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

Abstract 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 Summary Long 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.