Abstract
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/SignificanceAnalysis 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.
Highlights
Streptococcus iniae is a significant finfish pathogen responsible for annual losses in aquaculture exceeding $100 million [1]
To date only three S. iniae virulence factors have been characterized in the context of fish virulence: the capsular polysaccharide which contributes to phagocyte resistance [5,6]; the cytolysin streptolysin S which contributes to host cell injury [7,8]; and phosphoglucomutase, which is required for cell wall rigidity and resistance to cationic antimicrobial peptides [9]
BLAST analysis groups SiMA closest to the S. uberis lactoferrin binding protein, Lbp (32% identity, 49% positive) [30] and the S. dysgalactiae subsp. dysgalactiae (GCS) M-like protein, DemA (31% identity, 51% positive) [31], though SiMA has near comparable similarity to a number of other streptococcal M family proteins (Fig. 1A)
Summary
Streptococcus iniae is a significant finfish pathogen responsible for annual losses in aquaculture exceeding $100 million [1]. There are no commercial vaccines approved for prevention of S. iniae infection in US aquaculture. Our understanding of S. iniae pathogenesis is limited. The identified S. iniae virulence determinant shared homology with counterparts expressed by other major streptococcal pathogens of humans and/ or animals. In an effort to identify additional genes involved in S. iniae pathogenesis, we have used pyrosequencing [10] 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)
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