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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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Interactions between sarA and saeRS.Top: Production of SarA was assessed by western blot using SarA antibody in the indicated strains (WT) and their isogenic sarA mtuants (S). Bottom: Impact of sarA on transcription of saeR in post-exponential cultures (OD560 = 3.0) was assessed by qRT-PCR. Results are shown relative to those observed with FPR3757, which were set to a value of 1.0. Asterisks indicate statistical significance (p<0.05) by comparison to the parent strain.
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pone-0038453-g008: Interactions between sarA and saeRS.Top: Production of SarA was assessed by western blot using SarA antibody in the indicated strains (WT) and their isogenic sarA mtuants (S). Bottom: Impact of sarA on transcription of saeR in post-exponential cultures (OD560 = 3.0) was assessed by qRT-PCR. Results are shown relative to those observed with FPR3757, which were set to a value of 1.0. Asterisks indicate statistical significance (p<0.05) by comparison to the parent strain.

Mentions: Finally, we investigated the interaction between sarA and saeRS by examining the impact of mutating one on the other. The relative activity of saeRS had no impact on the production of SarA, but mutation of sarA resulted in reduced transcription of saeRS even in the context of the otherwise constitutive activation of saeRS in Newman (Fig. 8). However, even with decreased, but not elimination of, saeR transcription, constitutive activation of the saePQRS operon can be achieved by constitutive phosphorylation of SaeR by SaeS. This transcriptional downregulation of saeR by SarA was true in the USA300 isolate FPR3757 as well. Moreover, mutation of saeRS in FPR3757 was correlated with a reduced capacity to form a biofilm (Fig. 9). Importantly, while this biofilm-deficient phenotype was not apparent in a comparison of pFnbA Newman and its pFnbA saeRS mutant, it was apparent in a comparison of pFnbA P18L and the pFnbA saeRS mutant, in which the functional status of saeRS and fnbA are similar to FPR3757 and its isogenic saeRS mutant (Fig. 1). While the biofilm defect in the FPR3757 saeRS mutant was modest, particularly by comparison to mutation of sarA, it was nevertheless statistically significant, and this phenotype could be “complemented” by mutation the genes encoding specific extracellular proteases (Fig. 9). This, along with the observation that mutation of saeRS has no effect on biofilm formation in a FPR3757 sarA mutant, suggests that mutation of saeRS resulting in inactivation would not jeopardize therapy with a sarA inhibitor. Furthermore, these results further demonstrate the correlation between reduced saeRS expression, increased protease production, and a reduced capacity to form a biofilm, and these correlations are independent of, but synergistic with, the impact of sarA on these same phenotypes.


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)

Interactions between sarA and saeRS.Top: Production of SarA was assessed by western blot using SarA antibody in the indicated strains (WT) and their isogenic sarA mtuants (S). Bottom: Impact of sarA on transcription of saeR in post-exponential cultures (OD560 = 3.0) was assessed by qRT-PCR. Results are shown relative to those observed with FPR3757, which were set to a value of 1.0. Asterisks indicate statistical significance (p<0.05) by comparison to the parent strain.
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Related In: Results  -  Collection

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

pone-0038453-g008: Interactions between sarA and saeRS.Top: Production of SarA was assessed by western blot using SarA antibody in the indicated strains (WT) and their isogenic sarA mtuants (S). Bottom: Impact of sarA on transcription of saeR in post-exponential cultures (OD560 = 3.0) was assessed by qRT-PCR. Results are shown relative to those observed with FPR3757, which were set to a value of 1.0. Asterisks indicate statistical significance (p<0.05) by comparison to the parent strain.
Mentions: Finally, we investigated the interaction between sarA and saeRS by examining the impact of mutating one on the other. The relative activity of saeRS had no impact on the production of SarA, but mutation of sarA resulted in reduced transcription of saeRS even in the context of the otherwise constitutive activation of saeRS in Newman (Fig. 8). However, even with decreased, but not elimination of, saeR transcription, constitutive activation of the saePQRS operon can be achieved by constitutive phosphorylation of SaeR by SaeS. This transcriptional downregulation of saeR by SarA was true in the USA300 isolate FPR3757 as well. Moreover, mutation of saeRS in FPR3757 was correlated with a reduced capacity to form a biofilm (Fig. 9). Importantly, while this biofilm-deficient phenotype was not apparent in a comparison of pFnbA Newman and its pFnbA saeRS mutant, it was apparent in a comparison of pFnbA P18L and the pFnbA saeRS mutant, in which the functional status of saeRS and fnbA are similar to FPR3757 and its isogenic saeRS mutant (Fig. 1). While the biofilm defect in the FPR3757 saeRS mutant was modest, particularly by comparison to mutation of sarA, it was nevertheless statistically significant, and this phenotype could be “complemented” by mutation the genes encoding specific extracellular proteases (Fig. 9). This, along with the observation that mutation of saeRS has no effect on biofilm formation in a FPR3757 sarA mutant, suggests that mutation of saeRS resulting in inactivation would not jeopardize therapy with a sarA inhibitor. Furthermore, these results further demonstrate the correlation between reduced saeRS expression, increased protease production, and a reduced capacity to form a biofilm, and these correlations are independent of, but synergistic with, the impact of sarA on these same phenotypes.

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