Project description:Mycobacterium marinum is a pathogenic bacterium that causes infections in both fish and humans. The PE/PPE protein family is unique to mycobacteria and has been implicated in various aspects of mycobacterial pathogenicity. Understanding the presence and potential function of these proteins in M. marinum is essential for unraveling the mechanisms underlying its virulence. In this study, we conducted a proteomics analysis to investigate the secretion and potential role of the PE/PPE protein family in the culture filtrate of M. marinum. To investigate the presence of PE/PPE proteins in the culture filtrate, we employed a proteomics approach combining Asp-N and trypsin based digestion. Cultures of M. marinum were grown under laboratory conditions, and the culture filtrate was collected and subjected to digestion and LC-MS injection. Our proteomics analysis revealed the secretion of about half PE/PPE proteins in the culture filtrate of M. marinum. Majority of these proteins are confirmed to be involved in EspG5 interaction. The identification of these proteins in the culture filtrate suggests their secretion and potential role in M. marinum. Our proteomics analysis highlights the presence of PE/PPE proteins in the culture filtrate of M. marinum, suggesting their secretion and potential involvement in the pathogenicity of this bacterium.
Project description:Mycobacterium tuberculosis is the world’s leading bacterial infectious agent. The absence of an effective vaccine and the rise of multi-drug resistant M. tuberculosis strains emphasize the need to identify novel targets for prevention and intervention strategies. Previously, the M. marinum orthologue of the aspartic protease PecA (PE_PGRS35) was found on the bacterial cell surface of the pathogenic strain M. marinum, after secretion by the specialized type VII secretion system. PecA was shown to be responsible for the processing of secreted PE_PGRS proteins, including itself. Both M. marinum and M. tuberculosis encode two additional predicted aspartic proteases with similar secretion domains (PE_PGRS16 and PE26). In this study, the roles of the M. marinum PecA, PecB and PecC were studied using frameshift mutants generated by CRISPR-Cas9 technology. Numerous PecA substrates were identified using analysis of semi-tryptic peptides detected by proteomics of secreted, surface-associated protein fractions. The most abundant substrates are members of the PE and PPE protein families. In addition, analysis of semi-tryptic peptides revealed consensus cleavage sites. Interestingly, these cleavage sites were often located within the Type VII secretion motif (YxxxD/E) and thereby effectively removing the entire PE domain. PecB and PecC were also predicted to process PE and PPE proteins, although to a lesser extent than PecA. Follow-up experiments revealed that also PecC is involved in processing of a small subset of the PPE protein family. Finally, our evidence indicates that PecA plays a similar role in M. tuberculosis, as PecA is also responsible for cleavage of PE_PGRS proteins in this species.
Project description:In this study we investigate the molecular physiology of the main S. cerevisiae commercial strain (PE-2) used on Brazilian bioethanol process under two distinct conditions: typical (TF) and flocculated (co-aggregated - FL) fermentation. Transcriptional machinery of PE-2 was assessed by high throughput sequencing-based methods (RNA-seq) during industrial fed-batch fermentations. Data from comparative analysis revealed distinct transcriptional profiles among conditions, characterized mainly by a deep gene repression on FL process.
Project description:We observed through Western blot that Mycobacterium marinum secreted more highly abundant PE-PGRS proteins that are above 130 kDa when treated with compound 36 than mock-treated bacteria. We would like to explore which PE-PGRS proteins are more secreted in this condition and whether other proteins are also differentially secreted between two conditions.
