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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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Comparison of putative Mga-like binding motifs upstream of sim genes.The 51 bp upstream regions of S. iniae sim genes with high similarity to GAS emm gene Mga binding sites are identical in strains K288, 9117, and 02161A. A 47 bp sequence sharing similarity to Mga-like binding sites located upstream of the gene encoding the S. uberis lactoferrin binding protein (Lbp, a close phylogenetic relative of SiMA) is also included for comparison. S. iniae and S. uberis putative binding motifs are aligned with the established 45 bp Mga binding site found in M6 GAS upstream of the emm6.1 M protein gene. Abbreviations: SIn–S. iniae, SUb–S. uberis, SPy–S. pyogenes.
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pone-0002824-g004: Comparison of putative Mga-like binding motifs upstream of sim genes.The 51 bp upstream regions of S. iniae sim genes with high similarity to GAS emm gene Mga binding sites are identical in strains K288, 9117, and 02161A. A 47 bp sequence sharing similarity to Mga-like binding sites located upstream of the gene encoding the S. uberis lactoferrin binding protein (Lbp, a close phylogenetic relative of SiMA) is also included for comparison. S. iniae and S. uberis putative binding motifs are aligned with the established 45 bp Mga binding site found in M6 GAS upstream of the emm6.1 M protein gene. Abbreviations: SIn–S. iniae, SUb–S. uberis, SPy–S. pyogenes.

Mentions: Almost exactly halfway between the divergently transcribed mgx and simA genes (162 bp upstream from the simA start codon ) lies a highly conserved 51 bp region, identical in isolates K288, 9117, 02161A and QMA00131 [29] (Fig. 4). This region has similarity to the established 45 bp Mga binding site for the emm6.1 gene of M6 GAS [14] and a 47 bp region upstream of the S. uberis lactoferrin binding protein gene [30] (Fig. 4). Downstream from simA is a putative tellurite resistance protein gene, telX, encoding a gene product with 99% identical amino acid composition to the TelX protein of S. iniae strain QMA0076 [29]. The chromosomal arrangement of mgx, simA, and telX was identical in S. iniae strains K288, 9117, 02161A, and QMA0076 [29]. Aside from insertion and deletion mutations in 02161A, nucleotide level analysis of the remainder of the Mgx locus reveals high conservation between strains (Fig. 3).


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)

Comparison of putative Mga-like binding motifs upstream of sim genes.The 51 bp upstream regions of S. iniae sim genes with high similarity to GAS emm gene Mga binding sites are identical in strains K288, 9117, and 02161A. A 47 bp sequence sharing similarity to Mga-like binding sites located upstream of the gene encoding the S. uberis lactoferrin binding protein (Lbp, a close phylogenetic relative of SiMA) is also included for comparison. S. iniae and S. uberis putative binding motifs are aligned with the established 45 bp Mga binding site found in M6 GAS upstream of the emm6.1 M protein gene. Abbreviations: SIn–S. iniae, SUb–S. uberis, SPy–S. pyogenes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002824-g004: Comparison of putative Mga-like binding motifs upstream of sim genes.The 51 bp upstream regions of S. iniae sim genes with high similarity to GAS emm gene Mga binding sites are identical in strains K288, 9117, and 02161A. A 47 bp sequence sharing similarity to Mga-like binding sites located upstream of the gene encoding the S. uberis lactoferrin binding protein (Lbp, a close phylogenetic relative of SiMA) is also included for comparison. S. iniae and S. uberis putative binding motifs are aligned with the established 45 bp Mga binding site found in M6 GAS upstream of the emm6.1 M protein gene. Abbreviations: SIn–S. iniae, SUb–S. uberis, SPy–S. pyogenes.
Mentions: Almost exactly halfway between the divergently transcribed mgx and simA genes (162 bp upstream from the simA start codon ) lies a highly conserved 51 bp region, identical in isolates K288, 9117, 02161A and QMA00131 [29] (Fig. 4). This region has similarity to the established 45 bp Mga binding site for the emm6.1 gene of M6 GAS [14] and a 47 bp region upstream of the S. uberis lactoferrin binding protein gene [30] (Fig. 4). Downstream from simA is a putative tellurite resistance protein gene, telX, encoding a gene product with 99% identical amino acid composition to the TelX protein of S. iniae strain QMA0076 [29]. The chromosomal arrangement of mgx, simA, and telX was identical in S. iniae strains K288, 9117, 02161A, and QMA0076 [29]. Aside from insertion and deletion mutations in 02161A, nucleotide level analysis of the remainder of the Mgx locus reveals high conservation between strains (Fig. 3).

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