11 research outputs found
saeRS and sarA Act Synergistically to Repress Protease Production and Promote Biofilm Formation in Staphylococcus aureus
Mutation of the staphylococcal accessory regulator (sarA) limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L) or a Newman saeRS mutant (ΔsaeRS) resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level
Impact of <i>saeRS</i> and surface-associated FnbA on biofilm formation in Newman.
<p>Surface-anchored FnbA was restored in Newman (New), its <i>saeS</i>-repaired derivative (P18L), and its isogenic <i>saeRS</i> mutant (<i>sae</i>) by introduction of a plasmid-borne copy of <i>fnbA</i>. Biofilm formation was assessed using a microtiter plate assay, with UAMS-1 (U1) and its <i>sarA</i> mutant included as positive and negative controls, respectively. <i>sarA</i> mutants are designated as “S.” Asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain (WT).</p
Impact of protein A on biofilm formation in Newman.
<p>Biofilm formation was assessed using a microtiter plate assay in Newman and its <i>sarA</i> and <i>spa</i> derivatives without the introduction of surface-anchored FnbA. Single asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain. Double asterisk indicates significance by comparison to the isogenic <i>sarA</i> mutant.</p
Impact of endogenous fibronectin-binding proteins on biofilm formation in Newman.
<p>Biofilm formation was assessed using a microtiter plate assay in Newman with and without introduction of surface-anchored FnbA (pFnbA) and/or mutation of its endogenous <i>fnbA</i> and <i>fnbB</i>. Asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain (WT).</p
Interactions between <i>sarA</i> and <i>saeRS</i>.
<p>Top: Production of SarA was assessed by western blot using SarA antibody in the indicated strains (WT) and their isogenic <i>sarA</i> mtuants (S). Bottom: Impact of <i>sarA</i> on transcription of <i>saeR</i> in post-exponential cultures (OD<sub>560</sub> = 3.0) was assessed by qRT-PCR. Results are shown relative to those observed with FPR3757, which were set to a value of 1.0. Asterisks indicate statistical significance (p<0.05) by comparison to the parent strain.</p
Impact of <i>saeRS</i> and <i>sarA</i> on protease production.
<p>Production of extracellular proteases in derivatives of Newman as a function of <i>saeRS</i> and <i>sarA</i> was assessed by zymography using gelatin as the substrate. The presumed identity of individual proteases is indicated to the right. The graph illustrates relative expression levels the <i>sspA</i> promoter as assessed using an <i>sspA::lux</i> reporter. Differences between the Newman <i>sarA</i> mutant, the P18L <i>sarA</i> mutant, and the <i>saeRS/sarA</i> mutant were all statistically significant (p<0.05) by comparison to Newman. Differences between the <i>sarA/saeRS</i> and the P18L <i>sarA</i> mutants, and between the P18L <i>sarA</i> mutant and the Newman <i>sarA</i> mutant, were also significant.</p
Impact of <i>sarA</i>, <i>saeRS</i>, and extracellular proteases on accumulation of FnbA and biofilm formation.
<p>Top: Relative amounts of surface-anchored FnbA were assessed in Newman (New), its <i>saeS</i>-repaired derivative (P18L), and its <i>saeRS</i> mutant (<i>sae</i>) after introduction of an intact copy of <i>fnbA</i> on a plasmid. Newman without this plasmid was included as a negative control. The impact of mutating <i>sarA</i> was assessed in each of these strains together with the impact of mutating the gene encoding aureolysin (<i>aur</i>), <i>sspABC</i> (<i>ssp</i>) or <i>sae</i> on the phenotype of the <i>sarA</i> mutants. Bottom: Biofilm formation was assessed by microtiter plate assay in Newman and P18L as well as their <i>sarA</i> and <i>sarA/ssp</i> derivatives after the introduction of pFnbA.</p
PCR Primers and Probes Used in This Study.
1<p>Underlined sequences correspond to attB and attP sites, as indicated.</p
Impact of aureolysin on <i>saeRS</i> and <i>sarA</i>-dependent biofilm formation.
<p>Biofilm formation was assessed in Newman, its P18L derivative, and their <i>sarA</i>, <i>sarA/aur</i> and <i>sarA/ssp</i> mutants with (left) and without (right) the introduction of an intact copy of <i>fnbA</i>. A single asterisk indicates statistical significance (p<0.05) by comparison to the isogenic parent strain, while the double asterisk indicates statistical significance (p<0.05) by comparison to the isogenic <i>sarA</i> mutant.</p