Comparative Genomic Analysis of Clinical Strains of Campylobacter jejuni from South Africa

BACKGROUND: Campylobacter jejuni is a common cause of acute gastroenteritis and is also associated with the post-infectious neuropathies, Guillain-Barré and Miller Fisher syndromes. In the Cape Town area of South Africa, C. jejuni strains with Penner heat-stable (HS) serotype HS:41 have been observed to be overrepresented among cases of Guillain-Barré syndrome. The present study examined the genetic content of a collection of 32 South African C. jejuni strains with different serotypes, including 13 HS:41 strains, that were recovered from patients with enteritis, Guillain-Barré or Miller Fisher syndromes. The sequence-based typing methods, multilocus sequence typing and DNA microarrays, were employed to potentially identify distinguishing features within the genomes of these C. jejuni strains with various disease outcomes. METHODOLOGY/PRINCIPAL FINDINGS: Comparative genomic analyses demonstrated that the HS:41 South African strains were clearly distinct from the other South African strains. Further DNA microarray analysis demonstrated that the HS:41 strains from South African patients with the Guillain-Barré syndrome or enteritis were highly similar in gene content. Interestingly, the South African HS:41 strains were distinct in gene content when compared to HS:41 strains from other geographical locations due to the presence of genomic islands, referred to as Campylobacter jejuni integrated elements (CJIEs). Only the integrated element CJIE1, a Campylobacter Mu-like prophage, was present in the South African HS:41 strains whereas this element was absent in two closely-related HS:41 strains from Mexico. A more distantly-related HS:41 strain from Canada possessed both integrated elements CJIE1 and CJIE2. CONCLUSION/SIGNIFICANCE: These findings demonstrate that CJIEs may contribute to the differentiation of closely-related C. jejuni strains. In addition, the presence of bacteriophage-related genes in CJIE1 may contribute to the genomic diversity of C. jejuni strains. This comparative genomic analysis of C. jejuni provides fundamental information that potentially could lead to improved methods for analyzing the epidemiology of disease outbreaks

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

Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni▿ †

The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in the absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA nonspecific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated elements 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modeling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophage-encoded DNA/RNA nonspecific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA

Patterns of presence, absence or divergence in the <i>Campylobacter jejuni</i>-integrated elements.

<p>A detailed genomic analysis of the genomic integrated elements CJIE1 (top) and CJIE2 (bottom) was compiled in GeneSpring version 7.3 with the standard correlation and bootstrapping (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#s2" target="_blank">Materials and Methods</a>). Each column corresponds to a <i>C. jejuni</i> strain designated vertically across the bottom, as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#pone-0002015-g002" target="_blank">Figure 2</a>. The gene status based on cutoff values of absence and presence predictions is shown color-coded: blue, present; light blue, divergent; red, highly divergent or absent; white, no data.</p

Dendrogram of <i>Campylobacter jejuni</i> sequence types, including clinical strains from South Africa, Mexico and Canada.

<p>The dendrogram was constructed by using the neighbor-joining algorithm and the Kimura two-parameter distance estimation method. Bootstrap values of >75%, generated from 500 replicates, are shown at the nodes. The scale bar represents substitutions per site.</p

Genome comparison of <i>Campylobacter jejuni</i> clinical strains by DNA microarrays analysis.

<p>An average linkage hierarchical clustering of the <i>C. jejuni</i> strains with a distance score scale bar was compiled in GeneSpring version 7.3 with the standard correlation and bootstrapping (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#s2" target="_blank">Materials and Methods</a>). The gene status based on cutoff values of absence and presence predictions is shown color-coded: blue, present; light blue, divergent; red, highly divergent or absent; white, no data. <i>C. jejuni</i> strains from South Africa with HS∶41 serotype (yellow), with other serotypes (green), or strains with HS∶41 serotype from Mexico and Canada (white) are designated vertically across the bottom. GBS-associated strains are annotated with stars; MFS-associated strains are annotated with diamonds. The four <i>C. jejuni</i>-integrated elements (CJIEs) and the assigned MLST sequence type (ST) for each strain cluster is indicated.</p

Intraspecies hypervariable regions in <i>Campylobacter jejuni</i>.

a<p>The start and end of each region is shown for genes in strain RM1221 (strain NCTC 11168) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#pone.0002015-Parker3" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#pone.0002015-Taboada1" target="_blank">[42]</a>.</p

MLST analysis of the <i>Campylobacter jejuni</i> strains used in this study.

a<p>Original strain number is designated in parenthesis.</p>b<p>Unk, unknown.</p>c<p>UK, United Kingdom; USA, United States of America; SA, South Africa; MEX, Mexico; CAN, Canada.</p>d<p>GBS, Guillain-Barré syndrome; MFS, Miller Fisher syndrome.</p>e<p>Penner heat-stable (HS) serotypes.</p

Patterns of presence, absence or divergence in the 18 hypervariable regions in <i>Campylobacter jejuni</i> strains.

<p>A detailed genomic analysis of the 18 hypervariable regions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#pone-0002015-t002" target="_blank">Table 2</a>) was compiled in GeneSpring version 7.3 with the standard correlation and bootstrapping (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#s2" target="_blank">Materials and Methods</a>). Each panel represents a hypervariable region, and each column corresponds to a <i>C. jejuni</i> strain designated vertically across the bottom, as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002015#pone-0002015-g002" target="_blank">Figure 2</a>. The gene status based on cutoff values of absence and presence predictions is shown color-coded: blue, present; light blue, divergent; red, highly divergent or absent; white, no data.</p