Rgg144/SHP144-controlled streptolancidin D mediates intra-species competition in Streptococcus pneumoniae with cumulative effect from other bacteriocins and fratricide.
UNLABELLED: Streptococcus pneumoniae is a major colonizer of the human nasopharynx, where inter- and intra-strain competition plays a critical role in shaping population structure and influencing vaccine outcomes. Bacteriocins are key mediators of intra-species competition, yet many of their functions and regulatory mechanisms remain poorly understood. Here, we identify and characterize streptolancidin D, a previously uncharacterized bacteriocin encoded by the sldA-T locus, and demonstrate its contribution to pneumococcal competition. Using isogenic streptolancidin-producing and non-producing variants of a naturally colonizing strain, we show that sldA-T contributes to the inhibition of competitor strains in in vitro biofilms and during murine co-colonization. Importantly, streptolancidin D also inhibited in vitro a subset of genetically diverse pneumococcal isolates representing multiple serotypes, whereas non-producing variants showed no activity. This indicates that its effect is broad and not restricted to isogenic interactions. Genomic analysis of over 7,500 pneumococcal genomes revealed that sldA-T is present in ~12% of isolates, with lineage-associated distribution patterns, and is consistently encoded downstream of the Rgg144/SHP144 quorum sensing system. We further demonstrate that sldA-T is regulated by this system, with sldA-T promoter activity abolished in a SHP-deficient background and partially restored by exogenous peptide stimulation. Finally, we show that streptolancidin D acts in concert with other bacteriocin systems and competence-mediated fratricide, highlighting a multifactorial antimicrobial strategy that enhances pneumococcal competitiveness. Overall, our findings identify a quorum sensing-regulated bacteriocin that contributes to pneumococcal competition and helps shape population dynamics. IMPORTANCE: Bacteriocins are central to bacterial competition and niche occupation, particularly in structured environments like the human nasopharynx. While several pneumococcal bacteriocins have been characterized, the functions of many remain unknown, limiting our understanding of how these systems shape strain fitness and population dynamics. We characterize streptolancidin D, a bacteriocin that enhances intraspecies competitiveness in vitro and in vivo and contributes to the inhibition of genetically diverse pneumococcal strains. We demonstrate that its expression is tightly regulated by the conserved Rgg144/SHP144 quorum sensing system and that the locus is distributed and shows synteny across multiple pneumococcal lineages. Our findings reveal that streptolancidin D operates within a broader network of bacteriocins and competence-associated mechanisms that collectively shape competitive interactions. By integrating genomic, functional, and regulatory analyses, this work expands the known repertoire of pneumococcal antimicrobial systems and provides new insights into the mechanisms underpinning competition and population structure in S. pneumoniae.