Pathogenesis of Campylobacter fetus
Campylobacter fetus is traditionally recognized as a significant pathogen of livestock. Its occurrence as an opportunistic human pathogen is uncommon, yet this is believed to be underestimated and is apparently increasing (Blaser, 1998; Blaser et al., 2008). Campylobacter fetus belongs to the group of ε-proteobacteria and is highly adapted to mucosal surfaces (Hu and Kopecko, 2000). Two subspecies of C. fetus have been designated, C. fetus ssp. fetus and C. fetus ssp. venerealis. Nearly all C. fetus infections in humans are systemic and arise due to C. fetus ssp. fetus. This subspecies is the predominant Campylobacter species isolated from human blood (Blaser, 1998) and is considered to be an emerging pathogen placing infants, elderly, immunocompromised and debilitated persons at risk (Skirrow and Blaser, 2000; Thompson and Blaser, 2000). Both C. fetus ssp. fetus and C. fetus ssp. venerealis cause disease in cattle. The subspecies show distinct niche preferences, yet these are not strictly exclusive. In ruminants C. fetus ssp. venerealis colonizes the genital tract while C. fetus ssp. fetus is largely confined to the gut. However, both subspecies can be recovered from the genital tract and are a major cause of abortion and infertility causing substantial losses in bovine, ovine and caprine herds worldwide. Despite this pathogen's global economic and rising clinical significance, the molecular mechanisms underlying infection of its human and animal hosts remain largely unknown. Until very recently, the absence of genetic tools to manipulate C. fetus (Kienesberger et al., 2007) and the lack of tractable infection models to investigate the molecular basis of host–pathogen interactions has slowed research with this organism. In this review we describe recent progress in molecular approaches applicable in the study of C. fetus.
Genome analyses and the mobile gene pool
The first complete genome sequence of a Campylobacter species, the C. jejuni clinical isolate NCTC 11168, was published in 2000 (Parkhill et al., 2000). Up to now 11 complete and 24 unfinished genome sequences have been deposited in the public databases (Parkhill et al., 2000; Fouts et al., 2005; Pearson et al., 2007; Poly et al., 2007; Miller, 2008). Similar to other campylobacters the genome size of C. fetus is rather small (∼1.8 Mb). Genome sizing by pulsed field gel electrophoresis revealed size variation between strains (Salama et al., 1992). Multilocus sequence typing (MLST) is a procedure for characterizing isolates of bacterial species using the sequences of internal fragments of genes (Maiden et al., 1998; http://pubmlst.org/). Typically, seven housekeeping genes are used in this analysis. The profile of allele sequences for a given bacterial isolate defines the sequence type. The MLST analysis has shown a clonal population structure within C. fetus, wherein C. fetus ssp. venerealis represents a bovine clone (van Bergen et al., 2005; Dingle et al., 2010). This is in contrast to other Campylobacter species, which show extensive genetic variation (e.g. C. jejuni). This variability is thought to continuously improve the capacity to colonize and persist in various habitats (de Boer et al., 2002; Wiesner et al., 2003).
Current efforts to obtain complete genome sequences of representatives of both C. fetus subspecies are expected to bring rapid advances. The complete sequence of C. fetus ssp. fetus 82-40 was finished in 2006, revealing that 90% of the genome constitutes coding sequence (GenBank Accession number NC_008599). A draft genome sequence of C. fetus ssp. venerealis 84-112 has just become available (EBI Project ID: 42511). Access to both genome sequences provides the resources for detailed analysis of C. fetus physiology as well as subspecies-specific adaptations, and will stimulate efforts to identify mechanisms contributing to pathogen–host interactions and virulence. The comparative approach will certainly shed light on the niche preferences displayed by the C. fetus subspecies. In addition, the C. fetus sequences are expected to open new perspectives for subtyping methods as well as for improving or establishing novel diagnostic approaches for this emerging pathogen.
The process of horizontal gene transfer (HGT) in bacteria drives genetic diversity and evolution providing also a basis for variation in the virulence repertoire as well as resistance to antibiotics and host defences (Hacker et al., 1997; Gogarten et al., 2002; Koonin and Wolf, 2008; Boto, 2010; Juhas et al., 2009). DNA acquired by these mechanisms apparently accounts for 5–6% of the genome of certain campylobacters (Eppinger et al., 2004). Comparative genomic analysis is expected to reveal further species- or strain-variation due to evolutionary gene mobility. An initial comparison of the C. fetus genomes confirmed that C. fetus ssp. venerealis DNA presumably acquired by horizontal mechanisms indeed represents the major difference between the two subspecies.
An important technique in functional and comparative genomics is representational difference analysis (RDA). The methodology was developed to compare the differences in complex genomes as well as to obtain clones of those differentiating genes (Lisitsyn and Wigler, 1993). Before the availability of the C. fetus genome sequences RDA was applied to reveal genes uniquely or predominantly present in just one C. fetus subspecies (Gorkiewicz et al., 2010). Consistent with the hypothesis that distinguishing physiological and virulence properties of the subspecies relate to the existence of different sets of genes, homology searches revealed that multiple genes uniquely or predominantly associated with a given subspecies indeed encode virulence-related attributes ranging from motility to lipopolysaccharide production to bacterial secretion. A large genomic island unique to C. fetus ssp. venerealis was shown to encode a conjugation-related type IVa macromolecular secretion system (T4SS) (for system classifications see Christie and Vogel, 2000). Mutational analyses confirmed that the secretion machinery is involved in the ability of C. fetus ssp. venerealis to infect and induce cytopathic effects in cultured human epithelial and placenta cells (see below; Gorkiewicz et al., 2010). Moreover, the T4SS was shown to be active in intra- and interspecies conjugative mobilization of plasmid DNA (S. Kienesberger, G. Gorkiewicz, A. Fauster and E.L. Zechner, unpublished) derived from the cryptic C. coli plasmid pIP1455 (Lambert et al., 1985). This finding marks the first experimental demonstration of HGT in C. fetus. Evidence for natural transformation is still lacking (Blaser et al., 2008). Characterization of gene transfer by conjugation will certainly lead to the application of tools for DNA delivery among campylobacters (S. Kienesberger, G. Gorkiewicz, A. Fauster and E.L. Zechner, unpublished).
Knowledge of gene mobilization via conjugative mechanisms is also important in assessing the contribution of HGT to the evolution of the C. fetus subspecies and campylobacters generally. Conversely analysis of the genomes will provide insights to the presence and activities of clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, which have been identified in a variety of different bacteria including campylobacters (Miller, 2008). These hypervariable genetic loci capture incoming DNA acquired by multiple routes of HGT and provide sequence-directed immunity to invasive phage and plasmids (Marraffini and Sontheimer, 2008; Horvath and Barrangou, 2010). The CRISPR interference thus limits HGT. It is reasonable to predict that future studies will demonstrate a continued proficiency for mobility for some of the horizontally acquired elements in C. fetus as well as direct evidence for CRISPR-directed counteraction of lateral gene spread.