Subspecies I Signature Genes

While the genomic features that differentiate Salmonella serotypes and their closest relative E. coli nicely fit the differences in metabolism and pathogenicity that have been historically described in the literature, the relationship between genetic and phenotypic differences within the species S. enterica are less clearly defined. The species S. enterica contains six subspecies, and all of these are capable of causing disease in mammals [1]. However, the overwhelming majority of cases of disease in mammals and birds are the result of infection with S. enterica subspecies I; the remaining subspecies are commonly isolated from cold-blooded animals [1, 37]. These findings suggest that genetic factors exclusive to S. enterica subspecies I may influence the epidemiological success of this subspecies among mammals and birds [38-40]. Understanding the relationship between the genetics and pathogenesis of S. enterica subspecies I is complicated by the fact that it contains 1454 serotypes [1], and there is considerable genetic diversity and diversity in disease syndromes and host range within this group [41]. Nevertheless, the completion of four genomes of S. enterica subspecies I serotypes [4, 42-44], with 12 more underway [45], and microarray analysis [5, 7, 46] have provided a list of genes exclusive to subspecies I, and there is preliminary information about the function of several of these genes.

Understanding the emergence of S. enterica subspecies I as the dominant subspecies infecting mammals and birds has been the subject of recent genomic analysis using microarrays [5]. This study revealed that 216 genes were gained by S. enterica subspecies I after its divergence from other subspecies. Of these genes, 128, or 59%, are currently unnamed, which illustrates that our understanding of factors that contribute to the epidemiological success of Salmonella serotypes is still in its infancy. Microarray analysis further revealed that 74 of these genes were present or possibly present across all S. enterica subspecies I strains tested and can thus be considered subspecies I signature genes. Subspecies I signature genes with known function or sequence homology can be grouped into three categories, as follows.

One group of subspecies I signature genes encode products that are located in the bacterial outer membrane. These include envF, sinI, yfeN, STM0280, STM2816, STM2423, and STM3026, whose products are predicted to be located in the outer membrane but whose function is poorly characterized [5, 47]. Furthermore, S. enterica subspecies I serotypes have gained three fimbrial gene clusters, including stfACDEFG, safABCD, and stcABCD [32, 48]. The saf operon encodes Salmonella atypical fimbria and is located on SPI6 [48], a DNA region that also contains the putative regulatory gene sinR [49]. The saf operon is present in the vast majority of S. enterica subspecies I isolates (195 of 198 tested), and deletion of safA or the sinR region does not result in virulence defects in genetically susceptible mice (BALB/c) [48, 49]. The effect of deleting the saf operon on long-term intestinal persistence has not been studied. In addition to fimbrial gene sequences, S. enterica subspecies I serotypes gained a nonfimbrial adhesin, termed ShdA [50]. The shdA gene encodes an outer membrane autotransporter protein that binds fibronectin and type I collagen on the bacterial surface [51, 52] and contributes to long-term intestinal persistence of S. enterica serotype Typhi-murium in genetically resistant mice (CBA) [53]. The shdA gene is located on the CS54 genetic island, adjacent to another subspecies I signature gene termed ratB [53]. Although its mode of action is not known, the product of the ratB gene has recently also been shown to be critical for intestinal persistence in a murine model of infection [53]. Based on mathematical models that combine epidemiology with population biology, persistent intestinal carriage is predicted to be a factor that will enhance the ability of a pathogen to circulate in populations of mammals and birds [50]. Acquisition of intestinal colonization factors such as shdA and ratB by S. enterica subspecies I may have contributed to the epidemiological success of this group of pathogens within populations of warm-blooded vertebrates.

A second group of subspecies I signature genes encode products that affect the properties of the bacterial cell surface. Microarray analysis suggests that the O-antigen biosynthesis (rfb) gene cluster of S. enterica subspecies I contains the subspecies I signature genes rfbP (wbaP), rfbK, rfbU, rffbI, rfbC, and rfbM (manC) [5]. The O-antigen comprises the portion of the bacterial lipopolysaccharide (LPS) that is exposed on the surface of the bacterium and is composed of repeating oli-

gosaccharide units (O-repeat). The rfbP gene encodes an enzyme with two functions: a galactosyltransferase function necessary for the first step of O-antigen synthesis, and a flippase function necessary for flipping the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic to the periplasmic face of the cytoplasmic membrane [54]. The product of the rfbK gene is important for completion of outer core synthesis and subsequent attachment of the O-antigen [55]. The rfbU gene encodes a mannosyl transferase that is necessary for the incorporation of GDPmannose into the oligosaccharide backbone of the O-antigen repeat unit [56]. The rfbC gene product is involved in production of an activated precursor (dTDP-L-rhamnose) necessary for the incorporation of rhamnose moieties into the oligosaccharide backbone of the O-antigen [57]. The rfbI gene encodes an enzyme involved in adding a dideoxyhexose branch to the oligosaccharide backbone of the O-antigen [57]. Finally, the product of the rfbM gene is required to form GDPmannose, the activated precursor necessary for the incorporation of mannose residues into the oligosaccharide backbone of the O-antigen [58]. Comparative sequence analysis of rfbM from Salmonella serotypes belonging to subspecies I, II, and VI shows that although this gene contains considerable nucleotide polymorphisms, it is not a subspecies I signature gene [59]. Thus, the list of 74 subspecies I signature genes identified by microarray analysis [5] may be further reduced as more information becomes available for individual genes.

Mutational analysis shows that subspecies I signature genes involved in O-anti-gen biosynthesis are required for host-pathogen interaction, because a S. enterica serotype Typhimurium rfbK mutant is rough and shows a reduced ability to colonize the intestine of 3-week-old chicks [55]. However, comparison of a bacterial strain expressing intact LPS with its isogenic derivative that is defective for O-anti-gen biosynthesis provides little insight into the selective advantage conferred by subspecies I signature genes located in the rfb gene cluster, because the difference between S. enterica subspecies I serotypes and serotypes belonging to other S. enterica subspecies is not the presence or absence of O-antigen, but the presence of O-antigen biosynthesis gene clusters that differ genetically. The biological significance of genetic differences between O-antigen biosynthesis gene clusters of S. enterica subspecies I and other S. enterica subspecies is not obvious, because most of the O-antigens used to distinguish Salmonella serotypes serologically occur in two or more subspecies. Since most O-antigens expressed by S. enterica subspecies I serotypes can also be detected in serotypes that do not belong to this subspecies, it is not clear which selective forces are responsible for the presence of subspecies I signature genes in the rfb gene cluster.

A third group of subspecies I signature genes encode those involved in transport and utilization of nutrients. These include subspecies I signature genes STM3251-STM3256 encoding components of a putative sugar phosphotransfer-ase transport system. Additional subspecies I signature genes of this group are located in the phn operon, which confers the ability to utilize phosphonate as a sole phosphorus source by mediating phosphonate transport and breakdown [60]. The phn operon is absent from E. coli but present in K. pneumoniae, and mutations in the S. enterica serotype Typhimurium phn operon do not seem to affect viru lence in mice [60]. The selective advantage conferred upon S. enterica subspecies I serotypes by possessing the ability to utilize the above nutrients remains to be identified.

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