Bacteria
Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based ProteotypingPeptide biomarker candidates for the respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus are presented. First, bacterial cultures representing the genetic variability in each of the four species, were analyzed. The peptide biomarker candidates were then experimentally verified to be present in a clinical situation by analyzing true positive clinical samples. The most promising peptide biomarkers were used in a targeted MS mode, demonstrating their use for future clinical implementation.
Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated PeptidesThe citrullinome of P. gingivalis outer membrane vesicles (OMV) has been explored by a novel two-dimensional separation system combined with high resolution mass spectrometry and in-house build software. Analysis of OMVs from wild-type and two PPAD mutants resulted in confident discrimination based on citrullinated peptides. In the wild-type citrullinome 78 proteins were identified having a total of 161 validated citrullination sites. A single citrullination was identified in the C351A mutant and none in the ΔPPAD mutant.
Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid ExtractionSample Preparation by Easy Extraction and Digestion (SPEED) is a universal method for peptide generation from various sources. Because of its detergent-free chemistry, SPEED offers some inherent benefits for bottom-up proteomics sample preparation, including improved quantitative reproducibility, simple applicability and enhanced proteome coverage for lysis-resistant sample types as well as very rapid sample processing. SPEED has high automatization potential and can contribute to simplify and standardize sample preparation and thus enhance reproducibility in proteomics.
Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion SystemKCl stimulation triggers assembly of the Francisella T6SS in culture. Differential whole cell proteomics reveals that the amounts of the T6SS proteins remain unchanged upon KCl stimulation. A phosphoproteomic analysis identifies a phosphorylation on the T6SS sheath, at site Y139 of IglB. Our data demonstrate that site Y139 of IglB plays a critical role in T6SS biogenesis, providing novel mechanistic insight into the role of sheath phosphorylation in T6SS biogenesis.
Fast and Accurate Bacterial Species Identification in Urine Specimens Using LC-MS/MS Mass Spectrometry and Machine LearningWe have developed a new method for the identification of bacterial species causing Urinary Tract Infections. The first training step used DIA analysis on multiple replicates of bacterial inoculates to define a peptide signature by machine learning classifiers. In a second identification step, the signature is monitored by targeted proteomics on unknown samples. This fast, culture-free and accurate method paves the way of the development of new diagnostic approaches limiting the emergence of antimicrobial resistances.
ScCobB2-mediated Lysine Desuccinylation Regulates Protein Biosynthesis and Carbon Metabolism in Streptomyces coelicolorLysine succinylation is a prevalent protein modification that regulates multiple critical cellular processes. Here, we performed a quantitative succinylome analysis in the model soil bacterium Streptomyces coelicolor after characterization of a specific desuccinylase ScCobB2. Comparison of the ΔScCobB2 to the wild-type succinylome identified a total of 673 unique succinylated sites, and among which, 144 protein sites are statistically hypersuccinylated in ΔScCobB2 cells. Analyses of these hypersuccinylated proteins suggested they are enriched in two major pathways, protein biosynthesis and carbon metabolism. We propose that ScCobB2 has critical regulatory roles in S. coelicolor cellular physiology.
Virulence Factors Produced by Staphylococcus aureus Biofilms Have a Moonlighting Function Contributing to Biofilm IntegrityWe comprehensively profiled intracellular and ECM proteomes of S. aureus flow biofilms and complemented these data by metabolic footprint analysis and phenotypic assays. We show that moonlighting, secreted virulence factors and ribosomal proteins within the ECM contribute to biofilm stabilization. Mechanistically, we propose that these alkaline proteins get protonated in an acidified ECM (because of the release of acids upon fermentation) mediating electrostatic interactions with anionic cell surface components, eDNA, and metabolites, which leads to cell aggregation and ECM stabilization.
Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways*Many Gram-negative pathogens employ type three secretion systems to translocate specialised bacterial effector proteins into the host cell. Endogenous levels of arginine-GlcNAcylation by two Salmonella effectors, SseK1 and SseK3, were determined by quantitative MS-based proteomics. The effectors modified different host death domain containing signaling proteins required for inflammatory and cell death signaling. The study developed new tools to study host-effector interactions and established the importance of native levels of effector expression in distinguishing between authentic and misleading interactions and activities.
Metabolic Cross-talk Between Human Bronchial Epithelial Cells and Internalized Staphylococcus aureus as a Driver for InfectionStaphylococcus aureus invades bronchial epithelial cells to reach underlying lung tissue and to escape from the human immune defenses or antibiotic therapy. The internalized pathogen achieves these objectives by differentiation into growing and dormant subpopulations. Here we tracked the dynamic interactions between internalized bacteria and their host over four days by quantitative proteomics. The results highlight metabolic cross-talk between host and pathogen as a key driver for mutual adaptation and the outcome of infection.