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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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Model for the synergistic impact of saeRS and sarA on biofilm formation.Both sarA and saeRS repress the production of extracellular proteases, with sarA having the greater effect owing to both direct repression and activation of saeRS transcription. This repression relieves the protease-mediated “repression” of specific surface proteins arising from degradation. This in turn promotes accumulation of these proteins and an enhanced capacity to form a biofilm. The accessory gene regulator (agr) has the opposite effects on all of these phenotypes, but, as previously described, the impact of sarA occurs independently of agr, and sarA is epistatic to agr in this context (Beenken et al., 2010).
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pone-0038453-g010: Model for the synergistic impact of saeRS and sarA on biofilm formation.Both sarA and saeRS repress the production of extracellular proteases, with sarA having the greater effect owing to both direct repression and activation of saeRS transcription. This repression relieves the protease-mediated “repression” of specific surface proteins arising from degradation. This in turn promotes accumulation of these proteins and an enhanced capacity to form a biofilm. The accessory gene regulator (agr) has the opposite effects on all of these phenotypes, but, as previously described, the impact of sarA occurs independently of agr, and sarA is epistatic to agr in this context (Beenken et al., 2010).

Mentions: This accounts for our overall focus on limiting the regulatory functions of sarA as a means of limiting biofilm formation and thereby enhancing the therapeutic response in the context of S. aureus biofilm-associated infection. It also accounts for our focus on Newman in these studies in that mutation of sarA has a limited impact on biofilm formation in this strain by comparison to contemporary clinical isolates of S. aureus. The results we present demonstrate that saeRS and sarA work in concert with each other to limit the production of extracellular proteases and promote biofilm formation in S. aureus. Our studies employing an sspA::lux reporter suggest that this occurs at the transcriptional level, although it remains unknown whether this effect on proteases occurs via a direct or indirect mechanism. The production of SarA was unaffected by the functional status of saeRS, while expression of saeRS was reduced in a sarA mutant. This was previously reported to be the case in a COL sarA mutant [23], although it was not found to be the case in the clinical isolate UAMS-1 [24]. This suggests that this effect is strain-dependent. Nevertheless, based on this, we propose a model in which sarA represses the production of extracellular proteases via both saeRS dependent and saeRS independent pathways (Fig. 10). At the same time, activation of saeRS promotes transcription of fnbA. When taken together, this promotes the accumulation of critical proteins that promote biofilm formation, including FnbA and Spa. While the saeRS-independent pathway of sarA-mediated regulation has the greater overall effect, the saeRS-dependent pathway plays a significant role in that constitutive activation of saeRS can compromise the impact of sarA on protease production and biofilm formation. Both sarA and saeRS also modulate the production of surface adhesins at the transcriptional level, but in the absence of the reduced production of extracellular proteases owing to constitutive activation of saeRS, the phenotypic impact of this is overridden by the degradation of these adhesins due to the increased production of specific extracellular proteases, including aureolysin, SspA and/or SspB.


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)

Model for the synergistic impact of saeRS and sarA on biofilm formation.Both sarA and saeRS repress the production of extracellular proteases, with sarA having the greater effect owing to both direct repression and activation of saeRS transcription. This repression relieves the protease-mediated “repression” of specific surface proteins arising from degradation. This in turn promotes accumulation of these proteins and an enhanced capacity to form a biofilm. The accessory gene regulator (agr) has the opposite effects on all of these phenotypes, but, as previously described, the impact of sarA occurs independently of agr, and sarA is epistatic to agr in this context (Beenken et al., 2010).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038453-g010: Model for the synergistic impact of saeRS and sarA on biofilm formation.Both sarA and saeRS repress the production of extracellular proteases, with sarA having the greater effect owing to both direct repression and activation of saeRS transcription. This repression relieves the protease-mediated “repression” of specific surface proteins arising from degradation. This in turn promotes accumulation of these proteins and an enhanced capacity to form a biofilm. The accessory gene regulator (agr) has the opposite effects on all of these phenotypes, but, as previously described, the impact of sarA occurs independently of agr, and sarA is epistatic to agr in this context (Beenken et al., 2010).
Mentions: This accounts for our overall focus on limiting the regulatory functions of sarA as a means of limiting biofilm formation and thereby enhancing the therapeutic response in the context of S. aureus biofilm-associated infection. It also accounts for our focus on Newman in these studies in that mutation of sarA has a limited impact on biofilm formation in this strain by comparison to contemporary clinical isolates of S. aureus. The results we present demonstrate that saeRS and sarA work in concert with each other to limit the production of extracellular proteases and promote biofilm formation in S. aureus. Our studies employing an sspA::lux reporter suggest that this occurs at the transcriptional level, although it remains unknown whether this effect on proteases occurs via a direct or indirect mechanism. The production of SarA was unaffected by the functional status of saeRS, while expression of saeRS was reduced in a sarA mutant. This was previously reported to be the case in a COL sarA mutant [23], although it was not found to be the case in the clinical isolate UAMS-1 [24]. This suggests that this effect is strain-dependent. Nevertheless, based on this, we propose a model in which sarA represses the production of extracellular proteases via both saeRS dependent and saeRS independent pathways (Fig. 10). At the same time, activation of saeRS promotes transcription of fnbA. When taken together, this promotes the accumulation of critical proteins that promote biofilm formation, including FnbA and Spa. While the saeRS-independent pathway of sarA-mediated regulation has the greater overall effect, the saeRS-dependent pathway plays a significant role in that constitutive activation of saeRS can compromise the impact of sarA on protease production and biofilm formation. Both sarA and saeRS also modulate the production of surface adhesins at the transcriptional level, but in the absence of the reduced production of extracellular proteases owing to constitutive activation of saeRS, the phenotypic impact of this is overridden by the degradation of these adhesins due to the increased production of specific extracellular proteases, including aureolysin, SspA and/or SspB.

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