Streptococcus iniae M-Like Protein Contributes to Virulence in Fish and Is a Target for Live Attenuated Vaccine Development

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.

The novel polysaccharide deacetylase homologue Pdi contributes to virulence of the aquatic pathogen Streptococcus iniae

“…The analysis and study of mutants from this library, along with candidate gene approaches, has previously served to elucidate virulence factors contributing to S. iniae pathogenicity in fish, including phosphoglucomutase (Buchanan et al, 2005), streptolysin S (Fuller et al, 2002;Locke et al, 2007a) and capsular polysaccharide (Barnes et al, 2003;Locke et al, 2007a;Lowe et al, 2007;Miller & Neely, 2005). In addition, recent whole-genome pyrosequencing of S. iniae strain K288 has uncovered an mga-like pathogenicity locus (Locke et al, 2008), which includes an M-like protein virulence factor similar to the fibrinogen-binding proteins discovered in S. iniae strains QMA0076 and QMA0131 (Baiano et al, 2008).…”

“…Contiguous sequences used for ORF determination and bioinformatics analysis were created from an automated assembly of pyrosequencing results (454 Life Sciences, Roche). This primary assembly was generated by the Phred/Phrap/Consed suite (Gordon et al, 1998) and resulted in 1865 contigs ranging in size from 51 bp to 22 kb (Locke et al, 2008). Without further assembly, the 1865 contigs were used for building a local S. iniae K288 BLAST database, and then a local copy of BLAST version 2.2.14 (Altschul et al, 1997) was used for finding the contiguous sequences matching the pdi-deficient transposon mutant, TnM7, in S. iniae K288.…”

“…We termed this putative gene and its predicted product pdi and Pdi, respectively. The full sequence and chromosomal context of pdi were identified using BLASTN and a genomic library of contiguous sequence data generated from the pyrosequencing of the parent S. iniae strain K288 genome (Locke et al, 2008). …”

Comparative genome analysis of Streptococcus iniae DX09 reveals new insights into niche adaptation and competitive host colonisation ability

“…To date, S. iniae has been characterized as a global zoonotic pathogen that mainly infects a broad range of marine and freshwater fish, including bream, trout, tilapia, salmon, barramundi, yellowtail, Japanese flounder, hybrid striped bass, channel catfish and Amazon dolphin [2,3]. The common histopathologic characteristics of infected fish are septicaemia and meningitis, resulting in high mortality in farmed fish and enormous economic losses in aquaculture [3].…”

“…The pathogen could also opportunistically infect elderly humans associated with the handling and preparation of infected fish [4]. While several studies have been conducted on S. iniae, attention to date has mainly focused on the so-called virulence factors SiM protein [2,5]. interleukin-8 protease [6], streptolysin S [7].…”