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

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

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

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

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 (OD₅₆₀ = 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

Bacterial Strains Used in This Study.

<p>Bacterial Strains Used in This Study.</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

Impact of <i>saeRS</i> and <i>sarA</i> on biofilm formation in clinical isolates.

<p>Biofilm formation was assessed in USA300 strain FPR3757 and its isogenic <i>sarA</i> and <i>saeRS</i> (<i>sae</i>) mutants. A single asterisk indicates statistical significance (p<0.05) by comparison to the isogenic parent strain. Differences between the FPR3757 <i>saeRS</i> mutant and the <i>saeRS/aur</i> and <i>saeRS/ssp</i> mutants were not significant.</p