Genomic comparisons between pathogens and related nonpathogenic relatives have played an important role in identifying the mechanisms responsible for acquisition of virulence in the natural environment [3
]. Through these analyses, a general picture is now emerging where different pathogens appear to have employed slightly different evolutionary mechanisms to develop virulence. For example, genomic comparisons between pathogenic and laboratory strains of E. coli
have revealed evidence of a common core chromosome interrupted by the horizontal introduction of multiple segments of virulence related genes (pathogenicity islands) [37
]. In contrast, the loss of ancestral genomic DNA may play an important role for generating virulence in Listeria
]. In the case of Yersinia pestis
, it has also been proposed that extensive chromosomal rearrangements and massive gene inactivation can also act as a driving force for pathogen evolution [5
In the case of Bp, our comparative analysis indicates gene mutation, gene deletion, and gene acquisition are likely to represent the major evolutionary drivers of Bp virulence, and that other proposed mechanisms of pathogen evolution, including chromosomal rearragement and bacteriophage-mediated recombination [5
] may thus a less relevant role in the pathogenic evolution of Bp. Our results are broadly consistent and support the findings of a previous study where genes found to be transcriptionally regulated in B. mallei
upon infection were compared to their counterparts in Bp and Bt [13
]. In that study, the investigators found that the three organisms all possessed the same genome structure of two chromosomes and high levels of conserved nucleotide identity. However, down-regulated genes, which were related to cell growth, were more well conserved while up-regulated potential virulence encoding genes were less well conserved or absent in Bt. Besides confirming these findings on a genome-wide scale, our study also possesses a number of novel features. Specifically, these include I) the discovery and validation of GIs in the Bt genome as genomic elements of lateral transfer, II) that unlike B. mallei
, the Bp and Bt genomes are highly syntenic, III) the increased divergence of virulence genes, especially those associated with Type III secretion, between Bp and Bt, IV) functional biases in inactivated genes for membrane-associated proteins in Bt and transcription factors in Bp, V) effects of species-specific genes on metabolism and virulence, and VI) evidence that the cis
-transcriptional regulatory machineries of Bp and Bt are likely to be broadly similar.
To develop as a successful pathogen, virulent bacteria need to evolve both offensive (eg. adherence, invasion, toxin, secretion systems) and defensive pathways (eg. antiphagocytosis, anti-proteolysis, phase variation, serum resistance). Bp and Bt share a large proportion of both offensive and defensive virulence factors (~71%), including adhesion factors, type IV pili, and two Type III secretion systems (TTS2 and TTS3). However, when treated collectively, these virulence genes appear to be significantly more divergent between Bp and Bt compared to the core metabolic genes or the rest of the genome. In contrast to this proteomic comparison, our analysis of the promoters of these genes failed to demonstrate an increased rate of divergence in cis-acting loci that might affect the transcriptional regulation of these genes. This result is unlikely to be caused by a lack of sensitivity in our comparison, as we were able to detect a significantly increased rate of conservation in the promoters of genes associated with core metabolism. Thus, at present, we favor the possibility that the cis-acting loci responsible for the regulation of these genes are likely to be fairly similar between Bp and Bt. However, we note that a different scenario may pertain to the trans-acting loci, since a significant enrichment of transcription factors appear to have been mutationally inactivated or altered in Bp. A close comparison of the transcriptomes of Bp and Bt, which is currently underway in our laboratory, should prove valuable in addressing this issue.
Our results also support key roles for large-scale gene loss, acqusition, and replacement in the development of Bp virulence. For example, both Bp and Bt share the TTS3 Type III secretion system, which is required for the full virulence of Bp in a hamster model of infection [42
]. However, it has been recently shown that arabinose exposure may downregulate TTS3 expression and activity [25
]. The absence of an arabinose assimilation operon in Bp might thus have contributed to the increased virulence of this species. Besides gene deletion, several gene clusters related to fimbriae and capsular polysaccharides synthesis have also been horizontally transferred to Bp, potentially contributing to the variation of surface components between the two organisms. It has long been recognized that bacterial surface components can play an indispensable role in the pathogenesis of infectious disease, and surface components of Bp may serve as virulence factors by playing a role in the attachment of bacteria to the host cell surface [43
]. One striking result from our analysis was our discovery that the polysaccharide capsule gene cluster, which has been shown to be an essential virulence determinant [29
], is likely to be non-randomly transferred into the Bp genome by replacing a pre-existing gene cluster in Bt already dedicated to polysaccharide synthesis. Polysaccharide coats play an important role in bacterial survival and persistence in the environment and evasion of host immune response, but may not constitute offensive attack. The fact that Bp has higher invasion, adherence capacity, and resistance to phagocytosis are thought to be related to the ability of Bp to produce exopolysaccharides [28
We conclude this report by noting that B. pseudomallei
is listed as a category B agent on the Centers for Disease Control Bioterrorism Agents/Diseases list [9
], and experimental manipulation of Bp is mandated by law in several countries to be conducted under biosafety level 3 (BSL3) laboratory requirements. As many centers in Bp-endemic areas do not have BSL3 facilities, this requirement has somewhat hampered the progress of research in basic Bp biology. By contrast, Bt is considered a risk group 1 agent, and although not considered clinically significant, Bt has been shown to be lethal to the model system C. elegans
]. Thus, although it is undoubtedly essential to be cautious when extrapolating findings from one species to another, the high degree of similarity between the Bt and Bp genomes raises the possibility that Bt could be used as a model system for studying certain aspects of Bp biology, similar to B. cereus
and B. anthracis
. The availability of an easily tractable experimental organism, which can be manipulated under standard laboratory conditions, could thus prove useful in accelerating research in the pathogenesis of melioidosis.