Limits...
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.

Show MeSH

Related in: MedlinePlus

Production of RNAIII as a function of strain, growth phase, and growth medium.RNA was extracted from each strain grown in biofilm medium (BM) during the exponential (E, OD560 = 1.0), post-exponential growth phase (PE, OD560 = 3.0) and stationary (S) phases and the amount of RNAIII determined by qRT-PCR. RNA was also isolated from stationary-phase cultures grown in TSB. The value observed with UAMS-1 during the exponential growth phase was set at 1.0 with the results observed for other strains shown relative to this value. Results are shown as the mean ± the standard deviation of triplicate samples. Statistical analysis of the results observed in stationary-phase samples grown in BM confirmed a significant difference between RN6390 and all other strains and between UAMS-1782 and UAMS-1790 by comparison to both UAMS-1 and UAMS-1625.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2875390&req=5

pone-0010790-g001: Production of RNAIII as a function of strain, growth phase, and growth medium.RNA was extracted from each strain grown in biofilm medium (BM) during the exponential (E, OD560 = 1.0), post-exponential growth phase (PE, OD560 = 3.0) and stationary (S) phases and the amount of RNAIII determined by qRT-PCR. RNA was also isolated from stationary-phase cultures grown in TSB. The value observed with UAMS-1 during the exponential growth phase was set at 1.0 with the results observed for other strains shown relative to this value. Results are shown as the mean ± the standard deviation of triplicate samples. Statistical analysis of the results observed in stationary-phase samples grown in BM confirmed a significant difference between RN6390 and all other strains and between UAMS-1782 and UAMS-1790 by comparison to both UAMS-1 and UAMS-1625.

Mentions: The levels of RNAIII produced by each strain were dependent on both growth phase and growth medium. RNAIII levels were higher in TSB than in biofilm medium (BM) in all strains except RN6390, which produced comparable levels of RNAIII whether grown in BM or TSB (Fig. 1). The reduced production of RNAIII in BM is consistent with reports demonstrating that RNAIII production is repressed in medium supplemented with glucose [7], [24]. Nevertheless, all clinical isolates produced RNAIII in the expected growth-phase dependent pattern when grown in BM in that, by comparison to exponential growth, the production of RNAIII was increased in all strains in the post-exponential growth phase while, by comparison to the post-exponential growth phase, RNAIII levels were decreased in all strains in stationary-phase cultures (Fig. 1). RN6390 was once again an exception in that the levels of RNAIII observed in this strain were generally consistent across all growth phases.


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 RNAIII as a function of strain, growth phase, and growth medium.RNA was extracted from each strain grown in biofilm medium (BM) during the exponential (E, OD560 = 1.0), post-exponential growth phase (PE, OD560 = 3.0) and stationary (S) phases and the amount of RNAIII determined by qRT-PCR. RNA was also isolated from stationary-phase cultures grown in TSB. The value observed with UAMS-1 during the exponential growth phase was set at 1.0 with the results observed for other strains shown relative to this value. Results are shown as the mean ± the standard deviation of triplicate samples. Statistical analysis of the results observed in stationary-phase samples grown in BM confirmed a significant difference between RN6390 and all other strains and between UAMS-1782 and UAMS-1790 by comparison to both UAMS-1 and UAMS-1625.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010790-g001: Production of RNAIII as a function of strain, growth phase, and growth medium.RNA was extracted from each strain grown in biofilm medium (BM) during the exponential (E, OD560 = 1.0), post-exponential growth phase (PE, OD560 = 3.0) and stationary (S) phases and the amount of RNAIII determined by qRT-PCR. RNA was also isolated from stationary-phase cultures grown in TSB. The value observed with UAMS-1 during the exponential growth phase was set at 1.0 with the results observed for other strains shown relative to this value. Results are shown as the mean ± the standard deviation of triplicate samples. Statistical analysis of the results observed in stationary-phase samples grown in BM confirmed a significant difference between RN6390 and all other strains and between UAMS-1782 and UAMS-1790 by comparison to both UAMS-1 and UAMS-1625.
Mentions: The levels of RNAIII produced by each strain were dependent on both growth phase and growth medium. RNAIII levels were higher in TSB than in biofilm medium (BM) in all strains except RN6390, which produced comparable levels of RNAIII whether grown in BM or TSB (Fig. 1). The reduced production of RNAIII in BM is consistent with reports demonstrating that RNAIII production is repressed in medium supplemented with glucose [7], [24]. Nevertheless, all clinical isolates produced RNAIII in the expected growth-phase dependent pattern when grown in BM in that, by comparison to exponential growth, the production of RNAIII was increased in all strains in the post-exponential growth phase while, by comparison to the post-exponential growth phase, RNAIII levels were decreased in all strains in stationary-phase cultures (Fig. 1). RN6390 was once again an exception in that the levels of RNAIII observed in this strain were generally consistent across all growth phases.

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