Biology subjects

Robert-Genthon, M.

Publications and source records attributed to Robert-Genthon, M..

5 recordsLinked to original sources

bioRxiv2025

Avirulent Pseudomonas aeruginosa T3SS-negative strains belonging to Clade 5 produce variable quantities of secondary metabolites

Pseudomonas species are ubiquitous in the environment and serve as valuable source of enzymes and secondary metabolites for industrial applications. P. aeruginosa secretes metalloproteases such as elastase LasB and produces bioactive small molecules, including pyocyanin, rhamnolipids, and pyoverdine, with potential biotechnological applications. However, the interest in P. aeruginosa for industrial use has been limited due to the virulence-associated Type III Secretion System (T3SS), a key factor in host-pathogen interactions. In this study, we genotypically and phenotypically characterized a collection of P. aeruginosa strains naturally lacking T3SS-encoding genes. Phylogenetic analysis revealed that these strains belong to two distinct clades. Several strains exhibited low or no cytotoxicity on epithelial cell lines and were avirulent in the Galleria infection model. The level of LasB and the three metabolites -- pyocyanin, rhamnolipids, and pyoverdine -- varied independently of virulence profiles. Notably, we identified avirulent strains capable of producing at least two secondary metabolites, including monorhamnolipids, highlighting their potential for biotechnological applications.

microbiology↗

bioRxiv2025

Strain-Specific Variation in the Complement Resistome of Pseudomonas aeruginosa

Bloodstream infections caused by Pseudomonas aeruginosa are associated with the highest mortality rates. The complement system, key component of the innate immune response, is mostly responsible for P. aeruginosa elimination in human blood. Nevertheless, the plasma sensitivity of P. aeruginosa strains varies, ranging from highly sensitive, persistent to fully resistant. Whereas most studies rely on model strains, given the species high genomic and phenotypic diversity, the interaction between P. aeruginosa and the complement system may be highly complex. In this work, we characterized the plasma resistome of P. aeruginosa in three strains displaying variable plasma sensitivity using Tn-seq. The gain-of-function screen performed on the sensitive strain PA14 highlighted numerous bacterial factors impacting plasma resistance, including members of the RetS-LadS/Gac/Rsm regulatory system and outer membrane porins. Tn-seq in plasma-resistant strains CHA and YIK, suggested that each strain utilizes a specific and limited set of proteins to evade complement-dependent killing. Despite this strain-specific response, we identified common strategies across all strains, including the production of exopolysaccharides (EPSs), the presence of surface appendages and modification in the O-specific antigen. We evidence Ssg and Crc as common factors contributing to plasma resistance. However, whereas mutants lacking ssg and/or crc in plasma had reduced survival, a subpopulation of these mutants persisted over prolonged exposure to human plasma. Overall, this work provides valuable insights into the complex interplay between P. aeruginosa and the complement system in the context of bloodstream infections and raises concerns for the development of therapies targeting individual virulence factors.

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↗

bioRxiv2021

Molecular features underlying Pseudomonas aeruginosa persistence in human plasma

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma and blood. However, the molecular mechanisms underlying the formation of evaders remained unknown. Here, using a gain-of-function genetic screen, we examined the molecular determinants of P. aeruginosa persistence in plasma. We found that, among other factors, ATP and biotin availability greatly influence bacterial survival in plasma; mutants in pur and bio genes display higher tolerance and persistence, respectively. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate granules upon incubation in plasma in several clinical strains, implying the bacterial response to a low-energy stress signal. Indeed, mutants with transposon insertions in ppk genes were eliminated in the plasma. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Through this study, we shed light on P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC that provides a comprehensive picture of the complex interplay between P. aeruginosa and the human complement system. Author summaryPersistence of bacterial pathogens is a main cause of treatment failure and establishment of chronic bacterial infection. Despite innate immune responses, some bacteria may persist in human blood and plasma. Here we used a genome-wide screen to investigate the molecular determinants influencing Pseudomonas aeruginosa persistence in human plasma facing the complement system. Alongside a multifactorial strategy, we found intracellular levels of ATP and biotin to significantly influence bacterial capacity to deal with membrane attack complex (MAC)-dependent killing. These results underline the need to understand the complex interplay between bacterial pathogens and the human immune system when seeking to develop efficient antibacterial strategies.

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↗