Manganese Homeostasis Supports Stenotrophomonas maltophilia Oxidative Stress Defense and Replication in Acanthamoeba castellanii Phagosomes

• The S. maltophilia MntH importer contributes to H 2 O 2 resistance and intracellular survival • The Mn exporter MntP limits toxicity even at sub-inhibitory Mn²⁺ concentrations • The S. maltophilia MntR regulated genes include the extracytoplasmic TBDR-pTFP module • The TBDR-pTFP locus is specifically induced under combined Mn 2+ and Fe 2+ limitation • S. maltophilia Mn homeostasis is linked to iron availability and oxidative stress responses Manganese homeostasis is essential for the environmental adaptability and pathogenic potential of Stenotrophomonas maltophilia , a bacterium that thrives across diverse and fluctuating environments. Here, we characterize the manganese homeostasis network of S. maltophilia strain Sm18, identifying a coordinated system that integrates conserved transporters with previously unrecognized candidate components. Central to this system is an MntR-controlled gene module that includes the canonical Mn²⁺ importer MntH and exporter MntP, together with a TonB-dependent receptor (TBDR) and a periplasmic thioredoxin-fold protein (pTFP), both representing novel protein families with restricted phylogenetic distribution. Transcriptomic analyses under varying Mn²⁺ and Fe²⁺ conditions revealed a tight interplay between these metals, highlighting the ferrophilic nature of S. maltophilia and the differential regulation of module components. Notably, the TBDR-pTFP locus is strongly induced under combined Mn 2+ and Fe 2+ limitation, suggesting a specialized role in metal acquisition under nutrient-restricted conditions. Functional analyses showed that MntP contributes to protection from Mn toxicity even at sub-inhibitory concentrations, whereas MntH supports growth under oxidative stress and promotes intracellular replication within Acanthamoeba castellanii phagosomes. Together, these findings identify a Mn-responsive module candidate associated with manganese homeostasis and provides new insight into mechanisms that support S. maltophilia adaptation to metal-limited and host-associated environments. (A) Manganese transport and regulation in S. maltophilia Sm18 axenic culture conditions, where MntH and MntP mediate uptake and efflux, respectively, under MntR control. (B) A novel TBDR-pTFP locus is specifically induced under combined Mn and Fe limitation via MntR and additional, as yet uncharacterized regulatory mechanisms. (C) During interaction with A. castellanii , MntH-dependent manganese uptake supports resistance to host-derived oxidative stress. MntH disruption reduces intracellular replication.