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saeRS and sarA act synergistically to repress protease production and promote biofilm formation in Staphylococcus aureus.

Mrak LN, Zielinska AK, Beenken KE, Mrak IN, Atwood DN, Griffin LM, Lee CY, Smeltzer MS - PLoS ONE (2012)

Bottom Line: The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon.These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm.Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

ABSTRACT
Mutation of the staphylococcal accessory regulator (sarA) limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L) or a Newman saeRS mutant (ΔsaeRS) resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level.

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Impact of saeRS and sarA on protease production.Production of extracellular proteases in derivatives of Newman as a function of saeRS and sarA was assessed by zymography using gelatin as the substrate. The presumed identity of individual proteases is indicated to the right. The graph illustrates relative expression levels the sspA promoter as assessed using an sspA::lux reporter. Differences between the Newman sarA mutant, the P18L sarA mutant, and the saeRS/sarA mutant were all statistically significant (p<0.05) by comparison to Newman. Differences between the sarA/saeRS and the P18L sarA mutants, and between the P18L sarA mutant and the Newman sarA mutant, were also significant.
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pone-0038453-g003: Impact of saeRS and sarA on protease production.Production of extracellular proteases in derivatives of Newman as a function of saeRS and sarA was assessed by zymography using gelatin as the substrate. The presumed identity of individual proteases is indicated to the right. The graph illustrates relative expression levels the sspA promoter as assessed using an sspA::lux reporter. Differences between the Newman sarA mutant, the P18L sarA mutant, and the saeRS/sarA mutant were all statistically significant (p<0.05) by comparison to Newman. Differences between the sarA/saeRS and the P18L sarA mutants, and between the P18L sarA mutant and the Newman sarA mutant, were also significant.

Mentions: When we examined the production of extracellular proteases in Newman and its saeRS and sarA derivatives, we found a direct correlation between the production of these proteases and the functional status of both saeRS and sarA. Specifically, protease production was lowest in Newman and increased progressively as the relative activity of both saeRS and sarA declined (Fig. 3). Most importantly, while mutation of sarA resulted in increased production of multiple extracellular proteases in all strains, this effect was moderated in a Newman sarA mutant. This was also evident in reporter assays using an sspA::luxABCDE reporter, suggesting that these changes occur at the transcriptional level.


saeRS and sarA act synergistically to repress protease production and promote biofilm formation in Staphylococcus aureus.

Mrak LN, Zielinska AK, Beenken KE, Mrak IN, Atwood DN, Griffin LM, Lee CY, Smeltzer MS - PLoS ONE (2012)

Impact of saeRS and sarA on protease production.Production of extracellular proteases in derivatives of Newman as a function of saeRS and sarA was assessed by zymography using gelatin as the substrate. The presumed identity of individual proteases is indicated to the right. The graph illustrates relative expression levels the sspA promoter as assessed using an sspA::lux reporter. Differences between the Newman sarA mutant, the P18L sarA mutant, and the saeRS/sarA mutant were all statistically significant (p<0.05) by comparison to Newman. Differences between the sarA/saeRS and the P18L sarA mutants, and between the P18L sarA mutant and the Newman sarA mutant, were also significant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369899&req=5

pone-0038453-g003: Impact of saeRS and sarA on protease production.Production of extracellular proteases in derivatives of Newman as a function of saeRS and sarA was assessed by zymography using gelatin as the substrate. The presumed identity of individual proteases is indicated to the right. The graph illustrates relative expression levels the sspA promoter as assessed using an sspA::lux reporter. Differences between the Newman sarA mutant, the P18L sarA mutant, and the saeRS/sarA mutant were all statistically significant (p<0.05) by comparison to Newman. Differences between the sarA/saeRS and the P18L sarA mutants, and between the P18L sarA mutant and the Newman sarA mutant, were also significant.
Mentions: When we examined the production of extracellular proteases in Newman and its saeRS and sarA derivatives, we found a direct correlation between the production of these proteases and the functional status of both saeRS and sarA. Specifically, protease production was lowest in Newman and increased progressively as the relative activity of both saeRS and sarA declined (Fig. 3). Most importantly, while mutation of sarA resulted in increased production of multiple extracellular proteases in all strains, this effect was moderated in a Newman sarA mutant. This was also evident in reporter assays using an sspA::luxABCDE reporter, suggesting that these changes occur at the transcriptional level.

Bottom Line: The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon.These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm.Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

ABSTRACT
Mutation of the staphylococcal accessory regulator (sarA) limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L) or a Newman saeRS mutant (ΔsaeRS) resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level.

Show MeSH
Related in: MedlinePlus