Craig T. Parker

Major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species.

Sequencing and comparative genome analysis of four strains of Campylobacter including C. lari RM2100, C. upsaliensis RM3195, and C. coli RM2228 has revealed major structural differences that are associated with the insertion of phage- and plasmid-like genomic islands, as well as major variations in the lipooligosaccharide complex. Poly G tracts are longer, are greater in number, and show greater variability in C. upsaliensis than in the other species. Many genes involved in host colonization, including racR/S, cadF, cdt, ciaB, and flagellin genes, are conserved across the species, but variations that appear to be species specific are evident for a lipooligosaccharide locus, a capsular (extracellular) polysaccharide locus, and a novel Campylobacter putative licABCD virulence locus. The strains also vary in their metabolic profiles, as well as their resistance profiles to a range of antibiotics. It is evident that the newly identified hypothetical and conserved hypothetical proteins, as well as uncharacterized two-component regulatory systems and membrane proteins, may hold additional significant information on the major differences in virulence among the species, as well as the specificity of the strains for particular hosts.

Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA.

ABSTRACT We describe a multiplex PCR assay to identify and discriminate between isolates of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis. The C. jejuni isolate F38011 lpxA gene, encoding a UDP-N-acetylglucosamine acyltransferase, was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 [lpxA(Ts)]. With oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA amplicons were amplified from an additional 11 isolates of C. jejuni, 20 isolates of C. coli, 16 isolates of C. lari, and five isolates of C. upsaliensis. The nucleotide sequence of each amplicon was determined, and sequence alignment revealed a high level of species discrimination. Oligonucleotide primers were constructed to exploit species differences, and a multiplex PCR assay was developed to positively identify isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis. We characterized an additional set of 41 thermotolerant isolates by partial nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The multiplex PCR assay was validated with 105 genetically defined isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis, 34 strains representing 12 additional Campylobacter species, and 24 strains representing 19 non-Campylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with biochemical typing methods.

Comparative genomic analysis of Campylobacter jejuni strains reveals diversity due to genomic elements similar to those present in C. jejuni strain RM1221.

ABSTRACT Analysis of the complete genomic sequence of Campylobacter jejuni strain RM1221 identified four large genomic elements, Campylobacter jejuni-integrated elements (CJIEs), that were absent from C. jejuni strain NCTC 11168. To further investigate the genomic diversity of Campylobacter, we conducted a comparative genomic analysis from a collection of 67 C. jejuni and 12 Campylobacter coli strains isolated from various geographical locations and clinical and veterinary sources. Utilizing PCR, we demonstrated that 55% of the C. jejuni strains examined were positive for at least one RM1221-like genomic element and 27% were positive for two or more of these CJIEs. Furthermore, many C. coli strains were positive for either genomic element CJIE1 or CJIE3. To simultaneously assess for the presence or absence of several genes that comprise the various CJIEs, we developed a multistrain C. jejuni DNA microarray that contained most of the putative coding sequences for strains NCTC 11168 and RM1221. A comparative genomic hybridization (CGH) analysis of 35 of the 67 C. jejuni strains confirmed the presence of genomic elements similar to those in strain RM1221. Interestingly, the DNA microarray analysis demonstrated that these genomic elements in the other C. jejuni strains often exhibited modular patterns with some regions of the CJIEs present and other regions either absent or highly divergent compared to strain RM1221. Our CGH method also identified 18 other intraspecies hypervariable regions, such as the capsule and lipooligosaccharide biosynthesis regions. Thus, the inclusion of genes from these integrated genomic elements and the genes from the other intraspecies hypervariable regions contributes to a better assessment of the diversity in C. jejuni and may increase the usefulness of DNA microarrays as an epidemiological genotyping tool. Finally, we also showed that in CJIE1, a Campylobacter Mu-like phage, is located differentially in other strains of C. jejuni, suggesting that it may integrate essentially randomly.

Comparison of Campylobacter jejuni Lipooligosaccharide Biosynthesis Loci from a Variety of Sources

ABSTRACT Campylobacter jejuni strains exhibit significant variation in the genetic content of the lipooligosaccharide (LOS) biosynthesis loci with concomitant differences in LOS structure. The C. jejuni LOS loci have been grouped into six classes based on gene content and organization. Utilizing PCR amplifications of genes from these loci, we were able to classify a majority (80%) of the LOS biosynthesis loci from 123 strains of C. jejuni that included 39 of the Penner serotype reference strains. We found that a particular LOS class was not always associated with a specific Penner serotype, and 14 of 16 Guillain-Barré syndrome-associated isolates tested in this study shared the same LOS class. The remaining isolates that could not be classified were often distinguishable from each other based on the results of gene-specific PCR and the lengths of their LOS biosynthesis loci as determined by long (XL) PCR. Sequence analysis of two of these unique XL PCR products demonstrated two new LOS classes. These results support the hypothesis that the LOS locus is a hot spot for genetic exchange and rearrangements. Analysis of the LOS biosynthesis genes by PCR assays can be used for typing C. jejuni and offers the advantage of inferring potential LOS structures.

Transcriptome Analysis of Escherichia coli O157:H7 Exposed to Lysates of Lettuce Leaves

ABSTRACT Harvesting and processing of leafy greens inherently cause plant tissue damage, creating niches on leaves that human pathogens can exploit. We previously demonstrated that Escherichia coli O157:H7 (EcO157) multiplies more rapidly on shredded leaves than on intact leaves (M. T. Brandl, Appl. Environ. Microbiol. 74:5285-5289, 2008). To investigate how EcO157 cells adapt to physicochemical conditions in injured lettuce tissue, we used microarray-based whole-genome transcriptional profiling to characterize gene expression patterns in EcO157 after 15- and 30-min exposures to romaine lettuce lysates. Multiple carbohydrate transport systems that have a role in the utilization of substrates known to be prevalent in plant cells were activated in EcO157. This indicates the availability to the human pathogen of a variety of carbohydrates released from injured plant cells that may promote its extensive growth in leaf lysates and, thus, in wounded leaf tissue. In addition, microarray analysis revealed the upregulation of numerous genes associated with EcO157 attachment and virulence, with oxidative stress and antimicrobial resistance (including the OxyR and Mar regulons), with detoxification of noxious compounds, and with DNA repair. Upregulation of oxidative stress and antimicrobial resistance genes in EcO157 was confirmed on shredded lettuce by quantitative reverse transcription-PCR. We further demonstrate that this adaptation to stress conditions imparts the pathogen with increased resistance to hydrogen peroxide and calcium hypochlorite. This enhanced resistance to chlorinated sanitizers combined with increased expression of virulence determinants and multiplication at sites of injury on the leaves may help explain the association of processed leafy greens with outbreaks of EcO157.

Culture of Campylobacter jejuni with Sodium Deoxycholate Induces Virulence Gene Expression

Campylobacter jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human food-borne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion, as shown by gentamicin protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC secreted the Cia proteins, as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene, as determined by beta-galactosidase reporter, real-time reverse transcription-PCR, and microarray analyses. Microarray analysis further revealed that DOC induced the expression of virulence genes (ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrated that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation for identifying genes expressed by C. jejuni in response to in vivo-like culture conditions.

Characterization of Genetically Matched Isolates of Campylobacter jejuni Reveals that Mutations in Genes Involved in Flagellar Biosynthesis Alter the Organism's Virulence Potential

ABSTRACT Phenotypic and genotypic evidence suggests that not all Campylobacter jejuni isolates are pathogenic for humans. We hypothesized that differences in gene content or gene expression alter the degree of pathogenicity of C. jejuni isolates. A C. jejuni isolate (Turkey) recovered from a turkey and a second C. jejuni isolate (CS) recovered from a chicken differed in their degrees of in vitro and in vivo virulence. The C. jejuni Turkey isolate invaded INT 407 human epithelial cells and secreted the Cia (Campylobacter invasion antigen) proteins, while the C. jejuni CS isolate was noninvasive for human epithelial cells and did not secrete the Cia proteins. Newborn piglets inoculated with the C. jejuni Turkey isolate developed more severe clinical signs of campylobacteriosis than piglets inoculated with the C. jejuni CS isolate. Additional work revealed that flagellin was not expressed in the C. jejuni CS isolate. Microarray and real-time reverse transcription-PCR analyses revealed that all flagellar class II genes were significantly downregulated in the C. jejuni CS isolate compared to the C. jejuni Turkey isolate. Finally, nucleotide sequencing of the flgR gene revealed the presence of a single residue that was different in the FlgR proteins of the C. jejuni Turkey and CS isolates. Complementation of the C. jejuni CS isolate with a wild-type copy of the flgR gene restored the isolates motility. Collectively, these findings support the hypothesis that critical differences in gene content or gene expression can alter the pathogenic potential of C. jejuni isolates.

Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays

ABSTRACT To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter and the presence of Campylobacter in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of probe specificity; the signal intensities detected for A. butzleri, C. coli, or C. jejuni probes were at least 10-fold higher than the background levels. Specific identification of A. butzleri, C. coli, and C. jejuni was achieved without the need for a PCR amplification step. By adapting an isolation method that employed membrane filtration and selective media, C. jejuni isolates were recovered from package liquid from whole chicken carcasses prior to enrichment. Increasing the time of enrichment resulted in the isolation of A. butzleri and increased the recovery of C. jejuni. C. jejuni isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of the C. jejuni isolates likely possess class B, C, or H LOS. Validation experiments demonstrated that the DNA microarray had a detection sensitivity threshold of approximately 10,000 C. jejuni cells. Interestingly, the use of C. jejuni sequence-specific primers to label genomic DNA improved the sensitivity of this DNA microarray for detection of C. jejuni in whole chicken carcass samples. C. jejuni was efficiently detected directly both in package liquid from whole chicken carcasses and in enrichment broths.

The Salmonella Transcriptome in Lettuce and Cilantro Soft Rot Reveals a Niche Overlap with the Animal Host Intestine

ABSTRACT Fresh vegetables have been recurrently associated with salmonellosis outbreaks, and Salmonella contamination of retail produce has been correlated positively with the presence of soft rot disease. We observed that population sizes of Salmonella enterica serovar Typhimurium SL1344 increased 56-fold when inoculated alone onto cilantro leaves, versus 2,884-fold when coinoculated with Dickeya dadantii, a prevalent pathogen that macerates plant tissue. A similar trend in S. enterica populations was observed for soft-rotted lettuce leaves. Transcriptome analysis of S. enterica cells that colonized D. dadantii-infected lettuce and cilantro leaves revealed a clear shift toward anaerobic metabolism and catabolism of substrates that are available due to the degradation of plant cells by the pectinolytic pathogen. Twenty-nine percent of the genes that were upregulated in cilantro macerates were also previously observed to have increased expression levels in the chicken intestine. Furthermore, multiple genes induced in soft rot lesions are also involved in the colonization of mouse, pig, and bovine models of host infection. Among those genes, the operons for ethanolamine and propanediol utilization as well as for the synthesis of cobalamin, a cofactor in these pathways, were the most highly upregulated genes in lettuce and cilantro lesions. In S. Typhimurium strain LT2, population sizes of mutants deficient in propanediol utilization or cobalamin synthesis were 10- and 3-fold lower, respectively, than those of the wild-type strain in macerated cilantro (P < 0.0002); in strain SL1344, such mutants behaved similarly to the parental strain. Anaerobic conditions and the utilization of nutrients in macerated plant tissue that are also present in the animal intestine indicate a niche overlap that may explain the high level of adaptation of S. enterica to soft rot lesions, a common postharvest plant disease.

The Campylobacter jejuni PhosS/PhosR operon represents a non-classical phosphate-sensitive two-component system

The bacterial pathogen Campylobacter jejuni carries several putative two‐component signal transduction systems of unknown function. Here we report that the PhosS (Cj0889) and PhosR (Cj0890) proteins constitute a two‐component system that is activated by phosphate limitation. Microarray analysis, real‐time RT‐PCR, and primer extension experiments indicated that this system regulates 12 genes (including the pstSCAB genes) present in three transcriptional units. Gel shift assays confirmed that recombinant PhosR protein bound DNA fragments containing the promoter regions upstream of these three transcriptional units. Although functionally similar, the PhosS/PhosR does not exhibit sequence homology with the classical PhoBR systems, has a different pho box (5′‐GTTTCNAAAANGTTTC‐3′) recognized by the C. jejuni response regulator, and is not autoregulated. Because of these atypical properties, we designated the Cj0889‐Cj0890 operon as the C. jejuni PhosS/PhosR system (phosphate sensor/phosphate response regulator) and the phosphate‐regulated genes as the pho regulon of C. jejuni.