Biology subjects

Job, V.

Publications and source records attributed to Job, V..

5 recordsLinked to original sources

bioRxiv2026

Elongasome Dysfunction Triggers Dependence on MepM-Mediated Peptidoglycan Recycling

The bacterial peptidoglycan (PG) layer is responsible for maintaining cell shape and protecting the cell against both external stress and internal turgor pressure. In rod-shaped bacteria, PG synthesis is orchestrated by two major multi-protein complexes: the elongasome and the divisome, which drive lateral and septal cell wall synthesis, respectively. The membrane protein MreD plays a central role in regulating the elongasome activity. Here, we leveraged a viable Pseudomonas aeruginosa transposon mutant, with reduced mreD expression, to dissect cellular adaptations to impaired elongasome function. A genome-wide synthetic lethality screen revealed that cell division and PG recycling pathways become essential in the context of an impaired elongasome machinery. Moreover, Tn-seq analysis identified the endopeptidase MepM as essential in the mreD downregulated background, whereas its overexpression promoted cell elongation in the parental strain, supporting a conserved role in lateral wall biogenesis. We demonstrate that MepM sustains viability during elongasome impairment by maintaining an active MurU-dependent PG recycling pathway. Together, our findings uncover a functional coupling between PG hydrolysis, recycling, and synthesis. We also identify MepM and PG recycling as critical determinants of bacterial survival when elongasome activity is compromised, positioning these pathways as attractive targets for the development of next-generation or combinatory antibacterial therapies.

microbiology↗

bioRxiv2023

Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxin

Due to the rising incidence of antibiotic resistant infections, the last-line antibiotics polymyxins have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd (arn) operon. In this work, we characterized a polymyxin-induced operon, named mipBA, notably present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by ParR/ParS two-component regulatory system in response to polymyxin. MipA and MipB localize to bacterial outer membrane and form a complex in vitro. Structural modeling revealed that the lipoprotein MipB adopts a {beta}-lactamase fold with two additional C-terminal domains,while MipA folds as an outer-membrane {beta}-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule. Nano differential scanning fluorimetry (DSF) showed that polymyxin stabilized MipA protein in vitro. Mass spectrometry-based quantitative proteomics on whole bacterial membranes demonstrated that the {Delta}mipBA mutant synthesized less MexXY-OprA proteins in response to polymyxin compared to the wild-type strain, as a consequence of impaired transcriptional activation of the mex operon. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pomp, MexXY-OprA.

microbiology↗

bioRxiv2023

Improved Quality Metrics for Association and Reproducibility in Chromatin Accessibility Data Using Mutual Information

BackgroundCorrelation metrics are widely utilized in genomics analysis and often implemented with little regard to assumptions of normality, homoscedasticity, and independence of values. This is especially true when comparing values between replicated sequencing experiments that probe chromatin accessibility, such as assays for transposase-accessible chromatin via sequencing (ATAC-seq). Such data can possess several regions across the human genome with little to no sequencing depth and are thus non-normal with a large portion of zero values. Despite distributed use in the epigenomics field, few studies have evaluated and benchmarked how correlation and association statistics behave across ATAC-seq experiments with known differences or the effects of removing specific outliers from the data. Here, we developed a computational simulation of ATAC-seq data to elucidate the behavior of correlation statistics and to compare their accuracy under set conditions of reproducibility. ResultsUsing these simulations, we monitored the behavior of several correlation statistics, including the Pearsons R and Spearmans{rho} coefficients as well as Kendalls{tau} and Top-Down correlation. We also test the behavior of association measures, including the coefficient of determination R2, Kendalls W, and normalized mutual information. Our experiments reveal an insensitivity of most statistics, including Spearmans{rho} , Kendalls{tau} , and Kendalls W, to increasing differences between simulated ATAC-seq replicates. The removal of co-zeros (regions lacking mapped sequenced reads) between simulated experiments greatly improves the estimates of correlation and association. After removing co-zeros, the R2 coefficient and normalized mutual information display the best performance, having a closer one-to-one relationship with the known portion of shared, enhanced loci between simulated replicates. When comparing values between experimental ATAC-seq data using a random forest model, mutual information best predicts ATAC-seq replicate relationships. ConclusionsCollectively, this study demonstrates how measures of correlation and association can behave in epigenomics experiments. We provide improved strategies for quantifying relationships in these increasingly prevalent and important chromatin accessibility assays.

bioinformatics↗

bioRxiv2020

The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis

Pseudomonas aeruginosa is a causative agent in nosocomial infections, notably in ventilated or cystic fibrosis patients. P. aeruginosa isolates expressing the phospholipase ExoU, an effector of the type III secretion system, are the most pathogenic in clinics. Here, using a genome-wide screen, we discovered that ExoU toxicity requires DNAJC5, a host chaperone, to exert its necrotic activity. DNAJC5 is involved in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. DNAJC5-deficient human cells or Drosophila flies knocked-down for the DNAJC5 orthologue were largely resistant to ExoU virulence. ExoU colocalized with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking - identified in adult neuronal ceroid lipofuscinosis, a human congenital disease - inhibited ExoU-dependent cell lysis. These results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where its phospholipase activity is triggered by binding to PI(4,5)P2.

microbiology↗

bioRxiv2019

Pseudomonas two-partner secretion toxin Exolysin contributes to insect killing

Pseudomonas chlororaphis is a promising biocontrol agent promoting plant-growth and providing protection against pest insects and phytopathogenic fungi. We have identified in the genome of P. chlororaphis PA23 an operon encoding the toxin Exolysin (ExlA) and its outer-membrane transporter, ExlB. We found that P. chlororaphis producing ExlA (ExlAPch) is cytotoxic towards murine macrophages and human epithelial cells at 30 {degrees}C. P. chlororaphis PA23 provoked shrinkage of epithelial cell, leakage of cytoplasmic components and subsequent cell death. During infection, ExlAPch incorporated into epithelial cell membranes within detergent-resistant lipid rafts, suggesting the same mechanisms of cell destruction by pore-formation as reported for P. aeruginosa toxin. ExlAPch was not involved in the capacity of the strain to kill fungi, amoeba or other bacteria. The contribution of ExlA in insecticidal activity of P. chlororaphis was evaluated in the wax moth larvae Galleria mallonella and in Drosophila melanogaster flies. The impact of the deletion of a gene encoding exlA homologue was tested in the natural fly pathogen P. entonomophila. In both models, the ExlA absence delayed killing, suggesting the contribution of the toxin in bacteria-insect pathogenic interactions.

microbiology↗