Exploring glycopeptide-resistance in Staphylococcus aureus: a combined proteomics and transcriptomics approach for the identification of resistance-related markers.

BACKGROUND: To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. RESULTS: In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. CONCLUSION: Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.

Correlation of proteomic and transcriptomic profiles of Staphylococcus aureus during the post-exponential phase of growth.

A combined proteomic and transcriptomic analysis of Staphylococcus aureus strain N315 was performed to study a sequenced strain at the system level. Total protein and membrane protein extracts were prepared and analyzed using various proteomic workflows including: 2-DE, SDS-PAGE combined with microcapillary LC-MALDI-MS/MS, and multidimensional liquid chromatography. The presence of a protein was then correlated with its respective transcript level from S. aureus cells grown under the same conditions. Gene-expression data revealed that 97% of the 2'596 ORFs were detected during the post-exponential phase. At the protein level, 23% of these ORFs (591 proteins) were identified. Correlation of the two datasets revealed that 42% of the identified proteins (248 proteins) were amongst the top 25% of genes with highest mRNA signal intensities, and 69% of the identified proteins (406 proteins) were amongst the top 50% with the highest mRNA signal intensities. The fact that the remaining 31% of proteins were not strongly expressed at the RNA level indicates either that some low-abundance proteins were identified or that some transcripts or proteins showed extended half-lives. The most abundant classes identified with the combined proteomic and transcriptomic approach involved energy production, translational activities and nucleotide transport, reflecting an active metabolism. The simultaneous large-scale analysis of transcriptomes and proteomes enables a global and holistic view of the S. aureus biology, allowing the parallel study of multiple active events in an organism.

Shotgun proteomics: a qualitative approach applying isoelectric focusing on immobilized pH gradient and LC-MS/MS.

Shotgun proteomics is a rapid and near universal strategy to identify proteins in complex protein mixtures. After protein digestion, the resulting peptide mixture is submitted to two orthogonal techniques: peptides are first separated according to their isoelectric point (pI) by isoelectric focusing (IEF) on immobilized pH gradient (IPG); after peptide extraction, they are then separated in the second dimension according to their hydrophobic properties by reverse phase liquid chromatography (RPLC). Finally, they are detected by tandem mass spectrometry (MS/MS) and proteins are matched by means of bioinformatics software.

Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes.

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins.

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.