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 {Delta}lctO mutants exhibited significantly greater translocation than {Delta}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 {Delta}lctO pneumococci localized within N-acetylglucosamine/sialic acid (GN/SA)-containing compartments as early as 1 h post-infection. In murine macrophages, {Delta}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. ImportanceThis study identifies lactate oxidase (LctO) as a critical metabolic checkpoint that restrains Streptococcus pneumoniae invasion of respiratory epithelial barriers. By linking pyruvate node metabolism to the control of transmigration, these findings reveal a novel mechanism by which central carbon metabolism regulates pneumococcal virulence and the transition from colonization to invasive disease.