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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

Production of extracellular proteases in USA300 isolates.Supernatants were harvested from overnight (15 hr) cultures grown in TSB (left) or biofilm medium (right) and standardized with respect to each other prior to zymographic analysis using both casein (top) and gelatin gels (bottom).
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pone-0010790-g005: Production of extracellular proteases in USA300 isolates.Supernatants were harvested from overnight (15 hr) cultures grown in TSB (left) or biofilm medium (right) and standardized with respect to each other prior to zymographic analysis using both casein (top) and gelatin gels (bottom).

Mentions: Production of these proteases has a negative impact on the presence of several surface-associated adhesins. These include the fibronectin-binding proteins (FnbA and FnbB) and protein A [24], [46], both of which contribute to biofilm formation in at least some clinical isolates of S. aureus [45], [47], [48]. Additionally, expression of agr both represses the production of these adhesins and induces the production of extracellular proteases [21], [49], either or both of which could contribute to the negative correlation between agr and biofilm formation. At the same time, transcription of the genes encoding extracellular proteases, including aureolysin (aur) and sspA, is directly repressed by both SarA and Rot [22]. Moreover, the regulatory impact of sarA, rot and agr appears to be dependent on the relative concentrations of their products with respect to each other rather than the concentration of any individual product alone [50]. This suggests that USA300 isolates may not produce extracellular proteases at the levels that might be expected based on their relatively high overall levels of agr expression, and we found that this was in fact the case. Specifically, by comparison to RN6390, all three USA300 isolates produced reduced amounts of all extracellular proteases that could be detected using either casein or gelatin zymography (Fig. 5). In fact, USA300 protease levels were more comparable to UAMS-1 than RN6390 despite the relatively low levels of RNAIII production in UAMS-1. As was observed in assays examining the production of RNAIII (Fig. 1), this was true whether protease production was assessed using supernatants from cultures grown in TSB or in biofilm medium (Fig. 5).


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)

Production of extracellular proteases in USA300 isolates.Supernatants were harvested from overnight (15 hr) cultures grown in TSB (left) or biofilm medium (right) and standardized with respect to each other prior to zymographic analysis using both casein (top) and gelatin gels (bottom).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010790-g005: Production of extracellular proteases in USA300 isolates.Supernatants were harvested from overnight (15 hr) cultures grown in TSB (left) or biofilm medium (right) and standardized with respect to each other prior to zymographic analysis using both casein (top) and gelatin gels (bottom).
Mentions: Production of these proteases has a negative impact on the presence of several surface-associated adhesins. These include the fibronectin-binding proteins (FnbA and FnbB) and protein A [24], [46], both of which contribute to biofilm formation in at least some clinical isolates of S. aureus [45], [47], [48]. Additionally, expression of agr both represses the production of these adhesins and induces the production of extracellular proteases [21], [49], either or both of which could contribute to the negative correlation between agr and biofilm formation. At the same time, transcription of the genes encoding extracellular proteases, including aureolysin (aur) and sspA, is directly repressed by both SarA and Rot [22]. Moreover, the regulatory impact of sarA, rot and agr appears to be dependent on the relative concentrations of their products with respect to each other rather than the concentration of any individual product alone [50]. This suggests that USA300 isolates may not produce extracellular proteases at the levels that might be expected based on their relatively high overall levels of agr expression, and we found that this was in fact the case. Specifically, by comparison to RN6390, all three USA300 isolates produced reduced amounts of all extracellular proteases that could be detected using either casein or gelatin zymography (Fig. 5). In fact, USA300 protease levels were more comparable to UAMS-1 than RN6390 despite the relatively low levels of RNAIII production in UAMS-1. As was observed in assays examining the production of RNAIII (Fig. 1), this was true whether protease production was assessed using supernatants from cultures grown in TSB or in biofilm medium (Fig. 5).

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