Regulation of PBP2x Surface Localization by aliD and clpL Alters β-Lactam Resistance in Pneumococcus

Streptococcus pneumoniae is a leading cause of respiratory infections and is often treated with β-lactam antibiotics despite frequent resistance. The β-lactam resistance is through mutations in penicillin-binding proteins (PBPs), but the impact of PBP regulation remains poorly understood. Here, we investigate the role of gene regulation by the oligopeptide-binding protein aliD on β-lactam susceptibility. aliD-expressing strains exhibit elevated minimum inhibitory concentrations (MICs) to the β-lactam antibiotics amoxicillin and cefdinir compared to isogenic aliD mutants. In contrast, aliD had minimal effects on susceptibility to vancomycin, suggesting a mechanism specific to β-lactam antibiotics. We demonstrate that aliD increases clpL expression with or without antibiotics present. Inducible expression of clpL resulted in stepwise increases in amoxicillin MIC, demonstrating that elevated clpL expression directly contributes to decreased β-lactam susceptibility. Furthermore, we show that increased clpL expression enhances PBP2x surface exposure in a dose-dependent manner. Additionally, aliD-expressing strains exhibited significantly increased cell wall cross-linking relative to aliD mutants. Together, these findings identify a novel regulatory pathway in which aliD enhances clpL expression, promotes PBP2x surface exposure, and increases peptidoglycan cross-linking, ultimately reducing β-lactam susceptibility. This work expands current understanding of pneumococcal antibiotic resistance and suggests that peptide sensing through oligopeptide transport systems may influence antibiotic susceptibility in host-specific environments.