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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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The impact of protease inhibitors on biofilm formation in S. aureus sarA mutants.Biofilm formation was assessed in each of the indicated strains (WT) and their isogenic sarA mutants with (SPI) and without (S) the inclusion of protease inhibitors. Results are shown as the mean ± the standard deviation of 6 replicate samples. Statistical analysis confirmed a significant difference between each wild-type strain and its sarA mutant and, with the exception of UAMS-1625, between the sarA mutants assayed in the presence or absence of protease inhibitors. In RN6390 and UAMS-1782, the difference between the wild-type strain and its isogenic sarA mutant assayed in the presence of protease inhibitors was also significant.
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pone-0010790-g007: The impact of protease inhibitors on biofilm formation in S. aureus sarA mutants.Biofilm formation was assessed in each of the indicated strains (WT) and their isogenic sarA mutants with (SPI) and without (S) the inclusion of protease inhibitors. Results are shown as the mean ± the standard deviation of 6 replicate samples. Statistical analysis confirmed a significant difference between each wild-type strain and its sarA mutant and, with the exception of UAMS-1625, between the sarA mutants assayed in the presence or absence of protease inhibitors. In RN6390 and UAMS-1782, the difference between the wild-type strain and its isogenic sarA mutant assayed in the presence of protease inhibitors was also significant.

Mentions: While suggestive, the inverse relationship between biofilm formation and the production of extracellular proteases does not prove a cause-and-effect relationship. However, we subsequently demonstrated that a cocktail of three protease inhibitors that is capable of limiting the activity of multiple S. aureus extracellular proteases [23] enhanced biofilm formation in sarA mutants generated in UAMS-1, RN6390, and in two of the three USA300 isolates, the only exception being the USA300 isolate UAMS-1625 (Fig. 7). The overall levels of both RNAIII (Fig. 1) and extracellular proteases (Fig. 8) were comparable between UAMS-1625 and the other USA300 isolates, and the impact of mutating sarA on the production of extracellular proteases was comparable in all strains other than RN6390 (Fig. 8). This suggests that one possible explanation for the differential impact of protease inhibitors in UAMS-1625 is that this strain produces either a unique protease that was not inhibited by any component of the inhibitor cocktail or a common protease that is produced in elevated amounts by comparison to the other strains such that the inhibitor cocktail had a reduced effect in this strain. In this respect it is important to note that the inclusion of protease inhibitors did not fully restore biofilm formation (Fig. 7) or protease production (Fig. 8) in any of the sarA mutants. However, the relative impact of the inhibitor cocktail also appeared to be comparable among all USA300 isolates including UAMS-1625 (Fig. 8).


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)

The impact of protease inhibitors on biofilm formation in S. aureus sarA mutants.Biofilm formation was assessed in each of the indicated strains (WT) and their isogenic sarA mutants with (SPI) and without (S) the inclusion of protease inhibitors. Results are shown as the mean ± the standard deviation of 6 replicate samples. Statistical analysis confirmed a significant difference between each wild-type strain and its sarA mutant and, with the exception of UAMS-1625, between the sarA mutants assayed in the presence or absence of protease inhibitors. In RN6390 and UAMS-1782, the difference between the wild-type strain and its isogenic sarA mutant assayed in the presence of protease inhibitors was also significant.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010790-g007: The impact of protease inhibitors on biofilm formation in S. aureus sarA mutants.Biofilm formation was assessed in each of the indicated strains (WT) and their isogenic sarA mutants with (SPI) and without (S) the inclusion of protease inhibitors. Results are shown as the mean ± the standard deviation of 6 replicate samples. Statistical analysis confirmed a significant difference between each wild-type strain and its sarA mutant and, with the exception of UAMS-1625, between the sarA mutants assayed in the presence or absence of protease inhibitors. In RN6390 and UAMS-1782, the difference between the wild-type strain and its isogenic sarA mutant assayed in the presence of protease inhibitors was also significant.
Mentions: While suggestive, the inverse relationship between biofilm formation and the production of extracellular proteases does not prove a cause-and-effect relationship. However, we subsequently demonstrated that a cocktail of three protease inhibitors that is capable of limiting the activity of multiple S. aureus extracellular proteases [23] enhanced biofilm formation in sarA mutants generated in UAMS-1, RN6390, and in two of the three USA300 isolates, the only exception being the USA300 isolate UAMS-1625 (Fig. 7). The overall levels of both RNAIII (Fig. 1) and extracellular proteases (Fig. 8) were comparable between UAMS-1625 and the other USA300 isolates, and the impact of mutating sarA on the production of extracellular proteases was comparable in all strains other than RN6390 (Fig. 8). This suggests that one possible explanation for the differential impact of protease inhibitors in UAMS-1625 is that this strain produces either a unique protease that was not inhibited by any component of the inhibitor cocktail or a common protease that is produced in elevated amounts by comparison to the other strains such that the inhibitor cocktail had a reduced effect in this strain. In this respect it is important to note that the inclusion of protease inhibitors did not fully restore biofilm formation (Fig. 7) or protease production (Fig. 8) in any of the sarA mutants. However, the relative impact of the inhibitor cocktail also appeared to be comparable among all USA300 isolates including UAMS-1625 (Fig. 8).

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