Proteomic Profiling of Urine From Hospitalized Patients With Severe Pneumonia due to SARS-CoV-2 vs Other Causes: A Preliminary Report.

The pathogenesis of coronavirus disease 2019 (COVID-19) pneumonia remains poorly understood. The urine proteome of hospitalized patients with severe COVID-19 pneumonia, compared with severe non-COVID-19 pneumonia controls, was distinct and associated with lower abundance of several host proteins. Protein-specific machine learning analysis outlined biomarker combinations able to differentiate COVID-19 pneumonia from non-COVID-19 pneumonia controls.

Cell Wall Proteomics Reveal Phenotypic Adaption of Drug-Resistant Mycobacterium smegmatis to Subinhibitory Rifampicin Exposure.

Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase ß subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 µg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.

Associating H2O2-and NO-related changes in the proteome of Mycobacterium smegmatis with enhanced survival in macrophage.

Mycobacterium manages to evade the host cell immune system, partially owing to its ability to survive redox stress after macrophage engulfment. Exposure to redox stress has been linked to later replication, persistence, and latent infection. In this work, mass spectrometry was used to elucidate the cell-wide changes that occur in response to sublethal doses of hydrogen peroxide and nitric oxide over time, with Mycobacterium smegmatis being used as a model organism. A total of 3135 proteins were confidently assigned, of which 1713, 1674, and 1713 were identified under NO, H2O2, and control conditions, respectively. Both treatment conditions resulted in changes of protein expression from the DosR regulon as well as those related to lipid metabolism. Complementary to the changes in the proteome, sublethal exposure to NO and H2O2 improved the survival of the bacteria after macrophage infection. Our data indicate that pre-exposure to sublethal doses of these redox stressors causes an alteration in the expression of proteins related to lipid metabolism, suggesting a link between altered lipid metabolism and enhanced survival in macrophages.

Global proteome and phosphoproteome dynamics indicate novel mechanisms of vitamin C induced dormancy in Mycobacterium smegmatis.

Vitamin C has been found to affect mycobacteria in multiple ways, including increasing susceptibility to antimicrobial drugs, inducing dormancy, and having a bactericidal effect. However, the regulatory events mediating vitamin C related adaptations remain largely elusive. Ser/Thr/Tyr protein phosphorylation plays an important regulatory role in mycobacteria, contributing to environmental adaptation, including dormancy and drug resistance. This study utilised the model organism, Mycobacterium smegmatis, and TiO2 phosphopeptide enrichment combined with mass spectrometry-based proteomics methods to elucidate the mycobacterial signalling and regulatory response to sub-lethal concentrations of vitamin C. After initial validation of peptide spectra, 224 non-redundant phosphosites in 154 proteins were retained with high confidence. Data analysis revealed that 30 peptides were differentially phosphorylated with Vitamin C treatment, including novel phosphosites found on both PknG and GarA. Of these significant proteins, we validated 11 by parallel reaction monitoring of high-confidence phosphopeptides. Interestingly, 17/30 phosphopeptides were annotated as part of transmembrane proteins, suggesting that it is likely vitamin C triggers typical signal transduction events in which the protein periplasmic domain perceives environmental signals and the cytoplasmic domain is then phosphorylated. Finally, the diverse nature of phosphorylated proteins involved in signalling, transport, and carbohydrate biosynthesis indicates the extent of such regulatory phosphorylation events. BIOLOGICAL SIGNIFICANCE: Our findings provide new mechanistic insight into a coordinated network of signalling and regulatory responses to sub-lethal vitamin C in Mycobacterium smegmatis and provide evidence that vitamin C is able to act as a novel extracellular signalling molecule. Vitamin C treatment caused changes in both the proteome and phosphoproteome associated with response to oxidative stress, a shift in metabolic regulation and progression toward dormancy, as well as phospho-dependent activation of specific secretory pathways and activation of specific two component and Ser/Thr/Tyr protein kinase activities. This study confirms the potential of vitamin C as convenient means to study aspects of mycobacterial dormancy, including those regulated at post-translational level.