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Previous Article | Next Article 
Journal of Bacteriology, January 2004, p. 503-517, Vol. 186, No. 2
0021-9193/04/$08.00+0 DOI: 10.1128/JB.186.2.503-517.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
* Shaun Cawthraw,2, Georgina Manning,2, Joanna K. MacKichan,1 Stanley Falkow,1 and Diane G. Newell2
Department of Microbiology and Immunology, Stanford University, Stanford, California,1 Veterinary Laboratories Agency (Weybridge), Addlestone, Surrey, United Kingdom2
Received 22 July 2003/ Accepted 10 October 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human campylobacteriosis is considered primarily a food-borne disease. C. jejuni is a common gut commensal in most food-producing animals and birds, and fecal contamination of meat during processing is a well-recognized route of transmission to humans. Epidemiological studies indicate that a major source of human infection is the consumption and handling of meat from broiler chickens (19). C. jejuni colonizes the cecum and colon of such birds readily and in extremely high numbers (54). Thus, strategies for the control and prevention of poultry colonization at the farm level are considered an important approach to the reduction or elimination of campylobacteriosis in humans.
The mechanisms by which C. jejuni colonizes the intestinal tract are poorly understood. Models of colonization in orally challenged chickens have enabled some colonization factors to be identified by using defined mutants; these factors include flagellin (78), superoxide dismutase (60), and GalE (20). However, such studies are highly dependent on the presumed properties of genes and thus do not generally lead to the identification of C. jejuni-specific colonization factors. Recently, with the advent of genome sequencing and the development of strategies, such as signature-tagged mutagenesis, for the mass screening of mutants, the identification of bacterial factors important for virulence has become more rapid and independent of known gene functions (9, 48, 62).
In 2000, the genome sequence of C. jejuni NCTC 11168 was completed (58). This appeared to provide a unique database for postgenomic investigations, including the potential for identification of C. jejuni-specific colonization factors. However, initial studies demonstrated that the genome-sequenced variant of NCTC 11168, unlike other C. jejuni strains, was deficient in its ability to colonize 1-day-old chicks (1) or SCID mice (28) following oral challenge. Previous reports have indicated that the virulence properties of some bacteria, including campylobacters, may be reduced by laboratory subculture over time (7, 17, 40, 52, 72). However, none of these studies identified discernible molecular changes or differences that might lend insight into possible mechanisms underlying this phenomenon.
C. jejuni NCTC 11168 was originally isolated from the feces of a diarrheic patient in 1977 by Martin Skirrow (http://cphl.phls.org.uk/divisions/cdmssd/nctc/qcrc.htm). Fortuitously, this original clinical isolate (strain number 5636/77), archived by Skirrow in 1977 and frozen ever since, was donated to the campylobacter strain collection of the Veterinary Laboratories Agency (VLA) in 1999. In stark contrast to the genome-sequenced variant, we have found that the original isolate is an excellent colonizer of chickens. To identify other potential phenotype differences and to explore possible mechanisms underlying the colonization disparity, we undertook a detailed phenotypic, genotypic, and transcriptional comparison of the genome-sequenced and original variants of C. jejuni NCTC 11168 (henceforth designated 11168-GS and 11168-O, respectively). Our findings include a likely role for bacterial metabolism and adaptation to different oxygen tensions in colonization potential. This study also highlights the remarkable capacity of C. jejuni to evolve rapidly as a result of storage, culture, and passage conditions. Finally, our work demonstrates the potential of microarray-based transcriptional profiling in screening for differences in bacterial variants that have adapted to new environments, probably via small but accumulative vertical evolutionary events, without having incurred more readily identifiable genomic changes such as gene loss, gene gain, or horizontal gene transfer.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To avoid damage due to laboratory passage (particularly for 11168-O), C. jejuni strains were subcultured on plates no more than twice prior to initiating any experiment. The C. jejuni strains were either cultured on 10% (vol/vol) sheep blood agar plates with actidione (250 µg/ml) and Skirrow's supplement (10 µg of vancomycin/ml, 2.5 IU of polymyxin B/ml, and 5 µg of trimethoprim/ml) (BASA) at 42°C in a microaerobic environment (7.5% [vol/vol] CO2, 7.5% [vol/vol] O2, and 85% [vol/vol] N2) for 24 to 48 h or cultured on Mueller Hinton (MH) agar and in MH broth with 10 µg of vancomycin/ml and 5 µg of trimethoprim/ml at 37°C with an Oxoid CampyGen pack to generate microaerobic conditions (6% O2 and 12% CO2). To generate severely O2-limited conditions, bacteria were cultured with Oxoid AnaeroGen packs. According to the manufacturer's specifications, these packs result in an O2 environment of <1% within 30 min. Escherichia coli strain TOPO 10F' (Invitrogen) was grown on Luria-Bertani agar or in Luria-Bertani broth at 37°C under atmospheric conditions. For one experiment, 11168-O was passaged aerobically on BASA plates. The plates were not dried prior to inoculation and were placed in an unsealed gas jar with a moist tissue at the bottom and incubated at 37°C for 24 to 48 h. This process was repeated 13 times. As a control, 11168-O was also passaged 13 times microaerobically, again on undried BASA plates at 37°C for 24 to 48 h. A CampyPak (BBL, Becton Dickinson) was used in a sealed gas jar to generate the microaerobic environment.
Chicken colonization model. A 1-day-old chick model of colonization (78) was used to determine colonization potential. Briefly, eggs from specific-pathogen-free chickens (Lohmann's) were hatched in isolators. Groups of chicks (n = 10) were maintained in separate isolators with unlimited food and water. At 1 day old, chicks were each dosed orally by gavage with approximately 102 to 109 CFU of C. jejuni in 0.1 ml of phosphate-buffered saline (PBS). Bacteria were grown overnight on blood agar plates under microaerobic conditions at 42°C. Chicks were killed 5 days after dosing, and bacterial colonization levels were determined by plating out dilutions of cecal contents. The detection limit for colonization was 102 CFU/g of cecal content. Further colonization studies were carried out in which birds were dosed with (i) 11168-GS grown as above and then placed in anaerobic conditions at 37°C for 24 h by using an anaerobic cabinet and (ii) 11168-O that had been subcultured 13 times under aerobic conditions at 37°C.
In vitro phenotypic studies. The morphologies of the two strains were compared by transmission electron microscopy (TEM). Briefly, bacteria were grown under microaerobic conditions, suspended in water, allowed to sediment onto Formvar-carbon-coated grids, negatively stained with 2% (wt/vol) phosphotungstic acid, and examined with a Phillips CM 10 TEM. Motility was measured by swarming on motility agar plates. Each strain was grown as described above, and the concentration was adjusted to approximately 108 CFU/ml in PBS. One microliter of this suspension was then stabbed into a 0.4% MH agar plate. Following microaerobic growth at 42°C for 24 to 48 h, the diameter of each halo of growth was measured.
Bacterial invasion into INT407 cells was studied by a gentamicin protection assay (6, 16, 42, 76) with confluent INT407 monolayers seeded 24 h prior to infection. The starting bacterial inoculum for each strain was 
4 x 108 to 6 x 108 CFU. Invasion was assayed in minimum essential medium (MEM) without serum, with triplicate wells seeded for each strain for each time point. Bacteria were allowed to invade for 30 min or 2 h, then the cells were washed five times with prewarmed MEM prior to the addition of 250 µg of gentamicin/ml in MEM. After 2 h, cells were washed five times again with MEM and lysed with water and a 25-gauge syringe. For experiments in which the bacteria were first centrifuged onto the INT407 cells prior to initiating invasion, centrifugation was carried out at 450 x g for 15 min at room temperature. Translocation assays were carried out by using 18-day differentiated Caco-2 cells, grown in transwells (Corning Costar Corp.) with a pore size of 3 µm, as previously described (6, 42). Separate transwells were used for each time point, and the assay was performed in triplicate for each strain at each time point. The starting bacterial inoculum for each strain was 2.5 x 108 CFU. Translocation was performed in MEM plus 10% fetal bovine serum and allowed to proceed for 2 or 6 h. The entire basolateral fraction was collected, and bacteria were enumerated by serial dilution plate counts. Prior to initiating each assay, bacteria were inoculated in biphasic MH flasks (5 ml of broth over 10 ml of agar) at an optical density at 600 nm (OD600) of 0.002 and grown at 37°C microaerobically for 16 h to an OD600 of 0.3 before starting the experiment.
Electron microscopy of Caco-2 sections. Polarized Caco-2 cells infected with C. jejuni were washed gently three times with PBS and fixed at 4°C for 90 min with 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Samples were postfixed in 1% OsO4 (Polysciences) for 90 min at 4°C and stained with 0.25% uranyl acetate (Polysciences) at 4°C for 1 h. After dehydration with a series of ethanol and water solutions, samples were embedded in firm Spurr's resin (Polysciences). Samples were then sectioned, stained with uranyl acetate and lead citrate, and examined on a Philips 201c electron microscope.
Molecular genotyping. Amplified fragment length polymorphism (AFLP) was performed according to the method of Duim et al. (14). The final products were separated on a 7.3% denaturing acrylamide sequencing gel with an ABI 377 automated DNA sequencer. Pulsed-field gel electrophoresis (PFGE) was performed by adjusting a bacterial suspension to an OD500 of 0.5 in PBS prior to preparation of agar plugs. Cells were lysed by two consecutive 24-h incubations in lysis buffer and proteinase K at 55°C prior to digestion of genomic DNA with SmaI for 48 h. The DNA fragments were separated by using a Chef-DR 111 system (Bio-Rad) with a ramped pulse of 10 to 35 s for 21 h at 200 V and 14°C. Fla typing was performed as described previously (53).
Microarray-based genotyping (genomotyping) studies were performed by using a C. jejuni DNA microarray consisting of amplicons representing the largest nonoverlapping, nonhomologous regions of 99% of the open reading frames (ORFs) in the 11168-GS genome (44). Each array contains two spots per gene. Four hybridizations were performed, each of which used 11168-GS as a reference. Two hybridizations used 11168-GS as the test DNA (as a control for labeling and dye variability); two hybridizations used 11168-O as the test DNA. Hybridizations were performed essentially as described previously (44). Briefly, 500 ng of genomic DNA was incubated with amino-allyl dUTP (Sigma) and Klenow fragment (exo-) (NEB) at 37°C for 2 h to generate labeled probes. The probes were coupled to monofunctional Cy dyes (Amersham), with Cy3 for the reference dye and Cy5 for the test dye. Arrays were hybridized at 55°C for 16 h and scanned with an Axon 4000A scanner and GenePix software. Data were analyzed by using GACK (39) and Cluster (15).
Gene expression analyses.
After a single passage on MH plates, bacteria were inoculated into MH broth at an OD600 of 0.002 and grown microaerobically with the Oxoid CampyGen system at 37°C for 14 h with shaking at 200 rpm to mid-log phase (OD600 of 0.3). Bacteria were diluted back to an OD600 of 0.1, split into microaerobic or severely O2-limited cultures (with the CampyGen or AnaeroGen Oxoid system, respectively), and shaken at 200 rpm at 37°C in an airtight 2.5-liter BBL Campyjar. Starting cultures were 25 ml in a 150-ml flask. At various time points, 3-ml samples were harvested from the same flask to determine the OD600 and number of CFU per milliliter and to prepare RNA, after which the flask was returned to the jar with a fresh pack for further incubation. Flasks were exposed to atmospheric conditions for <30 s at each time point. For RNA preparation, samples were harvested into a 1/10 volume of 5% phenol-95% ethanol, spun, and frozen at -80°C. The samples were resuspended in 0.4 mg of lysozyme/ml, incubated at room temperature for 5 min, and lysed in a 20x volume of Trizol reagent (Invitrogen). Following chloroform extraction, the samples were combined with an equal volume of 70% ethanol, loaded onto an RNeasy Mini column (Qiagen), and processed according to manufacturer's procedures (including on-column DNase treatment). RNA was confirmed as DNA-free by reverse transcription (RT)-PCR. For hybridizations, RT was performed with 0.6 pmol of C. jejuni gene-specific primer mix (Sigma-Genosys) and 1.5 µg of C. jejuni RNA per hybridization. cDNA labeling and array hybridization were performed essentially as described above for genomic DNA labeling and in reference 44, except that the Klenow labeling and probe-generating reactions were performed for 16 h at 37°C prior to dye coupling. For the RNA-based hybridizations, reference cDNA was generated by performing RT reactions on a pool of RNA comprised of equimolar aliquots of RNA from each sample in the experiment. Probes generated from this reference cDNA were labeled with Cy3 and hybridized against probes generated from individual samples labeled with Cy5. Data were entered into the Stanford Microarray Database, and data were retrieved only for those spots exhibiting a regression correlation of >0.6 and which contained >75% good data. To identify spots with the most pronounced differences between the variants, we employed SAM (significance analysis of microarrays), a statistical program which uses repeated permutations of the data to determine whether the expression of any gene (or spot) is significantly related to the response (71). Spots were identified on the basis of an overall (i.e., over the four time points) >1.5x signal intensity difference and a low false-discovery rate. Because expression levels change over time, for a given spot, the signal intensity difference is likely to be much greater than 1.5x between the variants at certain time points and slightly less than 1.5x different at others (see Fig. 5C and D and 6C and D for examples). The spots identified were confirmed as significant by using ORF order and Cluster analyses to investigate operon organization and coexpression with known genes of likely similar function. The original data for the spots identified were mean centered and hierarchically clustered with Cluster and visualized by Treeview (15). Quantitative real-time RT-PCR (QRT-PCR) was performed with a Bio-Rad iCycler, primers designed to produce 70-bp amplimers, and recombinant Tth enzyme, buffer, and SYBR Green (Perkin-Elmer/Molecular Probes). Standard curves were generated for each primer pair in every reaction mixture. Reaction conditions were as follows: (i) RT, 50°C for 5 min and 60°C for 30 min; (ii) PCR, 50 cycles of 94°C for 10 s, 50°C for 30 s, and 60°C for 30 s; (iii) melt curve, 100 to 60°C in 1°C increments every 30 s.
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Subtractive hybridization. The method used for subtractive hybridization was similar to that described previously (1), except that both primary and secondary hybridizations were carried out at 58°C. Subtracted products were cloned into pCR-2.1TOPO, and a selection, based on the dot blot results, were sequenced with M13 forward and reverse primers.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Growth data and growth stage and gene expression controls. OD600 and CFU/ml data indicated that during microaerobic culture, 11168-GS grew faster, reached a higher number of CFU per milliliter at stationary phase, and maintained a high level of viability for a longer period of time than 11168-O (Fig. 4). Under severely O2-limited conditions, neither variant grew well, as expected given previous reports on the inability of C. jejuni to grow during anoxia (64). Nevertheless, both variants retained a significant proportion of viable cells at the 26-h time point under both conditions.
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Gene expression data. Using the time points as technical replicates, we first employed SAM (see Materials and Methods) to identify genes that differed significantly between the variants regardless of culture condition (Fig. 5). Whole-genome analyses indicated that some differences observed only under severe O2 limitation were not identified by this method; thus, we also used SAM to identify genes with significant differences specifically under these conditions (Fig. 6). In both Fig. 5 and 6, panel A shows genes with higher expression in 11168-O compared to 11168-GS, with the log2 of the red/green ratios shown numerically for one example in panel C. Panel B shows genes with lower expression in 11168-O than in 11168-GS, with the log2 of the red/green ratios shown numerically for one example in panel D. Where possible, both array spots for a given gene are shown (i.e., when both spots passed the filtering and SAM criteria).
(i) Respiration and metabolism clusters. (a) TCA cycle, C4-dicarboxylate, and electron transport chain genes.
The tricarboxylic acid (TCA) cycle plays a dual role in cell metabolism: it generates biosynthetic starting compounds that are used in amino acid metabolism and it provides a metabolic energy source by creating intermediates that feed into the electron transport chain. In facultative anaerobes like E. coli, the TCA cycle is primarily oxidative during aerobic growth while during anaerobiosis it branches into oxidative (biosynthetic) and reductive (energy-generating) arms. Half of the TCA cycle is comprised of C4-dicarboxylic intermediates (e.g., fumarate, succinate, malate, and aspartate [during anaerobiosis]); the other half is comprised of classic biosynthetic starting compounds (e.g., 
-ketoglutarate, succinyl-coenzyme A [succinyl-CoA], and oxaloacetate). The genome sequence of C. jejuni suggests that it contains a complete TCA cycle and a complex, branched electron transport chain that is likely to allow both aerobic and anaerobic respiration with a variety of electron acceptors (38, 58, 66). Apart from the genome information and a few studies on enzyme activity (64, 67), little is known about the TCA cycle in C. jejuni.
Several genes likely to be involved in uptake or metabolism of C4-dicarboxylate TCA cycle intermediates were expressed at significantly higher levels in 11168-O than in 11168-GS under both culture conditions (Fig. 5A, cluster 1). One of these genes, dcuA, is also shown in Fig. 5C, where a >9-fold difference in signal intensity is observed at the 6-h time point for 11168-O (microaerobic) compared to 11168-GS (either condition). dcuA and dcuB have been characterized in other bacteria as mediating anaerobic succinate-fumarate antiport and succinate uptake for use as a carbon source, whereas dctA catalyzes aerobic uptake of succinate (34). aspA, in a two-gene operon with dcuA, encodes an aspartate ammonia-lyase that catalyzes the conversion of aspartate to fumarate, which can feed into the anaerobic reductive branch of the TCA cycle. Up-regulation of several of these genes in 11168-O, although faster under severely O2-limited conditions, was somewhat higher during microaerobic growth. This cluster also contains an operon (sdhABC) annotated as encoding succinate dehydrogenase, an enzyme that has been well-characterized in E. coli and several other bacteria as a key component of the aerobic oxidative TCA cycle that converts succinate to fumarate and also connects the TCA cycle with the aerobic respiratory chain (8, 64). In those bacteria, succinate dehydrogenase is repressed during anaerobiosis so that the reverse reaction, reduction of fumarate to succinate by fumarate reductase, can prevail. In our experiments, expression of the frdCAB operon likely to encode fumarate reductase was modestly higher in 11168-O than in 11168-GS but only at the 6- and 10-h time points under microaerobic conditions (data not shown). Other interesting genes in cluster 1 of Fig. 5A include Cj0358, which has been suggested to be an oxygen-independent terminal oxidase in the C. jejuni electron transport chain (73), and Cj0069 and Cj0021c, which are annotated as hypothetical proteins but contain conserved domains potentially involved in glutathione synthesis and coenzyme modification and conversion of tyrosine to fumarate and acetoacetate, respectively.
In contrast, several other TCA cycle and putative electron transport chain genes exhibited significantly lower expression levels in 11168-O than in 11168-GS but only under severely O2-limited conditions (Fig. 6B). Eight of these genes are contained in a single operon (Cj0531 to Cj0538). The first four of these genes encode homologs of enzymes that participate in the more biosynthetic intermediate-generating half of the TCA cycle: the conversion of isocitrate to -ketoglutarate (icd), the interconversion of succinate and succinyl-CoA (sucCD), and the interconversion of malate and oxaloacetate (mdh). Several other genes in the Fig. 6B cluster are likely to be involved in general biosynthetic processes, e.g., glyA, pheT, purE, moeB, and thiG. This cluster also contains genes likely to encode two oxygen-labile oxidoreductases that have been characterized in Helicobacter pylori as mediating electron transport to NADP (29, 30) (see also Fig. 6D). OOR, a 2-oxoglutarate-acceptor oxidorecuctase, interconverts 2-oxoglutarate and succinyl-CoA using a ferredoxin; POR, a pyruvate-flavodoxin oxidoreductase, acts together with the fldA flavodoxin (Fig. 6B) to convert pyruvate to acetyl-CoA.
(b) Gluconeogenesis genes. C. jejuni cannot use glucose as a primary carbon source; however, genome sequence and experimental data indicate that C. jejuni possesses an Embden-Meyerhof (glycolysis and gluconeogenesis) pathway that is likely to be largely gluconeogenic (49, 58, 73). C. jejuni requires gluconeogenesis to generate glucose-derived polysaccharides and can also incorporate glycolysis end products like pyruvate into the TCA cycle via anaplerotic enzymes like pyruvate carboxylase (49, 73). A four-gene operon (Cj1403c to Cj1400c) likely to encode three key bidirectional enzymes of the glycolysis-gluconeogenesis pathway (gapA, pgk, and tpiA) and one fatty acid synthesis gene (fabI) were expressed at significantly higher levels in 11168-O than in 11168-GS (Fig. 5A). Most of these genes fall into cluster 2 of Fig. 5A and were expressed even more highly under microaerobic conditions. The expression of other genes in this pathway (e.g., pgm, eno) (73) was similar in both variants (data not shown).
(ii) Flagellar export, motility, and other gene clusters.
A number of likely flagellar export apparatus genes were expressed significantly more highly in 11168-O under severely O2-limited conditions than in any of the other samples (Fig. 6A). This cluster contains several putative hook and basal body structures and one putative flagellin. flgB, for example, exhibits an 7-fold-higher signal intensity in 11168-O at the 6-h time point under severely O2-limited conditions than in either strain cultured microaerobically (Fig. 6C). Other flagellar genes and the major flagellins flaA and flaB were expressed at similar levels in the two variants (data not shown). Figure 6A also contains one spot representing Cj1465; the three other spots representing Cj1464 and Cj1465 are found in Fig. 5A, cluster 3. As with the flagellar genes in Fig. 6A, these genes were expressed much more highly in 11168-O during severe O2 limitation and fall into a likely operon (Cj1462 to Cj1466) with flgK (Cj1466), flgI (Cj1462, a putative flagellar P-ring protein), and Cj1463. Although flgI and Cj1463 did not pass the SAM cutoff employed, both spots for each gene exhibited expression profiles similar to those of the other genes in the operon (data not shown). Cj1463, Cj1464, and Cj1465 have no homology to any other proteins and no conserved domains. Several other genes also found in cluster 3 of Fig. 5A also have no known homologies or conserved domains (e.g., Cj0056c and Cj0931c) but were strikingly coexpressed with Cj1464 and Cj1465. Other interesting genes with higher expression in 11168-O under severely O2-limited conditions (Fig. 6A) include Cj0062c, which was recently shown to play a role in motility (27), Cj1631c, which contains an EF hand Ca2+ binding domain, and Cj0604, a close homolog of the recently identified ppk2 gene from Pseudomonas aeruginosa (31, 80), a polyphosphate kinase that preferentially synthesizes GTP from polyphosphate.
Finally, a number of genes were expressed at significantly higher levels in 11168-GS than in 11168-O under both culture conditions (Fig. 5B and D). Apart from the putative cytochrome C (Cj0037c), which exhibits a >10-fold-higher signal intensity in 11168-GS (microaerobic) than 11168-O (microaerobic) at the 6- and 26-h time points (Fig. 5D), none of these genes have homology to any other translated ORFs in the National Center for Biotechnology Information microbial genome database.
DNA sequencing of the three major sigma factors reveals amino acid differences between the two variants. Although many of the genes exhibiting significant expression differences are functionally related, they are physically scattered throughout the 11168 genome, suggesting that the two variants may possess differences in global regulatory molecules. To investigate this, and to explore the relative frequency with which single-nucleotide polymorphisms (SNPs) may occur between the otherwise clonal variants, we sequenced the three C. jejuni sigma factors from 11168-O and compared them with the known 11168-GS sequences. We found that each sigma factor contains SNPs resulting in at least one amino acid change in 11168-GS relative to 11168-O (Table 3). There are two amino acid changes in the RpoN sequence. The serine at amino acid 84 in 11168-O is replaced by a glycine in 11168-GS, and the lysine at amino acid 411 in 11168-O is replaced by an asparagine in 11168-GS. RpoD and FliA each have a single amino acid change. In RpoD, the alanine at amino acid 40 in 11168-O is replaced by a threonine in 11168-GS, and in FliA, the threonine at amino acid 91 in 11168-O is replaced by a methionine in 11168-GS.
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DISCUSSION |
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In this work, we report that the genome-sequenced variant of C. jejuni NCTC 11168 (11168-GS) and the original clinical isolate from which it was derived (11168-O) exhibit dramatic differences in numerous virulence-associated phenotypes, including colonization, invasion, translocation, and motility. Despite these differences, these clonally derived strains appear indistinguishable by multiple high-resolution molecular genotyping techniques, although targeted sequencing detected SNPs in each sigma factor. Unlike previous studies on C. jejuni attenuation or gain of virulence associated phenotypes due to culture condition, the availability of the precise DNA sequence of 11168-GS, together with technical advances such as microarray technology, has allowed us to take a whole-genome approach that includes transcriptional profiling as a platform for exploring the molecular and biological bases of the phenotypic alterations. The identification of several unexpected differences in gene expression has provided a heretofore unavailable window of insight into what might have changed in these variants and perhaps also how and why these changes occurred.
In nature, C. jejuni encounters a range of environments. Only in the intestinal tracts of temperate animal species does this organism naturally grow. However, long-term survival can occur in such diverse environments as fecal matter, water, and retail meats. These environments encompass a range of nutrient availability and oxygen tension, from largely aerobic to microaerobic to largely anaerobic. C. jejuni possesses genes and pathways that can allow survival and often growth under many different oxygen tensions (58, 64, 66). Efficient regulation of these genes and pathways is thus vital to the success of this organism in traversing these different niches. As 11168-O became 11168-GS, it was frozen, passaged in the laboratory, freeze-dried, and then passaged in the laboratory again. Our genotyping data strongly suggest that the original and genome-sequenced variants are clonal and that 11168-GS evolved vertically from 11168-O. Thus, it is likely that selective pressure to survive and thrive under laboratory growth and culture conditions (possibly including exposure to a largely aerobic environment) resulted in small genetic changes that allowed more robust in vitro growth (Fig. 4) while concomitantly compromising the strain's ability to readapt readily to the environment of the intestinal tract (Fig. 1).
Indeed, our gene expression and animal data suggest that the ability of C. jejuni to shift its metabolism and respiration strategies to accommodate the lower-oxygen microaerobic and largely anaerobic environments encountered in the gut is likely to be important for in vivo survival and colonization. Figure 6 clearly illustrates that many genes exhibit differential expression in 11168-O but not 11168-GS as a result of severe O2 limitation, suggesting that 11168-O may be better able to sense and respond to such an environment than 11168-GS. Many of the affected genes are involved in respiration and metabolism. For instance, the expression of several TCA cycle enzymes likely to be involved in generating biosynthetic starting compounds (e.g., icd, mdh, and sucCD) was lower in 11168-O cultured under severe O2 limitation than in any of the other samples, as was expression of several putative biosynthesis genes (Fig. 6B). In contrast, a number of genes likely to be involved in transport and metabolism of C4-dicarboxylates were expressed much more highly in 11168-O than 11168-GS under both conditions tested (Fig. 5A); many of these genes have also been characterized as participating in anaerobic metabolism (e.g., dcuA, dcuB, aspA, and Cj0358). It is thus possible that, upon sensing oxygen depletion, 11168-O (but not 11168-GS) efficiently shifts the TCA cycle towards transport, production, and utilization of C4-dicarboxylate intermediates at the expense of other TCA intermediates and biosynthetic processes. This may have important implications for energy generation during intestinal colonization. The putative OOR and POR complexes and the POR-associated flavodoxin also exhibited a relative down-regulation in 11168-O cultured under severe O2 limitation (Fig. 6B and D). Both enzymes are oxygen labile in other bacteria; if transcription of their genes is closely linked to protein levels in the cell, 11168-O may down-regulate these genes by sensing and responding either to their presence or to the O2 environment. Finally, several genes likely to operate in both directions of the gluconeogenesis-glycolysis pathway were preferentially up-regulated in 11168-O under microaerobic conditions (Fig. 5A). Glycolysis-gluconeogenesis and TCA cycle enzymes have been shown to participate in colonization and virulence in several other bacteria (47, 68, 69); as such, exploration of these pathways in C. jejuni should yield new insight into colonization requirements for this pathogen.
The chick colonization data presented in Fig. 1 also support our hypothesis that adaptation to different O2-containing environments is a crucial and heretofore unappreciated colonization determinant for C. jejuni. Previous work indicated that aerobically adapted C. jejuni can colonize mice; however, in that study, not every animal was colonized and colonization levels were not presented (35). In our experiments, repeated aerobic passage of 11168-O significantly attenuated its ability to colonize chicks, whereas anaerobically priming 11168-GS for 24 h prior to infection significantly increased its colonization potential (Fig. 1). The fact that neither strain could be induced to mimic the other completely in colonization potential indicates that the changes incurred at the genetic level as 11168-O became 11168-GS are more significant than can be rescued solely by preadapting each strain to a different oxygen tension. Indeed, targeted sequencing demonstrated that the 11168-GS variant has undergone genetic changes in all three sigma factors, possibly as a result of selective pressure (Table 3). The functional consequences of these changes remain to be established. However, these investigations will need to be closely monitored, since as the evidence in this study demonstrates, genetic manipulations to test the outcome of single amino acid changes in a highly plastic organism like C. jejuni may well invoke additional undocumented genetic alterations. Furthermore, given the abundance of differences between these variants and the relative frequency with which SNPs have already been identified, it is possible that other regulatory molecules such as two-component systems might also be altered, that specific promoter regions might have undergone change, and/or that a combination of SNPs are responsible for the transcriptional and phenotype variations presented. Significant further work will be required to dissect this issue.
As noted in Results, expression patterns for the sdh and frd operons are somewhat paradoxical given the abundance of other data suggesting that 11168-GS is better adapted for higher-O2 laboratory culture and 11168-O is better adapted for the very low-O2 environment of the intestinal tract. In E. coli, succinate dehydrogenase is only present in aerobically growing cells; during anaerobiosis, the sdh genes are strongly repressed by ArcA (32, 57) while fumarate reductase expression is enhanced by the fumarate nitrate reductase regulator FNR (63). In C. jejuni, literally nothing is known about the regulation and function of the sdh and frd genes. Several putative arcA-like two-component regulators are present in the C. jejuni genome, but a functional homolog has not yet been identified. One FNR-CRP (catabolite repressing protein) homolog is also present in the C. jejuni genome (Cj0466); however, its deletion did not impair in vitro survival under either microaerobic or severely O2-limited conditions (J. K. MacKichan and S. Falkow, unpublished data). Furthermore, although C. jejuni possesses fumarate reductase activity (67), and fumarate reductase is essential for H. pylori colonization of the mouse stomach (21), a C. jejuni frdA mutant was not defective for colonizing mice (J. K. MacKichan, S. Falkow, C. Chang, and J. Miller, unpublished data). It is also possible that the gene expression differences observed are due to the specific culture conditions employed in our experiments. Unraveling the regulation and function of the C. jejuni sdh and frd operons will require significant biochemical and transcriptional analyses and should, in the future, lend considerable insight into the roles of these very similar enzyme complexes in C. jejuni biology and colonization potential.
Another striking phenotype change in 11168-GS compared to 11168-O is the near-complete loss of motility. This is likely to contribute to the colonization and translocation differences observed as well as to the marked difference in invasion speed and efficiency, although 11168-O still invaded more efficiently than 11168-GS when the bacteria were centrifuged onto the monolayer (Table 1) (G. Manning and D. G. Newell, unpublished data). Phase variation in flagellar and other genes has previously been shown to affect C. jejuni motility and shape (11, 37, 46, 55, 56). However, all genes thus far identified as phase variable affect flagellar biosynthesis; as both 1168 variants have full-length polar flagella (Fig. 2) and similar flaA and flaB expression profiles, it seems unlikely that phase variation is responsible for the motility differences. Both the FliA and RpoN sigma factors have been implicated as important regulators of C. jejuni motility (23, 24, 27, 55, 75), and each contains 1 to 2 amino acid differences in the variants. Complete regulons for these sigma factors have not been identified for C. jejuni; however, several flagellar export genes shown to be part of the H. pylori RpoN regulon (36) were identified in our experiments as exhibiting significantly higher expression in 11168-O cultured under severely O2-limited conditions than in any other samples (Fig. 6A). As noted in Results, this cluster also contains several uncharacterized genes that, by virtue of their coexpression with known motility-related genes, might lend insight into their function. For instance, Cj1631c contains a likely calcium-binding domain, and Cj0604 is a likely polyphosphate kinase. Both calcium and inorganic polyphosphate are known to participate in motility in other bacteria (50, 61, 70), but their roles in C. jejuni have not yet been established. It will also be interesting to explore the function of genes with no database homologies, such as Cj1464 and Cj1465, and genes that are strikingly coexpressed with them, such as Cj0056c, in the context of their coexpression with known motility genes.
Is there a connection between the motility and morphology phenotypes and respiration and metabolism gene expression differences between these variants? Mechanical energy for flagellar rotation is provided by a proton motive force across the membrane that can be generated by the electron transport chain. Many genes exhibiting expression differences between the variants in our study are involved in metabolism and respiration; at least one, Cj0358, has been directly implicated in energy generation via the electron transport chain (64). Furthermore, in 11168-O, up-regulation of flagellar export genes occurred specifically under severely O2-limited conditions, suggesting that a low-oxygen environment may trigger these expression changes. It was recently found that the aerobically passaged 11168-O strain is, like 11168-GS, both markedly attenuated for motility and appears as a straight rod by electron microscopy (Manning and Newell, unpublished). Preliminary data also suggest that severe O2 limitation enhances 11168-O motility and can induce a small fraction of 11168-GS within a population to become motile (Gaynor and Falkow, unpublished). These observations highlight the remarkable vertical plasticity of C. jejuni and are consistent with the existence of intermediate NCTC 11168 variants such as the motile strain used by Jagannathan et al. for motility studies (33). We are currently pursuing the intriguing possibility of a genetic link between adaptation to different oxygen tensions, morphological change, and motility in C. jejuni and are exploring these phenomena in a controlled stepwise evolutionary manner by assaying phenotype and transcriptional profiles as adaptation to a higher O2 tension occurs. Studies such as these may also lend insight into whether genes exhibiting significantly higher expression in 11168-GS, such as a putative cytochrome c (Cj0037c) and coexpressed periplasmic and membrane-bound genes, might functionally interact to enhance the growth capacity of C. jejuni in laboratory and/or higher-O2 conditions.
In summary, C. jejuni is a remarkably malleable zoonotic bacterium that, despite its stringent in vitro growth conditions, is able to survive a diverse range of ecological niches. As it adapts to new environments, it is also likely to undergo some degree of genetic evolution, resulting in changes in gene expression and phenotype to accommodate its surroundings. Evidence of its large-scale genetic plasticity has recently been provided by several whole-genome DNA comparisons (13, 44), which demonstrated that different C. jejuni isolates exhibit marked variation in genes modifying the capsule, lipooligosaccharide, and flagella. One interpretation of those studies is that a possible defense mechanism for C. jejuni may be a constant remodeling of surface structures, via genome evolution, selective pressure, and/or natural selection, to survive a variety of natural host environments and bottlenecks. Here, we have used whole-genome approaches, only recently made accessible by virtue of the 11168 genome sequence and advances such as microarray technology, to gain insight into how and why two supposedly isogenic C. jejuni strains, one of which descended directly from the other via clonal propagation, might exhibit such different colonization and other virulence-related phenotypes. Unlike the studies mentioned above, these strains do not contain large variant regions of the genome or genes present in one strain but not the other, nor are the differences observed in surface structure genes. Rather, these strains contain very subtle genetic changes, detectable only by direct sequencing, but which clearly engender marked differences in transcription (primarily affecting respiration, metabolism, motility, and unknown genes) and phenotype. Because the 11168 variants are likely clonal, one important outcome of this study is that the NCTC 11168 genome sequence can be considered an accurate scaffold for exploring colonization and other virulence-associated phenotypes, especially if used in conjunction with the 11168-O variant. Furthermore, the use of microarray technology to explore not only genomic but also gene expression differences between isolates (or indeed clones) should be considered a powerful new tool for identifying possible mechanisms underlying phenotypic variation or change. Finally, this work underscores the need to sequence multiple isolates of the same pathogenic bacterial species and to appreciate that even inadvertent in vitro manipulations have the potential for altering the genome and biology of pathogens studied in the laboratory.
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ACKNOWLEDGMENTS |
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E.C.G. is funded by the Life Sciences Research Foundation and the Burroughs Wellcome Fund, S.F. is funded by grants from the NIH, and G.M., S.C., and D.G.N. are funded by the Department of Environmental Food and Rural Affairs (DEFRA), United Kingdom.
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FOOTNOTES |
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E.C.G., S.C., and G.M. contributed equally to the work.
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