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Fig. 8.1 GAS Genome alignments. The seven available GAS genomes were compared using the software package Mauve, a method that identifies conserved genomic regions, rearrangements and inversions in conserved regions, and the exact sequence breakpoints of such rearrangements across multiple genomes [11]. Corresponding regions share the same color and are connected by lines. Collinear regions are positioned above the central axis (relative to M1) while inverted regions are drawn below the axis. Light gray regions were too divergent in at least one genome to be meaningfully aligned. (This figure also appears with the color plates.)

Fig. 8.1 GAS Genome alignments. The seven available GAS genomes were compared using the software package Mauve, a method that identifies conserved genomic regions, rearrangements and inversions in conserved regions, and the exact sequence breakpoints of such rearrangements across multiple genomes [11]. Corresponding regions share the same color and are connected by lines. Collinear regions are positioned above the central axis (relative to M1) while inverted regions are drawn below the axis. Light gray regions were too divergent in at least one genome to be meaningfully aligned. (This figure also appears with the color plates.)

noted for emm, sic [14], scl [15], ska [16], speA [17], and speB [18]. This is especially the case with surface antigen genes such as emm, which has undergone significant gene duplication, recombination, and point mutations to form 158 different emm genes and the M family of genes [19]. These kinds of changes result in great genetic diversity of the GAS and represent a common survival strategy of micro-bial pathogens to avoid detection or to escape a host's immune system.

8.2.1.1.1 Virulence Factors

A comprehensive review of GAS virulence factors, including cell-associated and extracellular proteins as well as genes that regulate their expression, has been compiled by Hynes [20]. The genes for these virulence factors are either chromo-somally located or associated with a bacteriophage. Well-known and well-characterized virulence factor genes found on the bacterial chromosome include those for M protein, hyaluronic acid capsule, C5a peptidase, M-like proteins, fibronectin binding proteins, cysteine proteinase, streptolysin O, streptolysin S, streptokinase, CAMP factor, and hyaluronidases. Phage-associated GAS virulence factors (discussed below) include superantigens (pyrogenic exotoxins), DNases (streptodor-nases), and phospholipases.

More than 40 putative virulence factors were originally identified in the Ml genome, and since that time additional newly described virulence factors have been identified in the recently sequenced genomes: e.g., phospholipase A2 (Sla) [21], EndoS (endoglycosidase activity on IgG) [22], SpyA (ADP-ribosyltransferase) [23], Lbp (laminin binding protein [24], IdeS, immunoglobulin G degrading enzyme [25], and R6 surface protein [8]. Since approximately one-third of the genes in each genome remain to be characterized in terms of function, additional virulence factors will most certainly be identified in the future.

Approximately 50% of GAS strains obtained in population-based surveillance studies in the United States have the ability to opacify sera. These strains are said to be OF+ and contain the gene for serum opacity factor (sof) [26]. McShan and colleagues (personal communication) have recently sequenced an M49 strain, the first complete sequence of a GAS strain known to contain sof. The M49 genome contains only two complete bacteriophage genomes and, as in other strains, each phage has genes located near the insertion site which specify superantigens SpeH and MF3.

8.2.1.1.2 Horizontal Gene Transfer

Horizontal gene transfer mediated by bacteriophages is perhaps the most important phenomenon occurring in the GAS, where they have a major impact on pathogenicity as well as bacterial genome diversity and evolution. These bacterio-phages have been shown to contain genes termed "morons," because they add more DNA to the phage genome [27]. Virtually all of these bacteriophage-asso-ciated genes specify proteins with signal peptides essential for secretion into the extracellular environment where they may enhance the survival of the bacterial cell in that particular environment.

Phage-associated GAS virulence factors, many of which were unknown prior to genome sequencing, include SpeA, SpeC, SpeG, SpeH, Spel, SpeJ, SpeK, SpeL, SpeM, MF2. MF3, MF4, Sla, Ssa, Sda, Sdn, as well as HylP. Most of these factors are superantigens, also known as streptococcal pyrogenic exotoxins and erythro-genic toxins, which are potent activators of T cells that release cytokines and cause toxic shock (see Ref. [28]]). All of the streptodornases identified to date, with the exception of MF (SpeF or DNaseB), are phage encoded [29]. Proteomic analysis showed that these proteins shared a mosaic pattern, suggestive of multiple recombination events between phage virulence genes occurring during phage excision and integration. As in many of the chromosomal genes, considerable allelic variation exists in the associated virulence genes [13]. Taken together, these facts emphasize the important contribution phages make via horizontal gene transfer to the generation of new strains with increased virulence.

The primary mechanism of horizontal gene transfer among the GAS has been by bacteriophages, though it is clear that conjugative transfer of plasmids containing antibiotic resistance genes occurs [30]. Natural transformation of GAS has never been observed; however, Hanski and colleagues [31] discovered a streptococcal invasion locus (sil) that appears to be involved with DNA transfer in vitro. The sil locus is contained on one of two notable complex transposable elements that have been recently identified in GAS. The IS1562 transposon was found associated with many strains causing necrotizing fasciitis, and inactivation of this transposable element caused decreased lethality in a mouse model. Identified in an M14 strain, the transposon contains at least five genes (silA-E) that are highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two-component system whereas silD and silE encode two putative ABC transporters. silC is a small open reading frame (ORF) of unknown function preceded by a combox promoter [31]. The authors speculate that the sil locus may have been obtained by horizontal gene transfer since it has a lower G+C content (32%) than the 38.5% found in the Ml genome. Among sequenced GAS genomes, the sil locus is absent from the Ml, M3, and M5 genomes, but is found in the M18 and M49 genomes.

IS1562 has been suggested to be responsible for facilitating recombination events in GAS [32] and is also found in genome strains MGAS8232 and NZ131 (unpublished results). The genome of the M6 strain MGAS10394 contains a genetic element (MGAS10394.4) that confers macrolide resistance (mefA) [8]. This element is very closely related to Tn1207.3 [33], having a portion of another IS element incorporated into the distal end. MGAS10394 also has a gene encoding a novel surface-exposed protein that elicits an antibody response detectable in convalescent sera [8].

Thus, horizontal gene transfer, as well as an active recombination system, contributes to overall genome plasticity and GAS genetic diversity. Genome decay by the loss of genes and gene function is thought to counterbalance the acquisition of new sequences by horizontal gene transfer [34, 35], and both of these processes contribute to GAS evolution.

8.2.2.1 Streptococcus agalactiae

Streptococcus agalactiae, a group B streptococcus (GBS), is a human pathogen responsible for neonatal sepsis and meningitis, as well as for severe invasive disease in adults. Additionally, GBS are also animal pathogens responsible for bovine mastitis. Nine serotypes of GBS have been identified based on the differences in polysaccharide capsule [36]. The sequences of GBS serotypes III and V genomes [37, 38] have been completed, and the genome of a serotype Ia strain is in progress (Table8.2). The two complete genomes were approximately 2.2 Mbp in size with a G+C content of 35.6%. Although no complete bacteriophages were found in any of the sequenced genomes, bacteriophages have long been used in various GBS typing systems [39] and epidemiological studies [40-42].

The most prominent observation in the two genomes was a clustering of virulence factors in regions or islands encoding at least five contiguous genes and of sizes up to 81kbp. The majority of putative virulence factors were found in these islands, 14 islands in the type II strain and 15 in the type V strain, as well as mobile genetic element sequences such as phage and insertion sequences, transposons, and plasmids. Although the role of these sequences in gene acquisition is not clear because of extensive rearrangements among the islands, many of the virulence genes had G+C contents differing from that of the entire genome [38]. As in the GAS, there is a strong association of genetic transfer elements and virulence factors contributing to the common theme that pathogenicity islands play an important role in increased virulence and genetic diversity.

Both of the sequenced GBS strains contained a larger number of two-component regulatory systems than other streptococcal species, e.g., 17-21 in GBS vs 14 in S. pneumoniae, and 13 in both GAS and S.mutans. A number of other differences were also observed, including differences in metabolic pathways and transport systems, as well as a larger number of transcriptional regulators. These observations suggest that the GBS have a wider range of life style opportunities than other gram-positive streptococci, which allows them to survive in response to variations in the external environment.

A central question relates to the differences which exist in GBS strains that are preferentially associated with humans or animals. Spellerberg and colleagues [43] identified a putative composite transposon that contains a gene encoding the surface protein Lmb (mediates binding of GBS to human laminin) and ScpB (C5a peptidase), both factors which have been identified as important GBS virulence factors. All GBS strains of human origin, as well as group C and G strains isolated from humans, contained this putative composite transposon. However, the majority of GBS strains from animal origin contained little or none of the scpB or lmb sequences.

8.2.2.2 Group C (GCS) and Group G Streptococci

The streptococci of groups C and G are found in humans and animals, where they may cause serious diseases similar to those described for GAS disease (see Ref. [44] for a comprehensive review). These organisms include Streptococcus equi subsp. equi, Streptococcus dysgalactiae subsp. equisimilis, and Streptococcus equi subsp. zooepidemicus. These three species are "large" colony forming streptococci and resemble the GAS in many biochemical and clinical characteristics. Other, typically small colony forming streptococcal species can also carry the group C antigen, including Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus milleri [45]. Streptococcus canis is a member of the group G streptococci. The genome sequences of S. equi and S. zooepidemicus are in progress (Table8.2). GCS, in contrast to the other pathogenic streptococci, are remarkable in that they are commonly associated with both human and animal disease. S. equi is the causative agent of strangles, an acute upper respiratory infection of horses. S. equisimilis is the most common member of the GCS associated with human disease, causing pharyngitis, bacteremia, skin infections, and puerperal sepsis. Like GAS, GCS often carry genes encoding superantigens [46-48]. For example, evidence from the in-progress S. equi genome reveals that two streptococcal superantigen genes (speL and speM) are present; remarkably, these superantigens are also found in a subpopulation of GAS isolates (15% and 5%, respectively) [48]. The carriage of these genes by separate streptococcal species suggests that these genes are avail-

Fig. 8.2 GAS vs S. uberis alignment. The corresponding regions in the M1 and S. uberis genomes identified by Mauve analysis are shown. Several large regions ofcollinearity are evident between the two species. Corresponding regions share the same color and are connected by lines. (This figure also appears with the color plates.)

Fig. 8.2 GAS vs S. uberis alignment. The corresponding regions in the M1 and S. uberis genomes identified by Mauve analysis are shown. Several large regions ofcollinearity are evident between the two species. Corresponding regions share the same color and are connected by lines. (This figure also appears with the color plates.)

able for horizontal transfer, perhaps via a mobile element that enables gene transfer across phylogenetic borders.

8.2.2.3 Streptococcus uberis

The species Streptococcus uberis is heterogeneous with respect to Lancefield typing and is another important animal pathogen, which causes bovine mastitis. The genome sequence of this organism has just been completed (J. Leigh and P. Ward, personal communication) (Table8.2). S. uberis 0140J has a genome size of 1.85 Mbp and a G+C content of 36.6%. Several proposed virulence-associated genes have been characterized in S. uberis, including the capsule, neutrophil toxin, M-like protein, R-like protein, plasminogen activator PauA, and the CAMP factor [49-51]. Two substantial tracts of bacteriophage-associated DNA are present, as are paralogues of several superantigens found in GAS. The alignment of the S. uberis and GAS Ml genomes is shown in Fig. 8.2, where several rearrangements are apparent, but with substantial homology in the overall genome order between the two genomes, suggesting a close evolutionary relatedness between the two organisms. As with several of the other streptococci that infect animals of agricultural importance, this genetic relatedness with GAS may provide a pool of genes that could be horizontally transferred from the animal to the human pathogen in situations where humans and animals are in close contact, such as on farms or in food processing facilities.

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