Comparative impact of diverse regulatory loci on Staphylococcus aureus biofilm formation

Abstract

The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin-resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur- and mgrA-enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin-sensitive strain UAMS-1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS-1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin-resistant versus methicillin-sensitive strains.

Keywords: Biofilm; Staphylococcus aureus; protease; regulation; sarA.

Figure 1

Biofilm phenotypes as a function of assay conditions. Biofilm formation was assessed in LAC and its isogenic mutants under four different assay conditions. To allow direct comparisons between conditions, the results shown in all panels are shown as raw data and represent the average ± standard error of the mean (SEM) from a minimum of two experiments, each of which was repeated with at least three replicates. Mutants that exhibit a statistically significant difference under each assay condition (asterisk; P < 0.05) are indicated in each panel. For every individual strain, including the sarA mutant, the results observed with BFM and plasma coating were statistically significant by comparison to the same strain assayed under all other conditions. Overall order in all panels is LAC followed by isogenic strains with mutations in sarA, agr, arl, atl, clpP, codY, fur, lyt, mgrA, msa, rot, rsbU, rsr, sae, sarS, sarT, sarU, sarV, sarX, sarY, sarZ, sigB, and srr.

Figure 2

Relative impact of Staphylococcus aureus regulatory loci on biofilm formation in vitro. Biofilm formation was assessed in LAC (WT) and its isogenic regulatory mutants using a microtiter plate assay with BFM and plasma coating of the substrate. Results shown represent the average ± SEM from a minimum of six experiments, each of which was repeated with at least three technical replicates. Single asterisk indicates statistical significance by comparison to the parent strain (P < 0.05). Double asterisks indicate statistical significance by comparison to the isogenic sarA mutant (P < 0.05).

Figure 3

Relative impact of sarA versus other regulatory loci in LAC. (A) Biofilm formation was assessed in each regulatory mutant found to have a significant impact on biofilm formation with (+) and without (−) plasmid-based genetic complementation. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least three replicates. Single asterisk indicates that the results observed with the indicated mutant were significantly different from those observed with the LAC parent strain (P < 0.05). Double asterisks indicate that the results observed with the complemented strain were significantly different by comparison to those observed with the uncomplemented isogenic mutant (P < 0.05). (B) Biofilm formation was assessed in each regulatory mutant found to have a significant impact on biofilm formation with (−) and without (+) concomitant mutation of sarA. Single asterisk indicates statistical significance by comparison to the LAC parent strain (P < 0.05). Double asterisks indicate significance of the double mutant relative to the corresponding isogenic single mutant (P < 0.05).

Figure 4

Impact of LAC regulatory mutations on accumulation of SarA. Relative amounts of SarA were assessed by western blot. Graphs illustrate quantitative results from three separate blots. Single asterisk indicates statistical significance by comparison to the LAC parent strain (P < 0.05).

Figure 5

Impact of extracellular proteases in LAC. (A) Total protease activity was assessed in LAC mutants with (−) and without (+) concomitant mutation of sarA. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least four replicates. Single asterisk indicates statistical significance of the individual mutants by comparison to the LAC parent strain (P < 0.05). Double asterisks indicate significance of the double mutant relative to the appropriate isogenic single mutant (P < 0.05). (B) Biofilm formation was assessed in LAC and its regulatory mutants as a function of the relative capacity to produce extracellular proteases. Protease positive refers to strains with the capacity to produce all extracellular proteases. Protease deficient refers to strains unable to produce aureolysin, SspA, SspB, and ScpA. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least six replicates. Asterisk indicates statistical significance of protease-deficient derivatives relative to the respective isogenic protease-positive strains (P < 0.05).

Figure 6

Impact of extracellular nucleases in LAC. (A) Total nuclease activity was assessed in LAC regulatory mutants with (−) and without (+) concomitant mutation of sarA using a FRET-based assay. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least four replicates. Single asterisk indicates statistical significance of the individual mutants by comparison to the LAC parent strain (P < 0.05). Double asterisks indicate significance of the double mutant relative to the corresponding isogenic single mutant (P < 0.05). (B) Relative levels of nuclease activity were assessed as above, but as a function of the production of extracellular proteases. Single asterisk indicates statistical significance of the individual mutants by comparison to the LAC parent strain (P < 0.05). Double asterisks indicate significance of the sarA/ΔProtease mutant relative to the corresponding isogenic sarA mutant (P < 0.05). (C) For comparison, relative levels of nuclease activity were also assessed using DNase Agar assay.

Figure 7

Impact of PIA in LAC. Biofilm formation was assessed using a microtiter plate assay with and without the addition of Dispersin B. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least three replicates. Asterisks indicate mutants in which the addition of Dispersin B had a statistically significant impact by comparison to the same strain in the absence of Dispersin B (P < 0.05). Inset illustrates levels of PIA production in LAC and its indicated isogenic mutants in the absence of Dispersin B.

Figure 8

Relative impact of sarA versus other regulatory loci in UAMS-1. (A) Biofilm formation was assessed in each regulatory mutant found to have a significant impact on biofilm formation with (+) and without (−) plasmid-based genetic complementation. Results shown represent the average ± SEM from a minimum of three experiments, each of which was repeated with at least three replicates. Single asterisk indicates that the results observed with the indicated mutant were significantly different from those observed with the UAMS-1 (U1) parent strain (P < 0.05). Double asterisks indicate that the results observed with the complemented strain were significantly different by comparison to those observed with the uncomplemented isogenic mutant (P < 0.05). (B) Biofilm formation was assessed in each regulatory mutant found to have a significant impact on biofilm formation with (−) and without (+) concomitant mutation of sarA. Single asterisk indicates statistical significance of the individual mutants by comparison to the UAMS-1 parent strain (P < 0.05). Double asterisks indicate significance of the double mutant relative to the corresponding isogenic single mutant (P < 0.05).

Figure 9

Impact of extracellular proteases in UAMS-1. Total protease activity was assessed in UAMS-1 mutants with (−) and without (+) concomitant mutation of sarA. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least four replicates. Single asterisk indicates statistical significance by comparison to the UAMS-1 parent strain (P < 0.05). Double asterisks indicate significance of the double mutant relative to the corresponding isogenic single mutant (P < 0.05).

Figure 10

Impact of extracellular nucleases in UAMS-1. Total nuclease activity was assessed in UAMS-1 and its isogenic mutants with (−) and without (+) concomitant mutation of sarA using a FRET-based assay. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least four replicates. Single asterisk indicates statistical significance by comparison to the isogenic parent strain (P < 0.05). Double asterisks indicate significance of the double mutant relative to the respective isogenic single mutant (P < 0.05). Inset illustrates results observed using DNase agar.

Figure 11

Impact of PIA in UAMS-1. (A) PIA production as assessed by dot blot. Graph illustrates quantitative results obtained from three independent blots, with a representative dot blot shown below the graph. Single asterisk indicates statistical significance by comparison to the isogenic UAMS-1 (U1) parent strain (P < 0.05). Double asterisks indicate significance of the double mutant relative to the appropriate isogenic single mutant (P < 0.05). (B) Biofilm formation was assessed with and without Dispersin B. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least three replicates. Asterisk indicates mutants in which the addition of Dispersin B had a statistically significant impact by comparison to the same strain in the absence of Dispersin B (P < 0.05).

Figure 12

Impact of sarA mutants on wild-type biofilm phenotypes. (A) Biofilm formation was assessed in LAC or UAMS-1 (U1) after coculture with the indicated sarA mutants, with each parent strain and its isogenic sarA mutant included as controls. Results shown represent the average ± SEM from a minimum of three experiments, each of which was repeated with at least three replicates. Single asterisk indicates statistical significance by comparison to the respective parent strain (P < 0.05). Double asterisks indicate statistical significance by comparison to the isogenic sarA mutant (P < 0.05). Triple asterisks indicate a statistically significant difference between results observed with the LAC sarA mutant by comparison to the UAMS-1 sarA mutant (P < 0.05). (B) Total protease activity was assessed in LAC, its isogenic sarA mutant, UAMS-1, and its isogenic sarA mutant. WT parent strains, LAC, and UAMS-1, were set to 1.0 with the respective sarA mutants set relative to that. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least four replicates. Single asterisk indicates statistical significance by comparison to the respective parent strain (P < 0.05).

Figure 13

Strain-dependent impact of codY on biofilm formation. The impact of mutating codY on biofilm formation was assessed as a function of methicillin resistance status, with the strains shown in the upper panel being MRSA isolates, and those shown in the lower panel being MSSA isolates. Results shown represent the average ± SEM from a minimum of two experiments, each of which was repeated with at least six replicates. Asterisk indicates statistical significance by comparison to the respective parent strain (P < 0.05).