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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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M-like protein contributes to S. iniae adherence to and invasion of cultured fish epithelial cells and resistance to killing by fish macrophages.(A) Adherence and (B) invasion characteristics of WT K288, the isogenic ΔsimA allelic mutant, and the naturally M-deficient WT 02161A S. iniae strain for the fish epithelial cell line WBE27. (C) Survival of WT K288, WT 02161A, and the ΔsimA mutant upon co-incubation with CLC fish macrophage/monocytes for 2, 4, or 18 h. Significance indicated as: * P<0.05, ** P<0.005, *** P<0.0005. Data are presented as mean±SEM from two-tailed t-tests.
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pone-0002824-g007: M-like protein contributes to S. iniae adherence to and invasion of cultured fish epithelial cells and resistance to killing by fish macrophages.(A) Adherence and (B) invasion characteristics of WT K288, the isogenic ΔsimA allelic mutant, and the naturally M-deficient WT 02161A S. iniae strain for the fish epithelial cell line WBE27. (C) Survival of WT K288, WT 02161A, and the ΔsimA mutant upon co-incubation with CLC fish macrophage/monocytes for 2, 4, or 18 h. Significance indicated as: * P<0.05, ** P<0.005, *** P<0.0005. Data are presented as mean±SEM from two-tailed t-tests.

Mentions: The ability to adhere to and invade epithelial layers is proposed to play a role in S. iniae virulence [44]. We used cultured monolayers of the white bass epithelial cell line WBE27 to assess the adherence and intracellular invasive properties of S. iniae strains in vitro [45]. Compared to the WT parent strain K288, the S. iniae ΔsimA mutant demonstrated significantly less adherence to (∼40% reduction, P<0.005) and invasion (∼20% reduction, P<0.02) of WBE27 cells (Fig. 7A, B). The levels of adherence and invasion associated with the S. iniae WT 02161A strain (harboring a frameshift/truncation mutation in the simA gene) had a similar trend (P = 0.0067, P<0.0001, respectively) to those of the S. iniae K288 ΔsimA mutant (Fig. 7A, B).


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)

M-like protein contributes to S. iniae adherence to and invasion of cultured fish epithelial cells and resistance to killing by fish macrophages.(A) Adherence and (B) invasion characteristics of WT K288, the isogenic ΔsimA allelic mutant, and the naturally M-deficient WT 02161A S. iniae strain for the fish epithelial cell line WBE27. (C) Survival of WT K288, WT 02161A, and the ΔsimA mutant upon co-incubation with CLC fish macrophage/monocytes for 2, 4, or 18 h. Significance indicated as: * P<0.05, ** P<0.005, *** P<0.0005. Data are presented as mean±SEM from two-tailed t-tests.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002824-g007: M-like protein contributes to S. iniae adherence to and invasion of cultured fish epithelial cells and resistance to killing by fish macrophages.(A) Adherence and (B) invasion characteristics of WT K288, the isogenic ΔsimA allelic mutant, and the naturally M-deficient WT 02161A S. iniae strain for the fish epithelial cell line WBE27. (C) Survival of WT K288, WT 02161A, and the ΔsimA mutant upon co-incubation with CLC fish macrophage/monocytes for 2, 4, or 18 h. Significance indicated as: * P<0.05, ** P<0.005, *** P<0.0005. Data are presented as mean±SEM from two-tailed t-tests.
Mentions: The ability to adhere to and invade epithelial layers is proposed to play a role in S. iniae virulence [44]. We used cultured monolayers of the white bass epithelial cell line WBE27 to assess the adherence and intracellular invasive properties of S. iniae strains in vitro [45]. Compared to the WT parent strain K288, the S. iniae ΔsimA mutant demonstrated significantly less adherence to (∼40% reduction, P<0.005) and invasion (∼20% reduction, P<0.02) of WBE27 cells (Fig. 7A, B). The levels of adherence and invasion associated with the S. iniae WT 02161A strain (harboring a frameshift/truncation mutation in the simA gene) had a similar trend (P = 0.0067, P<0.0001, respectively) to those of the S. iniae K288 ΔsimA mutant (Fig. 7A, B).

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