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Abstract Facing the antibiotic resistance crisis, bacteriocins are considered as a promising alternative to treat bacterial infections. In the human commensal Streptococcus salivarius, the production of unmodified bacteriocins (or salivaricins) is directly controlled at the transcriptional level by quorum-sensing. To discover hidden bacteriocins, we harnessed here the unique molecular signatures of salivaricins not yet used in available computational pipelines and performed genome mining followed by orthogonal reconstitution and expression. From 100 genomes of S. salivarius, we identified more than 50 bacteriocin candidates clustered into 21 groups. Strain-based analysis of bacteriocin combinations revealed significant diversity, reflecting the plasticity of seven independent loci. Activity tests showed both narrow and broad-spectrum bacteriocins with overlapping activities against a wide panel of Gram-positive bacteria, including notorious multidrug-resistant pathogens. Overall, this work provides a search-to-test generic pipeline for bacteriocin discovery with high impact for bacterial ecology and broad applications in the food and biomedical fields.

Abstract Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins of the ABC transporter Ami-AliA/AliB oligopeptide permease. Exposure to these peptides alters pneumococcal phenotypes such as growth. Using a mutant in which a permease domain of the transporter was disrupted, by growth analysis and epifluorescence microscopy, we confirmed peptide uptake via the Ami permease and intracellular location in the pneumococcus. By RNA-sequencing we found that the peptides modulated expression of genes involved in metabolism, as pathways affected were mostly associated with energy or synthesis and transport of amino acids. Both peptides downregulated expression of genes involved in branched-chain amino acid metabolism and the Ami permease; and upregulated fatty acid biosynthesis genes but differed in their regulation of genes involved in purine and pyrimidine biosynthesis. The transcriptomic changes are consistent with growth suppression by peptide treatment. The peptides inhibited growth of pneumococcal isolates of serotypes 3, 8, 9N, 12F and 19A, currently prevalent in Switzerland, and caused no detectable toxic effect to primary human airway epithelial cells. We conclude that pneumococci take up K. pneumoniae peptides from the environment via binding and transport through the Ami permease. This changes gene expression resulting in altered phenotypes, particularly reduced growth.

Abstract Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.

Abstract Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

Peptide CSP regulates natural competence in pneumococci and has been proposed as a quorum-sensing signal or a probe for sensing environmental cues. Here, the authors show that CSP levels can indeed act as an indicator of cell density and also incorporate information on environmental factors or cell history.