Metabolic Rewiring at the Pyruvate Node Drives Severe Pneumonia and T-Cell Suppression in Serotype 3 Streptococcus pneumoniae Infection

Abstract Background Serotype 3 (ST3) Streptococcus pneumoniae remains a major cause of invasive pneumococcal disease and pneumonia despite PCV13 introduction, in part due to potent immune evasion properties. The contribution of the pyruvate metabolic node (SpxB/LctO pathways) to ST3 pathogenesis is poorly defined. Methods We investigated oxygen-dependent fitness, virulence, and lung pathology in the ST3 strain WU2 and isogenic Δ spxB , Δl ctO , and Δ spxB Δ lctO mutants. In vitro growth was assessed under nasopharyngeal (21% O₂) and alveolar (14% O₂) conditions. Murine pneumonia models evaluated survival, bacterial burdens, histopathology (H&E, confocal microscopy), and lung transcriptomics (RNA-seq). Results ST3-strain exhibited a unique oxygen-sensitive growth defect at 21% O₂, alleviated by spxB deletion, indicating metabolic burden from pyruvate flux. In mice, wild-type WU2 caused high mortality with severe suppurative bronchopneumonia, alveolar consolidation, hemorrhage, and perivascular inflammation. The Δ spxB mutant accelerated lethality with enhanced distal lung damage, uncontrolled dissemination, and amplified inflammation. Wild-type infection uniquely induced targeted reorganization of bronchial epithelial membranes, forming prominent bacterium-laden blebs—host-derived membrane protrusions encapsulating intact pneumococci. These novel structures facilitated organized bacterial translocation into tissue without overt cytotoxicity and were largely absent in spxB -deficient mutants despite comparable lung burdens. RNA-seq analysis revealed SpxB-dependent suppression of T cell activation (e.g., Rag1 and Themis ) and acute inflammatory pathways, consistent with immune sequestration via blebs. Conclusions The SpxB-dependent pathway orchestrates ST3 virulence by enabling metabolic adaptation and driving bleb-mediated epithelial invasion and immune evasion in the lung. These bacterium-laden blebs represent a novel hallmark mechanism in pneumococcal pathogenesis, offering new insights and potential therapeutic targets.