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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

Activity of UAMS-1 nuclease genes.Nuclease activity was assessed using DNase agar in UAMS-1 (WT) and its sarA mutant and in derivatives of each of these strains carrying mutations in SA0746 (nuc1) and/or SA1160 (nuc2).
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pone-0010790-g012: Activity of UAMS-1 nuclease genes.Nuclease activity was assessed using DNase agar in UAMS-1 (WT) and its sarA mutant and in derivatives of each of these strains carrying mutations in SA0746 (nuc1) and/or SA1160 (nuc2).

Mentions: At the same time, our previous results focusing on the impact of nuclease production on biofilm formation in UAMS-1 were done using a mutant generated in the nuc gene designated as open-reading frame SA0746 in the N315 genome, mutation of which was shown to abolish nuclease production even in a UAMS-1 sarA as assessed using DNase agar [23]. However, a recent report demonstrated the existence of two functional nuclease genes in S. aureus, one of which is encoded by SA0746 while the other is encoded by SA1160 [51]. These were designated nuc1 and nuc2 respectively. In these experiments we confirmed that mutation of nuc1 in UAMS-1 essentially eliminated the production of extracellular nuclease even in a sarA mutant and that mutation of nuc2 had no effect as assessed using DNase agar (Fig. 12). Even so, mutation of either or both of these genes was shown to enhance biofilm formation in UAMS-1 albeit to a limited degree and only in the absence of plasma coating (Fig. 13). This was also true in a UAMS-1 sarA mutant although in this case the effect was statistically significant only when both nuc1 and nuc2 were mutated and only when the assay was done with plasma coating. These results suggest that our failure to detect a nuclease-deficient phenotype in our nuc2 mutant may have been due primarily to our use of a relatively insensitive assay. They also confirm that the increased production of nucleases contributes to biofilm formation in S. aureus at least under some circumstances and that the agr-independent, sarA-mediated repression of extracellular nuclease production may also be important in this regard.


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)

Activity of UAMS-1 nuclease genes.Nuclease activity was assessed using DNase agar in UAMS-1 (WT) and its sarA mutant and in derivatives of each of these strains carrying mutations in SA0746 (nuc1) and/or SA1160 (nuc2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010790-g012: Activity of UAMS-1 nuclease genes.Nuclease activity was assessed using DNase agar in UAMS-1 (WT) and its sarA mutant and in derivatives of each of these strains carrying mutations in SA0746 (nuc1) and/or SA1160 (nuc2).
Mentions: At the same time, our previous results focusing on the impact of nuclease production on biofilm formation in UAMS-1 were done using a mutant generated in the nuc gene designated as open-reading frame SA0746 in the N315 genome, mutation of which was shown to abolish nuclease production even in a UAMS-1 sarA as assessed using DNase agar [23]. However, a recent report demonstrated the existence of two functional nuclease genes in S. aureus, one of which is encoded by SA0746 while the other is encoded by SA1160 [51]. These were designated nuc1 and nuc2 respectively. In these experiments we confirmed that mutation of nuc1 in UAMS-1 essentially eliminated the production of extracellular nuclease even in a sarA mutant and that mutation of nuc2 had no effect as assessed using DNase agar (Fig. 12). Even so, mutation of either or both of these genes was shown to enhance biofilm formation in UAMS-1 albeit to a limited degree and only in the absence of plasma coating (Fig. 13). This was also true in a UAMS-1 sarA mutant although in this case the effect was statistically significant only when both nuc1 and nuc2 were mutated and only when the assay was done with plasma coating. These results suggest that our failure to detect a nuclease-deficient phenotype in our nuc2 mutant may have been due primarily to our use of a relatively insensitive assay. They also confirm that the increased production of nucleases contributes to biofilm formation in S. aureus at least under some circumstances and that the agr-independent, sarA-mediated repression of extracellular nuclease production may also be important in this regard.

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