SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates of Staphylococcus aureus LAC and UAMS-1

Abstract

Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.

Keywords: Staphylococcus aureus; mgrA; rot; sarA; sarR; sarS; sarZ; biofilm; extracellular protease; regulation.

Figure 1.

Examination of captured proteins by SDS-PAGE and Western blot. The region extending ~400 bp upstream of the genes and/or operons encoding extracellular proteases indicated on the left were used as DNA baits to capture proteins from whole cell lysates prepared from the USA300 strain LAC or its isogenic sarA mutant (ΔsarA). Proteins were eluted with increasing concentrations of salt and examined by SDS-PAGE (left side of each panel) and Western blot using an anti-SarA antibody (right side of each panel). Control samples included experiments done with a LAC lysate and no DNA ait (top)

Figure 2.

Relative impact of regulatory mutations on protease activity. Protease activity in conditioned medium (CM) from stationary phase cultures from LAC (top), UAMS-1(bottom), and each of the indicated isogenic regulatory mutants was assessed using a gelatin-based FRET assay. Results are reported as the average ± standard error of the mean from two biological replicates, each of which included three experimental replicates. Asterisk indicates statistical significance by comparison to the results observed with the isogenic parent strain. Doubles asterisks indicate statistical significance by comparison to the isogenic sarA mutant

Figure 3.

Relative impact of regulatory loci on expression of individual protease genes and operons in LAC and UAMS-1. The indicated pCM11 gfp reporter plasmids were introduced into LAC, UAMS-1 and each of the isogenic regulatory mutants. Results are reported as the average mean fluorescence intensity ± standard error of the mean from two biological replicates, each of which included three experimental replicates. Asterisk indicates statistical significance by comparison to the results observed with the isogenic parent strain. Doubles asterisks indicate statistical significance by comparison to the isogenic sarA mutant

Figure 4.

Relative impact of regulatory loci on protein abundance. Top: SDS-PAGE of conditioned medium (CM) from LAC (top), UAMS-1 (bottom) and isogenic regulatory mutants generated in each parent strain. Bottom: Western blots of CM from the same strains using antibodies for alpha toxin (Hla, top), protein A (Spa, left), and Nuc1 (right). Purified alpha toxin (Hla) was included as a control for alpha toxin blots (UAMS-1 does not produce alpha toxin), while protein A and nuc1 mutants (Δspa and Δnuc1, respectively) were included as controls for the protein A and Nuc1 blots

Figure 5.

Relative impact of regulatory loci on biofilm formation. Biofilm formation was assessed in LAC (top), UAMS-1 (bottom) and their isogenic regulatory mutants. Results are reported as the average ± standard error of the mean from two biological replicates, each of which included six experimental replicates. Asterisk indicates statistical significance by comparison to the results observed with the isogenic parent strain. Doubles asterisks indicate statistical significance by comparison to the isogenic sarA mutant

Figure 6.

Impact of the functional status of regulatory mutations on protease production in isogenic sarA mutants. Protease activity in conditioned medium (CM) from stationary phase cultures from LAC, UAMS-1, and each of the indicated isogenic regulatory mutants was assessed using a gelatin-based FRET assay. Results are reported as the average ± standard error of the mean from two biological replicates, each of which included three experimental replicates. Asterisk indicates statistical significance by comparison to the results observed with the isogenic parent strain. Doubles asterisks indicate statistical significance by comparison to the isogenic sarA mutant

Figure 7.

Impact of the functional status of regulatory mutations on expression of protease genes/operons in LAC sarA mutants and UAMS-1 sarA mutants. pCM11 gfp reporter plasmids were introduced into LAC or UAMS-1 and the indicated regulatory mutants. Results are reported as the average mean fluorescence intensity ± standard error of the mean from two biological replicates, each of which included three experimental replicates. Asterisk indicates statistical significance by comparison to the results observed with the isogenic parent strain. Doubles asterisks indicate statistical significance by comparison to the isogenic sarA mutant

Figure 8.

Relative impact of regulatory loci on protein abundance. Top: SDS-PAGE of conditioned medium (CM) from LAC (left), UAMS-1 (right), their isogenic sarA mutants, and isogenic sarA mutants with additional mutations in the genes encoding each of the other regulatory proteins examined in this study. Bottom: Western blots of CM from the same strains using antibodies for alpha toxin (Hla, top), protein A (Spa, left), and Nuc1 (right). Purified alpha toxin (Hla) was included as a control for alpha toxin blots (UAMS-1 does not produce alpha toxin), while protein A and nuc1 mutants (Δspa and Δnuc1, respectively) were included as controls for the protein A and Nuc1 blots

Figure 9.

Relative impact of regulatory loci on biofilm formation. Biofilm formation was assessed in LAC (top), UAMS-1 (bottom), their isogenic sarA mutants, and isogenic sarA mutants with additional mutations in the genes encoding each of the other regulatory proteins examined in this study. Results are reported as the average ± standard error of the mean from two biological replicates, each of which included six experimental replicates. Asterisk indicates statistical significance by comparison to the results observed with the isogenic parent strain. Doubles asterisks indicate statistical significance by comparison to the isogenic sarA mutant

Figure 10.

Differential protein binding to the spl (left) and the aureolysin (right) promoters in LAC versus the isogenic ΔsarA mutant. Volcano plots were generated based on fold-change of protein levels using the averaged spectral counts from biological triplicates. The x-axis indicates a log₂ fold-change, and the y-axis indicates −log₁₀ p-value based on the Student’s t-test. The horizontal line indicates a p-value <0.05, and the vertical lines represent a fold-change >2. Proteins in which the abundance was reduced to a statistically significant degree in ΔsarA lysates compared to wild-type lysates are shown as red dots in the upper right quadrant, whereas those that were present in increased amounts are shown as blue circles in the upper left quadrant. Black dots indicate proteins for which differences in abundance were not statistically significant

Figure 11.

Impact of individual proteases on accumulation of alpha toxin (top) and cytotoxicity (bottom). Top: Conditioned medium (CM) from the indicated strains was resolved by SDS-PAGE prior to Western blotting with an anti-alpha toxin antibody. The control consists of purified alpha toxin. As previously described, this commercially available antibody is cross-reactive with gamma toxin.^13,60 Bottom: Cytotoxicity was assessed using conditioned medium (CM) from stationary phase cultures of LAC (WT), its isogenic sarA mutant, and derivatives of the sarA mutant unable to produce the indicted proteases. Assays were done with RAW (left) or MC3T3 cells (right) as surrogates for primary osteoclasts and osteoblasts, respectively. Results are reported as relative viability. Single asterisk indicates a statistically significant increase in viability by comparison to CM from LAC (WT)

Figure 12.

Impact of individual specific proteases on biofilm formation. Biofilm formation was assessed using a microtiter plate assay as previously described.¹⁶ WT refers to LAC (left) or UAMS-1 (right). “Total” refers to a sarA mutant unable to produce any extracellular protease. “Total + spl” refers to a sarA mutant unable to produce any extracellular protease other than those encoded by the spl operon. All other designations indicate the genes encoding specific proteases that were mutated in the LAC or UAMS-1 sarA mutants. Single asterisk indicates statistical significance relative to the isogenic parent strain. Double asterisks indicate statistical significance relative to the isogenic sarA mutant

Figure 13.

Characterization of regions containing cis active elements that contribute to SarA-mediated repression of extracellular proteases. Constructs containing the indicated number of base pairs upstream of each protease gene/operon translational start site were used to generate truncated versions of the pCM11 gfp reporter plasmid indicated in each panel. These were then introduced into LAC and its sarA mutant and fluorescence assessed after ON growth. Results are reported as the average mean fluorescence intensity ± standard error of the mean from two biological replicates, each of which included three experimental replicates. The sequence below each panel illustrates the region upstream of each protease gene/operon with the region containing putative SarA binding sites (bold italics), putative −35, −10, and ribosome-binding sites (bold underlined), and transcriptional and translational start sites (underlined)