in Crohn’s disease patients than in con-
trols were recently detected. In this study, 1D and 2D gel electrophoresis
coupled with LTQ FT-MS and QStar tandem MS, respectively, were per-
formed to characterize the secretome of a C. concisus strain isolated from a
Crohn’s disease patient. Two hundred and one secreted proteins were iden-
tified, of which 86 were bioinformatically predicted to be secreted. Searches
were performed on the genome of C. concisus strain 13826, and 25 genes
that have been associated with virulence or colonization in other organisms
were identified. The zonula occludens toxin was found only in C. concisus
among the Campylobacterales, although expanded searches revealed that
this protein was present in two e-proteobacterial species from extreme mar-
ine environments. Alignments and structural threading indicated that this
toxin shared features with that of other virulent pathogens, including
Neisseria meningitidis and Vibrio cholerae. Further comparative analyses
identified several associations between the secretome of C. consisus and
putative virulence factors of this bacterium. This study has identified
several factors putatively associated with disease outcome, suggesting that
C. concisus is a pathogen of the gastrointestinal tract.
Abbreviations
OMP, outer membrane protein; TCA, trichloroacetic acid; Zot, zonula occludens toxin.
1606 FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS
C. concisus has recently been suggested to be a
putative agent of diarrheal diseases [10–12]. A recent
study in our laboratory resulted in the culture of
several non-jejuni Campylobacter species from biopsy
samples obtained from children newly diagnosed with

Crohn’s disease [13]. Using a species-specific PCR,
C. concisus was shown to be present in 51% of
children with Crohn’s disease, a significantly higher
frequency than in controls (2%) [13]. Investigation of
the IgG antibody response in sera from children shown
to be PCR-positive showed a significantly higher level
of C. concisus antibodies to be present in patients with
Crohn’s disease than in controls [13], suggesting that
children infected with C. concisus mounted an IgG
response to this bacterium.
To date, there is limited information regarding the
molecular basis of the pathogenesis of C. concisus.
A study by Engberg et al. [14] has shown that C. concisus
is able to produce a toxin similar to cytolethal distending
toxin, and that cell lysates from C. concisus are able to
induce cytopathic effects in a monkey kidney epithelial
cell line. Two further studies have shown that C. concisus
isolates possess cell-bound and secreted hemolytic
activities [15,16]. In relation to animal models, a study
conducted in 2008 showed that some strains of
C. concisus have the ability to colonize the mouse
intestinal tract, and that C. concisus can be cultured
from the liver, ileum and jejunum of infected mice [17].
In this study, proteomics coupled with MS and
comparative bioinformatic searches were performed to
identify the secreted proteins and putative virulence
factors of C. concisus.
Results and Discussion
The secretome of C. concisus
The secreted proteins of a pathogen can be divided

into three major groups on the basis of their function
within the cell: those involved in cell survival; those
involved in protection against stresses; and those asso-
ciated with virulence and ⁄ or colonization of the host.
The secretome is an essential component of a patho-
gen’s arsenal, and can ultimately reflect its invasive-
ness. As such, the characterization of the secretome of
C. concisus can assist in unraveling the pathogenic
potential of this bacterium (391 proteins were pre-
dicted to be secreted by C. concisus strain 13826 using
the signalp 3.0 server). In addition, given that the
flagellar secretion system has a significant role in the
virulence of C. jejuni and that similarities are present
between the flagellar systems of C. jejuni and C. conci-
sus, detection and identification of the secreted pro-
teins of C. concisus was undertaken.
The secreted proteins of C. concisus strain
UNSWCD, which was isolated from a cecal biopsy
sample from a child with Crohn’s disease [13], were
purified from liquid cultures and separated using
1D-PAGE and 2D-PAGE (pI 4–7 and 7–10) (Fig. 1).
Bands or spots were excised and digested, and proteins
were identified using the appropriate MS protocol. The
use of two independent methods, each with its own
advantages, ensured the identification of the highest
number of proteins within the purified sample.
1D-PAGE coupled with LTQ FT-MS allows for the
209
124
80

49.1
34.8
28.9
20.6
7.1
kDa
AB
C
4
7
7
10
Fig. 1. One-dimensional (A) and two-dimen-
sional (B, C) PAGE on C. concisus UNSWCD
secreted proteins. The fragment of the gel
in (A) bordered by a dashed line was
sectioned into 25 gel slices and processed
for MS analyses. Proteins were also run on
2D-PAGE gels at pI 4–7 (B) and pI 7–10 (C),
and all spots on both gels were extracted
for MS analyses.
N. O. Kaakoush et al. Campylobacter concisus secretome
FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS 1607
identification of a high percentage of proteins from a
complex fraction. In contrast, 2D-PAGE coupled with
QStar tandem MS allows for the identification of
lower-abundance proteins that could be overlooked in
the process of analyzing complex fractions. The purifi-
cation of secreted proteins from large volumes of med-
ium may result in relatively higher quantities of

contaminants (e.g. salts) within the protein fraction.
Together with the presence of extracellular proteases
that will degrade proteins, this may explain the streak-
ing observed in the 2D-PAGE gels. The combined
results from both methods consisted of 201 identified
proteins within the purified fraction (PRIDE accession
number: 11363) (Table S1; Figs S1 and S2).
As cellular lysis and degradation during the growth
of bacterial cultures and high-abundance proteins may
result in nonsecretory contaminants within the identi-
fied proteins, the 201 proteins were analyzed for the
presence of a signal peptide, using the signalp 3.0 ser-
ver. Signal peptides interact with signal peptidases to
cleave proteins at specific sites, allowing proteins to be
folded and exported via secretion. Of the 201 proteins,
69 were predicted to have a signal peptide, confirming
that they are secreted proteins (Table S1; Table 1).
Seventeen of the remaining 132 proteins were found,
using the secretomep 2.0 server, to be nonclassically
secreted (Table S1; Table 1). Functional classification
of the 115 remaining proteins revealed that the major-
ity of proteins, bioinformatically predicted to be non-
secretory were involved in cellular survival (Table 2).
Enriched functions included amino acid metabolism
(n = 21), carbohydrate metabolism (n = 16), and
elongation factors and chaperones (n = 15) (Table 2).
It is possible that the high abundance of metabolic
proteins, elongation factors and chaperones within
cells may result in many of these proteins contaminat-
ing the secretory fraction; however, owing to their high

stringency, the bioinformatic predictio n processes
employed may have also overlooked true-positives.
The 86 confirmed secreted proteins of C. concisus
UNSWCD were also grouped on the basis of their
functions (Table 1). The proteins identified were either:
(a) related to bacterial physiology, such as metabolic
and solute-binding⁄ transport proteins; (b) involved in
host-related functions, such as virulence factors; or (c)
associated with protection against environmental stres-
ses, such as oxidative stress response proteins. An
additional 12 proteins previously annotated as putative
or hypothetical were identified in the secretome of
C. concisus.
The first major group of proteins identified was
involved in bacterial physiology and survival. For
example, seven solute-binding proteins were identified.
The presence of these proteins is to be expected, as
many of them have functions linked to cellular
metabolic processes. The proteins identified were two
solute-binding family 1 proteins, two C4-dicarboxy-
late-binding periplasmic proteins, extracellular tung-
state-binding protein, glutamine-binding periplasmic
protein, and d-methionine-binding lipoprotein MetQ.
Each protein is specific for a different substrate, which
it transports into or out of the cell across the periplas-
mic membrane. For example, the C4-dicarboxylate-
binding protein is a high-affinity transporter of
C4-dicarboxylates such as fumarate. Bacteria such as
C. concisus, which can respire anaerobically, are able
to utilize fumarate as a terminal electron acceptor for

this process. Similarly, MetQ binds d-methionine for
bacterial utilization, and the tungstate-binding protein
has been shown to protect cytochrome c oxidase from
tungsten inhibition by binding free tungsten in the
plasma membrane [18], thus allowing cytochrome c to
function uninhibited. Cytochrome c is involved in an
electron transfer system in which cytochrome c oxidase
is the terminal electron acceptor, helping to establish a
proton gradient, allowing the cell to synthesize ATP. It
is interesting to note that both the cytochrome c
assembly protein and cytochrome c oxidase were also
identified as secreted proteins, implying that their
function, as well as the function of tungstate-binding
protein, is critical to cell survival.
Another protein identified was the methyl-accepting
chemotaxis protein, which is involved in signal trans-
duction and chemotaxis. This protein is a member of a
family of signal transducers in which sensory adapta-
tion is mediated by the methylation of proteins. Stud-
ies have shown that these proteins are involved in the
general sensory control of both gliding and flagellar
motility [19]. Given that C. concisus may rely on its
flagella to both access and bind to the epithelial sur-
face of the gastrointestinal tract, the secretion of
proteins involved in the control of chemotaxis and
flagellar motility highlights not only the importance of
motility in cellular survival, but also the fact that
secreted proteins may play a role in the pathogenesis
of the bacterium.
Examples of proteins that were found to be

involved in the oxidative stress response are cop-
per ⁄ zinc superoxide dismutase, superoxide dismutase
(Fe), and protease Do. Superoxide dismutase is
involved in the catalysis of superoxides, such as
those produced by phagocytes during oxidative burst
killing of pathogens [20]. Protease Do is a serine
protease identical to the product of the high temper-
ature requirement A gene (htrA), which has been
described in Escherichia coli. HtrA protein has been
Campylobacter concisus secretome N. O. Kaakoush et al.
1608 FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS
Table 1. Functional classification of C. concisus UNSWCD secreted proteins bioinformatically predicted to be secreted (n = 86). Proteins
from Table S1 that contained a signal peptide in their amino acid sequence or were found to be nonclassically secreted were chosen for fur-
ther classification.
Classification GI number ORF Protein name
ABC transporter 157165101 CCC13826_1247 Nlpa lipoprotein
157165776 CCC13826_1793 Putative carbon storage regulator-like protein
157164930 CCC13826_2116 Hypothetical protein
157164768 CCC13826_2204 Periplasmic molybdate-binding protein
Protein transport 157164610 CCC13826_0922 Translocation protein TolB
157165665 CCC13826_1073 Outer membrane lipoprotein carrier protein
157165672 CCC13826_1131 Curli production component CsgG protein
Solute transport 158605017 CCC13826_0202 Extracellular tungstate-binding protein
157163977 CCC13826_0329 C4-dicarboxylate-binding periplasmic protein
157165691 CCC13826_0764 C4-dicarboxylate-binding periplasmic protein
158604975 CCC13826_1248
D-Methionine-binding lipoprotein MetQ
157165339 CCC13826_1924 Glutamine-binding periplasmic protein
157164859 CCC13826_1925 Solute-binding family 1 protein
157164888 CCC13826_1926 Solute-binding family 1 protein

Response regulator 157165180 CCC13826_1398 Putative cytochrome c-type periplasmic protein
157165649 CCC13826_2064 DNA-binding response regulator
157165183 CCC13826_2088 Methyl-accepting chemotaxis sensory transducer
Oxidoreduction 157164382 CCC13826_0059 Thioredoxin domain-containing protein
157165326 CCC13826_0161 Copper ⁄ zinc superoxide dismutase
157165242 CCC13826_0201 Molybdopterin oxidoreductase Fe
4
S
4
subunit
157165413 CCC13826_0328 Superoxide dismutase (Fe)
157163886 CCC13826_0758 Thiol peroxidase
157164029 CCC13826_1064 Protease Do
157164957 CCC13826_1400 Dyp-type peroxidase
157165527 CCC13826_1459 Hydrogenase-4 component I
157164172 CCC13826_1929 Flavodoxin FldA
Cytochrome c maturation 157164310 CCC13826_1461 Cytochrome c assembly protein
157165298 CCC13826_1472 Cytochrome c oxidase
Protein folding 157165622 CCC13826_0002 Disulfide isomerase DsbA
157164923 CCC13826_1633 Methionine sulfoxide reductase family protein
157164051 CCC13826_1670 TPR repeat-containing protein
Protein synthesis 157165109 CCC13826_0029
L-Asparaginase
157165495 CCC13826_0170 50S ribosomal protein L11
157164773 CCC13826_0171 50S ribosomal protein L1
157164220 CCC13826_0172 50S ribosomal protein L10
157164672 CCC13826_0341 Ribosome recycling factor
157164041 CCC13826_0468 Glutamyl-tRNA synthetase
157164944 CCC13826_1737 Sigma 54 modulation protein
157165065 CCC13826_1871 tRNA synthetase, class II

157165615 CCC13826_1872 Adenylosuccinate synthetase
157165454 CCC13826_1997 Amino acid carrier protein AlsT
157165048 CCC13826_2114 Diaminopimelate epimerase
Fatty acid synthesis 157164063 CCC13826_0560 3-Oxoacyl-(acyl carrier protein) synthase II
157164484 CCC13826_0562 3-Ketoacyl-(acyl carrier protein) reductase
157164819 CCC13826_1552 b-Ketoacyl-acyl carrier protein synthase II
157164251 CCC13826_2069 Holo-(acyl carrier protein) synthase
Nitrogen metabolism 157164023 CCC13826_0863 Carbon–nitrogen family hydrolase
157165227 CCC13826_0865 Periplasmic nitrate reductase
157165732 CCC13826_0868 Nitrate reductase
157164517 CCC13826_1729 Methyl-accepting chemotaxis protein
Sulfur metabolism 157164952 CCC13826_0231 Arylsulfotransferase
158604950 CCC13826_2236 Thiosulfate sulfurtransferase
Sugar metabolism 157165684 CCC13826_0411 N-acetylglucosamine deacetylase
157164467 CCC13826_0872 Phosphomannomutase
157165286 CCC13826_1509 Phosphoenolpyruvate carboxykinase
N. O. Kaakoush et al. Campylobacter concisus secretome
FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS 1609
found to play a role in the intramacrophagic replica-
tion of E. coli, with mutations in the htrA gene lead-
ing to reduced bacterial virulence in mice [21]. The
microaerophilic nature of C. concisus may explain
the secretion of proteins involved in combating oxi-
dative stress; however, the additional role of these
proteins in neutralizing the oxidative bursts produced
by polymorphonuclear cells in response to infection
may reflect the survival strategies of C. concisus
within its host.
In the group pertaining to host-related functions, an
outer membrane fibronectin-binding protein, known to

be involved in adhesion to the host cell,was identified.
Fibronectin is a large glycoprotein that is a component
of the extracellular matrix of the human intestinal
epithelium. Studies on C. jejuni have shown that the
bacterium binds to fibronectin on the basolateral sur-
face of human colonic cells [22]. The secretion of an
extracellular binding protein that is specific to recep-
tors in the intestinal epithelium is especially significant
if C. concisus plays a pathogenic role in humans, as
this protein assists in the adhesion to, and subsequent
colonization of, the host cells [22]. Other virulence
factors, CjaA and CjaC, were identified among the
secreted proteins analyzed. CjaA and CjaC are poten-
tially surface-exposed proteins that are homologs of
ABC-transport proteins and known to be highly
immunodominant in C. jejuni. Additionally, an
S-layer-RTX protein was also found to be secreted by
C. concisus UNSWCD. RTX proteins are pore-form-
ing toxins synthesized by a diverse group of Gram-neg-
ative pathogens. RTX-mediated cytotoxicity comprises
two phases: a passive phase of adsorption onto the tar-
get cell surface; and a membrane insertion phase [23].
The two forms of host cell death associated with
this type of toxin include apoptosis and necrosis
[23]. Finally, the flagellin-like protein FlaC, encoded
by ccc13826_2187, was secreted by C. concisus
UNSWCD. This protein is secreted by the flagellar
system of C. jejuni, and mutants in the flaC gene
showed a significantly reduced level of invasion into
HEp-2 cells [24].

Table 1. (Continued.)
Classification GI number ORF Protein name
157165707 CCC13826_2070 Fructose bisphosphate aldolase
Tricarboxylic acid cycle 157164353 CCC13826_1283 Succinate dehydrogenase flavoprotein subunit
Electron transfer 157164059 CCC13826_1119 Hypothetical protein
157165471 CCC13826_1395 Radical SAM domain-containing protein
DNA ⁄ RNA 157164945 CCC13826_0021 DNA-binding protein HU 1
157165374 CCC13826_0649 DNA primase
157165135 CCC13826_0888 ComEC ⁄ Rec2 family protein
Membrane and cell wall synthesis 157164040 CCC13826_0084 Glutamate racemase 2
157164709 CCC13826_0131 Peptidoglycan-associated lipoprotein
157164747 CCC13826_0576 ADP-glyceromanno-heptose-6-epimerase
157164370 CCC13826_0924 ADP-heptose-LPS heptosyltransferase II
157164830 CCC13826_1534 Lipopolysaccharide biosynthesis protein
Motility 157164686 CCC13826_2187 Flagellin-like protein FlaC
157164988 CCC13826_2297 Flagellin B
Virulence 157164816 CCC13826_0664 Surface antigen CjaA
157164740 CCC13826_0739 Outer membrane fibronectin-binding protein
157165273 CCC13826_0963 Antigen CjaC
157164622 CCC13826_1253 a-Macroglobulin family protein
157165243 CCC13826_1838 S-layer-RTX protein
Unknown 157164911 CCC13826_0243 Putative periplasmic protein
157164523 CCC13826_0601 Putative outer membrane protein
157165071 CCC13826_0859 Hypothetical protein
158604901 CCC13826_1186 Conserved hypothetical protein
157163941 CCC13826_1330 Hypothetical protein
157164030 CCC13826_1407 Hypothetical protein
157165213 CCC13826_1428 Hypothetical protein
157164793 CCC13826_1524 Hypothetical protein
157165368 CCC13826_1643 FAD-binding domain-containing protein

157164111 CCC13826_1707 Hypothetical protein
157164095 CCC13826_1803 FAD-binding domain-containing protein
157163930 CCC13826_2060 Outer membrane protein
Campylobacter concisus secretome N. O. Kaakoush et al.
1610 FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS
Putative virulence factors of C. concisus
Increasing reports suggesting a pathogenic role for
C. concisus in the intestinal tract of humans highlights
the importance of understanding the molecular mecha-
nisms by which this bacterium may cause disease in its
host. Therefore, blast searches of genes and proteins
previously associated with virulence or colonization in
other organisms were performed on the available gen-
ome of C. concisus 13826, to determine whether any of
these were present. This resulted in the identification of
25 potential candidates. These included known invasins,
adhesins, hemolysins and iron-associated virulence fac-
tors, such as invasin InvA, fibronectin-binding protein
CadF, hemolysin TlyA, and siderophore esterase IroE
(Table 3). Another virulence-associated factor identified
was outer membrane protein (OMP) 18, encoded by
ccc13826_0923 (Table 3). This protein is known to be
an immunodominant antigen in both C. jejuni and
Helicobacter pylori [25,26], and a study by Rathinavelu
et al. [27] demonstrated that OMP18 was capable of
inducing dendritic cell maturation and function, as well
as initiating a Th-1-mediated immune response.
Two proteins were found to be involved in twitching
motility (Table 3), a form of surface translocation that
enables the bacterium to crawl along surfaces [28].

This form of bacterial motility has been implicated in
virulence and cytotoxicity in E. coli, Pseudomo-
nas aeruginosa and Neisseria spp. [28].
Two genes encoding zonula occludens toxin (Zot)
were identified in C. concisus 13826 (Table 3). More-
over, two hypothetical proteins encoded by
ccc13826_0191 and ccc13826_1210 were identified with
Table 2. Functional classification of C. concisus UNSWCD identi-
fied proteins bioinformatically predicted to be nonsecretory
(n = 115).
Functional classification Number of proteins
Amino acid metabolism 21
Carbohydrate metabolism 16
Elongation factors and chaperones 15
Lipid metabolism 3
Metabolism of cofactors and vitamins 4
Nucleotide metabolism 6
Redox 8
Signal transduction and chemotaxis 8
Transcription 11
Translation 13
Transport and secretion 3
Unknown 7
Table 3. Putative virulence and colonization factors found in C. concisus 13826.
GI ORF Protein name Present in other Campylobacterales
158604981 CCC13826_0115 Integral membrane protein MviN Yes
157164228 CCC13826_0191 Hypothetical protein Yes
a
158605018 CCC13826_0222 Twitching motility protein Yes
112800340 CCC13826_0315 Symbiosis island integrase Yes

157101450 CCC13826_0608 Hemolysin activator-related protein HecB Yes
157164816 CCC13826_0664 Surface antigen, CjaA Yes
157164010 CCC13826_0706 Phage integrase family protein Yes
157164740 CCC13826_0739 Fibronectin-binding protein CadF Yes
157164577 CCC13826_0816 Acid membrane antigen A Yes
157164304 CCC13826_0923 Outer membrane protein 18 Yes
157165273 CCC13826_0963 Antigen CjaC Yes
157164212 CCC13826_1210 Hypothetical protein Yes
a
158604972 CCC13826_1235 Iron-regulated colicin 1 receptor Yes
158604973 CCC13826_1236 Siderophore esterase IroE Yes
158604983 CCC13826_1443 Peptidase U32 (collagenase) Yes
157164343 CCC13826_1584 Twitching motility protein Yes
157165243 CCC13826_1838 S-layer-RTX protein Yes
157164705 CCC13826_2000 RNase R (VacB homolog) Yes
157164282 CCC13826_2017 Invasin InvA Yes
157164345 CCC13826_2024 Hemolysin TlyA Yes
157164617 CCC13826_2075 Zonula occludens toxin No
157165505 CCC13826_2148 Invasion protein CiaB Yes
157164974 CCC13826_2191 Phospholipase PldA Yes
157163885 CCC13826_2197 Hsp12 variant C Yes
157164938 CCC13826_2276 Zonula occludens toxin No
a
Found only in C. jejuni ssp. doylei 269.97.
N. O. Kaakoush et al. Campylobacter concisus secretome
FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS 1611
47% and 46% similarity, respectively, to C. concisus
Zot. Zot is known to mimic a physiological modulator
of intercellular tight junctions [29], and is used by viru-
lent pathogens such as Vibrio cholerae and Neisse-

ria meningitidis to increase tissue permeability [30]. In
contrast to the activities of Clostridium difficile toxins
A and B, the changes in tight junctions after exposure
to Zot are reversible and are not associated with the
destruction of the tight junction complex [31]. Further
characterisation of Zot has indicated that its C-termi-
nal domain causes delocalization of occludin and ZO-1
from Caco-2 cell–cell contacts [32]. Furthermore, expo-
sure of Caco-2 cell monolayers to a peptide synthe-
sized on the basis of the active domain of V. cholerae
Zot caused the redistribution of ZO-1 away from cell
junctions [33]. The peptide also caused a reversible
reduction in transepithelial electrical resistance and an
increase in lucifer yellow permeability [33].
Searches for Zot homologs within the host-related
Campylobacterales order revealed this toxin to be only
present in C. concisus, suggesting that it may have an
important role in the pathogenesis of the bacterium. As
a result of its specificity, the possibility that C. concisus
acquired Zot from another pathogen through gene
transfer was strong. However, further expanded
searches against the genomes of all e-Proteobacteria
found Zot homologs within the genomes of the sulfur-
metabolizing bacteria Nautilia profundicola and
Caminibacter mediatlanticus. These bacterial species
have been isolated from extreme environments such as
deep sea vents [34,35]. It is unknown why bacteria from
these extreme environments would require a toxin that
targets tight junctions, but one possibility could be that
this toxin aids N. profundicola in penetrating the sheath

lining covering the worms that it colonizes.
Alignment of the Zot amino acid sequences of
C. concisus, Ne. meningitidis and V. cholerae demon-
strated that four highly conserved domains exist within
these proteins that are likely to be important for toxin
activity (Fig. 2). However, no domains within the
sequences of C. concisus Zot and Ne. meningitidis Zot
aligned with the previously identified active domain of
V. cholerae Zot (FCIGRL), suggesting that these
toxins may have different mechanisms of action. Ter-
tiary structure prediction indicated that Zot structures
from these three bacterial species were highly variable
(Fig. 3); however, high-scoring templates for structure
generation (mean sequence identity, 11.4%; mean
sequence length, 86%) were not available, and this
may have contributed to the differences observed in
the 3D structures. Analysis of the secondary structures
generated from the tertiary structure prediction showed
an overall similarity between the Zot secondary struc-
tures of C. concisus, Ne. meningitidis and V. cholerae,
with the exception of a few minor changes (Fig. 3).
These findings support the hypothesis that C. concisus
is capable of attaching to and invading host cells
through a paracellular mechanism in which it targets
the host cell tight junctions by expressing Zot.
Interactions between virulence factors and the
C. concisus secretome
Further analyses on the secretome of C. concisus
UNSWCD included the identification of interactions
between individual secreted proteins and the virulence

factors outlined in Table 3. Physical and functional
associations between proteins were searched for using
the string database for known and predicted protein
interactions, and six secreted proteins were recognized
to be putatively interacting with virulence factors.
The disulfide bond-forming protein DsbA, encoded by
ccc13826_0002, was secreted by C. concisus UNSWCD.
DsbA is reported to be essential for the pathogenic
process of many bacteria [36,37], where it plays a critical
role in the production of secreted virulence factors in
pathogens [38]. One such example is the secretion of the
pertussis toxin by Bordetella pertussis [39]. The inactiva-
tion of DsbA results in the perturbation of redox homeo-
stasis within the bacterial periplasm, and, as a result,
sulfydryl-containing proteins are not properly folded.
Translocation protein TolB, encoded by ccc1
3826_0922, and ADP-heptose-LPS heptosyltransferase
II, encoded by ccc13826_0924, were found within the
network of OMP18, an immunodominant antigen in
both C. jejuni and H. pylori [25,26]. These associations
would probably have resulted from the proximity of
the ORFs of the two secreted proteins to
ccc13826_0923, which encodes OMP18.
Holo-(acyl carrier protein) synthase, encoded by
ccc13826_2069, was found to interact with FlaC, a
flagellin-like protein. As previously discussed, this pro-
tein is secreted by C. jejuni and is capable of binding
host cells and modulating the invasion process [24]. The
carbon–nitrogen family hydrolase was putatively associ-
ated with the invasion protein CiaB. Approximately 14

Cia proteins have been shown to be synthesized when
C. jejuni is cocultured with epithelial cells, a subset of
which are secreted in the presence of eukaryotic cells.
Mutation of the ciaB gene has been shown to inhibit the
secretion of all other Cia proteins and significantly
reduce the number of internalized C. jejuni cells when
compared with the wild-type parent strain [3,40].
One further association between the secretome and
virulence was found between the peptidoglycan-associ-
ated lipoprotein encoded by ccc13826_0131 and invasin
Campylobacter concisus secretome N. O. Kaakoush et al.
1612 FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS
InvA. This invasin is an essential component of the
invasion-associated type III secretion system in Salmo-
nella spp. [41]. InvA-expressing bacteria enter the host
cell through the invasin-mediated pathway, and are
subsequently delivered to lysosomes [42]. Interestingly,
even though C. jejuni possesses InvA within its gen-
ome, Watson and Gala
´
n [42] have shown that it avoids
delivery into lysosomes after entering the cell via a
unique caveolae-dependent entry pathway.
Conclusions
Analysis of the secretome of C. concisus identified a
number of proteins that are involved in the general func-
tion of the bacterial cell, as well as a number of potential
virulence factors. The presence of virulence factors in the
secreted proteins of C. concisus, which are likely to come
into contact with the host cell more readily than mem-

brane-bound proteins, increases the likelihood that
C. concisus is a pathogen of the gastrointestinal tract.
Experimental procedures
Materials
Blood Agar Base No. 2, Brain Heart Infusion medium,
defibrinated horse blood and gas-generating CampyGen
packs were from Oxoid (Heidelberg West, Victoria, Austra-
lia). Bicinchoninic acid, BSA, Chaps, copper II sulfate,
b-cyclodextrin, dithiothreitol, iodoacetamide and trichloro-
acetic acid (TCA) were from Sigma (Castle Hill, NSW,
Australia). Vancomycin was from Eli Lilly (North Ryde,
Fig. 2. Sequence alignment of zonula occlu-
dens toxins found in the C. concisus 13826,
V. cholerae 86015 and Ne. meningitidis
MC58 genomes. Four highly conserved
domains are indicated by boxes.
N. O. Kaakoush et al. Campylobacter concisus secretome
FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS 1613
NSW, Australia). Tris base and SDS were from Amersham
Biosciences (Melbourne, Australia). All other reagents were
of analytical grade.
Bioinformatics and protein modeling
blastp searches were performed using complete protein
sequences available at the NCBI database (http://www.
ncbi.nlm.nih.gov/) against the genome of C. concisus 13826
(CP000792; GI:157101370). The Kyoto Encyclopedia of
Genes and Genomes [43], available at (o-
me.jp/kegg), was employed to determine the biochemical
pathways to which genes were assigned. The Search Tool
for the Retrieval of Interacting Proteins (string) is a data-

base of known and predicted protein–protein interactions
available at string was employed to
examine interactions between proteins. The presence
and location of signal peptide cleavage sites in the amino
acid sequences were predicted using the default settings
for Gram-negative bacteria on the signalp 3.0 server
( [44]. Nonclassical-
ly secreted proteins were predicted using the secretomep
2.0 server ( />MS data were submitted to the Proteomics Identifications
(PRIDE) database, available at />Protein structure files were compiled from the protein data
bank available at Comparative
modeling of proteins was performed using the loopp parallel
driver version 3.2, available at />loopp.aspx. Protein structures were viewed using deepview ⁄
swiss-pdbviewer [45].
Growth of C. concisus and preparation of
secreted proteins
C. concisus UNSWCD was grown on horse blood agar
supplemented with 6% defibrinated horse blood and
5.0 lgÆ mL
)1
vancomycin. Cultures were incubated at 37 °C
under microaerobic conditions generated using Campylo-
bacter gas-generating kits (Cat. no. BR0056A; Oxoid). The
purity of bacterial cultures was confirmed by motility and
morphology observed under phase contrast microscopy.
Secreted proteins were prepared using a modified version
of the method described by Bumann et al. [46]. Briefly, log
phase C. concisus was harvested and inoculated into 50 mL
of Brain Heart Infusion broth. Six 50 mL cultures were
grown overnight at 37 °C under microaerobic conditions.

Exponential cultures were centrifuged at 4 °C and 4000 g for
15 min, and the supernatant was filtered through a 0.45 lm
membrane filter to remove residual bacteria. Secreted pro-
teins were precipitated using a previously described modified
TCA method [47]. Three hundred milliliters of filtrate was
mixed with 95 mL of prechilled TCA and incubated on
ice–water for 15 min. The mixture was then centrifuged for
10 min at 4000 g at 4 °C before the pellet was resuspended in
10 mL of acetone, after which it was centrifuged, washed
C. concisus Zot Ne. meningitidis Zot V. cholerae Zot
A
C. concisus Zot
Ne.meningitidis Zot
V. cholerae Zot
B
Fig. 3. Predictions of the tertiary structures (A) and secondary structures (B) of zonula occludens toxins found in the C. concisus 13826,
V. cholerae 86015 and Ne. meningitidis MC58 genomes. Arrows represent strands, rectangles represent helices and lines represent loops
within the secondary structures (B).
Campylobacter concisus secretome N. O. Kaakoush et al.
1614 FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS
with acetone twice, and air-dried. Proteins were then resus-
pended in the appropriate buffer and stored at )80 °C.
Estimation of the protein content of the samples was
performed using the bicinchoninic acid method, employing
a microtiter protocol (Pierce, Rockford, IL, USA). Absor-
bances were measured using a Beckman Du 7500 spectro-
photometer.
One-dimensional PAGE
Secreted proteins (40 lg) were resuspended in 40 lLof
SDS ⁄ PAGE sample buffer (0.375 m Tris, pH 6.8, 0.01%

SDS, 20% glycerol, 40 mgÆmL
)1
SDS, 31 mgÆmL
)1
dith-
iothreitol, 1 lgÆmL
)1
bromophenol blue). For electropho-
retic analyses, proteins were further denatured by heating
at 95 °C for 5 min. Proteins were separated on 12%
SDS ⁄ PAGE gels by electrophoresis for 2 h at 100 V. Gels
were stained using Coomassie Brilliant Blue G-250
(Bio-Rad, Gladesville, Australia).
Two-dimensional PAGE
Strip rehydration, isoelectric focusing and SDS ⁄ PAGE were
performed according to the protocol supplied with the Ready-
Strip IPG strips (Bio-Rad). For each strip, protein aliquots
(200 lg) were suspended in 245 lL of a rehydration buffer
consisting of 8 m urea, 100 mm dithiothreitol, 65 mm Chaps,
40 mm Tris ⁄ HCl, pH 8.0, and 10 lL pH 4–7 IPG buffer.
Nuclease buffer (5 lL) was added, and the mixture was incu-
bated at 4 °C for 20 min. The sample was then centrifuged at
7230 g for 15 min at 4 °C, and the supernatant was loaded for
the first dimension of chromatography onto an 11 cm Ready-
Strip IPG (Bio-Rad) of the appropriate pI range, and left to
incubate, sealed, for 24 h at room temperature. Isoelectric
focusing was performed using an IsoeletrIQ Focusing System
(Proteome Systems, Sydney, NSW, Australia). The machine
was programmed to run at 300 V for 4 h, 10 000 V for 8 h,
and 10 000 V for 22 h, or until 80 000 Volt-hours was

reached. After focusing, strips were equilibrated sequentially
in two buffers of 6 m urea, 20% (w ⁄ w) glycerol, 2% (w ⁄ v)
SDS, and 375 mm Tris ⁄ HCl: the first one contained 130 mm
dithiothreitol, and the second one contained 135 mm iodoace-
tamide. Strips were rinsed briefly with 0.375 m (pH 8.0) Tris
before SDS ⁄ PAGE was performed using Criterion 12.5%
Tris ⁄ HCl Precast gels (Bio-Rad), run at 200 V for approxi-
mately 45 min. Gels were fixed individually in 0.1 L of fixing
solution [50% (v ⁄ v) methanol, 10% (v ⁄ v) acetic acid] for a
minimum of 1 h, and were subsequently stained using a sensi-
tive ammoniacal silver method based on silver nitrate [48].
MS
Protein-containing spots or bands excised from the gels were
digested according to a previously described method [49].
Digests originating from 2D-PAGE spots were analyzed on
an API QStar Pulsar I tandem MS instrument, using a previ-
ously described protocol [48]. Digests (2.5 lL) originating
from 1D-PAGE bands were separated by nano-LC using an
Ultimate 3000 HPLC and autosampler system (Dionex,
Amsterdam, The Netherlands). Samples were concentrated
and desalted onto a micro C18 precolumn (500 lm · 2 mm;
Michrom Bioresources, Auburn, CA, USA) with
H
2
O ⁄ CH
3
CN (98 : 2, 0.05% heptafluorobutyric acid) at
15 lLÆmin
)1
. After a 4 min wash, the precolumn was

switched (Valco 10 port valve; Dionex) in line with a fritless
nano column (75 lm ·  10 cm) containing C18 medium
(5 lm, 200 A
˚
; Magic, Michrom), manufactured according to
Gatlin et al. [50]. Peptides were eluted using a linear gradient
of H
2
O ⁄ CH
3
CN (98 : 2, 0.1% formic acid to 64 : 36, 0.1%
formic acid) at 250 nLÆmin
)1
over 30 min. High voltage
(1800 V) was applied to a low-volume tee (Upchurch Scien-
tific; Oak Harbor, WA, USA), and the column tip was posi-
tioned  0.5 cm from the heated capillary (T = 250 °C) of
an LTQ FT Ultra mass spectrometer (Thermo Electron,
Bremen, Germany). Positive ions were generated by electro-
spray, and the LTQ FT Ultra was operated in data-depen-
dent acquisition mode. A survey scan (m ⁄ z 350–1750) was
acquired in the Fourier transform ion cyclotron resonance
cell (resolution = 100 000 at m ⁄ z 400, with an accumulation
target value of 1 000 000 ions in the linear ion trap). Up to
six of the most abundant ions (> 3000 counts) with charge
states of > +2 were sequentially isolated and fragmented
within the linear ion trap, using collisionally induced dissoci-
ation with an activation of q = 0.25 and activation time of
30 ms at a target value of 30 000 ions. m ⁄ z ratios selected for
MS ⁄ MS were dynamically excluded for 30 s. Peak lists were

generated using mascot daemon ⁄ extract_msn (Matrix Sci-
ence, London, UK), using the default parameters, and sub-
mitted to the database search program mascot (version 2.2;
Matrix Science). Search parameters were as follows: precur-
sor tolerance was 4 p.p.m., and product ion tolerances were
±0.4 Da; Met(O) was specified as a variable modification,
enzyme specificity was trypsin, one missed cleavage was pos-
sible, and the NCBI
nr
(July, 2009) or C. concisus (strain
13826) databases were searched (Figs S1 and S2). A false-
positive rate of  2% was applied to searches from the LTQ
FT-MS data.
Acknowledgements
This work was made possible by the support of the
National Health and Medical Research Council, Aus-
tralia.
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Supporting information
The following supplementary material is available:
Table S1. Secreted proteins identified from cultures of
C. concisus UNSWCD (n = 201).
Fig. S1. Peptide summary reports for LTQ FT-MS
searches against the NCBI
nr
database (July, 2009).
Fig. S2. Peptide summary reports for LTQ FT-MS
searches against the C. concisus 13826 proteome.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
N. O. Kaakoush et al. Campylobacter concisus secretome
FEBS Journal 277 (2010) 1606–1617 ª 2010 The Authors Journal compilation ª 2010 FEBS 1617