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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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Impact of sarA and agr on biofilm formation in USA300 isolates.Biofilm formation was assessed in each of three USA300 isolates (WT) and their isogenic sarA (S), agr (A), and sarA/agr (SA) mutants using a static microtiter plate assay. 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 isogenic sarA and sarA/agr mutants but no difference between any wild-type strains and its agr mutant or between isogenic sarA and sarA/agr mutants.
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pone-0010790-g004: Impact of sarA and agr on biofilm formation in USA300 isolates.Biofilm formation was assessed in each of three USA300 isolates (WT) and their isogenic sarA (S), agr (A), and sarA/agr (SA) mutants using a static microtiter plate assay. 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 isogenic sarA and sarA/agr mutants but no difference between any wild-type strains and its agr mutant or between isogenic sarA and sarA/agr mutants.

Mentions: We also found that mutation of agr did not enhance biofilm formation in any of the USA300 isolates irrespective of whether plasma coating was employed (Fig. 4) and this is consistent with the results of Lauderdale et al. [8] who found that mutation of agr in LAC had little impact on biofilm formation as assessed using flow cells. However, this certainly does not preclude an important role for agr in S. aureus biofilm formation, particularly given the relatively low levels of RNAIII production observed in USA300 isolates under our biofilm growth conditions. For instance, O'Neill et al. [45] demonstrated that mutation of agr enhanced biofilm formation in 5 of 13 methicillin-resistant clinical isolates even when the medium was supplemented with glucose to a degree that has been associated with reduced expression of agr [7]. Additionally, mutation of sigB in LAC limited biofilm formation even in flow cells, and concomitant mutation of agr reversed this effect [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)

Impact of sarA and agr on biofilm formation in USA300 isolates.Biofilm formation was assessed in each of three USA300 isolates (WT) and their isogenic sarA (S), agr (A), and sarA/agr (SA) mutants using a static microtiter plate assay. 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 isogenic sarA and sarA/agr mutants but no difference between any wild-type strains and its agr mutant or between isogenic sarA and sarA/agr mutants.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010790-g004: Impact of sarA and agr on biofilm formation in USA300 isolates.Biofilm formation was assessed in each of three USA300 isolates (WT) and their isogenic sarA (S), agr (A), and sarA/agr (SA) mutants using a static microtiter plate assay. 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 isogenic sarA and sarA/agr mutants but no difference between any wild-type strains and its agr mutant or between isogenic sarA and sarA/agr mutants.
Mentions: We also found that mutation of agr did not enhance biofilm formation in any of the USA300 isolates irrespective of whether plasma coating was employed (Fig. 4) and this is consistent with the results of Lauderdale et al. [8] who found that mutation of agr in LAC had little impact on biofilm formation as assessed using flow cells. However, this certainly does not preclude an important role for agr in S. aureus biofilm formation, particularly given the relatively low levels of RNAIII production observed in USA300 isolates under our biofilm growth conditions. For instance, O'Neill et al. [45] demonstrated that mutation of agr enhanced biofilm formation in 5 of 13 methicillin-resistant clinical isolates even when the medium was supplemented with glucose to a degree that has been associated with reduced expression of agr [7]. Additionally, mutation of sigB in LAC limited biofilm formation even in flow cells, and concomitant mutation of agr reversed this effect [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