Biology subjects

Sa-Leao, R.

Publications and source records attributed to Sa-Leao, R..

7 recordsLinked to original sources

bioRxiv2026

Serotype-independent inhibition of S. pneumoniae by SMiTE, a commensal-derived bacteriocin

Streptococcus pneumoniae remains a leading cause of disease despite widespread vaccination, highlighting the need for serotype-independent strategies. We recently identified commensal streptococci that produce bacteriocins with anti-pneumococcal activity. Here, we evaluate these bacteriocins as candidates for pneumococcal control. Using cell-free protein synthesis, we screened 58 bacteriocins, the majority of which absent in available pneumococcal genomes, and identified SMiTE as the most potent. Purified SMiTE disrupted pneumococcal membrane integrity, as shown by confocal, transmission, and scanning electron microscopy, induced ATP leakage, and triggered transcriptional responses consistent with envelope stress and metabolic remodeling. In a mouse nasopharyngeal colonization model, intranasal SMiTE treatments reduced pneumococcal loads by 65-fold with no significant weight loss. SMiTE inhibited a broad range of serotypes, with strongest activity against serotype 3, which is poorly controlled by current vaccines, while sparing most oral and upper respiratory tract commensals. Repeated sub-inhibitory exposure did not select resistant mutants in line with the membrane-targeting mechanism. These findings establish SMiTE as a commensal-derived strategy for serotype-independent, microbiota-sparing pneumococcal decolonization.

microbiology↗

bioRxiv2025

Coexistence of two divergent TprA/PhrA cell-cell communication systems in Streptococcus mitis coordinate bacteriocin production, competence, oxidative stress responses, and interspecies competition with S. pneumoniae

Cell-cell communication (CCC) systems are key regulators of bacterial behaviors and adaptation. The human upper respiratory tract is co-colonized by commensal and pathogenic streptococci, but how CCC systems mediate their interactions remains unclear. Here, we investigated the TprA/PhrA quorum-sensing system, composed of the transcription factor TprA and its cognate signaling peptide PhrA, across Streptococcus mitis and S. pneumoniae. Comparative genomics showed that this system is broadly distributed across both species, with most strains sharing identical phrA alleles that enable interspecies signaling. In both species, the tprA/phrA module is commonly linked to the streptococcin E (sce) locus, encoding a putative bacteriocin. We show that activation of the sce operon enhances the competitive fitness of S. mitis in biofilm and infection models. In S. mitis strain C22, two diverse copies of tprA/phrA are present and differentially regulated, coordinating expression of tprA/phrA and the downstream sce locus through both shared and independent pathways. Transcriptomic analyses revealed redundancy, additivity, and cross-regulation between the two systems, linking them to the control of bacteriocin production, competence, and oxidative stress responses. Distinct promoter architectures and TprA-binding motifs underlie the functional divergence of these paralogues, highlighting how regulatory diversification can expand quorum-sensing outputs. Together, our findings show that S. mitis has evolved a flexible and layered communication network that integrates population sensing with antimicrobial and competence responses, providing a molecular basis for its competitive interactions with S. pneumoniae during colonization of the human respiratory tract.

microbiology↗

bioRxiv2025

Network rewiring of a pneumococcal communication system promotes stress adaptation in a globally successful lineage

Cell-cell communication (CCC) systems regulate bacterial behaviors and shape social interactions within microbial communities. In Streptococcus pneumoniae, several CCC systems coordinate intra- and inter-strain signaling, yet the genetic plasticity of their components and regulons remains underexplored. Here, we investigate a divergent allelic variant of the TprA/PhrA CCC system, a regulator-pheromone pair widely conserved in S. pneumoniae. We characterize PhrA1.2, the second most prevalent PhrA pheromone, as a non-signaling variant found in the globally distributed GPSC6 lineage, with presence in isolates dating back to the 1960s. In GPSC6, PhrA1.2 co-occurs with TprA1.2, a truncated transcriptional regulator carrying premature stop codons that eliminate the predicted pheromone-binding domain. Functional assays show that PhrA1.2 is non-functional, both when paired with the truncated TprA1.2 and when paired with the canonical regulator allele TprA1.1. Unexpectedly, despite its truncation, TprA1.2 activates genes involved in oxidative stress responses, including those related to iron-sulfur cluster formation and redox homeostasis. Loss of tprA1.2 impairs growth and increases hydrogen peroxide sensitivity, while in vivo experiments reveal a fitness defect during nasopharyngeal colonization. Together, our findings show that the TprA1.2 allele represents a regulator that has lost its communication component while modifying its regulon. This variant promotes adaptation to oxidative stress and contributes to colonization, demonstrating circuit rewiring of a peptide-regulator CCC system in an epidemiologically relevant S. pneumoniae lineage.

microbiology↗

bioRxiv2025

Streptolancidin D mediates intra-species competition in Streptococcus pneumoniae through the Rgg144/SHP144 quorum-sensing system

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 inhibition of competitor strains in in vitro biofilms and during murine co-colonization. Importantly, streptolancidin D also inhibited 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. IMPORTANCEBacteriocins 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 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.

microbiology↗

bioRxiv2025

Streptococcus mitis bacteriocins drive contact-dependent lysis of S. pneumoniae facilitating transformation in multispecies environments

Natural competence allows bacterial species like Streptococcus pneumoniae and S. mitis to acquire environmental DNA, driving horizontal gene transfer (HGT) and adaptation. In S. pneumoniae, a human pathogen, competence-induced predation is well characterized and involves the release of bacteriocins and a murein hydrolase to lyse noncompetent siblings and liberate DNA. In contrast, in the human commensal S. mitis, mechanisms mediating DNA acquisition remain poorly understood. Here, we identify a diverse set of competence-associated bacteriocins (cab) that are produced by S. mitis during the late phase of competence. We focus on one bacteriocin pair, CabAB, that triggers contact-dependent growth inhibition and lysis of S. pneumoniae through activation of the major pneumococcal autolysin LytA. We demonstrate that CabAB compromises S. pneumoniae membrane integrity, leading to formation of intracellular membrane aggregates and the release of cytoplasmatic content, thereby increasing available DNA, which enhances HGT from S. pneumoniae to S. mitis in biofilms. These findings uncover a mechanism of interspecies predation and gene acquisition, revealing a critical role for competence-associated bacteriocins in shaping evolutionary dynamics of streptococci. ImportanceMany streptococci are naturally competent, acquiring environmental DNA through transformation. This includes pathogens like S. pneumoniae and commensals like S. mitis, which can exchange genetic material through horizontal gene transfer (HGT). For example, S. mitis can acquire pneumococcal capsules, leading to its misidentification in polymicrobial samples such as those obtained from the upper respiratory tract. Understanding the drivers of HGT between these species is therefore critical. Here, we characterize a competence-induced bacteriocin cluster in S. mitis. These bacteriocins lyse pneumococci, promoting DNA release and enhancing gene transfer in dual-species biofilms. Our findings uncover a mechanism by which competence-associated predation promotes interspecies HGT, shaping the evolution and epidemiology of streptococcal populations.

microbiology↗

bioRxiv2025

Bacteriocin-mediated prevention of secondary pneumococcal pneumonia by a human commensal Streptococcus mitis strain

Streptococcus pneumoniae remains a major public health threat despite widespread use of vaccines and antibiotics. Its well-established synergy with influenza A virus (IAV) often results in secondary bacterial pneumonia, a condition associated with high morbidity and mortality. As pneumococcal pneumonia is invariably preceded by nasopharyngeal colonization, preventing colonization represents a promising intervention strategy. Current approaches, including pneumococcal conjugate vaccines and antibiotics, can drive serotype replacement and antimicrobial resistance, underscoring the need for novel, targeted, and serotype-independent alternatives. We previously identified seven commensal streptococcal strains (S. oralis A22 and S. mitis B22-G22) that robustly inhibit S. pneumoniae growth and biofilm formation in vitro. Here, using a murine model, we demonstrate that colonization with the commensal strain F22Ad significantly reduces pneumococcal density in the nasopharynx by day 10 post-infection and prevents pneumococcal dissemination to the lungs following IAV co-infection. This protective effect was dependent on bacteriocins encoded at the blp1 locus. Among these, Bac2v1 (alone or in combination with Bac1) significantly reduced pneumococcal colonization. Notably, Bac2v1 exerted serotype-independent inhibitory activity, impairing the growth of diverse pneumococcal strains. Our findings provide the first in vivo proof-of-concept that commensal streptococci, and specifically their bacteriocins, can disrupt S. pneumoniae colonization and prevent progression to secondary bacterial pneumonia. This work introduces a previously unrecognized precision-based approach to pneumococcal disease prevention, offering a potential complement to current strategies.

microbiology↗

bioRxiv2018

Comparative genomic analysis of the emerging pathogen Streptococcus pseudopneumoniae: novel insights into virulence determinants and identification of a novel species-specific molecular marker

Streptococcus pseudopneumoniae is a close relative of the major human pathogen S. pneumoniae. While initially considered as a commensal species, it has been increasingly associated with lower-respiratory tract infections and high prevalence of antimicrobial resistance (AMR). S. pseudopneumoniae is difficult to identify using traditional typing methods due to similarities with S. pneumoniae and other members of the mitis group (SMG). Using phylogenetic and comparative genomic analyses of SMG genomes, we identified a new molecular marker specific for S. pseudopneumoniae and absent from any other bacterial genome sequenced to date. We found that a large number of known virulence and colonization genes are present in the core S. pseudopneumoniae genome and we reveal the impressive number of known and new surface-exposed proteins encoded by this species. Phylogenetic analyses of S. pseudopneumoniae show that specific clades are associated with allelic variants of core proteins. Resistance to tetracycline and macrolides, the two most common resistances, were encoded by Tn916-like integrating conjugative elements and Mega-2. Overall, we found a tight association of genotypic determinants of AMR as well as phenotypic AMR with a specific lineage of S. pseudopneumoniae. Taken together, our results sheds light on the distribution in S. pseudopneumoniae of genes known to be important during invasive disease and colonization and provide insight into features that could contribute to virulence, colonization and adaptation.\n\nImportanceS. pseudopneumoniae is an overlooked pathogen emerging as the causative agent of lower-respiratory tract infections and associated with chronic obstructive pulmonary disease (COPD) and exacerbation of COPD. However, much remains unknown on its clinical importance and epidemiology, mainly due to the lack of specific means to distinguish it from S. pneumoniae. Here, we provide a new molecular marker entirely specific for S. pseudopneumoniae. Furthermore, our research provides a deep analysis of the presence of virulence and colonization genes, as well as AMR determinants in this species. Our results provide crucial information and pave the way for further studies aiming at understanding the pathogenesis and epidemiology of S. pseudopneumoniae.

microbiology↗