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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 surface-associated FnbA on biofilm formation in Newman.Surface-anchored FnbA was restored in Newman (New), its saeS-repaired derivative (P18L), and its isogenic saeRS mutant (sae) by introduction of a plasmid-borne copy of fnbA. Biofilm formation was assessed using a microtiter plate assay, with UAMS-1 (U1) and its sarA mutant included as positive and negative controls, respectively. sarA mutants are designated as “S.” Asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain (WT).
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pone-0038453-g001: Impact of saeRS and surface-associated FnbA on biofilm formation in Newman.Surface-anchored FnbA was restored in Newman (New), its saeS-repaired derivative (P18L), and its isogenic saeRS mutant (sae) by introduction of a plasmid-borne copy of fnbA. Biofilm formation was assessed using a microtiter plate assay, with UAMS-1 (U1) and its sarA mutant included as positive and negative controls, respectively. sarA mutants are designated as “S.” Asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain (WT).

Mentions: As in our previous studies [12], Newman was found to have a reduced capacity to form a biofilm by comparison to the clinical isolate UAMS-1, and mutation of sarA resulted in only a modest decrease in biofilm formation (Fig. 1). Introduction of an intact copy of fnbA on a plasmid (pFnbA) increased biofilm formation in Newman to levels that approached those observed with UAMS-1, suggesting that the inability to anchor FnbA to the cell surface contributes to the reduced capacity of Newman to form a biofilm. This effect was also apparent in a derivative of Newman in which the saeS defect was repaired (P18L), but it was moderated in an isogenic saeRS mutant, a phenotype that is consistent with the demonstration that activation of saeRS enhances transcription of fnbA[16]. More importantly, mutation of sarA in the pFnbA derivative of Newman resulted in an increased rather than decreased capacity to form a biofilm (Fig. 1). In contrast, mutation of sarA in both the P18L pFnbA derivative and the pFnbA saeRS mutant limited biofilm formation to a degree comparable to that observed in a UAMS-1 sarA mutant (Fig. 1). However, this was also true in sarA mutants generated in these strains in the absence of pFnbA, thus suggesting that the disparate sarA-dependent biofilm phenotypes observed in Newman vs. its saeRS derivatives involve something other than the impact of saeRS on the production of surface-associated FnbA.


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 surface-associated FnbA on biofilm formation in Newman.Surface-anchored FnbA was restored in Newman (New), its saeS-repaired derivative (P18L), and its isogenic saeRS mutant (sae) by introduction of a plasmid-borne copy of fnbA. Biofilm formation was assessed using a microtiter plate assay, with UAMS-1 (U1) and its sarA mutant included as positive and negative controls, respectively. sarA mutants are designated as “S.” Asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain (WT).
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Related In: Results  -  Collection

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pone-0038453-g001: Impact of saeRS and surface-associated FnbA on biofilm formation in Newman.Surface-anchored FnbA was restored in Newman (New), its saeS-repaired derivative (P18L), and its isogenic saeRS mutant (sae) by introduction of a plasmid-borne copy of fnbA. Biofilm formation was assessed using a microtiter plate assay, with UAMS-1 (U1) and its sarA mutant included as positive and negative controls, respectively. sarA mutants are designated as “S.” Asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain (WT).
Mentions: As in our previous studies [12], Newman was found to have a reduced capacity to form a biofilm by comparison to the clinical isolate UAMS-1, and mutation of sarA resulted in only a modest decrease in biofilm formation (Fig. 1). Introduction of an intact copy of fnbA on a plasmid (pFnbA) increased biofilm formation in Newman to levels that approached those observed with UAMS-1, suggesting that the inability to anchor FnbA to the cell surface contributes to the reduced capacity of Newman to form a biofilm. This effect was also apparent in a derivative of Newman in which the saeS defect was repaired (P18L), but it was moderated in an isogenic saeRS mutant, a phenotype that is consistent with the demonstration that activation of saeRS enhances transcription of fnbA[16]. More importantly, mutation of sarA in the pFnbA derivative of Newman resulted in an increased rather than decreased capacity to form a biofilm (Fig. 1). In contrast, mutation of sarA in both the P18L pFnbA derivative and the pFnbA saeRS mutant limited biofilm formation to a degree comparable to that observed in a UAMS-1 sarA mutant (Fig. 1). However, this was also true in sarA mutants generated in these strains in the absence of pFnbA, thus suggesting that the disparate sarA-dependent biofilm phenotypes observed in Newman vs. its saeRS derivatives involve something other than the impact of saeRS on the production of surface-associated FnbA.

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