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Epistatic relationships between sarA and agr in Staphylococcus aureus biofilm formation.

Beenken KE, Mrak LN, Griffin LM, Zielinska AK, Shaw LN, Rice KC, Horswill AR, Bayles KW, Smeltzer MS - PLoS ONE (2010)

Bottom Line: There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect.In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr.The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci.

Methodology/principal findings: We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to form a biofilm in the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard.

Conclusions and significance: The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.

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Related in: MedlinePlus

Biofilm formation in isolates of the USA300 clonal lineage.Biofilm formation was assessed using a microtiter plate assay as previously described [10]. Results are shown as the mean ± the standard deviation of 6 replicate samples from each strain. Statistical analysis confirmed a significant difference between RN6390 and each of the other strains.
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pone-0010790-g002: Biofilm formation in isolates of the USA300 clonal lineage.Biofilm formation was assessed using a microtiter plate assay as previously described [10]. Results are shown as the mean ± the standard deviation of 6 replicate samples from each strain. Statistical analysis confirmed a significant difference between RN6390 and each of the other strains.

Mentions: Of the planktonic growth conditions examined in this report, the most applicable by comparison to our biofilm assays is a stationary-phase culture in biofilm medium, and under these conditions the targeted strains could be divided into three groups. The first consisted solely of RN6390, which produced higher levels of RNAIII by comparison to all other strains. The second consisted of the USA300 isolates UAMS-1782 and UAMS-1790, both of which produced RNAIII at levels that exceeded those observed in UAMS-1 or the USA300 isolate UAMS-1625 to a statistically-significant degree (Fig. 1). These results demonstrate that, while USA300 isolates generally produced RNAIII at levels that exceeded those observed in UAMS-1, they were nevertheless below those observed in RN6390. More importantly, this difference appears to be biologically relevant in that, as defined by our microtiter plate assay, all USA300 isolates formed a biofilm that was comparable to that observed with UAMS-1 and significantly greater than that observed with RN6390 (Fig. 2).


Epistatic relationships between sarA and agr in Staphylococcus aureus biofilm formation.

Beenken KE, Mrak LN, Griffin LM, Zielinska AK, Shaw LN, Rice KC, Horswill AR, Bayles KW, Smeltzer MS - PLoS ONE (2010)

Biofilm formation in isolates of the USA300 clonal lineage.Biofilm formation was assessed using a microtiter plate assay as previously described [10]. Results are shown as the mean ± the standard deviation of 6 replicate samples from each strain. Statistical analysis confirmed a significant difference between RN6390 and each of the other strains.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875390&req=5

pone-0010790-g002: Biofilm formation in isolates of the USA300 clonal lineage.Biofilm formation was assessed using a microtiter plate assay as previously described [10]. Results are shown as the mean ± the standard deviation of 6 replicate samples from each strain. Statistical analysis confirmed a significant difference between RN6390 and each of the other strains.
Mentions: Of the planktonic growth conditions examined in this report, the most applicable by comparison to our biofilm assays is a stationary-phase culture in biofilm medium, and under these conditions the targeted strains could be divided into three groups. The first consisted solely of RN6390, which produced higher levels of RNAIII by comparison to all other strains. The second consisted of the USA300 isolates UAMS-1782 and UAMS-1790, both of which produced RNAIII at levels that exceeded those observed in UAMS-1 or the USA300 isolate UAMS-1625 to a statistically-significant degree (Fig. 1). These results demonstrate that, while USA300 isolates generally produced RNAIII at levels that exceeded those observed in UAMS-1, they were nevertheless below those observed in RN6390. More importantly, this difference appears to be biologically relevant in that, as defined by our microtiter plate assay, all USA300 isolates formed a biofilm that was comparable to that observed with UAMS-1 and significantly greater than that observed with RN6390 (Fig. 2).

Bottom Line: There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect.In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr.The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci.

Methodology/principal findings: We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to form a biofilm in the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard.

Conclusions and significance: The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.

Show MeSH
Related in: MedlinePlus