Douarre, P.-E. | bioRxiv Science

bioRxiv2026

MreB is dispensable for viability but critical for rod shape, motility and biofilm fitness in Pseudomonas aeruginosa

Despite growing interest in the MreBCD morphogenetic complex as a potential antimicrobial target, its function in Pseudomonas aeruginosa remains poorly understood. While previous studies using the MreB inhibitor A22 have established its role in cell shape maintenance and pilus regulation, the impact of mreB deletion has not been comprehensively investigated. Using genetic and microscopy-based approaches, we show that deletion of mreB is viable in P. aeruginosa, but results in spherical cells that lose all forms of motility despite retaining flagella. Importantly, we uncover a previously overlooked polar effect of the in-frame mreB deletion on the downstream mreCD genes and show, using CRISPRi-mediated silencing, that mreCD expression is essential for viability.{Delta} mreB mutants also display increased sensitivity to {beta}-lactam antibiotics and enhanced initial surface attachment, yet form more compact biofilms with reduced dispersal. In mixed-culture biofilms, spherical{Delta} mreB cells are outcompeted by rod-shaped wild-type cells and remain confined to the biofilm base. The identification of natural P. aeruginosa isolates carrying truncated mreB alleles further indicates that loss of MreB function can be tolerated in natural populations. Together, our findings reveal important contributions of the MreBCD system to viability, morphogenesis, motility and biofilm development in P. aeruginosa, providing new insights into bacterial adaptation and informing the development of targeted antimicrobial strategies.

microbiology↗

bioRxiv2025

Large-scale phenotyping and comparative genomics reveal genetic features of Listeria persistence in epithelial cells

During infection in epithelial cells, after invading the cytosol, multiplying, and spreading, Listeria monocytogenes (Lm) ceases to produce ActA and becomes trapped in Listeria-containing vacuoles (LisCVs). These persistence acidic vacuoles harbor bacterial subpopulations that resist to stress in a metabolically dormant state. Although LisCVs have been proposed as a hallmark of Lm persistence in epithelial cells, their prevalence across strains and the bacterial factors underlying their formation remain uncharacterized. Given the significant genetic diversity within the species, it is important to consider this variability when studying persistence phenotype. Therefore, we screened over one hundred Lm isolates spanning two major evolutionary lineages and belonging to 23 clonal complexes from diverse ecological origins. Strikingly, the vast majority of strains, including both clinical and environmental isolates, were capable of forming LisCVs, suggesting that vacuolar persistence is a widespread and conserved feature of Lm pathogenesis. Nevertheless, among the group of hypo-virulent strains mostly associated with food and carrying a truncated InlA, we identified four isolates with an altered persistence phenotype. Two of them showed defects in the early stages of infection and carried mutations in key virulence genes (hly and gshF). The other two, instead, were specifically affected in the persistence by showing a reduced ability to form LisCVs. Comparative genomic analysis revealed that a mutation in the folP gene, required for folate biosynthesis, was responsible for impaired persistence. Live-imaging and microscopy analysis highlighted a reduced bacterial motility and intercellular spreading of the folP mutant, although the level of ActA at the bacterial surface was increased. Together, our work identifies folate biosynthesis as a critical metabolic pathway governing Lm persistence by regulating ActA levels and activity. Downregulation of ActA at the bacterial surface is therefore a crucial event for the establishment of the intracellular persistent niche during long-term infection of epithelial cells. Author SummaryLong regarded as a strictly cytosolic pathogen, Listeria monocytogenes is now revealing a dual lifestyle that includes intracellular vacuoles. Recent evidence shows that in epithelial cells this bacterium can enter a dormant state within acidic vacuoles, termed LisCVs, potentially contributing to silent carriage and antibiotic treatment failure. Here, we reveal that vacuolar persistence is a widespread and conserved feature among diverse Listeria strains. Strikingly, we identify folate metabolism as a key regulator of this phenotypic switch, linking metabolic cues to the shutdown of bacterial motility. Our findings uncover a novel connection between central metabolism and intracellular niche adaptation, shedding new light on how Listeria survives and hides within the host.

microbiology↗