+ Determinant is present in a genome; - determinant is absent in a genome
+ Determinant is present in a genome; - determinant is absent in a genome majority of these genes are either of unknown function or they encode putative intestinal colonization factors, including ratB and the fimbrial genes lpfCDE, stbD, stfF, and stiB . Although ratB and stbD were scored as being absent in the microarray analysis, sequence analysis shows that these genes are present in the genomes of S. enterica serotypes Typhi and Paratyphi A (where ratB was identified as a pseudogene) [42-44]. Furthermore, sequence analysis shows that stfF is present in the genome of S. enterica serotype Paratyphi A .
Pseudogene formation comprises a second category of genetic differences between specialists and generalists that lead to loss of function mutations. Complete genome sequencing reveals that the generalist S. enterica serotype Typhimurium has far fewer pseudogenes than the host-restricted serotypes S. enterica serotypes Typhi and Paratyphi A. There are approximately 210 pseudogenes in the genome of S. enterica serotype Typhi (strains CT18 and Ty2) [42, 43] and 173 pseudogenes in the genome of S. enterica serotype Paratyphi A strain SARB42  compared to only 39 pseudogenes in the genome of S. enterica serotype Typhimurium strain LT2 . Of the 173 pseudogenes present in S. enterica serotype Paratyphi A, 166 have orthologues but only 28 are also pseudogenes in S. enterica serotype Typhi . One group of pseudogenes present in strictly human-adapted serotypes is formed by putative intestinal colonization factors, including autotransporter genes (misL, shdA, and sivH), fimbrial biosynthesis genes (fimI, csgF, sefAD, safD, bcfCF, stbC, stcC, steA, stfCF, stgC, and sthCE) and the intestinal colonization factor ratB (Table 6.1) [4, 32, 42-44]. These data show that genome degradation through deletion of genes or through pseudogene formation resulted in loss of putative adherence determinants in the genomes of strictly human-adapted serotypes. As a result, the genomes of S. enterica serotypes Typhi and Paratyphi A contain between 7 and 10 intact determinants, compared to 16 present in the genome of S. enterica serotype Typhimurium (Table 6.1). The only adherence determinants that are both present and intact in all three genomes of human-adapted serotypes are the fimbrial operons stdABCDE and tcfABCD. The extent of the genome degradation in the host specialist serotypes may correlate with increased specialization of these pathogens to one particular niche (i.e., host species) or, put another way, with the loss of ability of these serotypes to use multiple niches. Genome degradation may thus in part explain the host restriction to humans exhibited by S. enterica serotypes Typhi and Paratyphi A.
The majority of S. enterica subspecies I serotypes (i.e., nontyphoidal Salmonella serotypes) cause infections in humans that remain localized to the intestine and mesenteric lymph node and result in diarrhea. In contrast, S. enterica serotypes Typhi and Paratyphi A (i.e., typhoidal Salmonella serotypes) cause systemic infections in humans with diarrhea being an insignificant symptom [19, 62]. It is generally assumed that acquiring the ability to cause enteric fever (i.e., typhoid fever or paratyphoid fever) involved gain of function (i.e., acquisition of genes) by typhoidal Salmonella serotypes. Genomic analysis has thus focused on identifying a third category of genes, namely those that are present in the specialist genome but absent in the more generalist isolates. Phylogenetic analysis by multilocus enzyme electrophoresis shows that the ability to cause enteric fever evolved inde pendently in four lineages within S. enterica subspecies I, represented by S. enterica serotypes Typhi, Paratyphi A, Paratyphi B, and Paratyphi C . Recent analysis of S. enterica subspecies I serotypes using a S. enterica serotype Typhimurium LT2 microarray supplemented with open reading frames from S. enterica serotype Typhi CT18 did not identify any genes that are conserved among typhoidal serotypes but absent from all nontyphoidal serotypes . The absence of signature genes for typhoidal Salmonella serotypes may reflect the fact that the ability to cause enteric fever developed in four lineages independently, perhaps by acquisition of different genetic material in each lineage, which would suggest that sero-types of each lineage cause enteric fever by somewhat different mechanisms. It may thus be more revealing to identify gene acquisition events that accompanied the formation of an individual typhoidal Salmonella serotype by comparing its genome with that of nontyphoidal serotypes.
The genome of S. enterica serotype Typhi CT18 reveals 601 genes in 82 blocks that are unique compared to S. enterica serotype Typhimurium LT2 . Insertions unique to S. enterica serotype Typhi include four prophages (phages 10, 15, 18, 46), three pathogenicity islands (SPI7, SPI8, SPI10), four chaperone-usher fim-brial systems (staABACDEFG, tcfABCD, steABCDEF, stgABCDEF), a homologue of an E. coli hemolysin gene (STY1498), homologues of the Campylobacter toxin gene cdtB (STY1886), homologues of the Bordetella pertussis toxin genes ptxA and ptxB (STY1890 and STY1891), and a putative polysaccharide acetyltransferase gene (STY2629) [32, 42]. Unique genetic material in the S. enterica serotype Paratyphi A SARB42 genome includes three prophages, (SPA-1, SPA2554-2600, SPA-3-P2) and 12 other insertions containing two or more genes including the stkABCDEFG fimbrial operon . However, most unique insertions identified in the genomes of typhoidal serotypes by comparison with S. enterica serotype Typhi-murium LT2 are also present in genomes of at least some nontyphoidal Salmonella serotypes . These data suggest that the ability to cause enteric fever did not result from a single horizontal transfer event but may have required acquisition of a unique combination of new insertions in each lineage of typhoidal sero-types, including the incorporation of different genetic material into the genomes of S. enterica serotypes Typhi and Paratyphi A.
The final category of genomic difference between host generalist and host specialist serotypes of S. enterica subspecies I are large-scale genomic rearrangements. S. enterica serotype Paratyphi A (SARB42), for example, generally has conservation of orthologous gene order with respect to S. enterica serotype Typhimurium LT2. However, a recombination event between the rrnH and rrnG operons has inverted a large portion of the S. enterica serotype Paratyphi A genome with respect to the S. enterica serotype Typhimurium LT2 genome [44, 64]. A similar situation is apparent for S. enterica serotype Typhi TY2 and CT18, where overall the gene order is similar to that of S. enterica serotype Typhimurium, but genomic rearrangements have occurred around the rrn genes [42, 65]. Genome rearrangements due to homologous recombination between the rrn operons, resulting in translocations and inversions, are also observed in the genome of S. enterica serotype Gallinarum, a host-restricted pathogen consisting of two biotypes, Galli-
narum and Pullorum, that are strictly fowl-adapted [66, 67]. In contrast, the gene order in the S. enterica subspecies I genomes of host generalists is conserved with respect to that of S. enterica serotype Typhimurium LT2 . It is currently unknown why large genomic rearrangements are exclusively found in the genomes of host-restricted serotypes of S. enterica serotype subspecies I.
The above examples illustrate that the formation of host-restricted serotypes within S. enterica subspecies I was a complex process that involved numerous genetic changes whose significance has not been established, making it difficult to identifying the key events driving this evolutionary process. One strategy to gain further insight into the evolution of host-restricted pathogens has been the analysis of strains in which adaptation to one niche is incomplete, providing the opportunity to obtain a snapshot of genetic changes that can be observed when a host-restricted lineage is beginning to branch from that of its generalist relatives. A possible example of a pathogen with incomplete host restriction is the pigeon-associated variant of S. enterica serotype Typhimurium. While the majority of S. enterica serotype Typhimurium isolates (including the sequenced LT2 strain) are host generalists, one clone represented by the phage types DT2 and DT99 causes a systemic infection in pigeons but is rarely isolated from other host species . Host restriction in the pigeon-associated variant of S. enterica serotype Typhimurium is incomplete since it is still capable of causing disease in other host species, although virulence seems to be somewhat reduced compared to that of generalist isolates [69-71]. While genomic rearrangements are absent from the genomes of S. enterica serotype Typhimurium generalists and isolates of the phage type DT99, the genome of some S. enterica serotype Typhimurium DT2 isolates carry genomic inversions between rrn genes . Thus, the development of geno-mic rearrangements is still incomplete within the pigeon-associated variant of S. enterica serotype Typhimurium, suggesting that genomic rearrangements are a consequence rather than a cause of host restriction. Analysis of DT2 and DT99 isolates using a LT2 microarray identified no genes that were present in the genomes of S. enterica serotype Typhimurium generalists but absent from the specialist genome of the pigeon-associated variant, suggesting that genome degradation by loss of discrete genes is not a major driving force for the evolution of host restriction . Whether pseudogene formation or acquisition of genetic material by horizontal transfer has contributed to formation of the pigeon-associated variant of S. enterica serotype Typhimurium remains to be investigated.
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