M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsono-phagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp 4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.     

Characterization of the argA gene required for arginine biosynthesis and syringomycin production by Pseudomonas syringae pv. syringae

Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.      

Streptococcus iniae M-like protein contributes to virulence in fish and is a target for live attenuated vaccine development

Background

Streptococcus iniae is a significant pathogen in finfish aquaculture, though knowledge of virulence determinants is lacking. Through pyrosequencing of the S. iniae genome we have identified two gene homologues to classical surface-anchored streptococcal virulence factors: M-like protein (simA) and C5a peptidase (scpI).

Methodology/Principal Findings

S. iniae possesses a Mga-like locus containing simA and a divergently transcribed putative mga-like regulatory gene, mgx. In contrast to the Mga locus of group A Streptococcus (GAS, S. pyogenes), scpI is located distally in the chromosome. Comparative sequence analysis of the Mgx locus revealed only one significant variant, a strain with an insertion frameshift mutation in simA and a deletion mutation in a region downstream of mgx, generating an ORF which may encode a second putative mga-like gene, mgx2. Allelic exchange mutagenesis of simA and scpI was employed to investigate the potential role of these genes in S. iniae virulence. Our hybrid striped bass (HSB) and zebrafish models of infection revealed that M-like protein contributes significantly to S. iniae pathogenesis whereas C5a peptidase-like protein does not. Further, in vitro cell-based analyses indicate that SiMA, like other M family proteins, contributes to cellular adherence and invasion and provides resistance to phagocytic killing. Attenuation in our virulence models was also observed in the S. iniae isolate possessing a natural simA mutation. Vaccination of HSB with the ΔsimA mutant provided 100% protection against subsequent challenge with a lethal dose of wild-type (WT) S. iniae after 1,400 degree days, and shows promise as a target for live attenuated vaccine development.

Conclusions/Significance

Analysis of M-like protein and C5a peptidase through allelic replacement revealed that M-like protein plays a significant role in S. iniae virulence, and the Mga-like locus, which may regulate expression of this gene, has an unusual arrangement. The M-like protein mutant created in this research holds promise as live-attenuated vaccine.     

Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection

The pathogen group A Streptococcus (GAS) produces a wide spectrum of infections including necrotizing fasciitis (NF). Streptolysin S (SLS) produces the hallmark beta-haemolytic phenotype produced by GAS. The nine-gene GAS locus (sagA-sagI) resembling a bacteriocin biosynthetic operon is necessary and sufficient for SLS production. Using precise, in-frame allelic exchange mutagenesis and single-gene complementation, we show sagA, sagB, sagC, sagD, sagE, sagF and sagG are each individually required for SLS production, and that sagE may further serve an immunity function. Limited site-directed mutagenesis of specific amino acids in the SagA prepropeptide supports the designation of SLS as a bacteriocin-like toxin. No significant pleotrophic effects of sagA deletion were observed on M protein, capsule or cysteine protease production. In a murine model of NF, the SLS-negative M1T1 GAS mutant was markedly diminished in its ability to produce necrotic skin ulcers and spread to the systemic circulation. The SLS toxin impaired phagocytic clearance and promoted epithelial cell cytotoxicity, the latter phenotype being enhanced by the effects of M protein and streptolysin O. We conclude that all genetic components of the sag operon are required for expression of functional SLS, an important virulence factor in the pathogenesis of invasive M1T1 GAS infection.     

Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci

Primary infection of the human host by group A streptococci (GAS) most often involves either the epidermis of the skin or the oropharyngeal mucosa. A humanized in vivo model for impetigo was used to investigate the basis for host tissue tropism among GAS. Disruption of the speB gene (encoding for a secreted cysteine proteinase) led to a loss of virulence for two impetigo-derived strains (M-types 33 and 53), as evidenced by a diminution in tissue damage and a lack of reproductive growth. The level of cysteine proteinase activity in overnight cultures was associated with the extent of gross pathological changes induced by strains displaying varied degrees of virulence in the impetigo model. Moreover, high levels of secreted cysteine proteinase activity correlated with a genetic marker for preferred tissue site of infection at the skin (emm pattern D). The addition of exogenous SpeB to a speB mutant (emm pattern D) or to an avirulent throat-like strain (emm pattern A) led to increased bacterial reproduction at the skin. The data provide both experimental and epidemiological evidence for a critical role of a secreted bacterial protease in promoting host tissue-specific infection.     

Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes

The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.     

Two DNA-binding domains of Mga are required for virulence gene activation in the group A streptococcus

Mga is a DNA-binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix-turn-helix DNA-binding motifs (HTH1-HTH4) identified within the amino-terminus of Mga, alanine substitutions were introduced within each domain in a MBP-Mga fusion allele and purified proteins were assayed for binding to Mga-specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH-1 and HTH-2 mutations showed wild type DNA-binding activity, an altered HTH-3 domain resulted in reduced binding to the three promoters and an HTH-4 mutant was devoid of detectable binding activity. Plasmid-encoded expression of the HTH-3 and HTH-4 alleles from a constitutive promoter (Pspac) in the mga-deleted GAS strain JRS519 demonstrated that Mga-regulated emm expression correlated directly to the DNA-binding activity observed for each mutant protein in vitro. Single-copy expression of HTH-3 and HTH-4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA-binding domains (HTH-3 and HTH-4) that are required for direct activation of the Mga virulence regulon in vivo.