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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 sarA, saeRS, and extracellular proteases on accumulation of FnbA and biofilm formation.Top: Relative amounts of surface-anchored FnbA were assessed in Newman (New), its saeS-repaired derivative (P18L), and its saeRS mutant (sae) after introduction of an intact copy of fnbA on a plasmid. Newman without this plasmid was included as a negative control. The impact of mutating sarA was assessed in each of these strains together with the impact of mutating the gene encoding aureolysin (aur), sspABC (ssp) or sae on the phenotype of the sarA mutants. Bottom: Biofilm formation was assessed by microtiter plate assay in Newman and P18L as well as their sarA and sarA/ssp derivatives after the introduction of pFnbA.
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pone-0038453-g004: Impact of sarA, saeRS, and extracellular proteases on accumulation of FnbA and biofilm formation.Top: Relative amounts of surface-anchored FnbA were assessed in Newman (New), its saeS-repaired derivative (P18L), and its saeRS mutant (sae) after introduction of an intact copy of fnbA on a plasmid. Newman without this plasmid was included as a negative control. The impact of mutating sarA was assessed in each of these strains together with the impact of mutating the gene encoding aureolysin (aur), sspABC (ssp) or sae on the phenotype of the sarA mutants. Bottom: Biofilm formation was assessed by microtiter plate assay in Newman and P18L as well as their sarA and sarA/ssp derivatives after the introduction of pFnbA.

Mentions: When we examined the accumulation of surface-associated FnbA, we found that it was present in reduced amounts in the pFnbA Newman sarA mutant by comparison to pFnbA Newman, and that this effect was reversed by mutation of sspABC (Fig. 4). In contrast, mutation of the gene encoding aureolysin (aur) had little impact on the FnbA phenotype of the Newman sarA mutant. Surface-associated FnbA was also detected in pFnbA P18L, but it was reduced to almost undetectable levels in the isogenic sarA mutant, and concomitant mutation of sspABC had relatively little impact. This was surprising in that production of both SspA and SspB was higher in a P18L sarA mutant than a Newman sarA mutant (Fig. 3), thus suggesting that mutation of sspABC would have a greater impact on the accumulation of FnbA in the P18L sarA mutant. Nevertheless, these same relative levels of FnbA production were evident in the context of biofilm formation, with mutation of sspABC enhancing biofilm formation in a pFnbA Newman sarA mutant, albeit to a modest extent, but having no impact on biofilm formation in the pFnbA P18L sarA mutant (Fig. 4). This suggests that, while surface associated FnbA is important, some other difference(s) must also exist between these strains that is (are) both relevant to biofilm formation and moderated in an saeRS-dependent manner.


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 sarA, saeRS, and extracellular proteases on accumulation of FnbA and biofilm formation.Top: Relative amounts of surface-anchored FnbA were assessed in Newman (New), its saeS-repaired derivative (P18L), and its saeRS mutant (sae) after introduction of an intact copy of fnbA on a plasmid. Newman without this plasmid was included as a negative control. The impact of mutating sarA was assessed in each of these strains together with the impact of mutating the gene encoding aureolysin (aur), sspABC (ssp) or sae on the phenotype of the sarA mutants. Bottom: Biofilm formation was assessed by microtiter plate assay in Newman and P18L as well as their sarA and sarA/ssp derivatives after the introduction of pFnbA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369899&req=5

pone-0038453-g004: Impact of sarA, saeRS, and extracellular proteases on accumulation of FnbA and biofilm formation.Top: Relative amounts of surface-anchored FnbA were assessed in Newman (New), its saeS-repaired derivative (P18L), and its saeRS mutant (sae) after introduction of an intact copy of fnbA on a plasmid. Newman without this plasmid was included as a negative control. The impact of mutating sarA was assessed in each of these strains together with the impact of mutating the gene encoding aureolysin (aur), sspABC (ssp) or sae on the phenotype of the sarA mutants. Bottom: Biofilm formation was assessed by microtiter plate assay in Newman and P18L as well as their sarA and sarA/ssp derivatives after the introduction of pFnbA.
Mentions: When we examined the accumulation of surface-associated FnbA, we found that it was present in reduced amounts in the pFnbA Newman sarA mutant by comparison to pFnbA Newman, and that this effect was reversed by mutation of sspABC (Fig. 4). In contrast, mutation of the gene encoding aureolysin (aur) had little impact on the FnbA phenotype of the Newman sarA mutant. Surface-associated FnbA was also detected in pFnbA P18L, but it was reduced to almost undetectable levels in the isogenic sarA mutant, and concomitant mutation of sspABC had relatively little impact. This was surprising in that production of both SspA and SspB was higher in a P18L sarA mutant than a Newman sarA mutant (Fig. 3), thus suggesting that mutation of sspABC would have a greater impact on the accumulation of FnbA in the P18L sarA mutant. Nevertheless, these same relative levels of FnbA production were evident in the context of biofilm formation, with mutation of sspABC enhancing biofilm formation in a pFnbA Newman sarA mutant, albeit to a modest extent, but having no impact on biofilm formation in the pFnbA P18L sarA mutant (Fig. 4). This suggests that, while surface associated FnbA is important, some other difference(s) must also exist between these strains that is (are) both relevant to biofilm formation and moderated in an saeRS-dependent manner.

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