Proteomic and Transcriptomic Profiling of Staphylococcus aureus Surface LPXTG-proteins: Correlation with agr Genotypes and Adherence Phenotypes

Mathilde Ythier,Paul Majcherczyk,Aurélie Gfeller,Grégory Resch,Alexandre Panchaud,Manfredo Quadroni,Philippe Moreillon,Patrice Waridel

doi:10.1074/mcp.m111.014191

Mathilde Ythier, Paul Majcherczyk + Show 6 more

Open Access

https://doi.org/10.1074/mcp.m111.014191

Copy DOI

Abstract

Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureus physiology should integrate all three approaches.

Highlights

From the ‡Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland; §Protein Analysis Facility, University of Lausanne, Lausanne, Switzerland
Trypsin digestion released different sets of peptides when LPXTG-proteins were expressed in parent S. aureus or surrogate L. lactis, as exemplified by protein A (Spa), clumping factor A (ClfA), clumping factor B (ClfB) and fibronectin-binding protein A (FnBPA)
This was achieved thank to a preliminary analysis of each of these proteins expressed in L. lactis, which does not carry S. aureus proteins [35]

Summary

Introduction

From the ‡Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland; §Protein Analysis Facility, University of Lausanne, Lausanne, Switzerland. Trypsin digestion released different sets of peptides when LPXTG-proteins were expressed in parent S. aureus or surrogate L. lactis, as exemplified by protein A (Spa), clumping factor A (ClfA), clumping factor B (ClfB) and fibronectin-binding protein A (FnBPA).

Results

Conclusion