Staphylococcus aureus virulence factor expression matters: input from targeted proteomics shows Panton-Valentine leucocidin impact on mortality

Abstract

In the case of commensal bacteria such as Staphylococcus aureus, the transition from commensalism to invasion and disease as well as disease severity in the course of an infection remain poorly predictable on the sole basis of virulence gene content. To determine whether variations in the levels of expression of the numerous S. aureus virulence factors could affect disease occurrence and/or severity, we developed a targeted proteomic approach that monitored 149 peptides surrogates targeting 44 proteins. Semi-quantification was achieved by normalization on the signal of ribosomal proteins. We then evaluated this approach on a series of S. aureus strains from 136 patients presenting a severe community-acquired pneumonia, all admitted to an intensive care unit. After adjusting to the Charlson Comorbidity Index score the multivariate analysis of severity parameters found that HlgB, Nuc, and Tsst-1 were positively associated while BlaI and HlgC were negatively associated with leucopenia. BlaZ and HlgB were positively associated with hemoptysis and HlgC was negatively associated with hemoptysis. Regarding mortality, both the multivariate (1.28; 95%CI[1.02;1.60]) and survival (1.15; 95%CI[1.016;1.302]) analyses showed that only PVL was associated with death in a dose-dependent manner. Beyond highlighting the decisive role of PVL in community-acquired pneumonia severity, this study brings the proof of concept that "expression matters" and proposes a method that can be routinely implemented in laboratories, for any Staphylococcal disease, and which could be developed for other commensal bacteria.

Complex regulation of gamma-hemolysin expression impactsS. aureusvirulence

“…Consistently, although HlgCB targets the same receptors as LukS-PV, it interacts with different motifs, leading to better cell permeabilization (12). Since all S. aureus strains possess the hlg locus (20,21) but not all cause severe CAP (4), the present study strengthens the hypothesis that expression matters (16).…”

“…To validate the previous semi-quantitative proteomic analysis of exotoxins (16), the two virulence factors HlgC and HlgB were quantified using isotope-dilution targeted proteomics in a subset of 39 clinical isolates of the previous cohort. The USA300 SF8300 (USA300) and HT20020209-LUG1799 (ST80) strains were used as control strains because their exoprotein profile and virulence in various animal models are well-defined (18,19).…”

“…Recently, the semi-quantitative proteomic analysis of exotoxins applied to 136 strains isolated from severe CAP suggested that high HlgCB production in PVL-negative strains could be associated with higher mortality. It also showed that HlgB and HlgC levels were not correlated, contrarily to other operon-encoded virulence factors such as LukDE and LukSF-PV (16). We thus decided to investigate the molecular mechanisms leading to HlgC and HlgB expression variations among clinical isolates and assess whether HlgCB is a major determinant for pneumonia severity in a rabbit model (17).…”

Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence

“…Consistently, although HlgCB targets the same receptors as LukS-PV, HlgCB interacts with different motifs, leading to better cell permeabilization ( 12 ). Since all S. aureus strains possess the hlg locus ( 19 , 20 ), but not all strains cause severe CAP ( 4 ), the present study strengthens the hypothesis that the level of expression of this locus matters ( 15 ). However, as in the PVL-positive strain, the inactivation of the Hlg locus did not lead to a full attenuation of virulence, one could raise the hypothesis of mutual compensation between the pore-forming toxins.…”

“…To validate the results of a previous semiquantitative proteomic analysis of exotoxins ( 15 ), the two hemolysins HlgC and HlgB were quantified using isotope-dilution-targeted proteomics in a subset of 39 clinical isolates representative of the genomic diversity of the cohort (maintained proportions of clonal complexes and pvl -positive strains). The USA300 SF8300 (USA300) and HT20020209-LUG1799 (ST80) strains were used as control strains because their exoprotein profiles and virulence in various animal models are well defined ( 16 , 17 ).…”