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 The secre
Trang 1Nadeem O Kaakoush1, Si Ming Man1, Sarah Lamb1, Mark J Raftery2, Marc R Wilkins1, Zsuzsanna Kovach1and Hazel Mitchell1
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 ingastroin-testinal
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: H.Mitchell@unsw.edu.au
(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 concisusis a pathogen of the gastrointestinal tract
Abbreviations
OMP, outer membrane protein; TCA, trichloroacetic acid; Zot, zonula occludens toxin.
Trang 2C 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
C
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.
Trang 3identification 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, owcontaminat-ing to their high
stringency, the bioinformatic prediction 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 methylaadapta-tion 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
Trang 4Table 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.
157165776 CCC13826_1793 Putative carbon storage regulator-like protein
157165665 CCC13826_1073 Outer membrane lipoprotein carrier protein
157163977 CCC13826_0329 C4-dicarboxylate-binding periplasmic protein
157165691 CCC13826_0764 C4-dicarboxylate-binding periplasmic protein
157165183 CCC13826_2088 Methyl-accepting chemotaxis sensory transducer
157165242 CCC13826_0201 Molybdopterin oxidoreductase Fe 4 S 4 subunit
157164923 CCC13826_1633 Methionine sulfoxide reductase family protein
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
Trang 5found to play a role in the intramacrophagic
replica-tion of E coli, with mutareplica-tions 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.)
157165471 CCC13826_1395 Radical SAM domain-containing protein
157164709 CCC13826_0131 Peptidoglycan-associated lipoprotein
157164370 CCC13826_0924 ADP-heptose-LPS heptosyltransferase II
157164830 CCC13826_1534 Lipopolysaccharide biosynthesis protein
157164740 CCC13826_0739 Outer membrane fibronectin-binding protein
157164622 CCC13826_1253 a-Macroglobulin family protein
157165368 CCC13826_1643 FAD-binding domain-containing protein
157164095 CCC13826_1803 FAD-binding domain-containing protein
Trang 6Putative 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 aeruginosaand 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_0191and ccc13826_1210 were identified with
Table 2 Functional classification of C concisus UNSWCD
identi-fied proteins bioinformatically predicted to be nonsecretory
(n = 115).
Elongation factors and chaperones 15
Metabolism of cofactors and vitamins 4
Signal transduction and chemotaxis 8
Table 3 Putative virulence and colonization factors found in C concisus 13826.
a Found only in C jejuni ssp doylei 269.97.
Trang 747% 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 perturbainactiva-tion 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
Trang 8InvA This invasin is an essential component of the
invasion-associated type III secretion system in
Salmo-nellaspp [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 concisusis 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.
Trang 9NSW, 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
(http://www.geno-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 http://string.embl.de/ 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
(http://www.cbs.dtu.dk/services/SignalP/) [44]
Nonclassical-ly secreted proteins were predicted using the secretomep
2.0 server (http://www.cbs.dtu.dk/services/SecretomeP/)
MS data were submitted to the Proteomics Identifications
(PRIDE) database, available at http://www.ebi.ac.uk/pride/
Protein structure files were compiled from the protein data
bank available at http://www.rcsb.org/pdb Comparative
modeling of proteins was performed using the loopp parallel
driver version 3.2, available at http://cbsuapps.tc.cornell.edu/ 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 lgÆmL)1vancomycin Cultures were incubated at 37C 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 37C under microaerobic conditions Exponential cultures were centrifuged at 4C 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 4C before the pellet was resuspended in
10 mL of acetone, after which it was centrifuged, washed
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).
Trang 10with 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 lL of
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 95C 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 4C for 20 min The sample was then centrifuged at
7230 g for 15 min at 4C, 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;
H2O⁄ CH3CN (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 H2O⁄ CH3CN (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 activdissoci-ation of q = 0.25 and activdissoci-ation 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 NCBInr (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
References
1 Moore JE, Corcoran D, Dooley JS, Fanning S, Lucey
B, Matsuda M, McDowell DA, Megraud F, Millar BC,