Characterization of Two Campylobacter jejuni Strains for Use inVolunteer Experimental-Infection Studies�†
Frederic Poly,1 Timothy D. Read,1 Yu-Han Chen,2 Mario A. Monteiro,2 Oralak Serichantalergs,3Piyarat Pootong,3 Ladaporn Bodhidatta,3 Carl J. Mason,3 David Rockabrand,1 Shahida Baqar,1
Chad K. Porter,1 David Tribble,4 Michael Darsley,5 and Patricia Guerry1*Naval Medical Research Center, Silver Spring, Maryland1; University of Guelph, Guelph, Ontario, Canada2;
Armed Forces Research Institute for Medical Sciences, Bangkok, Thailand3; Uniformed Services University forthe Health Sciences, Bethesda, Maryland4; and ACE Biosciences, Odense, Denmark5
Received 20 June 2008/Returned for modification 23 June 2008/Accepted 12 September 2008
The development of vaccines against Campylobacter jejuni would be facilitated by the ability to perform phase IIchallenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most C. jejuni strains withhuman gangliosides presents safety concerns about the development of Guillain-Barre syndrome. Clinical isolatesof C. jejuni that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probeanalyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to expressthe LOS type containing N-acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSsof these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involvedin ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed ofgalactose (Gal), 6-deoxy-ido-heptose, and, in smaller amounts, D-glycero-D-ido-heptose, and the CPS of CG8421 wasobserved 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-specificzones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion relatedto the CJEI3 element of C. jejuni RM1221. The genomes of both strains shared a high degree of similarity to eachother 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 ofbacterial gastroenteritis worldwide and a major cause of traveler’sdiarrhea (1, 41). C. jejuni colonization of the human digestivetract induces a wide range of symptoms, from mild, watery diar-rhea to dysentery that can be accompanied by fever and abdom-inal cramps. The preferred antibiotic treatment for campylobac-teriosis is either erythromycin-azithromycin or ciprofloxacin,although resistance to both drugs is increasing (50, 53). C. jejuniinfection has been linked to the development of postinfectioussequelae including Guillain-Barre syndrome (GBS) (3). The de-velopment of GBS following C. jejuni infection is associated withganglioside mimicry between the bacterium and host tissues.Thus, strains associated with GBS contain lipooligosaccharide(LOS) structures that are decorated with N-acetyl neuraminicacid (Neu5Ac) such that they mimic human gangliosides (38).Antibodies generated to these LOS cores during gastrointestinalinfection can damage peripheral nerves. The mimic most oftenassociated with GBS is GM1, and the mimic most often associatedwith the Miller Fisher variant of GBS, which affects the ocularnerves, is GQ1b (4, 9, 61). Recently, it was demonstrated that theimmunization of rabbits with purified C. jejuni LOS containing aGM1 mimic resulted in high levels of anti-GM1 antibodies andsymptoms of GBS in the animals (2, 64).
The development of a C. jejuni vaccine would prevent the riskof diarrheal disease and help reduce the burden of travelers andmilitary troops deployed abroad. Vaccine development would beaccelerated by the existence of a human challenge model forsafety and efficacy testing. Although human volunteer studieshave been performed with two strains of C. jejuni (8, 49), thosestudies occurred before our current understanding of the associ-ation of C. jejuni LOS structure and GBS. Thus, strain 81-176,which has been used in two volunteer studies, has an LOS struc-ture that undergoes phase variation between GM2 and GM3
ganglioside mimics (21), and additional use of this strain in vac-cine challenge trials would pose an unacceptable GBS risk tovolunteers. There are reports of C. jejuni strains that lack bothsialylated LOS cores and genes for Neu5Ac biosynthesis. How-ever, one of these strains, RM1221, expresses an LOS core thatmimics human P blood group antigens (32). A single strain of C.jejuni has been shown to contain an LOS core that lacks allglycolipid mimicry. This is strain TGH9011, the type strain of theHS3 serogroup, which contains N-acetyl quinovosamine(QuiNAc) in place of Neu5Ac in its LOS core (5). DNA sequenceanalysis of the genes encoding LOS biosynthetic enzymes inTGH9011 confirmed the absence of Neu5Ac biosynthetic genesand sialyl transferases (43). TGH9011 was a clinical isolate fromCanada, but no information is available on the disease symptomsthat it caused, and the strain has been passaged in the laboratoryfor �25 years. Here, we describe the identification, characteriza-tion, and genome sequences of two strains of C. jejuni fromwell-defined cases of diarrhea in Southeast Asia. We demonstratethat both strains express LOS cores that are similar to that ofTGH9011 and that both strains lack the genetic potential to
* Corresponding author. Mailing address: Enteric Diseases Depart-ment, Naval Medical Research Center, 503 Robert Grant Avenue,Silver Spring, MD 20910. Phone: (301) 319-7662. Fax: (301) 319-7679.E-mail: [email protected].
† Supplemental material for this article may be found at http://iai.asm.org/.
synthesize glycolipid mimics. These studies were conducted withthe 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 processsought to identify two C. jejuni isolates from well-characterized clinical cases ofenteritis, with C. jejuni as a solitary pathogen, without safety concerns that wouldprohibit use in volunteer experimental infection studies. The Campylobactercollection at the Armed Forces Research Institute of Medical Sciences inBangkok, Thailand, has been archived during previous approved human subjectresearch protocols. These strains included isolates from the Cobra Gold militaryexercises from 1998 to 2003 (274 isolates) and a study of acute diarrhea inexpatriates and Thai adults at Bumrungrad Hospital in Bangkok from 2001 to2002 (60 isolates). The C. jejuni isolates obtained from the Cobra Gold exerciseswere cultured by Millipore filtration of stool suspensions on Brucella agar sup-plemented with sheep blood both before and after enrichment in Doyle’s en-richment broth. The stool suspensions from Bumrungrad Hospital were culturedby the same filtration technique but were inoculated both before and afterenrichment in Preston broth on Brucella agar supplemented with sheep bloodand modified charcoal cefoperazone deoxycholate agar. All isolates were storedat �70°C in glycerol medium. Doyle (Brucella broth-based) and Preston (meatextract/peptone-based) media are commercially available enrichment broths forcampylobacters that contain antibiotics to eliminate other bacteria. Search cri-teria to identify potential challenge strains among this collection were estab-lished. The strain should have been isolated from an individual with a clinicalillness consistent with Campylobacter enteritis without extraintestinal complica-tions, and C. jejuni must have been the only pathogen identified. The microbi-ological evaluations to eliminate other pathogens were as follows. Routine bac-teriology detection for Salmonella, Shigella, Vibrio spp., Aeromonas, Yersinia, andPlesiomonas was performed, and DNA probing was done for enteropathogenic,enteroaggregative, and enterotoxigenic Escherichia coli. Wet smears of stoolswere examined microscopically for parasites, and acid-fast staining of stools wasperformed to detect Cryptosporidium and Cyclospora. Virology testing varied atthe two sites of isolation. Enzyme-linked immunosorbent assay methods for stoolspecimens were used to eliminate rotavirus, adenovirus, and astrovirus infectionsfor isolates from Bumrungrad Hospital and rotavirus infections from Cobra Goldisolates. Reverse transcription-PCR assays eliminated noroviruses in specimensfrom both sites.
Screening of C. jejuni isolates. The 334 strains described above had beenpreviously 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 isolatewas selected from each pulsed-field gel electrophoresis cluster to give a total of98 strains for further study. Seventy-four of these isolates were from U.S. militarypersonnel, and 24 were from the Bumrungrad Hospital study. These 98 C. jejuniisolates 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 thatencode machinery for the synthesis of ganglioside mimics: cgtA, encoding anN-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 byDNA sequencing (Macrogen, Seoul, South Korea). Amplicons were labeled withdigoxigenin by random primed labeling (Roche Applied Sciences) and used incolony blot hybridization assays of the 98 isolates on nylon membranes undernonstringent conditions. C. jejuni isolates that did not hybridize to any of theseprobes were subjected to LOS genotyping using primers and PCR conditionsdescribed previously by Parker et al. (43).
Growth conditions. C. jejuni cultures were routinely grown in Mueller-Hintonbroth 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-01-0142and CG8421 were isolated by hot phenol-water extraction (60). In each case, thewater layer was dialyzed overnight and submitted to ultracentrifugation at105,000 � g overnight at 4°C, and the gel-like pellet containing the crude LOSwas solubilized in sterile water and lyophilized. The water-soluble CPSs re-mained in the supernatant after ultracentrifugation. The core oligosaccharides(OSs) were obtained by treatment of the LOSs with 1% acetic acid for 1 h at100°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), andthe supernatant was applied to a column (1 m by 1 cm) of Bio-Gel P2 forpurification. A single carbohydrate fraction containing the core OS was obtainedafter the void volume as detected by the phenol-sulfuric acid assay (12).
Monosaccharide composition analysis and linkage analysis. Monosaccharidecomposition analysis was performed by the alditol acetate method (52). Theglycosyl hydrolyzes were carried out with 2 M trifluoroacetic acid at 105°C for 5 hfollowed by reduction in H2O with NaBD4 overnight at room temperature andsubsequent acetylation by acetic anhydride at 100°C for 2 h. The alditol acetatederivatives were analyzed by gas chromatography (GC) using a Varian 3400 gaschromatograph equipped with a 30-m DB-17 capillary column (210°C [30min]3240°C at 2°C/min) and by GC-mass spectrometry (MS) in the electronimpact and chemical ionization modes in a ThermoFinigan PolarisQ instrument.Sugar linkage analysis was achieved by analysis of the permethylated alditolacetates, which were obtained by methylation (10), hydrolysis (52), reduction(52), and acetylation (52). The characterization of the permethylated alditolacetate derivatives was achieved by GC-MS in the electron impact mode (DB-17column 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 quantumcorrelation spectroscopy experiments were performed using the instrument’sBruker software. Prior to performing the NMR experiments, the samples werelyophilized three times with D2O (99.9%). The water peak was used as theinternal reference at a �H of 4.821 for 1H NMR spectroscopy, and orthophos-phoric acid (�P 0.0) was used as the external reference for 31P NMR experiments.Just before the NMR experiments were carried out, a D2O sample containingtrimethylsilyl (�H 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 platesfor 48 h at 37°C, as previously described (18).
Invasion assays. Invasion assays were done using INT407 cells and Caco-2cells 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. Eachmonolayer was washed twice in Hanks’ balanced salt solution, and fresh minimalessential medium supplemented with 100 �g/ml gentamicin was added to the wellfor an additional 2 h of incubation at 37°C to kill the extracellular bacteria. Themonolayer was washed four times in Hanks’ balanced salt solution and lysed with0.01% Triton X-100 on an orbital shaker. Released intracellular bacteria wereenumerated by plate counting. Invasion was expressed as the percentage of theinoculum surviving gentamicin treatment. Assays with INT407 cells were run induplicate 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 induplicate for all strains. Statistical analyses were done using the Kruskal-Wallistest with a Bonferroni-adjusted post-hoc Mann-Whitney test.
Genome sequencing, sequence analysis, and annotation. Genomic DNAs fromstrains BH-01-0142 and CG8421 were isolated according to methods describedpreviously by Sambrook et al. (51). Sequencing was performed using a GenomeSequencer 20 apparatus (454 Life Sciences, Branford, CT). Two rounds ofsequencing 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 New-bler Assembler software, version 1.0.52.06 (454 Life Sciences). The assemblyresulted in a total of 476 (1.62 Mbp) and 75 (1.7 Mbp) contigs for BH-01-0142and CG8421, respectively. Genome annotation was done using Artemis software(http://www.sanger.ac.uk/Software/Artemis) for the detection of open readingframes (ORFs). Overlapping ORFs were manually removed, and the remainingORFs were attributed an arbitrary locus tag. A total of 1,690 and 1,700 ORFswere found for BH-01-0142 and CG8421, respectively. Functional attributionwas made using local BLASTP by comparison to a database containing plasmidspTet and pVir of C. jejuni 81-176 (23) and the NCTC 11168 genome (45). Proteinsequences coded by ORFs with no significant similarities (threshold of an E valueof 0.0000001) were subsequently compared to the NCBI protein database for theidentification of a putative function.
Gap closure. After alignment on the C. jejuni NCTC 11168 genome, gapspresent in the LOS and CPS loci of the two strains were closed by conventionalsequencing with an Applied Biosystems model 3100 DNA sequencer. Primerswere designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and synthesized using an Applied Biosystems model 392DNA synthesizer.
Nucleotide sequence accession numbers. Data for the CG8421 Whole GenomeShotgun project have been deposited in the DDBJ/EMBL/GenBank databaseunder accession number ABGQ00000000. The version described in this paper is
the first version, under accession number ABGQ01000000. Data for theunique BH-01-0142 coding sequence under the Whole Genome Shotgunproject have been deposited in the DDBJ/EMBL/GenBank database underaccession no. ABKD00000000. The version described in this paper is the firstversion, under accession number ABKD01000000.
RESULTS
Strain identification and clinical history. Four strains out ofthe 98 C. jejuni isolates screened appeared to lack genes forganglioside mimicry. Two of these strains were selected forfurther evaluation. BH-01-142 was isolated in 2001 from a27-year-old tourist who had traveled to Myanmar. Diarrheaonset was 3 days prior to presentation at Bumrungrad Hospi-tal. The symptoms at initial presentation at clinic were waterystools (10 per day), abdominal cramps, nausea with vomiting,and no fever. There was no gross blood in the stools, but fecalleukocytes were observed microscopically (�10 leukocytes/high-power field). The primary assessment was acute waterydiarrhea. The strain was susceptible to nalidixic acid, cipro-floxacin, azithromycin, and tetracycline and serotyped asHS3,13,50. Genotyping using methods described previously byParker et al. (43) indicated that the LOS core was class H (datanot shown), consistent with the absence of Neu5Ac biosyn-thetic genes.
CG8421 was isolated from a 29-year-old U.S. soldier inNakhon Ratchisima (Khorat), Thailand, in 1999. Diarrhea on-set was 4 days prior to presentation at the field medical clinic(6 days after arrival in the country). The patient presented atthe clinic with the following symptoms: a total of 24 diarrhealstools, abdominal cramps, fever (documented aural tempera-ture of 38.0°C), nausea without vomiting, myalgias, arthralgias,and headache. Stools contained gross blood and fecal leuko-cytes (�10 leukocytes/microscopic field). The primary assess-ment was acute dysentery. The patient was also taking doxy-cycline daily for malaria prophylaxis during deployment. TheC. jejuni strain was susceptible to nalidixic acid, ciprofloxacin,and azithromycin and resistant to tetracycline. CG8421 wasserotyped as HS23,36. Genotyping using methods describedpreviously by Parker et al. (43) indicated that the LOS core wasalso class H (data not shown).
Invasion of intestinal epithelial cells in vitro. Strain 81-176,which has been used in human challenge studies, is highlyinvasive in vitro for INT407 cells (6, 23, 29, 42). StrainsCG8421 and BH-01-0142 were compared to reference strain81-176 for their abilities to invade INT407 and Caco-2 cells.The motility of all strains was confirmed using semisolid mo-tility plates prior to invasion assays. The results, shown inTable 1, indicated that the level of invasion of CG8421 and
BH-01-0142 for both INT407 and Caco-2 cells was significantlylower than the level of invasion of 81-176.
LOS structural analysis. Monosaccharide composition anal-yses performed on both the LOSs and liberated core OSs of C.jejuni strains BH-01-0142 and CG8421 revealed that they werecomposed 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). Thesesugar composition analyses showed that the BH-01-0142 andCG8421 LOSs contained the same monosaccharides as theLOS of the previously described strain TGH9011 (HS3) (5)(Fig. 1A).
The 1H NMR spectrum (Fig. 2) of the core OSs of C. jejunistrains BH-01-0142 and CG8421 showed resonances belongingto the 6-deoxy protons (�H 1.28) of the QuiNAc unit and theN-acetyl resonances of GlcNAc, GalNAc, and QuiNAc (�H
2.00 to 2.19). These 1H NMR results confirmed the data ob-tained by GC-MS, in which the presence of GlcNAc, GalNAc,and QuiNAc components in the LOSs was observed.
Sugar linkage analyses revealed the presence of the fol-lowing linkage types in both BH-01-0142 and CG8421 coreOSs: terminal Glc [Glc-(13]; 2,3-substituted Gal [32,3)-Gal-(13]; 3-substituted Gal [33)-Gal-(13] {from un-branched [32,3)-Gal-(13]}; 3-substitued LDHep [33)-LDHep-(13]; terminal GlcNAc [GlcNAc-(13]; 4-substitutedGalNAc [34)-GalNAc-(13]; 3,4-substituted GalNAc [33,4)-GalNAc-(13]; and traces of 3,4,7-substituted LDHep [33,4,7)-LDHep-(13]. Only small amounts of terminal QuiNAc[QuiNAc-(13) were detected. MS data, yet to be fully deci-phered, point toward the possibility that some of the QuiNAcunits may be structurally modified.
Figure 3 shows the 31P NMR spectrum of the C. jejuni strainCG8421 core OS, which revealed that there are two monoesterphosphate moieties in the core OS region, one for the 7 posi-tion of the innermost LDHep (�P 0.10) and the other for the 4position of the nonreducing end GalNAc (�P 1.62). Similarevidence was obtained for strain BH-01-0142, but in this case,
FIG. 1. LOS structure and genetic loci. (A) LOS structures ofCG8421 and BH-01-0142. The asterisks indicate moieties that werepresent in nonstoichiometric amounts. (B) LOS locus of CG8421.ORFs are labeled by both their gene names and the nomenclature ofParker et al. (43), in which “h” designates the H LOS class, exemplifiedby TGH9011, and “e” designates the E LOS class, exemplified by81116. Genes marked with an asterisk contain homopolymeric tracts ofG or C. The LOS locus of BH-01-0142 is identical to that of CG8421,with two exceptions. Orf28e (in gray) is missing in BH-01-0142, andOrf31h of CG8421, but not that of BH-01-0142, contains a frameshift,as discussed in the text.
TABLE 1. Invasion of C. jejuni strains into intestinal epithelial cells
the resonance at 1.62 ppm was weaker, which indicated a lowerdegree of phosphorylation of GalNAc in BH-01-0142.
The overall LOS structures of both strains were identical toeach other and to that of the type strain of HS3 (5) (Fig. 1A).No data were obtained, including that from two-dimensional1H-1H correlation spectroscopy and 1H-13C heteronuclear spin
quantum correlation NMR experiments carried out on intactLOSs, which suggested the presence of Neu5Ac.
Composition of CPSs. Monosaccharide composition anal-yses (Fig. 4) was carried out on the crude supernatantscontaining the CPS preparations and showed that the CPSof BH-01-0142 (Fig. 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 the1,6-anhydro-LD-ido-Hep tetra-acetate), and that of CG8421(Fig. 4B) contained Gal, 6-deoxy-altro-heptose (6d-altro-Hep), GlcNAc, and minor amounts of 6-deoxy-3-O-Me-al-tro-heptose (6d-3-O-Me-altro-Hep). 31P NMR showed thatboth CPSs carried O-methyl-phosphoramidate moieties,with resonances at �P values of 14.4 ppm (major), 14.7 ppm(minor), and 14.2 ppm (minor) for CG8421 CPS and at �P
values of 15.3 ppm (major), 14.5 and 14.2 ppm (minor), 14.3ppm (minor), and 13.9 ppm (minor) for BH-01-0142.
Genomic sequencing and annotation. Two sequencing runson DNA from CG8421 on a GS20 sequencer resulted in a totalof 75 contigs more than 800 bp in length, representing1,705,154 bp. The overall G�C content was 30.21% G�C. Incontrast, two sequencing runs on BH-01-0142 resulted in atotal of 457 contigs for a total of 1,622,085 bp and an overall30.41% G�C content. The striking difference in the numbersof contigs appears to arise from a greater number of “partiallyassembled” sequenced reads in the BH-01-0142 project. Par-tially assembled reads typically have a preponderance of low-quality bases at their 3 termini. These reads comprised 7.8%
FIG. 2. 1H NMR spectrum of C. jejuni BH-01-0142 core OS showing the deoxy resonances of QuiNAc and the characteristic N-acetylresonances of QuiNAc, GlcNAc, and GalNAc. HOD, water.
FIG. 3. 31P NMR spectrum of C. jejuni CG8421 core OS showingthe presence of two monoester phosphate moieties.
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 qual-ity scores in the BH-01-0142 and CG8241 projects were almostidentical.
Since the quality of the CG8421 assembly was higher thanthat of BH-01-0142, the main genetic analyses, as discussedbelow, focused on strain CG8421.
LOS biosynthetic gene loci. Since both strains expressed anLOS core similar to that of strain TGH9011 (43), the LOSlocus of TGH9011 was used to align BH-01-0142 and CG8421
contigs using BLASTN. A total of six and two LOS contigswere 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 completegaps and to correct any potential pyrosequencing artifacts byconventional sequencing. The LOS loci of both strains arerelated to each other and to TGH9011 (data not shown). TheLOS locus of BH-01-0142 is approximately 14 kb in size, andthat of CG8421 is about 14.7 kb in size. There are two differ-ences in the LOS loci of BH-01-0142 and CG8421. First, thereis an additional gene in CG8421 that is a homolog of Orf28e,
FIG. 4. GC-MS profile of the alditol acetate derivatives of BH-01-0142 CPS (A) and CG8421 CPS (B) showing the monosaccharide residuespresent in their respective CPSs.
VOL. 76, 2008 C. JEJUNI HUMAN CHALLENGE STRAINS 5659
a putative glycosyl transferase (Cj8421_1173), from the E classof LOS (43), inserted between the homologs of ORFs 27 h(Cj8421_1172) and 29 h (Cj8421_1174), as shown in Fig. 1Band Table 2. Secondly, the homolog of Parker Orf31h has beentruncated 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 ParkerOrf31h encodes a putative �-1,4-N-acetylgalactosaminyltrans-ferase and the LOS cores of both strains contain GalNAc in a�-1,4 linkage, it may be that Cj8421_1177 still retains thatactivity. Importantly, neither CG8421 nor BH-01-0142 con-tained genes for the synthesis of Neu5Ac (neuB, neuC, andneuA) or sialyl transferase (cst) genes, which is consistent withtheir LOS core structures.
Capsule biosynthesis locus. The capsule locus of CG8421 isvery similar to that of 81-176 (Table 3), which is consistent withboth the Penner serotype of both strains (HS23,36) and thesugar composition of the CPS of CG8421 reported above. Analignment of the two regions is shown in Fig. 5. The capsulelocus of BH-01-0142 was represented by eight contigs duringsequencing. The capsule locus of BH-01-0142 (HS3,13,50) ap-peared to be similar to the partial sequences of the TGH9011(HS3) CPS locus that are available (47). Both BH-01-0142 andCG8421 capsule loci contained genes that have been shown tobe involved in the synthesis of 0-methyl-phosphoramidate (37),consistent with the compositional analyses described above.
Flagellin glycosylation locus. The flagellin glycosylation lo-cus is one of the more complex and hypervariable regions ofthe C. jejuni chromosome (17). This region encodes enzymesnecessary for the addition of novel nonulosonate sugars(pseudaminic acid, or Pse5Ac7Ac, and legionaminic acid, orLeg5Am7Ac) to flagellin via O linkage to serines or threonines(30, 35, 58). Genes in this locus were originally misannotatedas Neu5Ac biosynthetic genes (45) because some of these geneproducts are homologous to NeuB, NeuC, and NeuA, enzymesinvolved in Neu5Ac biosynthesis. However, it has been estab-lished by genetic, metabolomic, and enzymatic studies thatthese “sialic acid-like genes” encode part of the pathways for
the synthesis of the bacterium-specific sugars Pse5Ac7Ac andLeg5Am7Ac (20, 30, 31, 35, 36, 58). The flagellin glycosylationlocus of CG8421 proved problematic in terms of the alignmentof contigs, likely because of the redundancy found in the re-gion. 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 shownto be part of the Pse5Ac7Ac pathway (20). Thus, the region fellinto seven contigs. However, despite the large number of con-tigs, the flagellin glycosylation locus of CG8421 appeared to bevery similar to that seen in NCTC 11168, as shown in Table 4.CG8421 contains all genes necessary for the synthesis ofPse5Ac7Ac (pseB, pseC, and pseEFG) and appears to containboth genes to convert Pse5Ac7Ac to an acetamidino form(Pse5Ac7Am) (pseA and pseD) (20, 30, 58). CG8421 also con-tains all the genes that have been shown to be involved in thesynthesis or transfer of Leg5Am7Ac and a methylated form(Leg5AmNMe7Ac) (ptmABCDEFG) (30, 31, 35). Figure 6compares the flagellin glycosylation locus of NCTC 11168 withthose of both CG8421 and BH-01-0142.
Unique regions in the genome of CG8421 compared to thatof NCTC 11168. There are a total of 20 unique/divergent re-gions on the chromosome of CG8421 compared to NCTC11168 (Table 5), which is more than that seen in another Thaiisolate, 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 with81-176 (zones 1, 6, and 8). Excluding the CPS and LOS bio-synthesis loci, a total of 103 ORFs for BH-01-0142 (see TableS1 in the supplemental material) and 158 ORFs for CG8421(Table 5 and see Table S2 in the supplemental material) thatwere absent from the NCTC 11168 genome were identified.Notable unique regions are described below.
R-M systems. As seen in other C. jejuni genomes, the CG8421genome contains additional/divergent restriction-modification(R-M) systems compared to NCTC 11168. CG8421 harbors avariable type I R-M locus between its homologs of Cj1546 andCj1555c (zone 18) (Table 5). Variation in this locus has been
TABLE 2. Gene content of the CG8421 LOS locus
Locus tag Protein size(residues) Putative function Closest gene Closest strain
relationship
No. of shared genes/total no. of genes(%) by BLASTP
Cj8421_1164 298 Lipid A biosynthesis acyltransferase htrB C. jejuni GB24 HS:31 293/297 (98)Cj8421_1165 210 Glycosyltransferase orf3h C. jejuni TGH9011 210/210 (100)Cj8421_1166 307 Glucose-1-phosphate thymidyltransferase rmlA C. jejuni TGH9011 305/307 (99)Cj8421_1167 343 dTDP-glucose-4,6-dehydratase rmlB C. jejuni TGH9011 343/343 (100)Cj8421_1168 141 Hypothetical protein orf23h C. jejuni TGH9011 141/141 (100)Cj8421_1169 134 Hypothetical protein orf24h C. jejuni TGH9011 134/134 (100)Cj8421_1170 147 Putative acetyltransferase orf25h C. jejuni ZX 146/147 (99)Cj8421_1171 177 Hypothetical protein orf39h C. jejuni TGH9011 177/177 (100)Cj8421_1172 336 Hypothetical protein orf27h C. jejuni TGH9011 332/332 (100)Cj8421_1173 350 Putative glycosyltransferase orf28e C. jejuni GB24 HS:31 348/350 (99)Cj8421_1174 360 Putative aminotransferase orf29h C. jejuni TGH9011 360/360 (100)Cj8421_1175 395 Hypothetical protein orf30h C. jejuni GB24 HS:31 385/386 (99)Cj8421_1176 91 �-1,4-N-Acetylgalactosaminyltransferase orf31h C. jejuni TGH9011 87/88 (98)Cj8421_1177 276 �-1,4-N-Acetylgalactosaminyltransferase orf31h C. jejuni TGH9011 253/255 (99)Cj8421_1178 257 Hypothetical protein orf32h C. jejuni TGH9011 255/257 (99)Cj8421_1179 318 Hypothetical protein orf33h C. jejuni TGH9011 318/318 (100)Cj8421_1180 319 Hypothetical protein orf34h C. jejuni TGH9011 318/319 (99)Cj8421_1181 272 Putative glucosyltransferase waaV C. jejuni TGH9011 271/272 (99)
shown in other strains by whole-genome microarray hybridizationstudies and in the recently sequenced C. jejuni genomes CG8486(46) and RM1221 (13). However, the R-M system found inCG8421 is different than the one found at the same chromo-somal location in RM1221, NCTC 11168, and 81-176. HsdR(Cj8421_1598) and HsdM (Cj8421_1602) from CG8421 areidentical to their counterparts in CG8486 (Cj8486_1586 andCj8486_1590, respectively), but HsdS (Cj8421_1600) shares only40% identity with the HsdS protein of CG8486 (Cj8486_1589).These results suggest different DNA methylation patterns amongCG8421, NCTC 11168, 81-176, and RM1221.
An additional type III R-M system is present in the genomeof CG8421 in the region corresponding to Cj0627 and Cj0630cof NCTC 11168 (zone 9) (Table 5). This inserted type III R-Mis located as the same physical location as the one found inRM1221. It appears that both the methylases and endonucle-ases in CG8421 are identical to those in RM1221.
Na/Pi cotransporter. Cj8421_0524 (zone 7) (Table 5) codesfor a sodium/inorganic phosphate (Na/Pi) cotransporter. Thisgene is also found in the genomes of RM1221 (CJE0628) (99%identical) and 81-176 (CJJ81176_0549) (99% identical) butappears to be a pseudogene in NCTC 11168 (Cj0522, Cj0523,and Cj0524). In C. jejuni, phosphate uptake is controlled by thetwo-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 mech-anism may increase the fitness of CG8421 in specific phos-phate-limited environments such as surface water (62).
Tetracycline resistance. A tetO gene is found on the chro-mosome of CG8421 between Cj0813 and Cj0814 (zone 11)(Table 5). Although there have been previous reports of chro-mosomally encoded tetO genes in C. jejuni as determined bySouthern blot hybridizations (14, 48), most often, the gene isfound on conjugative plasmids, two of which have been se-quenced, pCC31 and pTet (7). However, this is the first dem-onstration of the chromosomal insertion point of tetO and thefirst indication that other pTet genes can be integrated intothe chromosome. Thus, as shown in Table 5, homologs ofother pTet and pCC31 genes (Cj8421_0812, Cj8421_0816,Cj8421_0820, and Cj8421_0821) are also found in zone 11.
TABLE 3. Capsule biosynthesis genes of C. jejuni strain CG8421
Locus taga Protein size(residues) Closest relationship
a *, G�C tract present in CG8421 and 81-176; **, G�C tract absent from 81-176; ***, frameshift due to G�C tract in 81-176 and CG8421.
FIG. 5. Comparison of the DNA of the capsule loci of C. jejunistrains 81-176 and CG8421. A comparison was made using the Artemiscomparison tool software (http://www.sanger.ac.uk/Software/ACT/).The vertical block between CPS sequences represents conservation.MeOPN, methyl phosphoramidate.
VOL. 76, 2008 C. JEJUNI HUMAN CHALLENGE STRAINS 5661
Additionally, there are two transposase genes (Cj8421_0822and Cj8421_0823) at this locus that were found on a plasmidfrom another C. jejuni isolate from Thailand (40). This sug-gests that a transposition event was responsible for the inte-gration of part of a tetracycline plasmid into the chromosomeof CG8421.
Degenerate vacuolating cytotoxin. CG8421 contains a de-generated gene related to the Helicobacter pylori vacuolatingcytotoxin gene between Cj1359 and Cj1361 (zone 16) (Table5). This cytotoxic autotransporter is responsible for cell dam-age to epithelial and lymphatic cells in H. pylori (11). A similardegenerate VacA gene was previously observed in the CG8486genome (46).
Arsenic resistance. Similar to another clinical isolate fromThailand, CG8486 (46), and chicken isolate RM1221 (13),CG8421 also contains two highly conserved genes that appearto encode arsenic resistance in zone 19.
Putative phage insertions. There are two putative phageinsertions in the chromosome of CG8421 (Table 5). The first,which is located in zone 20, adjacent to the CG8421 homologof Cj1669c and near a tRNA, contains eight potential ORFs.This insertion has a lower G�C content (24.98%), and one ofthe ORFs encodes a predicted protein with some similarities tobacteriophage integrase proteins. This CG8421 element ap-pears unrelated to any of the previously described phage ele-ments in RM1221. Most of the putative ORFs (seven of eight)
TABLE 4. Flagellin glycosylation locus of C. jejuni CG8421
Locus tag NCTC 11168 gene % Identity/% similaritya Putative function
a Percent identity/percent similarity of the CG8421 protein with its homolog in NCTC 11168.b Similarity was not determined either due to a frameshift or because the sequence could not be determined due to the repetitive nature of the gene.c Cj1306c and Cj1333 homologs in the CG8421 genome are represented by incomplete ORFs (Cj8421_1347, Cj8421_1348, and Cj8421_1375/Cj8421_1376, respec-
tively) due to their presence on different contigs.
show significant similarities with sequences previously identi-fied in TGH9011 (47) and BH-01-0142, suggesting that thisinsertion is shared by these three strains. However, theseORFs, which were identified by microarray methods inTGH9011, have not been mapped in that strain (47).
The CJIE3 element was first characterized as plasmid inte-gration in the RM1221 genome between Cj1012c and Cj1013c(CJE1092 to CJE1155). This element in RM1221 encodes atotal of 62 putative proteins, and 70% of these ORFs showsimilarities with megaplasmid pCC178 of Campylobacter coli,suggesting a possible insertion of a related plasmid into thegenome of RM1221. A similar element in a genomic island(HHGI1) in Helicobacter hepaticus ATCC 51449 was describedpreviously (56). The large majority of the genes found in thiselement encode hypothetical proteins. CG8421 and BH-01-0142 both contain CJIE3-like insertions in their chromosomes.Parker et al. (44) previously demonstrated by whole-genomemicroarray that 65.7% (23/35) strains contained more than75% of the genes found in the RM1221 CJIE3 element. More-over, approximately 70% (16/23) of the strains examined con-tained the element in the same physical location as RM1221,and this element is found at the same position in CG8421(zone 14) (Table 5 and see Table S2 in the supplementalmaterial). This region was represented by a high number ofcontigs during analysis of the CG8421 raw sequencing data,suggesting the presence of repeated sequences that reducedthe assembly capability of the assembler software. Neverthe-less, based on the similarity of the proteins coded by the CJIE3element in RM1221, pCC178 of C. coli, and HHGI1 of H.hepaticus (56), the content of this element in CG8421 andBH-01-0142 was drafted (see Table S2 in the supplementalmaterial).
Compared to RM1221, the CG8421 CJIE3-like elementlacks the regions from CJE1093 to CJE1104 and CJE1118 toCJE1132. These regions encode small ORFs and ORFs encod-
ing hypothetical proteins. Other contigs potentially included inthis element are similar to genes in the CJIE3 elements ofpCC178, HHGI1, and C. jejuni TGH9011. These results areconsistent with a mosaic structure of CJIE3-like elements, assuggested by comparative genomics studies (44).
Regions of NCTC 11168 missing from CG8421. A total of 61ORFs (3.3% of the genome) from NCTC 11168 are missingfrom CG8421 (see Table S3 in the supplemental material),excluding the LOS (Table 2), CPS (Table 3), and flagellin(Table 4) glycosylation loci. This number is similar to previousfindings for other sequenced C. jejuni genomes (66 ORFs weremissing in CG8486, and 51 ORFs were missing in 81-176) (23,46). Sixty-nine percent of these missing ORFs (42/61) are lo-cated in regions of the NCTC 11168 genome that were previ-ously characterized as being hypervariable by Taboada et al.(57). The 19 remaining ORFs are, for the most part, scatteredon the genome, with two exceptions. Cj0416 and Cj0417 arereplaced by one ORF coding for a hypothetical protein inCG8421 (Cj8421_410), and Cj0522 to Cj0524 are replaced by aputative Na/Pi cotransporter, as discussed above. This regionmight represent a pseudogene in the NCTC 11168 genome, asnoted in a recent reannotation of that genome (22).
CG8421 and BH-01-0142 genome comparisons. Most of theunique ORFs in CG8421 and BH-01-0142 are common to bothstrains. Thus, 65 of 103 unique ORFs in BH-01-0142 (seeTable S1 in the supplemental material) are also found in theCG8421 genome. Seventy-nine of these 103 genes could not bemapped on the BH-01-0142 chromosome, but 54 appear to besimilar to ORFs of pCC178 and the CJIE3 element, suggestinga conservation of this element between CG8421 and BH-01-0142, as mentioned above. It appears that BH-01-0142 con-tains a divergent zone 20 (encoding a putative phage insertion)at the same location as CG8421. In addition, BH-01-0142 con-tains zones 1, 5, and 16 (Table 5).
DISCUSSION
Whole-genome sequencing facilitated the examination oftwo C. jejuni genomes for genes involved in molecular mimicryand, together with structural analyses of LOS cores and sugarcomposition analyses of the capsular polysaccharides, providedstrong evidence that these strains lack glycolipid mimics. Fast,accurate, and relatively cheap “next-generation” sequencingtechnologies have made a spectacular impact on biology sincetheir introduction at the end of 2004 (33, 34, 54). This studyillustrates yet another new application: sequencing of the ge-nomes of vaccine challenge strains to confirm that the strainslack molecular mimicry, a method that is more accurate anddefinitive than either microarray analyses or phenotypic test-ing. Similar sequence analyses of challenge strains or live at-tenuated vaccine strains could also advance vaccine develop-ment against other pathogens. Alignment of most regions ofthe genome to a scaffold of the known C. jejuni genome wasreadily accomplished, although the highly repetitive flagellinglycosylation locus proved problematic. We also cannot pointto a specific reason for the elevated number of partially assem-bled sequence reads in the BH-01-0142 project compared toCG8421 or the previously sequenced strain CG8486 (46), butprocedures for the extraction of genomic DNA or the con-struction of the nebulized sequencing libraries are likely can-
FIG. 6. Comparison of the DNA of the flagellin glycosylation lociof C. jejuni strains BH-01-0142, CG8421, and NCTC 11168. A com-parison was made using Artemis comparison tool software (http://www.sanger.ac.uk/Software/ACT/). The vertical block between sequencesrepresents conservation. Flagellin glycosylation sequences from BH-01-0142 and CG8421 are incomplete and are represented by 15 and 7contigs, respectively. The order of the contigs presented is based on thealignment with the NCTC 11168 locus and might not reflect the phys-ical order in BH-01-0142 and CG8421.
VOL. 76, 2008 C. JEJUNI HUMAN CHALLENGE STRAINS 5663
didates. Using a more recent release of Newbler software,version 1.1.03.24, de novo BH-01-0142 assembly produced 200large contigs (1.66 Mb), suggesting that this data problemmight be resolved with improved bioinformatics tools. Withthe addition of the sequence information reported here forCG8421 and BH-01-0142, the number of sequenced Campy-lobacter genomes totals 17 (with 10 being C. jejuni). One strik-ing feature of the sequences of CG8421 and BH-01-0142 is thatthe numbers of unique C. jejuni genes are low. Thus, therewere only 13 genes identified in CG8421, less than 1% of thegenome, that were not previously reported for other C. jejunistrains. The availability of a complete C. jejuni gene repertoirewill allow the development of more complete composite spe-cies-specific microarrays that could be used to quickly surveythe genome content and pedigree of other strains.
The primary feature required for a safe human challenge is theabsence of ganglioside or other glycolipid mimicry, and genomicand structural analyses unequivocally revealed that strainsCG8421 and BH-01-0142 expressed LOSs that lack all such mim-ics (Fig. 1A). Moreover, structural analyses also confirmed theabsence of Neu5Ac in the CPSs of both strains. Although bothstrains contain homologs of the genes encoding enzymes respon-sible for the synthesis of the bacterium-specific nonulosonate sug-ars Pse5Ac7Ac and Leg5Am7Ac, there were no genes forNeu5Ac synthesis identified in the genomes. Sialylation of thebacterial surface affects the virulence of numerous pathogens(59). The role of Neu5Ac-containing LOS cores in the pathogen-esis of GBS has been extensively studied, but the role of such
cores in the pathogenesis of diarrheal disease is less well under-stood. The effect of a truncation of the LOS core on invasion invitro has been studied using strain 81-176. There was no effect oninvasive ability until the core was truncated to only Kdo-lipid A(21, 24–26). However, a loss of Neu5Ac from the core of 81-176and strain MSC57360 (19, 21) increased the sensitivity of thestrains to complement-mediated killing. Thus, the use of anystrain in which the LOS core had been truncated could potentiallyresult in attenuation in a human model.
There have been limited reports of C. jejuni strains that lacksialylated LOS cores (5, 16, 32). However, strains that were probenegative for Neu5Ac were found at a surprisingly high frequencyin this study (4/98 isolates), suggesting that nonsialylated LOScores may be more common among C. jejuni strains than previ-ously recognized. Although the complete genome sequence ofTGH9011 is not available, numerous novel genes have been iden-tified in this strain by a microarray study (47). Given the generalheterogeneity among C. jejuni strains, it is striking that these twoAsian isolates, CG8421 and BH-01-0142, and Canadian isolateTGH9011, which were isolated decades apart, appear to be highlyrelated throughout most of their genomes.
The fully defined chemical structures of the CPSs will be re-ported in the future, but preliminary sugar composition analysisperformed here showed that BH-01-0142 contained a CPS com-posed of Gal, 6d-ido-Hep, and small amounts of LD-ido-Hep (Fig.4A). The previously reported structure (5) of the TGH9011 CPS(HS3 serotype) was shown to be composed of a disacchariderepeating unit of Gal and LD-ido-Hep, [LD-ido-Hep-(134)-Gal-
TABLE 5—Continued
Zone (region) Locus tagProtein
size(residues)
Closest relationship (GenBank accession no.)
No. ofidentical
proteins/totalno. of proteins
(%)
18 (divergent restriction-modificationlocus) (Cj1546 to Cj1555c)
Cj8421_1614 347 Arsenical resistance protein CJE1733(YP_179707.1)
302/347 (87)
20 (possible phage insertion) (Cj1668c toCj1669c)
Cj8421_1724 122 No hits -
Cj8421_1725 397 Tgh039, similar to replicative helicase RepA 249/250 (99)Cj8421_1726 371 Hypothetical protein Tgh066 235/238 (98)Cj8421_1727 145 Hypothetical protein Tgh063 99/101 (98)Cj8421_1728 284 Hypothetical protein Tgh139 151/151 (100)Cj8421_1729 122 Hypothetical protein Tgh087 122/122 (100)Cj8421_1730 111 Tgh085, similar to conserved hypothetical protein
HH0278110/111 (99)
Cj8421_1732 260 Tgh128, similar to bacteriophage P4 integrase 132/132 (100)
a See Table S2 in the supplemental material.b See Table 1.c See Table 2.
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(133)]n. Thus, the CPS of BH-01-0142 appears to be closelyrelated to the TGH9011 CPS, with the main difference being theexpression of 6d-ido-Hep (instead of LD-ido-Hep) in the CPS ofBH-01-0142. Moreover, the genes in the capsule loci of BH-01-0142 and TGH9011 appear to be very similar (Fig. 5 and Table 3).The capsule has been shown to be the serodeterminant capsule ofthe HS3 serogroup (28), but the relationship of the complexserogroup seen for BH-01-0142 (i.e., HS3,13,50) to capsule struc-ture remains to be determined. In the case of CG8421 (serotypedas 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. 4B). This CPS composition is similar to that of strain81-176 (27) (HS23,36 serotype) that is composed of a trisaccha-ride repeating unit with Gal, 6d-3-O-Me-Hep, and GlcNAc [Gal-(132)-6d-3-O-Me-altro-Hep-(133)-GlcNAc-(133)]n, with thesole difference being the replacement the 6d-3-O-Me-altro-Hepby its nonmethylated derivative, 6d-altro-Hep, in the CG8421CPS. Similarly, the capsule loci of both 81-176 and CG8421 arevery similar to each other. Preliminary 31P NMR studies revealedthe presence of O-methyl-phosphoramidate residues in the CPSsof both BH-01-0142 and CG8421, consistent with the presence ofgenes for O-methyl-phosphoramidate synthesis (37) in both cap-sule loci.
To our knowledge, CG8421 is the first reported HS23,36 strainthat lacks a sialylated LOS core. Thus, other than the CPSs,CG8421 and BH-01-0142 appear to be very similar to each otherdespite the fact that the original clinical presentations were quitedistinct. Both strains lack numerous ORFs found in the referenceNCTC 11168 genome and share ORFs not found in NCTC 11168with other sequenced C. jejuni strains (81-176, RM1221, andCG8486).
Strain 81-176 has been studied extensively because it has beenconfirmed 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 vivovirulence of C. jejuni, we sought strains that were invasive in vitrowith the expectation that such strains would cause disease involunteers. Compared to strain 81-176, both CG8421 and BH-01-0142 were noninvasive for intestinal epithelial cells in culture, aswas another clinical isolate from Thailand, CG8486 (46). Allthree of these strains were obtained from patients with definedclinical syndromes and were subjected to minimal in vitro passageprior to DNA sequence analyses. The significance of this lack ofinvasiveness in vitro remains to be determined, but it should bementioned that at the time of this writing, the virulence ofCG8421 has been confirmed by a human volunteer challengestudy, which will be reported separately (B. Kirkpatrick, personalcommunication).
ACKNOWLEDGMENTS
We thank Nicci Nolan, Shannon Lentz, Cheryl Ewing, Gary Majam,Julie Phan, and Lisa Applebee at the NMRC for their contributions tothis study; Helen Tabor of the Canadian Microbiology ReferenceLaboratory in Winnipeg for Penner serotyping; and Michael Prouty forhis comments on the manuscript.
Work at the NMRC was funded by the Military Infectious DiseasesResearch Program Work Unit 6000.RAD1.DA3.A308. M.A.M. wasfunded by the NSERC and ACE Biosciences.
The views expressed in this work are those of the authors and do notnecessarily reflect the official policy or position of the Navy, the De-partment of Defense, or the U.S. government.
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Editor: S. R. Blanke
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