Surface-Shaving of Staphylococcus aureus Strains and Quantitative Proteomic Analysis Reveal Differences in Protein Abundance of the Surfaceome.

Staphylococcus aureus is a pathogen known to cause a wide range of infections. To find new targets for identification and to understand host-pathogen interactions, many studies have focused on surface proteins. We performed bacterial-cell surface-shaving, followed by tandem mass tag for quantitative mass spectrometry proteomics, to examine the surfaceome of S. aureus. Two steps were performed, the first step including surface protein-deficient mutants of S. aureus Newman strain lacking important virulence genes (clfA and spa, important for adhesion and immune evasion and srtAsrtB, linking surface-associated virulence factors to the surface) and the second step including isolates of different clinical origin. All strains were compared to the Newman strain. In Step 1, altogether, 7880 peptides were identified, corresponding to 1290 proteins. In Step 2, 4949 peptides were identified, corresponding to 919 proteins and for each strain, approximately 20 proteins showed differential expression compared to the Newman strain. The identified surface proteins were related to host-cell-adherence and immune-system-evasion, biofilm formation, and survival under harsh conditions. The results indicate that surface-shaving of intact S. aureus bacterial strains in combination with quantitative proteomics is a useful tool to distinguish differences in protein abundance of the surfaceome, including the expression of virulence factors.

Biofilm formation and dispersal of Staphylococcus aureus wound isolates in microtiter plate-based 2-D wound model.

Biofilm-associated wound infections in diabetic and immunocompromised patients are an increasing threat due to rising antibiotic resistance. Various wound models have been used to screen for efficient antiinfection treatments. However, results from in vitro models do not always match in vivo results, and this represents a bottleneck for development of new infection treatments. In this study, a static 2-D microtiter plate-based biofilm model was tested for growing clinically relevant Staphylococcus aureus wound isolates in various operating conditions, seeking to identify an optimal setup that would yield physiologically relevant results. Specifically, the tested variables included wound-mimicking growth media, precoating of surface with different proteins, multiwell plates with various surface properties, and the effect of bacterial pre-attachment step. Our results indicated that protein precoating is a key factor for supporting biofilm growth. The same wound isolate responded with significant differences in biofilm formation to different wound-mimicking media. Biofilm dispersal, as a proxy for effectiveness of antibiofilm treatments, was also investigated in response to proteinase K. The dispersal effect of proteinase K showed that the biofilm dispersal is contingent upon the specific wound isolate, with isolates CCUG 35571 and ATCC 6538 showing considerable dispersal responses. In conclusion, this study observed a higher biofilm formation in isolates when a protein precoating of collagen type I was applied but being dependent on the growth media selected. That is why we recommend to use simulated wound fluid or a wound-mimicking growth media to perform similar studies. Furthermore, proteinase K is suggested as an important factor that could affect biofilm dispersal within such models, since biofilm dispersal was induced in isolates CCUG 35571 and ATCC 6538 in simulated wound fluid on precoated collagen type I plates.