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Infect Immun. Dec 2008; 76(12): 5655–5667.
Published online Sep 22, 2008. doi:  10.1128/IAI.00780-08
PMCID: PMC2583572

Characterization of Two Campylobacter jejuni Strains for Use in Volunteer Experimental-Infection Studies[down-pointing small open triangle]

Abstract

The development of vaccines against Campylobacter jejuni would be facilitated by the ability to perform phase II challenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most C. jejuni strains with human gangliosides presents safety concerns about the development of Guillain-Barré syndrome. Clinical isolates of C. jejuni that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probe analyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to express the LOS type containing N-acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSs of these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involved in ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed of galactose (Gal), 6-deoxy-ido-heptose, and, in smaller amounts, d-glycero-d-ido-heptose, and the CPS of CG8421 was observed to contain Gal, 6-deoxy-altro-heptose, N-acetyl-glucosamine, and minor amounts of 6-deoxy-3-O-Me-altro-heptose. Both CPSs were shown to carry O-methyl-phosphoramidate. The two genomes contained strain-specific zones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion related to the CJEI3 element of C. jejuni RM1221. The genomes of both strains shared a high degree of similarity to each other and, with the exception of the capsule locus of CG8421, to the type strain of the HS3 serotype, TGH9011.

Campylobacter jejuni is among the most common causes of bacterial gastroenteritis worldwide and a major cause of traveler's diarrhea (1, 41). C. jejuni colonization of the human digestive tract induces a wide range of symptoms, from mild, watery diarrhea to dysentery that can be accompanied by fever and abdominal cramps. The preferred antibiotic treatment for campylobacteriosis is either erythromycin-azithromycin or ciprofloxacin, although resistance to both drugs is increasing (50, 53). C. jejuni infection has been linked to the development of postinfectious sequelae including Guillain-Barré syndrome (GBS) (3). The development of GBS following C. jejuni infection is associated with ganglioside mimicry between the bacterium and host tissues. Thus, strains associated with GBS contain lipooligosaccharide (LOS) structures that are decorated with N-acetyl neuraminic acid (Neu5Ac) such that they mimic human gangliosides (38). Antibodies generated to these LOS cores during gastrointestinal infection can damage peripheral nerves. The mimic most often associated with GBS is GM1, and the mimic most often associated with the Miller Fisher variant of GBS, which affects the ocular nerves, is GQ1b (4, 9, 61). Recently, it was demonstrated that the immunization of rabbits with purified C. jejuni LOS containing a GM1 mimic resulted in high levels of anti-GM1 antibodies and symptoms of GBS in the animals (2, 64).

The development of a C. jejuni vaccine would prevent the risk of diarrheal disease and help reduce the burden of travelers and military troops deployed abroad. Vaccine development would be accelerated by the existence of a human challenge model for safety and efficacy testing. Although human volunteer studies have been performed with two strains of C. jejuni (8, 49), those studies occurred before our current understanding of the association of C. jejuni LOS structure and GBS. Thus, strain 81-176, which has been used in two volunteer studies, has an LOS structure that undergoes phase variation between GM2 and GM3 ganglioside mimics (21), and additional use of this strain in vaccine challenge trials would pose an unacceptable GBS risk to volunteers. There are reports of C. jejuni strains that lack both sialylated LOS cores and genes for Neu5Ac biosynthesis. However, one of these strains, RM1221, expresses an LOS core that mimics human P blood group antigens (32). A single strain of C. jejuni has been shown to contain an LOS core that lacks all glycolipid mimicry. This is strain TGH9011, the type strain of the HS3 serogroup, which contains N-acetyl quinovosamine (QuiNAc) in place of Neu5Ac in its LOS core (5). DNA sequence analysis of the genes encoding LOS biosynthetic enzymes in TGH9011 confirmed the absence of Neu5Ac biosynthetic genes and sialyl transferases (43). TGH9011 was a clinical isolate from Canada, but no information is available on the disease symptoms that it caused, and the strain has been passaged in the laboratory for >25 years. Here, we describe the identification, characterization, and genome sequences of two strains of C. jejuni from well-defined cases of diarrhea in Southeast Asia. We demonstrate that both strains express LOS cores that are similar to that of TGH9011 and that both strains lack the genetic potential to synthesize glycolipid mimics. These studies were conducted with the goal of the development of a human challenge model of C. jejuni diarrheal disease that minimizes the risk of GBS.

MATERIALS AND METHODS

Criteria for strain selection and source of strains.

The selection process sought to identify two C. jejuni isolates from well-characterized clinical cases of enteritis, with C. jejuni as a solitary pathogen, without safety concerns that would prohibit use in volunteer experimental infection studies. The Campylobacter collection at the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, has been archived during previous approved human subject research protocols. These strains included isolates from the Cobra Gold military exercises from 1998 to 2003 (274 isolates) and a study of acute diarrhea in expatriates and Thai adults at Bumrungrad Hospital in Bangkok from 2001 to 2002 (60 isolates). The C. jejuni isolates obtained from the Cobra Gold exercises were cultured by Millipore filtration of stool suspensions on Brucella agar supplemented with sheep blood both before and after enrichment in Doyle's enrichment broth. The stool suspensions from Bumrungrad Hospital were cultured by the same filtration technique but were inoculated both before and after enrichment in Preston broth on Brucella agar supplemented with sheep blood and modified charcoal cefoperazone deoxycholate agar. All isolates were stored at −70°C in glycerol medium. Doyle (Brucella broth-based) and Preston (meat extract/peptone-based) media are commercially available enrichment broths for campylobacters that contain antibiotics to eliminate other bacteria. Search criteria to identify potential challenge strains among this collection were established. The strain should have been isolated from an individual with a clinical illness consistent with Campylobacter enteritis without extraintestinal complications, and C. jejuni must have been the only pathogen identified. The microbiological evaluations to eliminate other pathogens were as follows. Routine bacteriology detection for Salmonella, Shigella, Vibrio spp., Aeromonas, Yersinia, and Plesiomonas was performed, and DNA probing was done for enteropathogenic, enteroaggregative, and enterotoxigenic Escherichia coli. Wet smears of stools were examined microscopically for parasites, and acid-fast staining of stools was performed to detect Cryptosporidium and Cyclospora. Virology testing varied at the two sites of isolation. Enzyme-linked immunosorbent assay methods for stool specimens were used to eliminate rotavirus, adenovirus, and astrovirus infections for isolates from Bumrungrad Hospital and rotavirus infections from Cobra Gold isolates. Reverse transcription-PCR assays eliminated noroviruses in specimens from both sites.

Screening of C. jejuni isolates.

The 334 strains described above had been previously separated into 98 clusters based on pulsed-field gel electrophoresis (O. Serichantalergs, P. Pootong, L. Bodhidatta, A. Dalsgaard, C. Pitarangsi, P. Guerry, D. R. Tribble, S. Anuras, and C. J. Mason, unpublished data). An isolate was selected from each pulsed-field gel electrophoresis cluster to give a total of 98 strains for further study. Seventy-four of these isolates were from U.S. military personnel, and 24 were from the Bumrungrad Hospital study. These 98 C. jejuni isolates were subsequently grown on blood agar (BD Diagnostics, Sparks, MD) at 37°C for 48 h, and genomic DNAs were extracted by use of DNeasy tissue kits (Qiagen, Valencia, CA). PCR was performed for the following LOS genes that encode machinery for the synthesis of ganglioside mimics: cgtA, encoding an N-acetyl-galactosamine (GalNAc) transferase; cgtB, encoding a Gal transferase (39); and cstI and cstII, encoding sialyltransferases from HS2 and HS19 strains, respectively (15). The identities of the amplification products were confirmed by DNA sequencing (Macrogen, Seoul, South Korea). Amplicons were labeled with digoxigenin by random primed labeling (Roche Applied Sciences) and used in colony blot hybridization assays of the 98 isolates on nylon membranes under nonstringent conditions. C. jejuni isolates that did not hybridize to any of these probes were subjected to LOS genotyping using primers and PCR conditions described previously by Parker et al. (43).

Growth conditions.

C. jejuni cultures were routinely grown in Mueller-Hinton broth or on Mueller-Hinton agar at 37°C under microaerobic conditions (5% O2, 10% CO2, and 85% N2). Cells for LOS and capsular polysaccharide (CPS) structural analysis were grown in brain heart infusion broth at 37°C.

Extraction of LOSs, core OSs, and CPSs.

The LOSs of C. jejuni BH-01-0142 and CG8421 were isolated by hot phenol-water extraction (60). In each case, the water layer was dialyzed overnight and submitted to ultracentrifugation at 105,000 × g overnight at 4°C, and the gel-like pellet containing the crude LOS was solubilized in sterile water and lyophilized. The water-soluble CPSs remained in the supernatant after ultracentrifugation. The core oligosaccharides (OSs) were obtained by treatment of the LOSs with 1% acetic acid for 1 h at 100°C to cleave the acid-labile ketosidic linkage that connects the core OS to lipid A. Insoluble lipid A was removed by mild centrifugation (3,000 × g), and the supernatant was applied to a column (1 m by 1 cm) of Bio-Gel P2 for purification. A single carbohydrate fraction containing the core OS was obtained after the void volume as detected by the phenol-sulfuric acid assay (12).

Monosaccharide composition analysis and linkage analysis.

Monosaccharide composition analysis was performed by the alditol acetate method (52). The glycosyl hydrolyzes were carried out with 2 M trifluoroacetic acid at 105°C for 5 h followed by reduction in H2O with NaBD4 overnight at room temperature and subsequent acetylation by acetic anhydride at 100°C for 2 h. The alditol acetate derivatives were analyzed by gas chromatography (GC) using a Varian 3400 gas chromatograph equipped with a 30-m DB-17 capillary column (210°C [30 min]→240°C at 2°C/min) and by GC-mass spectrometry (MS) in the electron impact and chemical ionization modes in a ThermoFinigan PolarisQ instrument. Sugar linkage analysis was achieved by analysis of the permethylated alditol acetates, which were obtained by methylation (10), hydrolysis (52), reduction (52), and acetylation (52). The characterization of the permethylated alditol acetate derivatives was achieved by GC-MS in the electron impact mode (DB-17 column isothermally at 190°C for 100 min).

NMR spectroscopy.

1H, 13C, and 31P nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AMX 400 spectrometer at 293 K. Two-dimensional NMR correlation spectroscopy and heteronuclear spin quantum correlation spectroscopy experiments were performed using the instrument's Bruker software. Prior to performing the NMR experiments, the samples were lyophilized three times with D2O (99.9%). The water peak was used as the internal reference at a δH of 4.821 for 1H NMR spectroscopy, and orthophosphoric acid (δP 0.0) was used as the external reference for 31P NMR experiments. Just before the NMR experiments were carried out, a D2O sample containing trimethylsilyl (δH 0.00) was run to aid in the reference of the water signal.

Motility assays.

Motility was tested by stabbing motility agar plates (Mueller-Hinton broth supplemented with 0.4% agar), followed by incubation of the plates for 48 h at 37°C, as previously described (18).

Invasion assays.

Invasion assays were done using INT407 cells and Caco-2 cells at a multiplicity of infection of approximately 20:1 as previously described (6, 24-26, 42, 63), and the assay mixtures were incubated at 37°C for 2 h. Each monolayer was washed twice in Hanks' balanced salt solution, and fresh minimal essential medium supplemented with 100 μg/ml gentamicin was added to the well for an additional 2 h of incubation at 37°C to kill the extracellular bacteria. The monolayer was washed four times in Hanks' balanced salt solution and lysed with 0.01% Triton X-100 on an orbital shaker. Released intracellular bacteria were enumerated by plate counting. Invasion was expressed as the percentage of the inoculum surviving gentamicin treatment. Assays with INT407 cells were run in duplicate and repeated either six times (for 81-176 and CG8421) or five times (for BH-01-0142). Invasion assays with Caco-2 cells were repeated five times in duplicate for all strains. Statistical analyses were done using the Kruskal-Wallis test with a Bonferroni-adjusted post-hoc Mann-Whitney test.

Genome sequencing, sequence analysis, and annotation.

Genomic DNAs from strains BH-01-0142 and CG8421 were isolated according to methods described previously by Sambrook et al. (51). Sequencing was performed using a Genome Sequencer 20 apparatus (454 Life Sciences, Branford, CT). Two rounds of sequencing were performed for each strain, resulting in a total of 63 Mbp (~39-times coverage) and 83 Mbp (~49-times coverage) for C. jejuni BH-01-0142 and CG8421, respectively. De novo contig assembly was done using Newbler Assembler software, version 1.0.52.06 (454 Life Sciences). The assembly resulted in a total of 476 (1.62 Mbp) and 75 (1.7 Mbp) contigs for BH-01-0142 and CG8421, respectively. Genome annotation was done using Artemis software (http://www.sanger.ac.uk/Software/Artemis) for the detection of open reading frames (ORFs). Overlapping ORFs were manually removed, and the remaining ORFs were attributed an arbitrary locus tag. A total of 1,690 and 1,700 ORFs were found for BH-01-0142 and CG8421, respectively. Functional attribution was made using local BLASTP by comparison to a database containing plasmids pTet and pVir of C. jejuni 81-176 (23) and the NCTC 11168 genome (45). Protein sequences coded by ORFs with no significant similarities (threshold of an E value of 0.0000001) were subsequently compared to the NCBI protein database for the identification of a putative function.

Gap closure.

After alignment on the C. jejuni NCTC 11168 genome, gaps present in the LOS and CPS loci of the two strains were closed by conventional sequencing with an Applied Biosystems model 3100 DNA sequencer. Primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and synthesized using an Applied Biosystems model 392 DNA synthesizer.

Nucleotide sequence accession numbers.

Data for the CG8421 Whole Genome Shotgun project have been deposited in the DDBJ/EMBL/GenBank database under accession number ABGQ00000000. The version described in this paper is the first version, under accession number ABGQ01000000. Data for the unique BH-01-0142 coding sequence under the Whole Genome Shotgun project have been deposited in the DDBJ/EMBL/GenBank database under accession no. ABKD00000000. The version described in this paper is the first version, under accession number ABKD01000000.

RESULTS

Strain identification and clinical history.

Four strains out of the 98 C. jejuni isolates screened appeared to lack genes for ganglioside mimicry. Two of these strains were selected for further evaluation. BH-01-142 was isolated in 2001 from a 27-year-old tourist who had traveled to Myanmar. Diarrhea onset was 3 days prior to presentation at Bumrungrad Hospital. The symptoms at initial presentation at clinic were watery stools (10 per day), abdominal cramps, nausea with vomiting, and no fever. There was no gross blood in the stools, but fecal leukocytes were observed microscopically (≥10 leukocytes/high-power field). The primary assessment was acute watery diarrhea. The strain was susceptible to nalidixic acid, ciprofloxacin, azithromycin, and tetracycline and serotyped as HS3,13,50. Genotyping using methods described previously by Parker et al. (43) indicated that the LOS core was class H (data not shown), consistent with the absence of Neu5Ac biosynthetic genes.

CG8421 was isolated from a 29-year-old U.S. soldier in Nakhon Ratchisima (Khorat), Thailand, in 1999. Diarrhea onset was 4 days prior to presentation at the field medical clinic (6 days after arrival in the country). The patient presented at the clinic with the following symptoms: a total of 24 diarrheal stools, abdominal cramps, fever (documented aural temperature of 38.0°C), nausea without vomiting, myalgias, arthralgias, and headache. Stools contained gross blood and fecal leukocytes (≥10 leukocytes/microscopic field). The primary assessment was acute dysentery. The patient was also taking doxycycline daily for malaria prophylaxis during deployment. The C. jejuni strain was susceptible to nalidixic acid, ciprofloxacin, and azithromycin and resistant to tetracycline. CG8421 was serotyped as HS23,36. Genotyping using methods described previously by Parker et al. (43) indicated that the LOS core was also class H (data not shown).

Invasion of intestinal epithelial cells in vitro.

Strain 81-176, which has been used in human challenge studies, is highly invasive in vitro for INT407 cells (6, 23, 29, 42). Strains CG8421 and BH-01-0142 were compared to reference strain 81-176 for their abilities to invade INT407 and Caco-2 cells. The motility of all strains was confirmed using semisolid motility plates prior to invasion assays. The results, shown in Table Table1,1, indicated that the level of invasion of CG8421 and BH-01-0142 for both INT407 and Caco-2 cells was significantly lower than the level of invasion of 81-176.

TABLE 1.
Invasion of C. jejuni strains into intestinal epithelial cells

LOS structural analysis.

Monosaccharide composition analyses performed on both the LOSs and liberated core OSs of C. jejuni strains BH-01-0142 and CG8421 revealed that they were composed of glucose (Glc), galactose (Gal), GalNAc, N-acetyl-glucosamine (GlcNAc), 3-N-acetyl-3,6-dideoxy-glucosamine (QuiNAc), and l-glycero-d-manno-heptose (LDHep). These sugar composition analyses showed that the BH-01-0142 and CG8421 LOSs contained the same monosaccharides as the LOS of the previously described strain TGH9011 (HS3) (5) (Fig. (Fig.1A1A).

FIG. 1.
LOS structure and genetic loci. (A) LOS structures of CG8421 and BH-01-0142. The asterisks indicate moieties that were present in nonstoichiometric amounts. (B) LOS locus of CG8421. ORFs are labeled by both their gene names and the nomenclature of Parker ...

The 1H NMR spectrum (Fig. (Fig.2)2) of the core OSs of C. jejuni strains BH-01-0142 and CG8421 showed resonances belonging to the 6-deoxy protons (δH 1.28) of the QuiNAc unit and the N-acetyl resonances of GlcNAc, GalNAc, and QuiNAc (δH 2.00 to 2.19). These 1H NMR results confirmed the data obtained by GC-MS, in which the presence of GlcNAc, GalNAc, and QuiNAc components in the LOSs was observed.

FIG. 2.
1H NMR spectrum of C. jejuni BH-01-0142 core OS showing the deoxy resonances of QuiNAc and the characteristic N-acetyl resonances of QuiNAc, GlcNAc, and GalNAc. HOD, water.

Sugar linkage analyses revealed the presence of the following linkage types in both BH-01-0142 and CG8421 core OSs: terminal Glc [Glc-(1→]; 2,3-substituted Gal [→2,3)-Gal-(1→]; 3-substituted Gal [→3)-Gal-(1→] {from unbranched [→2,3)-Gal-(1→]}; 3-substitued LDHep [→3)-LDHep-(1→]; terminal GlcNAc [GlcNAc-(1→]; 4-substituted GalNAc [→4)-GalNAc-(1→]; 3,4-substituted GalNAc [→3,4)-GalNAc-(1→]; and traces of 3,4,7-substituted LDHep [→3,4,7)-LDHep-(1→]. Only small amounts of terminal QuiNAc [QuiNAc-(1→) were detected. MS data, yet to be fully deciphered, point toward the possibility that some of the QuiNAc units may be structurally modified.

Figure Figure33 shows the 31P NMR spectrum of the C. jejuni strain CG8421 core OS, which revealed that there are two monoester phosphate moieties in the core OS region, one for the 7 position of the innermost LDHep (δP 0.10) and the other for the 4 position of the nonreducing end GalNAc (δP 1.62). Similar evidence was obtained for strain BH-01-0142, but in this case, the resonance at 1.62 ppm was weaker, which indicated a lower degree of phosphorylation of GalNAc in BH-01-0142.

FIG. 3.
31P NMR spectrum of C. jejuni CG8421 core OS showing the presence of two monoester phosphate moieties.

The overall LOS structures of both strains were identical to each other and to that of the type strain of HS3 (5) (Fig. (Fig.1A).1A). No data were obtained, including that from two-dimensional 1H-1H correlation spectroscopy and 1H-13C heteronuclear spin quantum correlation NMR experiments carried out on intact LOSs, which suggested the presence of Neu5Ac.

Composition of CPSs.

Monosaccharide composition analyses (Fig. (Fig.4)4) was carried out on the crude supernatants containing the CPS preparations and showed that the CPS of BH-01-0142 (Fig. (Fig.4A)4A) was composed of galactose (Gal), 6-deoxy-ido-heptose (6d-ido-Hep), and, in smaller amounts, l-glycero-d-ido-heptose (LD-ido-Hep) (also detected as the 1,6-anhydro-ld-ido-Hep tetra-acetate), and that of CG8421 (Fig. (Fig.4B)4B) contained Gal, 6-deoxy-altro-heptose (6d-altro-Hep), GlcNAc, and minor amounts of 6-deoxy-3-O-Me-altro-heptose (6d-3-O-Me-altro-Hep). 31P NMR showed that both CPSs carried O-methyl-phosphoramidate moieties, with resonances at δP values of 14.4 ppm (major), 14.7 ppm (minor), and 14.2 ppm (minor) for CG8421 CPS and at δP values of 15.3 ppm (major), 14.5 and 14.2 ppm (minor), 14.3 ppm (minor), and 13.9 ppm (minor) for BH-01-0142.

FIG. 4.
GC-MS profile of the alditol acetate derivatives of BH-01-0142 CPS (A) and CG8421 CPS (B) showing the monosaccharide residues present in their respective CPSs.

Genomic sequencing and annotation.

Two sequencing runs on DNA from CG8421 on a GS20 sequencer resulted in a total of 75 contigs more than 800 bp in length, representing 1,705,154 bp. The overall G+C content was 30.21% G+C. In contrast, two sequencing runs on BH-01-0142 resulted in a total of 457 contigs for a total of 1,622,085 bp and an overall 30.41% G+C content. The striking difference in the numbers of contigs appears to arise from a greater number of “partially assembled” sequenced reads in the BH-01-0142 project. Partially assembled reads typically have a preponderance of low-quality bases at their 3′ termini. These reads comprised 7.8% of the CG8241 assembly but 27.1% of the BH-01-0142 project. The reason for the elevated levels of these reads in the BH-01-0142 project is not clear, since the raw distribution of quality scores in the BH-01-0142 and CG8241 projects were almost identical.

Since the quality of the CG8421 assembly was higher than that of BH-01-0142, the main genetic analyses, as discussed below, focused on strain CG8421.

LOS biosynthetic gene loci.

Since both strains expressed an LOS core similar to that of strain TGH9011 (43), the LOS locus of TGH9011 was used to align BH-01-0142 and CG8421 contigs using BLASTN. A total of six and two LOS contigs were identified for BH-01-0142 and CG8421, respectively. ORFs on these contigs were identified using Artemis software, and primers were designed at the ends of contigs to complete gaps and to correct any potential pyrosequencing artifacts by conventional sequencing. The LOS loci of both strains are related to each other and to TGH9011 (data not shown). The LOS locus of BH-01-0142 is approximately 14 kb in size, and that of CG8421 is about 14.7 kb in size. There are two differences in the LOS loci of BH-01-0142 and CG8421. First, there is an additional gene in CG8421 that is a homolog of Orf28e, a putative glycosyl transferase (Cj8421_1173), from the E class of LOS (43), inserted between the homologs of ORFs 27 h (Cj8421_1172) and 29 h (Cj8421_1174), as shown in Fig. Fig.1B1B and Table Table2.2. Secondly, the homolog of Parker Orf31h has been truncated in CG8421 and appears as two ORFs (Cj8421_1176, encoding a predicted 91-amino-acid protein, and Cj8421_1177, encoding a predicted 276-amino-acid protein). Since Parker Orf31h encodes a putative β-1,4-N-acetylgalactosaminyltransferase and the LOS cores of both strains contain GalNAc in a β-1,4 linkage, it may be that Cj8421_1177 still retains that activity. Importantly, neither CG8421 nor BH-01-0142 contained genes for the synthesis of Neu5Ac (neuB, neuC, and neuA) or sialyl transferase (cst) genes, which is consistent with their LOS core structures.

TABLE 2.
Gene content of the CG8421 LOS locus

Capsule biosynthesis locus.

The capsule locus of CG8421 is very similar to that of 81-176 (Table (Table3),3), which is consistent with both the Penner serotype of both strains (HS23,36) and the sugar composition of the CPS of CG8421 reported above. An alignment of the two regions is shown in Fig. Fig.5.5. The capsule locus of BH-01-0142 was represented by eight contigs during sequencing. The capsule locus of BH-01-0142 (HS3,13,50) appeared to be similar to the partial sequences of the TGH9011 (HS3) CPS locus that are available (47). Both BH-01-0142 and CG8421 capsule loci contained genes that have been shown to be involved in the synthesis of 0-methyl-phosphoramidate (37), consistent with the compositional analyses described above.

FIG. 5.
Comparison of the DNA of the capsule loci of C. jejuni strains 81-176 and CG8421. A comparison was made using the Artemis comparison tool software (http://www.sanger.ac.uk/Software/ACT/). The vertical block between CPS sequences represents conservation. ...
TABLE 3.
Capsule biosynthesis genes of C. jejuni strain CG8421

Flagellin glycosylation locus.

The flagellin glycosylation locus is one of the more complex and hypervariable regions of the C. jejuni chromosome (17). This region encodes enzymes necessary for the addition of novel nonulosonate sugars (pseudaminic acid, or Pse5Ac7Ac, and legionaminic acid, or Leg5Am7Ac) to flagellin via O linkage to serines or threonines (30, 35, 58). Genes in this locus were originally misannotated as Neu5Ac biosynthetic genes (45) because some of these gene products are homologous to NeuB, NeuC, and NeuA, enzymes involved in Neu5Ac biosynthesis. However, it has been established by genetic, metabolomic, and enzymatic studies that these “sialic acid-like genes” encode part of the pathways for the synthesis of the bacterium-specific sugars Pse5Ac7Ac and Leg5Am7Ac (20, 30, 31, 35, 36, 58). The flagellin glycosylation locus of CG8421 proved problematic in terms of the alignment of contigs, likely because of the redundancy found in the region. In addition to the two tandem flagellin structural genes, CG8421 contains numerous related genes of the “maf” family (for motility accessory factor), some of which have been shown to be part of the Pse5Ac7Ac pathway (20). Thus, the region fell into seven contigs. However, despite the large number of contigs, the flagellin glycosylation locus of CG8421 appeared to be very similar to that seen in NCTC 11168, as shown in Table Table4.4. CG8421 contains all genes necessary for the synthesis of Pse5Ac7Ac (pseB, pseC, and pseEFG) and appears to contain both genes to convert Pse5Ac7Ac to an acetamidino form (Pse5Ac7Am) (pseA and pseD) (20, 30, 58). CG8421 also contains all the genes that have been shown to be involved in the synthesis or transfer of Leg5Am7Ac and a methylated form (Leg5AmNMe7Ac) (ptmABCDEFG) (30, 31, 35). Figure Figure66 compares the flagellin glycosylation locus of NCTC 11168 with those of both CG8421 and BH-01-0142.

FIG. 6.
Comparison of the DNA of the flagellin glycosylation loci of C. jejuni strains BH-01-0142, CG8421, and NCTC 11168. A comparison was made using Artemis comparison tool software (http://www.sanger.ac.uk/Software/ACT/). The vertical block between sequences ...
TABLE 4.
Flagellin glycosylation locus of C. jejuni CG8421

Unique regions in the genome of CG8421 compared to that of NCTC 11168.

There are a total of 20 unique/divergent regions on the chromosome of CG8421 compared to NCTC 11168 (Table (Table5),5), which is more than that seen in another Thai isolate, CG8486 (15 regions) (46), or 81-176 (13 regions) (23). Five of the CG8421 unique/divergent regions (zones 1, 2, 16, 18, and 19) are shared with CG8486, and three are shared with 81-176 (zones 1, 6, and 8). Excluding the CPS and LOS biosynthesis loci, a total of 103 ORFs for BH-01-0142 (see Table S1 in the supplemental material) and 158 ORFs for CG8421 (Table (Table55 and see Table S2 in the supplemental material) that were absent from the NCTC 11168 genome were identified. Notable unique regions are described below.

TABLE 5.
C. jejuni CG8421 ORFs absent from the NCTC 11168 genome

R-M systems.

As seen in other C. jejuni genomes, the CG8421 genome contains additional/divergent restriction-modification (R-M) systems compared to NCTC 11168. CG8421 harbors a variable type I R-M locus between its homologs of Cj1546 and Cj1555c (zone 18) (Table (Table5).5). Variation in this locus has been shown in other strains by whole-genome microarray hybridization studies and in the recently sequenced C. jejuni genomes CG8486 (46) and RM1221 (13). However, the R-M system found in CG8421 is different than the one found at the same chromosomal location in RM1221, NCTC 11168, and 81-176. HsdR (Cj8421_1598) and HsdM (Cj8421_1602) from CG8421 are identical to their counterparts in CG8486 (Cj8486_1586 and Cj8486_1590, respectively), but HsdS (Cj8421_1600) shares only 40% identity with the HsdS protein of CG8486 (Cj8486_1589). These results suggest different DNA methylation patterns among CG8421, NCTC 11168, 81-176, and RM1221.

An additional type III R-M system is present in the genome of CG8421 in the region corresponding to Cj0627 and Cj0630c of NCTC 11168 (zone 9) (Table (Table5).5). This inserted type III R-M is located as the same physical location as the one found in RM1221. It appears that both the methylases and endonucleases in CG8421 are identical to those in RM1221.

Na/Pi cotransporter.

Cj8421_0524 (zone 7) (Table (Table5)5) codes for a sodium/inorganic phosphate (Na/Pi) cotransporter. This gene is also found in the genomes of RM1221 (CJE0628) (99% identical) and 81-176 (CJJ81176_0549) (99% identical) but appears to be a pseudogene in NCTC 11168 (Cj0522, Cj0523, and Cj0524). In C. jejuni, phosphate uptake is controlled by the two-component system PhoS/PhoR, which regulates 12 genes (62). Although phosphate does not appear to be limited in vivo (55), the presence of this alternative phosphate uptake mechanism may increase the fitness of CG8421 in specific phosphate-limited environments such as surface water (62).

Tetracycline resistance.

A tetO gene is found on the chromosome of CG8421 between Cj0813 and Cj0814 (zone 11) (Table (Table5).5). Although there have been previous reports of chromosomally encoded tetO genes in C. jejuni as determined by Southern blot hybridizations (14, 48), most often, the gene is found on conjugative plasmids, two of which have been sequenced, pCC31 and pTet (7). However, this is the first demonstration of the chromosomal insertion point of tetO and the first indication that other pTet genes can be integrated into the chromosome. Thus, as shown in Table Table5,5, homologs of other pTet and pCC31 genes (Cj8421_0812, Cj8421_0816, Cj8421_0820, and Cj8421_0821) are also found in zone 11. Additionally, there are two transposase genes (Cj8421_0822 and Cj8421_0823) at this locus that were found on a plasmid from another C. jejuni isolate from Thailand (40). This suggests that a transposition event was responsible for the integration of part of a tetracycline plasmid into the chromosome of CG8421.

Degenerate vacuolating cytotoxin.

CG8421 contains a degenerated gene related to the Helicobacter pylori vacuolating cytotoxin gene between Cj1359 and Cj1361 (zone 16) (Table (Table5).5). This cytotoxic autotransporter is responsible for cell damage to epithelial and lymphatic cells in H. pylori (11). A similar degenerate VacA gene was previously observed in the CG8486 genome (46).

Arsenic resistance.

Similar to another clinical isolate from Thailand, CG8486 (46), and chicken isolate RM1221 (13), CG8421 also contains two highly conserved genes that appear to encode arsenic resistance in zone 19.

Putative phage insertions.

There are two putative phage insertions in the chromosome of CG8421 (Table (Table5).5). The first, which is located in zone 20, adjacent to the CG8421 homolog of Cj1669c and near a tRNA, contains eight potential ORFs. This insertion has a lower G+C content (24.98%), and one of the ORFs encodes a predicted protein with some similarities to bacteriophage integrase proteins. This CG8421 element appears unrelated to any of the previously described phage elements in RM1221. Most of the putative ORFs (seven of eight) show significant similarities with sequences previously identified in TGH9011 (47) and BH-01-0142, suggesting that this insertion is shared by these three strains. However, these ORFs, which were identified by microarray methods in TGH9011, have not been mapped in that strain (47).

The CJIE3 element was first characterized as plasmid integration in the RM1221 genome between Cj1012c and Cj1013c (CJE1092 to CJE1155). This element in RM1221 encodes a total of 62 putative proteins, and 70% of these ORFs show similarities with megaplasmid pCC178 of Campylobacter coli, suggesting a possible insertion of a related plasmid into the genome of RM1221. A similar element in a genomic island (HHGI1) in Helicobacter hepaticus ATCC 51449 was described previously (56). The large majority of the genes found in this element encode hypothetical proteins. CG8421 and BH-01-0142 both contain CJIE3-like insertions in their chromosomes. Parker et al. (44) previously demonstrated by whole-genome microarray that 65.7% (23/35) strains contained more than 75% of the genes found in the RM1221 CJIE3 element. Moreover, approximately 70% (16/23) of the strains examined contained the element in the same physical location as RM1221, and this element is found at the same position in CG8421 (zone 14) (Table (Table55 and see Table S2 in the supplemental material). This region was represented by a high number of contigs during analysis of the CG8421 raw sequencing data, suggesting the presence of repeated sequences that reduced the assembly capability of the assembler software. Nevertheless, based on the similarity of the proteins coded by the CJIE3 element in RM1221, pCC178 of C. coli, and HHGI1 of H. hepaticus (56), the content of this element in CG8421 and BH-01-0142 was drafted (see Table S2 in the supplemental material).

Compared to RM1221, the CG8421 CJIE3-like element lacks the regions from CJE1093 to CJE1104 and CJE1118 to CJE1132. These regions encode small ORFs and ORFs encoding hypothetical proteins. Other contigs potentially included in this element are similar to genes in the CJIE3 elements of pCC178, HHGI1, and C. jejuni TGH9011. These results are consistent with a mosaic structure of CJIE3-like elements, as suggested by comparative genomics studies (44).

Regions of NCTC 11168 missing from CG8421.

A total of 61 ORFs (3.3% of the genome) from NCTC 11168 are missing from CG8421 (see Table S3 in the supplemental material), excluding the LOS (Table (Table2),2), CPS (Table (Table3),3), and flagellin (Table (Table4)4) glycosylation loci. This number is similar to previous findings for other sequenced C. jejuni genomes (66 ORFs were missing in CG8486, and 51 ORFs were missing in 81-176) (23, 46). Sixty-nine percent of these missing ORFs (42/61) are located in regions of the NCTC 11168 genome that were previously characterized as being hypervariable by Taboada et al. (57). The 19 remaining ORFs are, for the most part, scattered on the genome, with two exceptions. Cj0416 and Cj0417 are replaced by one ORF coding for a hypothetical protein in CG8421 (Cj8421_410), and Cj0522 to Cj0524 are replaced by a putative Na/Pi cotransporter, as discussed above. This region might represent a pseudogene in the NCTC 11168 genome, as noted in a recent reannotation of that genome (22).

CG8421 and BH-01-0142 genome comparisons.

Most of the unique ORFs in CG8421 and BH-01-0142 are common to both strains. Thus, 65 of 103 unique ORFs in BH-01-0142 (see Table S1 in the supplemental material) are also found in the CG8421 genome. Seventy-nine of these 103 genes could not be mapped on the BH-01-0142 chromosome, but 54 appear to be similar to ORFs of pCC178 and the CJIE3 element, suggesting a conservation of this element between CG8421 and BH-01-0142, as mentioned above. It appears that BH-01-0142 contains a divergent zone 20 (encoding a putative phage insertion) at the same location as CG8421. In addition, BH-01-0142 contains zones 1, 5, and 16 (Table (Table55).

DISCUSSION

Whole-genome sequencing facilitated the examination of two C. jejuni genomes for genes involved in molecular mimicry and, together with structural analyses of LOS cores and sugar composition analyses of the capsular polysaccharides, provided strong evidence that these strains lack glycolipid mimics. Fast, accurate, and relatively cheap “next-generation” sequencing technologies have made a spectacular impact on biology since their introduction at the end of 2004 (33, 34, 54). This study illustrates yet another new application: sequencing of the genomes of vaccine challenge strains to confirm that the strains lack molecular mimicry, a method that is more accurate and definitive than either microarray analyses or phenotypic testing. Similar sequence analyses of challenge strains or live attenuated vaccine strains could also advance vaccine development against other pathogens. Alignment of most regions of the genome to a scaffold of the known C. jejuni genome was readily accomplished, although the highly repetitive flagellin glycosylation locus proved problematic. We also cannot point to a specific reason for the elevated number of partially assembled sequence reads in the BH-01-0142 project compared to CG8421 or the previously sequenced strain CG8486 (46), but procedures for the extraction of genomic DNA or the construction of the nebulized sequencing libraries are likely candidates. Using a more recent release of Newbler software, version 1.1.03.24, de novo BH-01-0142 assembly produced 200 large contigs (1.66 Mb), suggesting that this data problem might be resolved with improved bioinformatics tools. With the addition of the sequence information reported here for CG8421 and BH-01-0142, the number of sequenced Campylobacter genomes totals 17 (with 10 being C. jejuni). One striking feature of the sequences of CG8421 and BH-01-0142 is that the numbers of unique C. jejuni genes are low. Thus, there were only 13 genes identified in CG8421, less than 1% of the genome, that were not previously reported for other C. jejuni strains. The availability of a complete C. jejuni gene repertoire will allow the development of more complete composite species-specific microarrays that could be used to quickly survey the genome content and pedigree of other strains.

The primary feature required for a safe human challenge is the absence of ganglioside or other glycolipid mimicry, and genomic and structural analyses unequivocally revealed that strains CG8421 and BH-01-0142 expressed LOSs that lack all such mimics (Fig. (Fig.1A).1A). Moreover, structural analyses also confirmed the absence of Neu5Ac in the CPSs of both strains. Although both strains contain homologs of the genes encoding enzymes responsible for the synthesis of the bacterium-specific nonulosonate sugars Pse5Ac7Ac and Leg5Am7Ac, there were no genes for Neu5Ac synthesis identified in the genomes. Sialylation of the bacterial surface affects the virulence of numerous pathogens (59). The role of Neu5Ac-containing LOS cores in the pathogenesis of GBS has been extensively studied, but the role of such cores in the pathogenesis of diarrheal disease is less well understood. The effect of a truncation of the LOS core on invasion in vitro has been studied using strain 81-176. There was no effect on invasive ability until the core was truncated to only Kdo-lipid A (21, 24-26). However, a loss of Neu5Ac from the core of 81-176 and strain MSC57360 (19, 21) increased the sensitivity of the strains to complement-mediated killing. Thus, the use of any strain in which the LOS core had been truncated could potentially result in attenuation in a human model.

There have been limited reports of C. jejuni strains that lack sialylated LOS cores (5, 16, 32). However, strains that were probe negative for Neu5Ac were found at a surprisingly high frequency in this study (4/98 isolates), suggesting that nonsialylated LOS cores may be more common among C. jejuni strains than previously recognized. Although the complete genome sequence of TGH9011 is not available, numerous novel genes have been identified in this strain by a microarray study (47). Given the general heterogeneity among C. jejuni strains, it is striking that these two Asian isolates, CG8421 and BH-01-0142, and Canadian isolate TGH9011, which were isolated decades apart, appear to be highly related throughout most of their genomes.

The fully defined chemical structures of the CPSs will be reported in the future, but preliminary sugar composition analysis performed here showed that BH-01-0142 contained a CPS composed of Gal, 6d-ido-Hep, and small amounts of ld-ido-Hep (Fig. (Fig.4A).4A). The previously reported structure (5) of the TGH9011 CPS (HS3 serotype) was shown to be composed of a disaccharide repeating unit of Gal and ld-ido-Hep, [ld-ido-Hep-(1→4)-Gal-(1→3)]n. Thus, the CPS of BH-01-0142 appears to be closely related to the TGH9011 CPS, with the main difference being the expression of 6d-ido-Hep (instead of ld-ido-Hep) in the CPS of BH-01-0142. Moreover, the genes in the capsule loci of BH-01-0142 and TGH9011 appear to be very similar (Fig. (Fig.55 and Table Table3).3). The capsule has been shown to be the serodeterminant capsule of the HS3 serogroup (28), but the relationship of the complex serogroup seen for BH-01-0142 (i.e., HS3,13,50) to capsule structure remains to be determined. In the case of CG8421 (serotyped as HS23,36), the CPS was observed to be composed of Gal, 6d-altro-Hep, GlcNAc, and trace amounts of 6d-3-O-Me-altro-Hep (Fig. (Fig.4B).4B). This CPS composition is similar to that of strain 81-176 (27) (HS23,36 serotype) that is composed of a trisaccharide repeating unit with Gal, 6d-3-O-Me-Hep, and GlcNAc [Gal-(1→2)-6d-3-O-Me-altro-Hep-(1→3)-GlcNAc-(1→3)]n, with the sole difference being the replacement the 6d-3-O-Me-altro-Hep by its nonmethylated derivative, 6d-altro-Hep, in the CG8421 CPS. Similarly, the capsule loci of both 81-176 and CG8421 are very similar to each other. Preliminary 31P NMR studies revealed the presence of O-methyl-phosphoramidate residues in the CPSs of both BH-01-0142 and CG8421, consistent with the presence of genes for O-methyl-phosphoramidate synthesis (37) in both capsule loci.

To our knowledge, CG8421 is the first reported HS23,36 strain that lacks a sialylated LOS core. Thus, other than the CPSs, CG8421 and BH-01-0142 appear to be very similar to each other despite the fact that the original clinical presentations were quite distinct. Both strains lack numerous ORFs found in the reference NCTC 11168 genome and share ORFs not found in NCTC 11168 with other sequenced C. jejuni strains (81-176, RM1221, and CG8486).

Strain 81-176 has been studied extensively because it has been confirmed to cause diarrheal disease in human volunteers (8, 49) and because it is more invasive in vitro than other strains (6, 23, 29, 42). Since invasion is thought to correlate with the in vivo virulence of C. jejuni, we sought strains that were invasive in vitro with the expectation that such strains would cause disease in volunteers. Compared to strain 81-176, both CG8421 and BH-01-0142 were noninvasive for intestinal epithelial cells in culture, as was another clinical isolate from Thailand, CG8486 (46). All three of these strains were obtained from patients with defined clinical syndromes and were subjected to minimal in vitro passage prior to DNA sequence analyses. The significance of this lack of invasiveness in vitro remains to be determined, but it should be mentioned that at the time of this writing, the virulence of CG8421 has been confirmed by a human volunteer challenge study, which will be reported separately (B. Kirkpatrick, personal communication).

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Nicci Nolan, Shannon Lentz, Cheryl Ewing, Gary Majam, Julie Phan, and Lisa Applebee at the NMRC for their contributions to this study; Helen Tabor of the Canadian Microbiology Reference Laboratory in Winnipeg for Penner serotyping; and Michael Prouty for his comments on the manuscript.

Work at the NMRC was funded by the Military Infectious Diseases Research Program Work Unit 6000.RAD1.DA3.A308. M.A.M. was funded by the NSERC and ACE Biosciences.

The views expressed in this work are those of the authors and do not necessarily reflect the official policy or position of the Navy, the Department of Defense, or the U.S. government.

Notes

Editor: S. R. Blanke

Footnotes

[down-pointing small open triangle]Published ahead of print on 22 September 2008.

Supplemental material for this article may be found at http://iai.asm.org/.

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