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

Locke JB, Aziz RK, Vicknair MR, Nizet V, Buchanan JT - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

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 Delta simA 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.

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Bioinformatic analysis of SiMA.(A) Phylogenetic clustering of SiMA shows greatest similarity to other streptococcal M family proteins, most closely the S. uberis lactoferrin binding protein. (B) Amino acid sequence alignments of SiMA with other streptococcal M family proteins shows highest conservation in the C-terminal region which includes the LPXTG surface anchor motif. Strain abbreviations: SIn–S. iniae, SPy–S. pyogenes, SUb–S. uberis, SEq–S. equi, and SDy–S. dysgalactiae.
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pone-0002824-g001: Bioinformatic analysis of SiMA.(A) Phylogenetic clustering of SiMA shows greatest similarity to other streptococcal M family proteins, most closely the S. uberis lactoferrin binding protein. (B) Amino acid sequence alignments of SiMA with other streptococcal M family proteins shows highest conservation in the C-terminal region which includes the LPXTG surface anchor motif. Strain abbreviations: SIn–S. iniae, SPy–S. pyogenes, SUb–S. uberis, SEq–S. equi, and SDy–S. dysgalactiae.

Mentions: The 1,566 bp M-like protein gene simA, from S. iniae strain K288, encodes a 521 amino acid gene product, SiM (S. iniae M-like protein), with a predicted precursor protein mass of 57.5 kDa. This M-like protein gene is identical to the recently published simA gene sequences from S. iniae strains QMA0076 and QMA0131 [29]. BLAST (tblastn) 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). Amino acid sequence alignments between SiMA and related M family proteins, as expected, showed the highest degree of similarity in the C-terminus which includes the LPXTG Gram-positive surface anchor motif (Fig. 1B, S1) [32].


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

Locke JB, Aziz RK, Vicknair MR, Nizet V, Buchanan JT - PLoS ONE (2008)

Bioinformatic analysis of SiMA.(A) Phylogenetic clustering of SiMA shows greatest similarity to other streptococcal M family proteins, most closely the S. uberis lactoferrin binding protein. (B) Amino acid sequence alignments of SiMA with other streptococcal M family proteins shows highest conservation in the C-terminal region which includes the LPXTG surface anchor motif. Strain abbreviations: SIn–S. iniae, SPy–S. pyogenes, SUb–S. uberis, SEq–S. equi, and SDy–S. dysgalactiae.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483786&req=5

pone-0002824-g001: Bioinformatic analysis of SiMA.(A) Phylogenetic clustering of SiMA shows greatest similarity to other streptococcal M family proteins, most closely the S. uberis lactoferrin binding protein. (B) Amino acid sequence alignments of SiMA with other streptococcal M family proteins shows highest conservation in the C-terminal region which includes the LPXTG surface anchor motif. Strain abbreviations: SIn–S. iniae, SPy–S. pyogenes, SUb–S. uberis, SEq–S. equi, and SDy–S. dysgalactiae.
Mentions: The 1,566 bp M-like protein gene simA, from S. iniae strain K288, encodes a 521 amino acid gene product, SiM (S. iniae M-like protein), with a predicted precursor protein mass of 57.5 kDa. This M-like protein gene is identical to the recently published simA gene sequences from S. iniae strains QMA0076 and QMA0131 [29]. BLAST (tblastn) 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). Amino acid sequence alignments between SiMA and related M family proteins, as expected, showed the highest degree of similarity in the C-terminus which includes the LPXTG Gram-positive surface anchor motif (Fig. 1B, S1) [32].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

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 Delta simA 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.

Show MeSH
Related in: MedlinePlus