BACKGROUND
campylobacter jejuni is a curved, microaerophilic gram-negative bacterium that is an important human pathogen  <cit> .
the main reservoir of c. jejuni is the cecum of poultry, and humans can become infected through consumption of undercooked chicken that is contaminated with the bacterium  <cit> .
in humans, the bacterium colonizes both the small and large intestine resulting in fever, severe abdominal pain, and diarrhea, with possible autoimmune sequelae of infection including guillain-barré syndrome and reactive arthritis  <cit> .
expression of proteins is an energetically costly activity.
therefore, bacteria often express certain proteins only under conditions where those proteins are needed for growth, survival, or pathogenicity.
growth temperatures cause differential expression of proteins in a number of pathogenic bacteria including c. jejuni  <cit> , although the mechanisms of thermoregulation can be complex and result from overlapping regulatory systems.
in c. jejuni, the racrs two-component regulatory system is involved in the regulation of some proteins, although the majority of the targets have not been identified  <cit> .
because the body temperatures of humans and chickens differ , c. jejuni is likely to express different proteins when colonizing chickens than when colonizing humans.
we used proteomics to determine which c. jejuni proteins are more highly expressed at 37°c compared to 42°c, because such upregulation might suggest an importance of these proteins in colonization of humans.
one of the proteins identified was cj <dig>  which is annotated as playing a role in outer membrane protein folding and stabilization.
cj <dig> was previously identified as an immunogenic outer membrane protein and was named cell binding factor  <dig> ; it is also called peb <dig>  <cit> .
it was later suggested that peb <dig> is not surface exposed, but is periplasmically located in association with the inner membrane  <cit> .
cj <dig> shows homology to sura, a peptidyl-prolyl cis-trans isomerase  found in e. coli, and other orthologs in numerous bacteria including helicobacter pylori, bacilis subtilis, and lactococcus lactis  <cit> .
ppiases have been characterized as virulence factors in shigella flexneri, salmonella enterica, legionella pneumophila, chlamydia trachomatis, trypanosoma cruzi, and neisseria gonorrhoeae  <cit> .
asakura et al.
<cit>  recently characterized a cj <dig> mutant of c. jejuni strain nctc  <dig>  finding decreases in ability to adhere to int <dig> cells and to colonize mice, and an increase in biofilm formation.
however, this mutant was not complemented with a wild-type copy of cj <dig>  allowing some question of whether the observed phenotypes were specific for cj <dig>
in this study, we examine the effects of deletion of cj <dig> in a different, highly invasive c. jejuni strain  on phenotypes related to growth, protein expression, and pathogenicity.
our results show that deletion of cj <dig> alters several virulence-related phenotypes in vitro and decreases the ability of the bacterium to colonize mice, although the phenotypes of the 81– <dig> cj <dig> mutant differ significantly from those of an analogous mutant in strain nctc  <dig>
methods
bacterial strains and routine culture conditions
campylobacter jejuni strains derived from the parent 81– <dig>  <cit>   were routinely maintained with minimal passage on blood agar plates  at 37°c in sealed culture boxes  containing a microaerobic atmosphere generated by pack-micro aero .
liquid cultures of c. jejuni were grown in brucella broth or mueller-hinton  broth and cultured in microaerobic environments.
when appropriate, strains were cultured in the presence of chloramphenicol  or streptomycin  to select for antibiotic resistance markers.
escherichia coli jm <dig> was used as the host strain for cloning experiments and e. coli bl21plyss  <cit>  was used as the host strain for expression of the his-tagged cj <dig> protein.
e. coli strains were cultured in luria-bertani  broth or agar  <cit> , supplemented with the following antibiotics as appropriate for selection of plasmids: ampicillin,  <dig> μg/ml; chloramphenicol,  <dig> μg/ml; streptomycin,  <dig> μg/ml.
proteome analysis of c. jejuni strains
proteomics experiments were performed on c. jejuni cells grown at 37°c and 42°c as described  <cit> .
briefly, cells were grown overnight at 37°c in brucella broth, then diluted the following morning into two aliquots of fresh brucella broth , which were grown at 37°c and 42°c to mid-log phase .
chloramphenicol  was added to stop protein synthesis  <cit> , and the cells were harvested for proteome analysis as described  <cit> .
proteomics experiments were performed using differential in-gel electrophoresis  technology from ge biosystems , whole-cell protein lysates from the 37°c- and 42°c-grown c. jejuni  were labelled individually with cy <dig> and cy <dig> dyes according to the protocol supplied by the manufacturer , then mixed in equal mass and separated using two-dimensional  sds-page.
for comparison of wt, mutant and revertant proteomes , protein lysates  were labelled with cy <dig> , cy <dig> , or cy <dig>  dyes, mixed in equal mass, and separated by 2d sds-page.
following separation, gels were scanned on a typhoon fluorescent flatbed scanner , at the following wavelengths: cy <dig>   <dig> nm excitation,  <dig> nm emission, cy <dig>   <dig> nm excitation,  <dig> nm emission; cy <dig>   <dig> nm excitation,  <dig> nm emission.
images were analyzed with decyder differential in-gel analysis  software  for identification of proteins with higher or lower expression in different samples.
the identities of proteins of interest were determined using a matrix-assisted laser desorption ionization – time-of-flight/time-of-flight  spectrometer , using both tryptic fingerprint data and fragmentation-based ms/ms.
purification of cj <dig> protein and antibody production
to allow purification of the cj <dig> protein, a c-terminal his6-tag was added to cj <dig> lacking the n-terminal signal sequence by inserting the gene into pet-20b.
first, the cj <dig> gene without the signal sequence and stop codon was amplified from c. jejuni strain 81– <dig> and nde i and xho i sites were added using primers purprot-f and purprot-r .
the resulting pcr product was cloned into pcr ii-topo, creating plasmid pkr <dig> .
using nde i and xho i, the cj <dig> gene was excised from pkr <dig> and pet-20b was linearized.
the cj <dig> gene was ligated into the linearized pet-20b creating plasmid pkr <dig>  which was used to transform e. coli strain bl21plyss .
the plasmid-carrying strain was grown overnight in lb broth at 37°c.
the next morning the culture was diluted to od <dig> ~  <dig>  and incubated at 37°c until od <dig> ~  <dig> .
iptg was added to the culture to induce expression of the his6-tagged protein.
after  <dig> h, the cells were harvested by centrifugation, washed, and the supernatant passed through a nickel column to further purify the his6-tagged protein by standard methods  <cit> .
the purified protein was sent to cocalico biologicals, inc.  for production of anti-cj <dig> antibodies.
for use in the ppiase assay, the protein was refolded using the pro-matrix protein refolding kit  and dialyzed against pbs.
†bold nucleotides indicate restriction sites introduced for cloning purposes
to confirm the proteomics-implicated temperature regulation of cj <dig>  a western blot was performed on c. jejuni cells grown at 37°c or 42°c using anti-cj <dig> antibody  as the primary antibody and hrp conjugated goat anti-rabbit igg  as the secondary antibody.
a control western blot against cj <dig>  was performed using anti-cj <dig> antibody  as the primary antibody and hrp conjugated goat anti-rabbit igg  as the secondary antibody.
the blots were developed using a dab substrate kit .
densitometry measurements were conducted using imagej software  <cit> .
localization of the cj <dig> protein
to determine the cellular location of cj <dig>  c. jejuni cells grown at 37°c were separated into cytoplasmic, periplasmic, and inner membrane fractions  <cit> , and outer membrane fractions as described  <cit> .
western blots were performed on c. jejuni cell fractions using anti-cj <dig> antibody  as the primary antibody, along with control blots using anti-cj <dig> , anti-ceta , and anti-momp  polyclonal sera  as the primary antibodies.
hrp conjugated goat anti-rabbit igg  was used as the secondary antibody for all blots, which were then developed using a dab substrate kit .
ppiase assay
the ppiase activity of cj <dig> was determined in a coupled assay, which measures the ability of cj <dig> to convert the cis isomer of the oligopeptide substrate n-suc-ala-ala-pro-phe-p-nitroanilide into the trans form which is cleavable by α-chymotrypsin  <cit> .
chymotrypsin  and varying concentrations of cj <dig> were combined in  <dig> mm tris-hcl ph  <dig>  and incubated at 4°c.
the substrate  was added and the reaction was monitored at 10°c by the increase in absorbance at  <dig> nm .
the kobs value for each ppiase concentration was found by plotting ln  vs. time  and determining the slope.
the catalytic efficiency  of the ppiase activity was obtained by plotting kobs vs.  and determining the slope.
analysis of cj <dig> in additional campylobacter strains
the dna sequences of the cj <dig> locus  were determined from the campylobacter strains listed in table  <dig> <cit> .
primers cj0596-f <dig> and cj0596-r <dig>  were designed based on the published nctc  <dig> genome sequence and used in pcr reactions with template genomic dna prepared using a masterpure dna purification kit  to amplify the region encompassing cj <dig>  thermocycler parameters were  <dig> cycles of: 94°c for  <dig> seconds, 51°c for  <dig> seconds, and 72°c for  <dig> minutes.
pcr products were purified using qiaquick pcr purification kits  and were then sequenced directly , using an abi 3730xl sequencer .
vectornti  was used to analyze dna sequences.
the protein sequences were analyzed for motifs using the expasy prosite server http://au.expasy.org/prosite/ <cit> , and potential signal peptides were evaluated using signalp  <dig>  http://www.cbs.dtu.dk/services/signalp/ <cit> .
isolation of a c. jejuni cj <dig> mutant
a cj <dig> mutant of c. jejuni strain 81– <dig> was constructed using a streptomycin counterselection system similar to the heterologous h. pylori-c. jejuni method described by dailidiene et al.
<cit>  in which an rpsl gene from c. jejuni was used for counterselection in h. pylori, to decrease background gene conversion events.
in the heterologous rpslhp system reported here, cj <dig> was exactly replaced by the rpslhp  gene from h. pylori strain 84– <dig>  linked to a chloramphenicol acetyltransferase  cassette .
this strategy allows for selection of a mutant  based on chloramphenicol resistance and then allows for selection of a revertant strain  based on streptomycin resistance.
first, a pcr-amplified cj <dig> gene was amplified using primers cj0596-f <dig> and cj0596-r <dig> designed based on the published c. jejuni nctc  <dig> genome sequence  and the resulting product was cloned into pcr ii-topo creating pkr <dig> .
the plasmid pkr <dig> was subjected to inverse pcr  to remove the cj <dig> gene and add restriction sites.
the inverse pcr product was self-ligated to form plasmid pkr <dig>  the cat cassette was amplified from pry <dig>  <cit>  and age i, mfe i, and nhe i sites were added using primers cat-f <dig> and cat-r <dig>  the resulting pcr product was cloned into pcr ii-topo to create plasmid pkr <dig>  the rpslhp gene was amplified and age i and mfe i sites were added using primers rpslhp-f <dig> and rpslhp-r <dig> and the pcr product was cloned into pcr ii-topo creating plasmid pkr <dig>  age i and nhe i were used to excise the cat cassette from pkr <dig> and linearize pkr <dig>  the cat cassette was ligated into pkr <dig> creating plasmid pkr <dig>  using age i and mfe i, the rpslhp gene was removed from pkr <dig> and ligated with linearized pkr <dig>  creating plasmid pkr <dig>  with the rpslhp/cat construct oriented in the same direction as the cj <dig> and cj <dig> genes.
care was taken to ensure that this replacement would not produce polar effects by preserving the cj <dig> ribosome binding site and by leaving only  <dig> bp between the stop codon of cat and the start codon of cj <dig>  the mutagenized cj <dig> allele was introduced into a spontaneous strepr derivative of c. jejuni 81– <dig> by electroporation.
several cmr/streps transformants were verified as cj <dig> mutants by pcr with primers cj0596-f <dig> and cj0596-r <dig>  and dna sequencing , a representative of which was designated 81–176cj <dig>  and used for further analysis.
reversion of the cj <dig> mutation
a revertant of c. jejuni 81–176cj <dig> was isolated by replacing the mutated cj <dig> allele in 81–176cj <dig> with a wild-type cj <dig> gene using streptomycin counterselection.
c. jejuni strain 81–176cj0596+ was created by using electroporation to introduce pkr <dig> into 81–176cj <dig> cells, selecting for colonies on plates containing streptomycin .
putative revertants were identified by screening strr colonies for sensitivity to chloramphenicol  to ensure loss of the rpslhp/cat cassette.
chromosomal dna was isolated from these transformants and proper replacement of the rpslhp/cat cassette with wild-type cj <dig> was confirmed by pcr using primers cj0596-f <dig> and cj0596-r <dig>  and by dna sequencing of the region.
quantitative real-time reverse transcription pcr
cdna was prepared from rna samples of c. jejuni grown 81– <dig> and 81–176cj <dig> using a geneamp rna pcr kit .
an icycler iq real-time pcr detection system  was used to run qrt-pcr with iq sybr green super mix, and primers cj0597rt-f and cj0597rt-r .
data were analyzed using bio-rad icycler data analysis software.
control reactions used primers specific for 16s rdna , which allows amplification of a non-regulated rna  <cit> .
differences in transcript levels among samples were calculated from amplification profiles using the comparative threshold cycle  method, as previously described  <cit> .
growth experiments
the growth rates of c. jejuni wild-type 81– <dig>  mutant 81–176cj <dig>  and revertant 81–176cj0596+ were assessed by growing cells overnight in mh broth, then diluting the following morning in mh broth to od <dig> ~  <dig>   and shaking at 37°c under microaerobic conditions.
growth was monitored by measuring od <dig> and numbers of viable bacteria were determined by plating serial dilutions of the bacterial suspensions on mh agar and counting the resultant colonies.
motility
the motility of c. jejuni 81– <dig>  81–176cj <dig>  and 81–176cj0596+ was determined as previously described  <cit> .
briefly, cells were grown overnight in mh broth, then diluted the following morning in mh broth to od <dig> =  <dig> .
a pipet tip dipped into the suspension was used to stab the center of a mh motility plate .
the plates were incubated at 37°c and the diameter of the motility zone was measured every  <dig> h.
adherence/invasion/intracellular survival assay
a gentamicin protection assay  <cit>  was used to assess the ability of 81– <dig>  81–176cj <dig>  and 81–176cj0596+ to adhere to, invade, and survive within int <dig> human intestinal epithelial cells.
briefly, bacteria were grown in biphasic  cultures at 37°c under microaerobic conditions for ~ <dig> h. bacteria were harvested, resuspended in phosphate buffered saline , then added in triplicate to semi-confluent int <dig> cell monolayers  at a multiplicity of infection  of ~40: <dig> .
the number of bacteria added was quantified by determination of cfu/ml.
the cells were incubated for  <dig> h at 37°c under microaerobic conditions and were washed with hanks' balanced salt solution , lysed with triton x- <dig> and the number of adherent bacteria was quantified by viable counts.
for determination of invasion, cells were incubated for  <dig> h with bacteria and then gentamicin was added to a final concentration of  <dig> μg/ml to kill any extracellular bacteria.
after an additional  <dig> h of incubation, the cells were washed, lysed with triton x- <dig> and intracellular bacteria were quantified by viable counts.
the gentamicin and triton x- <dig> mics of the three strains were also determined.
for determination of intracellular survival, the cells were incubated for  <dig> h with bacteria,  <dig> h with gentamicin, and then the int <dig> cells were washed and incubated for  <dig> h in minimal essential media containing 3% fetal bovine serum and gentamicin  as described by candon et al.
<cit> .
after the incubation period, cells were washed and lysed with triton-x  <dig> and the number of bacteria that survived intracellularly was quantified by viable counts.
mouse colonization experiments
the in vivo relevance of cj <dig> was investigated by testing the ability of 81– <dig>  81–176cj <dig>  and 81–176cj0596+ to colonize mice as described  <cit> .
10-week old female balb/c-byj mice were given  <dig> μl of 5% sodium bicarbonate by oral gavage to neutralize stomach acid.
the mice were then given a dose of  <dig> ×  <dig> cfu in  <dig> ml of bhi/1% ye broth by oral gavage.
because there was an observed discrepancy between od <dig> and cfu for the mutant , we first performed pilot experiments correlating od <dig> and cfu for all of the strains.
after four repetitions, we found the mutant od <dig> that gave the same number of cfu as for the wt and revertant strain, and this is what we used for the mouse inocula.
we also verified that each mouse received equal cfu by plating the inocula for viable counts at the time of inoculation.
colonization was determined by viable counts of bacteria in fecal pellets, which were enumerated on selective cva media  <cit>  starting  <dig> days after the challenge and continuing every  <dig> days thereafter until  <dig> days post-inoculation.
in parallel, experiments were carried out to determine the ability of cj <dig> mutant bacteria to compete with wild-type bacteria in colonization.
for competition experiments, wild-type and mutant bacteria were mixed in equal amounts  immediately prior to inoculation.
colonization was determined by enumerating bacteria on selective media with or without chloramphenicol .
the number of bacteria counted on the plates containing chloramphenicol  was subtracted from the number of bacteria found on the plates without chloramphenicol  to obtain the number of viable wild-type bacteria.
control experiments showed that the plating efficiency of the cj <dig> mutant was equivalent on media containing or lacking chloramphenicol.
all vertebrate animal experiments were conducted in accordance with recommendations by the office of laboratory animal welfare, and were approved by the medical college of georgia institutional animal care and use committee .
RESULTS
expression of cj <dig> is slightly higher at 37°c than at 42°c
in a search to identify c. jejuni genes with differential response to steady-state growth temperature , several proteins were identified that were more highly expressed at 37°c than at 42°c.
c. jejuni 81– <dig> was grown overnight at 37°c and then diluted into fresh media.
the two cultures were grown in parallel at 37°c and 42°c to mid-log growth phase.
proteomics experiments were then performed on cultures of c. jejuni 81– <dig> grown at the two temperatures.
one protein that was upregulated at 37°c had the approximate pi and molecular mass of the predicted cj <dig> protein .
this protein was  <dig> -fold more highly expressed at 37°c, a result that was consistent in five different proteomics experiments.
the protein was excised from the polyacrylamide gel and subjected to maldi-tof/tof mass spectrometry.
this protein was identified with 100% confidence as cj <dig> .
in an attempt to confirm the proteomics results, we performed western blots using anti-cj <dig> antibodies and c. jejuni 81– <dig> grown at 37°c and 42°c.
while only semi-quantitative, in two separate experiments the western blots showed a more modest  <dig> – <dig> -fold greater expression of cj <dig> at 37°c .
therefore, we conclude that while cj <dig> appears to be expressed at a somewhat higher level at 37°c compared to 42°c, the effect of growth temperature on cj <dig> expression is not dramatic.
because proteins homologous to cj <dig> are involved in virulence in other pathogenic bacteria, we nevertheless characterized the role of this protein in c. jejuni physiology and pathogenesis.
similarity of cj <dig> sequences among campylobacter species
because campylobacter genomes are quite diverse  <cit> , we characterized the conservation of the cj <dig> gene in other campylobacter strains.
using pcr primers designed from the c. jejuni nctc  <dig> genomic sequence and located in the cj <dig> and cj <dig> genes , we amplified a  <dig> kb segment encompassing the cj <dig> locus from five additional c. jejuni strains and one c. coli strain.
pcr products of the expected size were obtained from each strain, and were subsequently sequenced .
a search of  <dig> additional campylobacter genome sequences  was also performed and showed that a cj <dig> ortholog was found in every strain.
the sequences of these orthologs were also included in the sequence comparison analysis.
the nucleotide sequences between pairs of c. jejuni strains or c. coli d <dig> were at least 98% identical.
the corresponding sequences from c. coli rm <dig> and other campylobacter species were somewhat lower .
the predicted cj <dig> protein was also highly similar in all c. jejuni strains and c. coli d <dig>  with an amino acid sequence identity of at least 99%.
as with the nucleotide sequences, the degree of identity of proteins from c. coli rm <dig> and other non-jejuni campylobacter strains was lower, with identities ranging from 87% to 45%.
together, these results indicate that cj <dig> is highly conserved in c. jejuni , c. coli , and one strain each of c. concisus, c. curvus, c. fetus, c. hominis, c. lari, and c. upsaliensis.
we focused on cj <dig> from c. jejuni strain 81– <dig>  for our subsequent work.
in silico analysis of cj <dig> protein features
in the nctc  <dig> genome, the predicted cj <dig> protein had a predicted molecular mass of  <dig>  kda and pi of  <dig>  and was annotated as a major antigenic peptide peb4\cell binding factor  <dig>  similar to peptidyl prolyl cis-trans isomerases found in a variety of organisms  <cit> .
because some peptidyl-prolyl cis-trans isomerases are located in the periplasm, the signalp algorithm  <cit>  was used to analyze the 81– <dig> cj <dig> protein for the presence of an n-terminal signal sequence.
a signal sequence with a probable cleavage site between amino acids  <dig> and  <dig> of the preprotein  was predicted.
cj <dig> was also analyzed for the presence of protein motifs  <cit>  and was found to contain a ppic-type peptidyl-prolyl cis-trans isomerase motif located between amino acids 131– <dig>  secondary structure predictions showed no transmembrane segments in the mature protein, suggesting that cj <dig> is likely to be a periplasmic protein.
cj <dig> is located in the periplasm of c. jejuni
the amino acid sequence of cj <dig> suggested that this protein is located in the periplasm.
to test this experimentally, western blots were performed on cytoplasmic, inner membrane, periplasmic, and outer membrane fractions of c. jejuni 81– <dig> using anti-cj <dig>  anti-cj <dig>  anti-ceta, and anti-momp antibodies .
as expected, anti-cj <dig> antibodies reacted with a ~ <dig> kda protein in the cytoplasmic fraction, anti-ceta antibodies reacted with a ~ <dig> kda protein in the inner membrane fraction, and anti-momp antibodies reacted with a ~ <dig> kda protein in the outer membrane.
anti-cj <dig> antibodies reacted with a ~ <dig> kda protein present primarily in the periplasmic fraction.
cj <dig> has ppiase activity
cj <dig> has one rotamase domain and is similar to e. coli sura, suggesting that it is a ppiase.
the ppiase activity of purified cj <dig> was determined using a coupled assay in which the cleavage of the trans isomer of n-suc-ala-ala-pro-phe-p-nitroanilide by α-chymotrypsin results in the release of p-nitroanilide, causing a colorimetric change over time.
conversion of the cis to the trans isomers of the substrate occurs spontaneously in solution, allowing chymotrypsin cleavage .
however, addition of cj <dig> accelerates this cis-trans conversion, indicative of ppiase activity .
by using varying concentrations of purified cj <dig>  and plotting calculated kobs vs. , the ppiase activity  was calculated to be  <dig>  mm-1sec- <dig>  an activity consistent with values published for other ppiases  <cit> .
creation of a non-polar cj <dig> mutation
to test the role of cj <dig> in c. jejuni physiology or pathogenesis, we created a non-polar cj <dig> mutant.
to facilitate mutant construction, we developed a modified streptomycin counter selection system based on a similar strategy used in h. pylori  <cit> .
the rpslhp/cat cassette  was used to precisely replace cj <dig>  maintaining the ribosome binding site of the downstream cj <dig> gene.
together with the lack of a transcriptional terminator on the rpslhp/cat cassette, this construct is expected to yield a non-polar mutation.
the resulting mutant strain was designated 81–176cj <dig>  to confirm that mutation of the cj <dig> gene did not alter the expression of the putative co-transcribed cj <dig> gene, quantitative real-time rt-pcr was performed on rna samples harvested from 81– <dig> and 81–176cj <dig>  these studies confirmed that mrna levels downstream of the mutation in 81–176cj <dig> were equivalent to those in 81– <dig>  and that the mutation was non-polar .
however, to ensure that any phenotypes of the cj <dig> mutant were specific for cj <dig>  we subsequently isolated a reversion of the mutation by replacing the rpslhp/cat cassette with a wild-type cj <dig> gene.
analogous to the counterselection system of dailidiene et al.
<cit> , use of the h. pylori rpsl gene decreased background gene conversion events and facilitated the recovery of cells in which the mutagenized cj <dig> allele was replaced with the wild-type cj <dig> gene.
this process was extremely efficient, as ~70% of the recovered streptomycin-resistant colonies contained the desired reversions of the cj <dig> mutation.
mutation of cj <dig> causes a moderate growth defect of c. jejuni in mh broth
the role of cj <dig> in campylobacter growth was studied by comparing the growth rates of c. jejuni 81– <dig>  81–176cj <dig>  and 81–176cj0596+ in mh broth .
when inoculated at od <dig> ~  <dig>  and growth rate was measured by od <dig> , the mutant initially appeared to have a significant growth defect illustrated both by a slower increase in od <dig> and a lower maximum od <dig>  however, we also found that a similar starting od <dig> resulted in a significantly lower starting cfu for the mutant, indicating that the od <dig> did not accurately reflect the number of cj <dig> mutant cfu.
we then grew the mutant under two starting conditions: one had the same starting od <dig> as the wild-type and revertant  and the other had approximately the same starting cfu  as the wild-type and the revertant.
the mutant inoculated at od <dig> ~  <dig>  showed a faster initial increase in od <dig>  but reached a similar maximum od <dig> as the mutant inoculated at od <dig> ~  <dig> .
however, when growth rate was monitored by plating for viable counts , the mutant had an initial growth rate more similar to that of wild-type, although both mutant cultures yielded final viable counts lower than wild-type.
wild-type growth characteristics were restored in the revertant strain.
together, these data suggest that mutation of cj <dig> resulted in a moderate growth defect, especially later in the growth curve, and that the cj <dig> mutant had apparent changes in cell characteristics such that the od <dig> had poor concordance with cfu measurements.
deletion of cj <dig> increases the motility of c. jejuni
because motility plays an important role in invasion of host intestinal cells and is required for animal colonization, the motility of c. jejuni 81– <dig>  81–176cj <dig>  and 81–176cj0596+ was compared at 37°c .
the average diameter of the zone of motility for the wild-type was  <dig>  mm ±  <dig>  at  <dig> h. the mutant was significantly more motile with a zone diameter of  <dig>  mm ±  <dig>  .
the revertant returned to wild-type motility levels with a zone diameter of  <dig>  mm ±  <dig> .
a similar increase in motility was seen when the assay was performed at 42°c .
thus, cj <dig> is involved in the expression of motility.
deletion of cj <dig> increases the ability of c. jejuni to invade int <dig> cells, but does not affect adherence or intracellular survival
the possibility that cj <dig> plays a role in interaction with host cells was studied by comparing the adherence and invasion abilities of c. jejuni 81– <dig>  81–176cj <dig>  and 81–176cj0596+ in an in vitro assay using int <dig> intestinal epithelial cells .
the mean percentages of the inoculum that adhered were  <dig>  ,  <dig>  , and  <dig>   for the wild-type, mutant, and revertant, respectively, demonstrating that deletion of cj <dig> does not significantly affect the ability of c. jejuni to adhere to int <dig> cells .
in contrast, mutation of cj <dig> had a significant effect on the invasion ability of c. jejuni.
while the percentages of the wild-type and revertant inocula invading int <dig> cells were  <dig>   and  <dig>  , respectively, the cj <dig> mutant showed a nearly 20-fold increase in invasion .
the gentamicin and triton x- <dig> sensitivities of the three strains were tested to ensure that the invasion results were not due to altered killing of a strain, and no significant difference was found for either compound.
compared to the number of bacteria that invaded the int <dig> cells, the percentages of wild-type, mutant, or revertant that were able to survive intracellularly at timepoints from  <dig> to  <dig> hours post-invasion were not significantly different .
81–176cj <dig> is defective in mouse colonization
to determine whether cj <dig> plays a role in mouse colonization, we used a balb/c model that has been used previously to assess colonization differences between wild-type and mutant bacteria  <cit> .
female balb/c-byj mice were given doses of c. jejuni 81– <dig>  81–176cj <dig>  and 81–176cj0596+ individually , as well as a mixture of wild-type and cj <dig> mutant  in a competition experiment, and colonization was measured by determining viable counts of bacteria in fecal pellets at weekly intervals .
on days  <dig> and  <dig>  viable bacteria were found in all seven mice receiving the wild-type, mutant, or the revertant .
following the peak in colonization at  <dig> days, viable mutant bacteria were recovered from only four mice on day  <dig>  and only three mice on days  <dig> and  <dig>  at these latter three timepoints, the wild-type and revertant were recovered from all but one mouse.
the mean colonization densities of the wild-type and revertant were  <dig>  ×  <dig> and  <dig>  ×  <dig> cfu/g, respectively, on day  <dig> and remained relatively consistent throughout the experiment.
the mean colonization level of the mutant was significantly lower than wild-type and revertant on days  <dig>  and  <dig> .
when placed in competition with the wild-type, the mutant showed an inability to compete for colonization .
wild-type bacteria were recovered from five mice on day  <dig>  four mice on day  <dig>  and then one mouse for the remainder of the experiment.
viable mutant bacteria were recovered from no mice on day  <dig> , two mice on day  <dig> , one mouse on day  <dig>  and then were not recovered on days  <dig> and  <dig>
deletion of cj <dig> alters c. jejuni protein expression
because cj <dig> is thought to be a periplasmic chaperone, its loss could result in compensatory changes in the expression of other proteins.
to determine the effect that deletion of cj <dig> had on the expression of other proteins, a comparison of total cell proteins from c. jejuni 81– <dig>  81–176cj <dig>  and 81–176cj0596+ was performed using 2d sds-page .
three proteins were found to be significantly upregulated in the mutant.
they were identified as htra , cj <dig> , and flaa .
as expected, the cat protein was found only in the cj <dig> mutant.
conversely, the cj <dig> protein was found in wild-type and revertant strains, but was absent in the cj <dig> mutant, as expected.
three proteins were found to be significantly downregulated in the mutant.
these proteins were ef-ts , superoxide dismutase  , and ef-tu .
all of the proteins that showed altered abundance in the mutant returned to near wild-type levels in the revertant.
discussion
c. jejuni is a major cause of human diarrheal infection worldwide, yet we have only limited knowledge regarding the mechanisms the bacterium uses to colonize humans and cause disease.
because c. jejuni inhabits two hosts with differing body temperatures, we became interested in proteins  that are more abundant when c. jejuni is grown at 37°c  compared to 42°c .
because of its homology to other ppiases that are involved in the virulence of other bacteria and the fact that it is highly conserved among campylobacter species, this protein may play an important role in human colonization.
in silico analyses of the gene and protein sequences suggest that cj <dig> is probably a periplasmic ppiase that is involved in folding integral outer membrane proteins.
among the changes that occur in bacterial cells when encountering lower growth temperatures are a decrease in membrane fluidity, and inefficient folding of some proteins  <cit> .
proper protein folding or refolding of cold-damaged proteins is important after cold shock, and certain chaperones may be upregulated during cold shock in an attempt to compensate for the decreased efficiency of protein folding  <cit> .
in e. coli, several molecular chaperones  were transiently induced upon cold shock  <cit> .
additionally, the chaperone clpb may renature and solubilize aggregated proteins at low temperatures at which translation is repressed  <cit> .
whether the modest induction of cj <dig> that seems to occur at 37°c has any biological relevance to protein folding in the periplasm or modulation of membrane fluidity is a topic for future study.
cj <dig> is similar to the e. coli protein sura, which is a peptidyl prolyl cis-trans isomerase located in the periplasm and which plays a role in folding outer membrane proteins, particularly lamb and ompa, and in pilus biogenesis  <cit> .
a upec strain in which sura was inactivated was less able to bind and invade bladder epithelial cells, in addition to showing a decreased ability to survive intracellularly  <cit> .
there are several other examples of ppiases, and sura orthologs in particular, having roles in bacterial pathogenesis.
in s. flexneri, sura is required for proper folding and insertion into the outer membrane of icsa, which is responsible for the ability of the bacterium to spread intercellularly  <cit> .
deletion of sura decreases the ability of s. enterica to adhere to and invade caco- <dig> and raw <dig>  cells in vitro, as well as reducing the capacity to colonize balb/c mice  <cit> .
a l. pneumophila mutant lacking the ppiase mip was defective in initiating macrophage infection in vitro, and less virulent when introduced into a guinea pig model  <cit> .
similarly, the c. trachomatis mip-like protein and the t. cruzi tcmip protein play roles in the early steps of intracellular infection by these bacteria  <cit> .
ng-mip, found in n. gonorrhoeae, is similar to these mips, but plays a role in intracellular survival rather than invasion  <cit> .
previously, a c. jejuni nctc  <dig> cj <dig> mutant was found to have a decreased growth rate when growth was measured by od <dig>  <cit> .
our measurements by od <dig> initially suggested that the mutant had a reduced growth rate, but when growth was monitored using viable counts, the mutant was found to grow initially at a rate more similar to the wild-type, although a modest growth defect was still apparent at later stages of growth .
the difference in the results obtained by od <dig> and viable counts might be the result of a change in cell size or the light scattering properties of the cj <dig> mutant, possibly caused by a change in the composition of the outer membrane of the bacterium.
future work, such as using electron microscopy to evaluate the shape and surface components of the mutant, might help explain the reason for the discrepancy between the results obtained by od <dig> measurements and viable counts.
c. jejuni has two polar flagella which play a major role in virulence.
flagella-mediated motility is responsible for colonization of the mucous lining of the mammalian and avian gastrointestinal tracts as well as invasion of gastrointestinal epithelial cells  <cit> .
we found that the cj <dig> mutant was significantly more motile than wild-type bacteria.
because we found that removal of cj <dig> increased the motility of the bacterium, we considered that the cj <dig> mutant might be more invasive than wild-type.
studies using int <dig> cells showed that mutation of cj <dig> did in fact increase the invasiveness of c. jejuni without altering the adherence and intracellular survival abilities.
previously, it was noted that a c. jejuni nctc  <dig> cj <dig> mutant was significantly deficient in its ability to adhere to host cells  <cit> .
the discrepancy in adherence results seen between the previous study and our current work could be due to strain differences, however, we cannot exclude the possibility that the previously obtained adherence phenotype was due to an unlinked mutation in the uncomplemented nctc  <dig> cj <dig> mutant.
the increased motility and invasiveness could be due to an increase in chemotaxis, or to increased flagellar function because of a change in outer membrane architecture or cell morphology that provides a motility advantage.
several proteins located on the cell surface play a role in the initial cell-to-cell contact that is a component of intestinal colonization by c. jejuni.
because cj <dig> is thought to be involved in folding outer membrane proteins, its mutation is likely to have an effect on surface-exposed proteins, which could affect the ability to colonize the host intestinal tract.
when mice were inoculated individually with the wild-type, mutant, or revertant, the cj <dig> mutant initially was able to colonize at mean levels comparable to the wild-type and revertant strains.
however, the mutant became increasingly colonization deficient over time.
the differences were statistically significant at days  <dig> and  <dig>  but not at day  <dig> due to increased clearance of the wild-type and revertant strains from some mice.
this colonization defect is likely not the result of the increased motility of the mutant, since motility typically correlates with better animal colonization.
one possible explanation for the decreased colonization ability of the mutant is that cj <dig> is required for the proper presentation of surface structures that are necessary for mouse colonization .
additionally, when the mutant was placed in direct competition with the wild-type, it demonstrated an inability to compete with the wild-type for colonization of the mice.
in competition experiments, curiously, colonization levels of both the wild-type and mutant were significantly lower , suggesting some sort of interference of these strains with each other.
the cj <dig> mutant shows elevated autoaggregation and biofilm formation , so it is possible that these or other features impacting c. jejuni community structure could be involved.
in an effort to determine some of the molecular causes of the altered virulence phenotypes discussed previously, we conducted a proteomic analysis comparing the whole-cell protein profiles of wild-type, mutant, and revertant.
as expected, cat was found only in the mutant and cj <dig> was absent in the mutant, confirming the replacement of cj <dig> with the cat cassette in the mutant, and restoration of cj <dig> expression in the revertant.
a total of six other proteins showed altered expression in the cj <dig> mutant.
all proteins that showed altered abundance in the mutant returned to near wild-type levels in the revertant.
three proteins were found to be significantly upregulated in the mutant.
they were identified as htra , cj <dig> , and flaa .
htra is a serine protease with homologs found in most bacteria.
in e. coli, htra is located on the periplasmic side of the inner membrane  <cit> , has protease activity  <cit> , and has some chaperone activity at low temperatures  <cit> .
it is possible that c. jejuni htra is upregulated in the mutant in a compensatory manner due to an increase in unfolded protein in the periplasm resulting from the loss of a major periplasmic ppiase.
brondsted et al.
<cit>  found that a c. jejuni htra mutant showed no change in motility or autoagglutination, but did have a decreased ability to adhere to and invade int <dig> cells and also exhibited altered cell morphology.
cj <dig> is annotated as 'putative periplasmic protein' and is restricted primarily to the epsilon proteobacteria, although the function of this protein is currently unknown.
flaa is the major subunit of the c. jejuni flagellum, and the upregulation of flaa is consistent with the increase in motility and invasion of int <dig> cells seen in the cj <dig> mutant.
three proteins were shown to be significantly downregulated in the cj <dig> mutant.
they were identified as ef-ts , sod , and ef-tu .
among proteins whose expression was lower in the cj <dig> mutant, ef-ts and ef-tu are involved in protein translation.
they may be downregulated in the mutant due to an increase in unfolded protein in the periplasm and this in turn may result in the late stage growth defect due to a general decrease in protein synthesis.
as c. jejuni lacks a sigma-e response  <cit> , the signalling mechanism that would be responsible is unknown.
sod plays a role in protecting c. jejuni against damage from oxidative stress and mutation of sod in c. coli was found to decrease the ability of the bacterium to colonize the intestines of 1-day-old chicks  <cit> .
the decreased levels of sod in the cj <dig> mutant may therefore play a role in the colonization defects seen in mice.
CONCLUSIONS
cj <dig> is a highly conserved protein whose expression in c. jejuni is induced at human body temperature.
bacteria lacking cj <dig> were found to exhibit changes in several virulence-related phenotypes, including motility and host cell invasion, as well as alterations in protein expression and a defect in mouse colonization.
authors' contributions
jeh carried out the proteomics experiments comparing 81– <dig> grown at 37°c and 42°c.
kmr carried out all other experiments and participated in the study design and drafting of the manuscript.
sat conceived the study and participated in the study design and drafting of the manuscript.
all authors read and approved the final manuscript.

 campylobacter jejuni is a curved, microaerophilic gram-negative bacterium that is an important human pathogen. the main reservoir of c. jejuni is the cecum of poultry, and humans can become infected through consumption of undercooked chicken that is contaminated with the bacterium. in humans, the bacterium colonizes both the small and large intestine resulting in fever, severe abdominal pain, and diarrhea, with possible autoimmune sequelae of infection including guillain-barré syndrome and reactive arthritis. expression of proteins is an energetically costly activity. therefore, bacteria often express certain proteins only under conditions where those proteins are needed for growth, survival, or pathogenicity. growth temperatures cause differential expression of proteins in a number of pathogenic bacteria including c. jejuni, although the mechanisms of thermoregulation can be complex and result from overlapping regulatory systems. in c. jejuni, the racrs two-component regulatory system is involved in the regulation of some proteins, although the majority of the targets have not been identified. because the body temperatures of humans and chickens differ, c. jejuni is likely to express different proteins when colonizing chickens than when colonizing humans. we used proteomics to determine which c. jejuni proteins are more highly expressed at 37°c compared to 42°c, because such upregulation might suggest an importance of these proteins in colonization of humans. one of the proteins identified was cj which is annotated as playing a role in outer membrane protein folding and stabilization. cj was previously identified as an immunogenic outer membrane protein and was named cell binding factor; it is also called peb. it was later suggested that peb is not surface exposed, but is periplasmically located in association with the inner membrane. cj shows homology to sura, a peptidyl-prolyl cis-trans isomerase found in e. coli, and other orthologs in numerous bacteria including helicobacter pylori, bacilis subtilis, and lactococcus lactis. ppiases have been characterized as virulence factors in shigella flexneri, salmonella enterica, legionella pneumophila, chlamydia trachomatis, trypanosoma cruzi, and neisseria gonorrhoeae. asakura et al. recently characterized a cj mutant of c. jejuni strain nctc finding decreases in ability to adhere to int cells and to colonize mice, and an increase in biofilm formation. however, this mutant was not complemented with a wild-type copy of cj allowing some question of whether the observed phenotypes were specific for cj in this study, we examine the effects of deletion of cj in a different, highly invasive c. jejuni strain on phenotypes related to growth, protein expression, and pathogenicity. our results show that deletion of cj alters several virulence-related phenotypes in vitro and decreases the ability of the bacterium to colonize mice, although the phenotypes of the 81– cj mutant differ significantly from those of an analogous mutant in strain nctc methods bacterial strains and routine culture conditions campylobacter jejuni strains derived from the parent 81– were routinely maintained with minimal passage on blood agar plates at 37°c in sealed culture boxes containing a microaerobic atmosphere generated by pack-micro aero. liquid cultures of c. jejuni were grown in brucella broth or mueller-hinton broth and cultured in microaerobic environments. when appropriate, strains were cultured in the presence of chloramphenicol or streptomycin to select for antibiotic resistance markers. escherichia coli jm was used as the host strain for cloning experiments and e. coli bl21plyss was used as the host strain for expression of the his-tagged cj protein. e. coli strains were cultured in luria-bertani broth or agar, supplemented with the following antibiotics as appropriate for selection of plasmids: ampicillin, μg/ml; chloramphenicol, μg/ml; streptomycin, μg/ml. proteome analysis of c. jejuni strains proteomics experiments were performed on c. jejuni cells grown at 37°c and 42°c as described. briefly, cells were grown overnight at 37°c in brucella broth, then diluted the following morning into two aliquots of fresh brucella broth, which were grown at 37°c and 42°c to mid-log phase. chloramphenicol was added to stop protein synthesis, and the cells were harvested for proteome analysis as described. proteomics experiments were performed using differential in-gel electrophoresis technology from ge biosystems, whole-cell protein lysates from the 37°c- and 42°c-grown c. jejuni were labelled individually with cy and cy dyes according to the protocol supplied by the manufacturer, then mixed in equal mass and separated using two-dimensional sds-page. for comparison of wt, mutant and revertant proteomes, protein lysates were labelled with cy, cy, or cy dyes, mixed in equal mass, and separated by 2d sds-page. following separation, gels were scanned on a typhoon fluorescent flatbed scanner, at the following wavelengths: cy nm excitation, nm emission, cy nm excitation, nm emission; cy nm excitation, nm emission. images were analyzed with decyder differential in-gel analysis software for identification of proteins with higher or lower expression in different samples. the identities of proteins of interest were determined using a matrix-assisted laser desorption ionization – time-of-flight/time-of-flight spectrometer, using both tryptic fingerprint data and fragmentation-based ms/ms. purification of cj protein and antibody production to allow purification of the cj protein, a c-terminal his6-tag was added to cj lacking the n-terminal signal sequence by inserting the gene into pet-20b. first, the cj gene without the signal sequence and stop codon was amplified from c. jejuni strain 81– and nde i and xho i sites were added using primers purprot-f and purprot-r. the resulting pcr product was cloned into pcr ii-topo, creating plasmid pkr. using nde i and xho i, the cj gene was excised from pkr and pet-20b was linearized. the cj gene was ligated into the linearized pet-20b creating plasmid pkr which was used to transform e. coli strain bl21plyss. the plasmid-carrying strain was grown overnight in lb broth at 37°c. the next morning the culture was diluted to od ~ and incubated at 37°c until od ~. iptg was added to the culture to induce expression of the his6-tagged protein. after h, the cells were harvested by centrifugation, washed, and the supernatant passed through a nickel column to further purify the his6-tagged protein by standard methods. the purified protein was sent to cocalico biologicals, inc. for production of anti-cj antibodies. for use in the ppiase assay, the protein was refolded using the pro-matrix protein refolding kit and dialyzed against pbs. †bold nucleotides indicate restriction sites introduced for cloning purposes to confirm the proteomics-implicated temperature regulation of cj a western blot was performed on c. jejuni cells grown at 37°c or 42°c using anti-cj antibody as the primary antibody and hrp conjugated goat anti-rabbit igg as the secondary antibody. a control western blot against cj was performed using anti-cj antibody as the primary antibody and hrp conjugated goat anti-rabbit igg as the secondary antibody. the blots were developed using a dab substrate kit. densitometry measurements were conducted using imagej software. localization of the cj protein to determine the cellular location of cj c. jejuni cells grown at 37°c were separated into cytoplasmic, periplasmic, and inner membrane fractions, and outer membrane fractions as described. western blots were performed on c. jejuni cell fractions using anti-cj antibody as the primary antibody, along with control blots using anti-cj, anti-ceta, and anti-momp polyclonal sera as the primary antibodies. hrp conjugated goat anti-rabbit igg was used as the secondary antibody for all blots, which were then developed using a dab substrate kit. ppiase assay the ppiase activity of cj was determined in a coupled assay, which measures the ability of cj to convert the cis isomer of the oligopeptide substrate n-suc-ala-ala-pro-phe-p-nitroanilide into the trans form which is cleavable by α-chymotrypsin. chymotrypsin and varying concentrations of cj were combined in mm tris-hcl ph and incubated at 4°c. the substrate was added and the reaction was monitored at 10°c by the increase in absorbance at nm. the kobs value for each ppiase concentration was found by plotting ln vs. time and determining the slope. the catalytic efficiency of the ppiase activity was obtained by plotting kobs vs. and determining the slope. analysis of cj in additional campylobacter strains the dna sequences of the cj locus were determined from the campylobacter strains listed in table. primers cj0596-f and cj0596-r were designed based on the published nctc genome sequence and used in pcr reactions with template genomic dna prepared using a masterpure dna purification kit to amplify the region encompassing cj thermocycler parameters were cycles of: 94°c for seconds, 51°c for seconds, and 72°c for minutes. pcr products were purified using qiaquick pcr purification kits and were then sequenced directly, using an abi 3730xl sequencer. vectornti was used to analyze dna sequences. the protein sequences were analyzed for motifs using the expasy prosite server http://au.expasy.org/prosite/, and potential signal peptides were evaluated using signalp http://www.cbs.dtu.dk/services/signalp/. isolation of a c. jejuni cj mutant a cj mutant of c. jejuni strain 81– was constructed using a streptomycin counterselection system similar to the heterologous h. pylori-c. jejuni method described by dailidiene et al. in which an rpsl gene from c. jejuni was used for counterselection in h. pylori, to decrease background gene conversion events. in the heterologous rpslhp system reported here, cj was exactly replaced by the rpslhp gene from h. pylori strain 84– linked to a chloramphenicol acetyltransferase cassette. this strategy allows for selection of a mutant based on chloramphenicol resistance and then allows for selection of a revertant strain based on streptomycin resistance. first, a pcr-amplified cj gene was amplified using primers cj0596-f and cj0596-r designed based on the published c. jejuni nctc genome sequence and the resulting product was cloned into pcr ii-topo creating pkr. the plasmid pkr was subjected to inverse pcr to remove the cj gene and add restriction sites. the inverse pcr product was self-ligated to form plasmid pkr the cat cassette was amplified from pry and age i, mfe i, and nhe i sites were added using primers cat-f and cat-r the resulting pcr product was cloned into pcr ii-topo to create plasmid pkr the rpslhp gene was amplified and age i and mfe i sites were added using primers rpslhp-f and rpslhp-r and the pcr product was cloned into pcr ii-topo creating plasmid pkr age i and nhe i were used to excise the cat cassette from pkr and linearize pkr the cat cassette was ligated into pkr creating plasmid pkr using age i and mfe i, the rpslhp gene was removed from pkr and ligated with linearized pkr creating plasmid pkr with the rpslhp/cat construct oriented in the same direction as the cj and cj genes. care was taken to ensure that this replacement would not produce polar effects by preserving the cj ribosome binding site and by leaving only bp between the stop codon of cat and the start codon of cj the mutagenized cj allele was introduced into a spontaneous strepr derivative of c. jejuni 81– by electroporation. several cmr/streps transformants were verified as cj mutants by pcr with primers cj0596-f and cj0596-r and dna sequencing, a representative of which was designated 81–176cj and used for further analysis. reversion of the cj mutation a revertant of c. jejuni 81–176cj was isolated by replacing the mutated cj allele in 81–176cj with a wild-type cj gene using streptomycin counterselection. c. jejuni strain 81–176cj0596+ was created by using electroporation to introduce pkr into 81–176cj cells, selecting for colonies on plates containing streptomycin. putative revertants were identified by screening strr colonies for sensitivity to chloramphenicol to ensure loss of the rpslhp/cat cassette. chromosomal dna was isolated from these transformants and proper replacement of the rpslhp/cat cassette with wild-type cj was confirmed by pcr using primers cj0596-f and cj0596-r and by dna sequencing of the region. quantitative real-time reverse transcription pcr cdna was prepared from rna samples of c. jejuni grown 81– and 81–176cj using a geneamp rna pcr kit. an icycler iq real-time pcr detection system was used to run qrt-pcr with iq sybr green super mix, and primers cj0597rt-f and cj0597rt-r. data were analyzed using bio-rad icycler data analysis software. control reactions used primers specific for 16s rdna, which allows amplification of a non-regulated rna. differences in transcript levels among samples were calculated from amplification profiles using the comparative threshold cycle method, as previously described. growth experiments the growth rates of c. jejuni wild-type 81– mutant 81–176cj and revertant 81–176cj0596+ were assessed by growing cells overnight in mh broth, then diluting the following morning in mh broth to od ~ and shaking at 37°c under microaerobic conditions. growth was monitored by measuring od and numbers of viable bacteria were determined by plating serial dilutions of the bacterial suspensions on mh agar and counting the resultant colonies. motility the motility of c. jejuni 81– 81–176cj and 81–176cj0596+ was determined as previously described. briefly, cells were grown overnight in mh broth, then diluted the following morning in mh broth to od =. a pipet tip dipped into the suspension was used to stab the center of a mh motility plate. the plates were incubated at 37°c and the diameter of the motility zone was measured every h. adherence/invasion/intracellular survival assay a gentamicin protection assay was used to assess the ability of 81– 81–176cj and 81–176cj0596+ to adhere to, invade, and survive within int human intestinal epithelial cells. briefly, bacteria were grown in biphasic cultures at 37°c under microaerobic conditions for ~ h. bacteria were harvested, resuspended in phosphate buffered saline, then added in triplicate to semi-confluent int cell monolayers at a multiplicity of infection of ~40:. the number of bacteria added was quantified by determination of cfu/ml. the cells were incubated for h at 37°c under microaerobic conditions and were washed with hanks' balanced salt solution, lysed with triton x- and the number of adherent bacteria was quantified by viable counts. for determination of invasion, cells were incubated for h with bacteria and then gentamicin was added to a final concentration of μg/ml to kill any extracellular bacteria. after an additional h of incubation, the cells were washed, lysed with triton x- and intracellular bacteria were quantified by viable counts. the gentamicin and triton x- mics of the three strains were also determined. for determination of intracellular survival, the cells were incubated for h with bacteria, h with gentamicin, and then the int cells were washed and incubated for h in minimal essential media containing 3% fetal bovine serum and gentamicin as described by candon et al.. after the incubation period, cells were washed and lysed with triton-x and the number of bacteria that survived intracellularly was quantified by viable counts. mouse colonization experiments the in vivo relevance of cj was investigated by testing the ability of 81– 81–176cj and 81–176cj0596+ to colonize mice as described. 10-week old female balb/c-byj mice were given μl of 5% sodium bicarbonate by oral gavage to neutralize stomach acid. the mice were then given a dose of × cfu in ml of bhi/1% ye broth by oral gavage. because there was an observed discrepancy between od and cfu for the mutant, we first performed pilot experiments correlating od and cfu for all of the strains. after four repetitions, we found the mutant od that gave the same number of cfu as for the wt and revertant strain, and this is what we used for the mouse inocula. we also verified that each mouse received equal cfu by plating the inocula for viable counts at the time of inoculation. colonization was determined by viable counts of bacteria in fecal pellets, which were enumerated on selective cva media starting days after the challenge and continuing every days thereafter until days post-inoculation. in parallel, experiments were carried out to determine the ability of cj mutant bacteria to compete with wild-type bacteria in colonization. for competition experiments, wild-type and mutant bacteria were mixed in equal amounts immediately prior to inoculation. colonization was determined by enumerating bacteria on selective media with or without chloramphenicol. the number of bacteria counted on the plates containing chloramphenicol was subtracted from the number of bacteria found on the plates without chloramphenicol to obtain the number of viable wild-type bacteria. control experiments showed that the plating efficiency of the cj mutant was equivalent on media containing or lacking chloramphenicol. all vertebrate animal experiments were conducted in accordance with recommendations by the office of laboratory animal welfare, and were approved by the medical college of georgia institutional animal care and use committee.  expression of cj is slightly higher at 37°c than at 42°c in a search to identify c. jejuni genes with differential response to steady-state growth temperature, several proteins were identified that were more highly expressed at 37°c than at 42°c. c. jejuni 81– was grown overnight at 37°c and then diluted into fresh media. the two cultures were grown in parallel at 37°c and 42°c to mid-log growth phase. proteomics experiments were then performed on cultures of c. jejuni 81– grown at the two temperatures. one protein that was upregulated at 37°c had the approximate pi and molecular mass of the predicted cj protein. this protein was -fold more highly expressed at 37°c, a result that was consistent in five different proteomics experiments. the protein was excised from the polyacrylamide gel and subjected to maldi-tof/tof mass spectrometry. this protein was identified with 100% confidence as cj. in an attempt to confirm the proteomics results, we performed western blots using anti-cj antibodies and c. jejuni 81– grown at 37°c and 42°c. while only semi-quantitative, in two separate experiments the western blots showed a more modest – -fold greater expression of cj at 37°c. therefore, we conclude that while cj appears to be expressed at a somewhat higher level at 37°c compared to 42°c, the effect of growth temperature on cj expression is not dramatic. because proteins homologous to cj are involved in virulence in other pathogenic bacteria, we nevertheless characterized the role of this protein in c. jejuni physiology and pathogenesis. similarity of cj sequences among campylobacter species because campylobacter genomes are quite diverse, we characterized the conservation of the cj gene in other campylobacter strains. using pcr primers designed from the c. jejuni nctc genomic sequence and located in the cj and cj genes, we amplified a kb segment encompassing the cj locus from five additional c. jejuni strains and one c. coli strain. pcr products of the expected size were obtained from each strain, and were subsequently sequenced. a search of additional campylobacter genome sequences was also performed and showed that a cj ortholog was found in every strain. the sequences of these orthologs were also included in the sequence comparison analysis. the nucleotide sequences between pairs of c. jejuni strains or c. coli d were at least 98% identical. the corresponding sequences from c. coli rm and other campylobacter species were somewhat lower. the predicted cj protein was also highly similar in all c. jejuni strains and c. coli d with an amino acid sequence identity of at least 99%. as with the nucleotide sequences, the degree of identity of proteins from c. coli rm and other non-jejuni campylobacter strains was lower, with identities ranging from 87% to 45%. together, these results indicate that cj is highly conserved in c. jejuni, c. coli, and one strain each of c. concisus, c. curvus, c. fetus, c. hominis, c. lari, and c. upsaliensis. we focused on cj from c. jejuni strain 81– for our subsequent work. in silico analysis of cj protein features in the nctc genome, the predicted cj protein had a predicted molecular mass of kda and pi of and was annotated as a major antigenic peptide peb4\cell binding factor similar to peptidyl prolyl cis-trans isomerases found in a variety of organisms. because some peptidyl-prolyl cis-trans isomerases are located in the periplasm, the signalp algorithm was used to analyze the 81– cj protein for the presence of an n-terminal signal sequence. a signal sequence with a probable cleavage site between amino acids and of the preprotein was predicted. cj was also analyzed for the presence of protein motifs and was found to contain a ppic-type peptidyl-prolyl cis-trans isomerase motif located between amino acids 131– secondary structure predictions showed no transmembrane segments in the mature protein, suggesting that cj is likely to be a periplasmic protein. cj is located in the periplasm of c. jejuni the amino acid sequence of cj suggested that this protein is located in the periplasm. to test this experimentally, western blots were performed on cytoplasmic, inner membrane, periplasmic, and outer membrane fractions of c. jejuni 81– using anti-cj anti-cj anti-ceta, and anti-momp antibodies. as expected, anti-cj antibodies reacted with a ~ kda protein in the cytoplasmic fraction, anti-ceta antibodies reacted with a ~ kda protein in the inner membrane fraction, and anti-momp antibodies reacted with a ~ kda protein in the outer membrane. anti-cj antibodies reacted with a ~ kda protein present primarily in the periplasmic fraction. cj has ppiase activity cj has one rotamase domain and is similar to e. coli sura, suggesting that it is a ppiase. the ppiase activity of purified cj was determined using a coupled assay in which the cleavage of the trans isomer of n-suc-ala-ala-pro-phe-p-nitroanilide by α-chymotrypsin results in the release of p-nitroanilide, causing a colorimetric change over time. conversion of the cis to the trans isomers of the substrate occurs spontaneously in solution, allowing chymotrypsin cleavage. however, addition of cj accelerates this cis-trans conversion, indicative of ppiase activity. by using varying concentrations of purified cj and plotting calculated kobs vs., the ppiase activity was calculated to be mm-1sec- an activity consistent with values published for other ppiases. creation of a non-polar cj mutation to test the role of cj in c. jejuni physiology or pathogenesis, we created a non-polar cj mutant. to facilitate mutant construction, we developed a modified streptomycin counter selection system based on a similar strategy used in h. pylori. the rpslhp/cat cassette was used to precisely replace cj maintaining the ribosome binding site of the downstream cj gene. together with the lack of a transcriptional terminator on the rpslhp/cat cassette, this construct is expected to yield a non-polar mutation. the resulting mutant strain was designated 81–176cj to confirm that mutation of the cj gene did not alter the expression of the putative co-transcribed cj gene, quantitative real-time rt-pcr was performed on rna samples harvested from 81– and 81–176cj these studies confirmed that mrna levels downstream of the mutation in 81–176cj were equivalent to those in 81– and that the mutation was non-polar. however, to ensure that any phenotypes of the cj mutant were specific for cj we subsequently isolated a reversion of the mutation by replacing the rpslhp/cat cassette with a wild-type cj gene. analogous to the counterselection system of dailidiene et al., use of the h. pylori rpsl gene decreased background gene conversion events and facilitated the recovery of cells in which the mutagenized cj allele was replaced with the wild-type cj gene. this process was extremely efficient, as ~70% of the recovered streptomycin-resistant colonies contained the desired reversions of the cj mutation. mutation of cj causes a moderate growth defect of c. jejuni in mh broth the role of cj in campylobacter growth was studied by comparing the growth rates of c. jejuni 81– 81–176cj and 81–176cj0596+ in mh broth. when inoculated at od ~ and growth rate was measured by od, the mutant initially appeared to have a significant growth defect illustrated both by a slower increase in od and a lower maximum od however, we also found that a similar starting od resulted in a significantly lower starting cfu for the mutant, indicating that the od did not accurately reflect the number of cj mutant cfu. we then grew the mutant under two starting conditions: one had the same starting od as the wild-type and revertant and the other had approximately the same starting cfu as the wild-type and the revertant. the mutant inoculated at od ~ showed a faster initial increase in od but reached a similar maximum od as the mutant inoculated at od ~. however, when growth rate was monitored by plating for viable counts, the mutant had an initial growth rate more similar to that of wild-type, although both mutant cultures yielded final viable counts lower than wild-type. wild-type growth characteristics were restored in the revertant strain. together, these data suggest that mutation of cj resulted in a moderate growth defect, especially later in the growth curve, and that the cj mutant had apparent changes in cell characteristics such that the od had poor concordance with cfu measurements. deletion of cj increases the motility of c. jejuni because motility plays an important role in invasion of host intestinal cells and is required for animal colonization, the motility of c. jejuni 81– 81–176cj and 81–176cj0596+ was compared at 37°c. the average diameter of the zone of motility for the wild-type was mm ± at h. the mutant was significantly more motile with a zone diameter of mm ±. the revertant returned to wild-type motility levels with a zone diameter of mm ±. a similar increase in motility was seen when the assay was performed at 42°c. thus, cj is involved in the expression of motility. deletion of cj increases the ability of c. jejuni to invade int cells, but does not affect adherence or intracellular survival the possibility that cj plays a role in interaction with host cells was studied by comparing the adherence and invasion abilities of c. jejuni 81– 81–176cj and 81–176cj0596+ in an in vitro assay using int intestinal epithelial cells. the mean percentages of the inoculum that adhered were,, and for the wild-type, mutant, and revertant, respectively, demonstrating that deletion of cj does not significantly affect the ability of c. jejuni to adhere to int cells. in contrast, mutation of cj had a significant effect on the invasion ability of c. jejuni. while the percentages of the wild-type and revertant inocula invading int cells were and, respectively, the cj mutant showed a nearly 20-fold increase in invasion. the gentamicin and triton x- sensitivities of the three strains were tested to ensure that the invasion results were not due to altered killing of a strain, and no significant difference was found for either compound. compared to the number of bacteria that invaded the int cells, the percentages of wild-type, mutant, or revertant that were able to survive intracellularly at timepoints from to hours post-invasion were not significantly different. 81–176cj is defective in mouse colonization to determine whether cj plays a role in mouse colonization, we used a balb/c model that has been used previously to assess colonization differences between wild-type and mutant bacteria. female balb/c-byj mice were given doses of c. jejuni 81– 81–176cj and 81–176cj0596+ individually, as well as a mixture of wild-type and cj mutant in a competition experiment, and colonization was measured by determining viable counts of bacteria in fecal pellets at weekly intervals. on days and viable bacteria were found in all seven mice receiving the wild-type, mutant, or the revertant. following the peak in colonization at days, viable mutant bacteria were recovered from only four mice on day and only three mice on days and at these latter three timepoints, the wild-type and revertant were recovered from all but one mouse. the mean colonization densities of the wild-type and revertant were × and × cfu/g, respectively, on day and remained relatively consistent throughout the experiment. the mean colonization level of the mutant was significantly lower than wild-type and revertant on days and. when placed in competition with the wild-type, the mutant showed an inability to compete for colonization. wild-type bacteria were recovered from five mice on day four mice on day and then one mouse for the remainder of the experiment. viable mutant bacteria were recovered from no mice on day, two mice on day, one mouse on day and then were not recovered on days and deletion of cj alters c. jejuni protein expression because cj is thought to be a periplasmic chaperone, its loss could result in compensatory changes in the expression of other proteins. to determine the effect that deletion of cj had on the expression of other proteins, a comparison of total cell proteins from c. jejuni 81– 81–176cj and 81–176cj0596+ was performed using 2d sds-page. three proteins were found to be significantly upregulated in the mutant. they were identified as htra, cj, and flaa. as expected, the cat protein was found only in the cj mutant. conversely, the cj protein was found in wild-type and revertant strains, but was absent in the cj mutant, as expected. three proteins were found to be significantly downregulated in the mutant. these proteins were ef-ts, superoxide dismutase, and ef-tu. all of the proteins that showed altered abundance in the mutant returned to near wild-type levels in the revertant. discussion c. jejuni is a major cause of human diarrheal infection worldwide, yet we have only limited knowledge regarding the mechanisms the bacterium uses to colonize humans and cause disease. because c. jejuni inhabits two hosts with differing body temperatures, we became interested in proteins that are more abundant when c. jejuni is grown at 37°c compared to 42°c. because of its homology to other ppiases that are involved in the virulence of other bacteria and the fact that it is highly conserved among campylobacter species, this protein may play an important role in human colonization. in silico analyses of the gene and protein sequences suggest that cj is probably a periplasmic ppiase that is involved in folding integral outer membrane proteins. among the changes that occur in bacterial cells when encountering lower growth temperatures are a decrease in membrane fluidity, and inefficient folding of some proteins. proper protein folding or refolding of cold-damaged proteins is important after cold shock, and certain chaperones may be upregulated during cold shock in an attempt to compensate for the decreased efficiency of protein folding. in e. coli, several molecular chaperones were transiently induced upon cold shock. additionally, the chaperone clpb may renature and solubilize aggregated proteins at low temperatures at which translation is repressed. whether the modest induction of cj that seems to occur at 37°c has any biological relevance to protein folding in the periplasm or modulation of membrane fluidity is a topic for future study. cj is similar to the e. coli protein sura, which is a peptidyl prolyl cis-trans isomerase located in the periplasm and which plays a role in folding outer membrane proteins, particularly lamb and ompa, and in pilus biogenesis. a upec strain in which sura was inactivated was less able to bind and invade bladder epithelial cells, in addition to showing a decreased ability to survive intracellularly. there are several other examples of ppiases, and sura orthologs in particular, having roles in bacterial pathogenesis. in s. flexneri, sura is required for proper folding and insertion into the outer membrane of icsa, which is responsible for the ability of the bacterium to spread intercellularly. deletion of sura decreases the ability of s. enterica to adhere to and invade caco- and raw cells in vitro, as well as reducing the capacity to colonize balb/c mice. a l. pneumophila mutant lacking the ppiase mip was defective in initiating macrophage infection in vitro, and less virulent when introduced into a guinea pig model. similarly, the c. trachomatis mip-like protein and the t. cruzi tcmip protein play roles in the early steps of intracellular infection by these bacteria. ng-mip, found in n. gonorrhoeae, is similar to these mips, but plays a role in intracellular survival rather than invasion. previously, a c. jejuni nctc cj mutant was found to have a decreased growth rate when growth was measured by od. our measurements by od initially suggested that the mutant had a reduced growth rate, but when growth was monitored using viable counts, the mutant was found to grow initially at a rate more similar to the wild-type, although a modest growth defect was still apparent at later stages of growth. the difference in the results obtained by od and viable counts might be the result of a change in cell size or the light scattering properties of the cj mutant, possibly caused by a change in the composition of the outer membrane of the bacterium. future work, such as using electron microscopy to evaluate the shape and surface components of the mutant, might help explain the reason for the discrepancy between the results obtained by od measurements and viable counts. c. jejuni has two polar flagella which play a major role in virulence. flagella-mediated motility is responsible for colonization of the mucous lining of the mammalian and avian gastrointestinal tracts as well as invasion of gastrointestinal epithelial cells. we found that the cj mutant was significantly more motile than wild-type bacteria. because we found that removal of cj increased the motility of the bacterium, we considered that the cj mutant might be more invasive than wild-type. studies using int cells showed that mutation of cj did in fact increase the invasiveness of c. jejuni without altering the adherence and intracellular survival abilities. previously, it was noted that a c. jejuni nctc cj mutant was significantly deficient in its ability to adhere to host cells. the discrepancy in adherence results seen between the previous study and our current work could be due to strain differences, however, we cannot exclude the possibility that the previously obtained adherence phenotype was due to an unlinked mutation in the uncomplemented nctc cj mutant. the increased motility and invasiveness could be due to an increase in chemotaxis, or to increased flagellar function because of a change in outer membrane architecture or cell morphology that provides a motility advantage. several proteins located on the cell surface play a role in the initial cell-to-cell contact that is a component of intestinal colonization by c. jejuni. because cj is thought to be involved in folding outer membrane proteins, its mutation is likely to have an effect on surface-exposed proteins, which could affect the ability to colonize the host intestinal tract. when mice were inoculated individually with the wild-type, mutant, or revertant, the cj mutant initially was able to colonize at mean levels comparable to the wild-type and revertant strains. however, the mutant became increasingly colonization deficient over time. the differences were statistically significant at days and but not at day due to increased clearance of the wild-type and revertant strains from some mice. this colonization defect is likely not the result of the increased motility of the mutant, since motility typically correlates with better animal colonization. one possible explanation for the decreased colonization ability of the mutant is that cj is required for the proper presentation of surface structures that are necessary for mouse colonization. additionally, when the mutant was placed in direct competition with the wild-type, it demonstrated an inability to compete with the wild-type for colonization of the mice. in competition experiments, curiously, colonization levels of both the wild-type and mutant were significantly lower, suggesting some sort of interference of these strains with each other. the cj mutant shows elevated autoaggregation and biofilm formation, so it is possible that these or other features impacting c. jejuni community structure could be involved. in an effort to determine some of the molecular causes of the altered virulence phenotypes discussed previously, we conducted a proteomic analysis comparing the whole-cell protein profiles of wild-type, mutant, and revertant. as expected, cat was found only in the mutant and cj was absent in the mutant, confirming the replacement of cj with the cat cassette in the mutant, and restoration of cj expression in the revertant. a total of six other proteins showed altered expression in the cj mutant. all proteins that showed altered abundance in the mutant returned to near wild-type levels in the revertant. three proteins were found to be significantly upregulated in the mutant. they were identified as htra, cj, and flaa. htra is a serine protease with homologs found in most bacteria. in e. coli, htra is located on the periplasmic side of the inner membrane, has protease activity, and has some chaperone activity at low temperatures. it is possible that c. jejuni htra is upregulated in the mutant in a compensatory manner due to an increase in unfolded protein in the periplasm resulting from the loss of a major periplasmic ppiase. brondsted et al. found that a c. jejuni htra mutant showed no change in motility or autoagglutination, but did have a decreased ability to adhere to and invade int cells and also exhibited altered cell morphology. cj is annotated as'putative periplasmic protein' and is restricted primarily to the epsilon proteobacteria, although the function of this protein is currently unknown. flaa is the major subunit of the c. jejuni flagellum, and the upregulation of flaa is consistent with the increase in motility and invasion of int cells seen in the cj mutant. three proteins were shown to be significantly downregulated in the cj mutant. they were identified as ef-ts, sod, and ef-tu. among proteins whose expression was lower in the cj mutant, ef-ts and ef-tu are involved in protein translation. they may be downregulated in the mutant due to an increase in unfolded protein in the periplasm and this in turn may result in the late stage growth defect due to a general decrease in protein synthesis. as c. jejuni lacks a sigma-e response, the signalling mechanism that would be responsible is unknown. sod plays a role in protecting c. jejuni against damage from oxidative stress and mutation of sod in c. coli was found to decrease the ability of the bacterium to colonize the intestines of 1-day-old chicks. the decreased levels of sod in the cj mutant may therefore play a role in the colonization defects seen in mice.  cj is a highly conserved protein whose expression in c. jejuni is induced at human body temperature. bacteria lacking cj were found to exhibit changes in several virulence-related phenotypes, including motility and host cell invasion, as well as alterations in protein expression and a defect in mouse colonization. authors' contributions jeh carried out the proteomics experiments comparing 81– grown at 37°c and 42°c. kmr carried out all other experiments and participated in the study design and drafting of the manuscript. sat conceived the study and participated in the study design and drafting of the manuscript. all authors read and approved the final manuscript. 
campylobacter jejuni is a gastrointestinal pathogen of humans, but part of the normal flora of poultry, and therefore grows well at the respective body temperatures of 37°c and 42°c. in either single or mixed infections, the mutant was less able to colonize mice than 81– the cj mutant also expressed altered levels of several proteins. proteomic studies on temperature regulation in c. jejuni strain 81– revealed the upregulation at 37°c of cj a predicted periplasmic chaperone that is similar to proteins involved in outer membrane protein folding and virulence in other bacteria. a revertant of this mutant was isolated by restoring the gene to its original chromosomal location using streptomycin counterselection. the cj mutant strain demonstrated a slightly decreased growth rate and lower final growth yield, yet was more motile and more invasive of human intestinal epithelial cells than wild-type. mutation of cj has an effect on phenotypes related to c. jejuni pathogenesis, probably due to its role in the proper folding of critical outer membrane proteins.
 campylobacter jejuni is a gastrointestinal pathogen of humans, but part of the normal flora of poultry, and therefore grows well at the respective body temperatures of 37°c and 42°c. proteomic studies on temperature regulation in c. jejuni strain 81– revealed the upregulation at 37°c of cj a predicted periplasmic chaperone that is similar to proteins involved in outer membrane protein folding and virulence in other bacteria.  the cj gene was highly conserved in strains and species of campylobacter, implying the importance of this gene. to study the role that cj plays in c. jejuni pathogenesis, a mutant derivative of strain 81– was constructed in which the cj gene was precisely deleted. a revertant of this mutant was isolated by restoring the gene to its original chromosomal location using streptomycin counterselection. the cj mutant strain demonstrated a slightly decreased growth rate and lower final growth yield, yet was more motile and more invasive of human intestinal epithelial cells than wild-type. in either single or mixed infections, the mutant was less able to colonize mice than 81– the cj mutant also expressed altered levels of several proteins.  mutation of cj has an effect on phenotypes related to c. jejuni pathogenesis, probably due to its role in the proper folding of critical outer membrane proteins. 