Lactate Oxidase (LctO) Acts as a Metabolic Checkpoint Restraining Streptococcus pneumoniae Invasion of Respiratory Epithelial Barriers

Streptococcus pneumoniae rapidly translocates across polarized human bronchial epithelial barriers, with viable bacteria recovered from the basolateral compartment within 1 h post-infection. Disruption of the pyruvate node through combined deletion of pyruvate oxidase ( spxB ) and lactate oxidase ( lctO ) markedly enhanced transmigration of S. pneumoniae across polarized Calu-3 monolayers without causing early cytotoxicity or loss of monolayer integrity. This hyper-invasive phenotype was conserved in the TIGR4 and EF3030 background and under air-liquid interface conditions. Importantly, single Δ lctO mutants exhibited significantly greater translocation than Δ spxB mutants or wild-type strains across bronchial (Calu-3), alveolar (A549), and pharyngeal (Detroit 562) epithelial models. Enhanced translocation correlated with increased bacterial adherence but was independent of capsule expression, extracellular H₂O₂ production, pneumolysin, or tight junction disruption, as evidenced by stable transepithelial electrical resistance (TEER), lack of caspase-3/7 activation, and minimal IL-18 release at early time points. High-resolution confocal microscopy revealed intracellular Δ lctO pneumococci localized within N-acetylglucosamine/sialic acid (GN/SA)-containing compartments as early as 1 h post-infection. In murine macrophages, Δ lctO mutants were phagocytosed at rates similar to wild-type bacteria but induced greater pneumolysin-dependent cytotoxicity at 24 h. These findings demonstrate that LctO functions as a metabolic checkpoint that restrains pneumococcal invasion of respiratory epithelia, revealing a previously unrecognized role for lactate oxidase in controlling the transition from colonization to invasive disease.