Lineage-specific macrophage programs dictate metabolic suppression and stress responses associated with VBNC-like states in Listeria monocytogenes

Listeria monocytogenes (Lm) is a significant cause of central nervous system (CNS) infection in humans and animals, yet the mechanisms governing its intracellular lifestyle in brain-resident and infiltrating macrophages remain unclear. Using dual RNA sequencing combined with host proteomics, we map host-pathogen interactions in bovine microglia and monocyte-derived macrophages (MDMs), the two principal macrophage populations encountered by Lm in the CNS. Although microglia and MDMs share core antimicrobial programs, they differ in their metabolic and immunological states, which dictate whether Lm adopts a replicative cytosolic lifestyle or a stress-tolerant intravacuolar state. In MDMs, nutrient restriction and phagolysosomal pressure drive metabolic suppression, robust stress and SOS responses, and induction of non-coding regulatory RNAs, shifting toward a dormant, viable but non-culturable phenotype. In microglia, the nutrient-rich cytosol supports bacterial growth, marked by upregulation of nucleotide salvage and carbohydrate and lipid metabolism. Functional analyses identify the stress-related genes recA and rtcB as contributors to intracellular persistence. Together, our findings show that the fate of Lm is shaped not solely by canonical virulence genes but also by the interplay between bacterial stress adaptation and lineage-specific macrophage environments, highlighting macrophage ontogeny as a critical determinant of infection outcome. Authors SummaryListeria monocytogenes (Lm) is a deadly foodborne pathogen and major cause of central nervous system (CNS) infection (neurolisteriosis) in humans and ruminants, underscoring the close interdependence of animal health, food safety, and human health central to the One Health paradigm. During neurolisteriosis, Lm encounters distinct macrophage populations in the brain, yet how these cells shape bacterial behaviour has remained poorly understood. Here, we simultaneously captured the responses of Lm and bovine brain-resident microglia and infiltrating monocyte-derived macrophages (MDMs) during infection. We show that although both macrophage populations activate core antimicrobial programs, their metabolic and immune responses diverge profoundly, creating distinct intracellular environments that elicit different bacterial responses. In microglia, cytosolic bacterial replication is accompanied by transcriptional upregulation of growth-associated metabolic pathways. In contrast, MDMs impose nutrient limitation and phagolysosomal stress, triggering a global bacterial transcriptional reprogramming marked by metabolic shutdown, activation of stress and SOS DNA repair responses, and induction of regulatory non-coding RNAs. This response drives Lm into a dormant, stress-tolerant state that enables intracellular persistence despite immune pressure. Among activated genes, the bacterial recombinase A (RecA) and the RNA ligase RtcB are key contributors to bacterial persistence. Together, our findings reveal that macrophage ontogeny governs infection outcome by shaping both host and bacterial transcriptional programs, demonstrating that Lm persistence in the CNS depends not only on classical virulence factors but also on adaptive stress responses tuned to lineage-specific macrophage environments.