Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus

International audienceStaphylococcus aureus is a dangerous human pathogen which antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Serine protease like proteins (Spl) encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins likely involved in virulence. Here we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolyzed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The likely physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel, significantly different mechanistic details

Specific protease activity indicates the degree of Pseudomonas aeruginosa infection in chronic infected wounds

Chronic non-healing wounds are a major health problem with resident bacteria strongly implicated in their impaired healing. A rapid-screen to provide detailed knowledge of wound bacterial populations would therefore be of value and help prevent unnecessary and indiscriminate use of antibiotics—a process associated with promoting antibiotic resistance. We analysed chronic wound fluid samples, which had been assessed for microbial content, using 20 different fluorescent labelled peptide substrates to determine whether protease activity correlated with the bacterial load. Eight of the peptide substrates showed significant release of fluorescence after reaction with some of the wound samples. Comparison of wound fluid protease activities with the microbiological data indicated that there was no correlation between bacterial counts and enzyme activity for most of the substrates tested. However, two of the peptide substrates produced a signal corresponding with the microbial data revealing a strong positive correlation with Pseudomonas aeruginosa numbers. This demonstrated that short fluorescent labelled peptides can be used to detect protease activity in chronic wound fluid samples. The finding that two peptides were specific indicators for the presence of P. aeruginosa may be the basis for a diagnostic test to determine wound colonisation by this organism

Genetic and Molecular Predictors of High Vancomycin MIC in Staphylococcus aureus Bacteremia Isolates

An elevated vancomycin MIC is associated with poor outcomes in Staphylococcus aureus bacteremia (SAB) and is reported in patients with methicillin-susceptible S. aureus (MSSA) bacteremia in the absence of vancomycin treatment. Here, using DNA microarray and phenotype analysis, we investigated the genetic predictors and accessory gene regulator (agr) function and their relationship with elevated vancomycin MIC using blood culture isolates from a multicenter binational cohort of patients with SAB. Specific clonal complexes were associated with elevated (clonal complex 8 [CC8] [P < 0.001]) or low (CC22 [P < 0.001], CC88 [P < 0.001], and CC188 [P = 0.002]) vancomycin MIC. agr dysfunction (P = 0.014) or agr genotype II (P = 0.043) were also associated with an elevated vancomycin MIC. Specific resistance and virulence genes were also linked to an elevated vancomycin MIC, including blaZ (P = 0.002), sea (P < 0.001), clfA (P < 0.001), splA (P = 0.001), and the arginine catabolic mobile element (ACME) locus (P = 0.02). These data suggest that inherent organism characteristics may explain the link between elevated vancomycin MICs and poor outcomes in patients with SAB, regardless of the antibiotic treatment received. A consideration of clonal specificity should be included in future research when attempting to ascertain treatment effects or clinical outcomes