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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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Role of sarA in production of the polysaccharide intracellular adhesin (PIA).PIA was isolated from each of the wild-type strains (WT) and their isogenic sarA mutants (S) and immunoblotted using anti-PIA serum. A UAMS-1 ica mutant was included as a negative control. Upper and lower rows are duplicate samples from each strain.
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pone-0010790-g009: Role of sarA in production of the polysaccharide intracellular adhesin (PIA).PIA was isolated from each of the wild-type strains (WT) and their isogenic sarA mutants (S) and immunoblotted using anti-PIA serum. A UAMS-1 ica mutant was included as a negative control. Upper and lower rows are duplicate samples from each strain.

Mentions: UAMS-1625 can also be distinguished from the other USA300 isolates included in this study by the absence of the arginine catabolite metabolic element (ACME) [38]. We are unaware of a correlation between ACME and biofilm formation, but there are reports describing the role of the arc operon itself in this context. Specifically expression of the arc operon was induced in UAMS-1 in a biofilm by comparison to both exponential and stationary-phase planktonic cultures [27]. Additionally, mutation of arcD, which encodes the arginine/ornithine antiporter of the arginine deiminase pathway, resulted in the reduced production of PIA in UAMS-1 [44]. However, as with the ica operon itself [27], this was not associated with a decreased capacity to form a biofilm. Nevertheless, the possibility that ACME contributes to biofilm formation by virtue of its impact on the production of PIA cannot be ruled out. Indeed, mutation of sarA results in reduced production of PIA [7], [11], and based on this one possible explanation for our results is that PIA plays a more important role in biofilm formation in UAMS-1625 than in the other USA300 isolates and that, in the absence of ACME, a UAMS-1625 sarA mutant cannot produce enough PIA to sustain biofilm formation irrespective of any other factor including extracellular proteases. To explore this possibility, we examined the relative levels of PIA produced by each strain using an anti-PIA immunoblot. These studies demonstrated that all of the USA300 isolates produced almost undetectable amounts of PIA irrespective of the presence of ACME or the functional status of sarA (Fig. 9). This not only suggests that the relative levels of PIA production do not account for the difference between UAMS-1625 and other USA300 isolates but also that PIA plays little role in USA300 biofilm formation. This is also consistent with previous reports demonstrating that mutation of ica had little effect on biofilm formation not only in LAC [8] but also in other methicillin-resistant S. aureus strains [45].


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)

Role of sarA in production of the polysaccharide intracellular adhesin (PIA).PIA was isolated from each of the wild-type strains (WT) and their isogenic sarA mutants (S) and immunoblotted using anti-PIA serum. A UAMS-1 ica mutant was included as a negative control. Upper and lower rows are duplicate samples from each strain.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010790-g009: Role of sarA in production of the polysaccharide intracellular adhesin (PIA).PIA was isolated from each of the wild-type strains (WT) and their isogenic sarA mutants (S) and immunoblotted using anti-PIA serum. A UAMS-1 ica mutant was included as a negative control. Upper and lower rows are duplicate samples from each strain.
Mentions: UAMS-1625 can also be distinguished from the other USA300 isolates included in this study by the absence of the arginine catabolite metabolic element (ACME) [38]. We are unaware of a correlation between ACME and biofilm formation, but there are reports describing the role of the arc operon itself in this context. Specifically expression of the arc operon was induced in UAMS-1 in a biofilm by comparison to both exponential and stationary-phase planktonic cultures [27]. Additionally, mutation of arcD, which encodes the arginine/ornithine antiporter of the arginine deiminase pathway, resulted in the reduced production of PIA in UAMS-1 [44]. However, as with the ica operon itself [27], this was not associated with a decreased capacity to form a biofilm. Nevertheless, the possibility that ACME contributes to biofilm formation by virtue of its impact on the production of PIA cannot be ruled out. Indeed, mutation of sarA results in reduced production of PIA [7], [11], and based on this one possible explanation for our results is that PIA plays a more important role in biofilm formation in UAMS-1625 than in the other USA300 isolates and that, in the absence of ACME, a UAMS-1625 sarA mutant cannot produce enough PIA to sustain biofilm formation irrespective of any other factor including extracellular proteases. To explore this possibility, we examined the relative levels of PIA produced by each strain using an anti-PIA immunoblot. These studies demonstrated that all of the USA300 isolates produced almost undetectable amounts of PIA irrespective of the presence of ACME or the functional status of sarA (Fig. 9). This not only suggests that the relative levels of PIA production do not account for the difference between UAMS-1625 and other USA300 isolates but also that PIA plays little role in USA300 biofilm formation. This is also consistent with previous reports demonstrating that mutation of ica had little effect on biofilm formation not only in LAC [8] but also in other methicillin-resistant S. aureus strains [45].

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