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Antibiotic resistance is a major challenge to modern medicine. Intraspecies and interspecies dissemination of antibiotic resistance genes among bacteria can occur through horizontal gene transfer. Competence-mediated gene transfer has been reported to contribute to the spread of antibiotic resistance genes in  Streptococcus pneumoniae . Induction of the competence regulon is mediated by a 17-amino acid peptide pheromone called the competence stimulating peptide (CSP). Thus, synthetic analogs that competitively inhibit CSPs may reduce horizontal gene transfer. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on CSP1 to screen for analogs that disable genetic transformation in  S. pneumoniae . Substitution of the glutamate residue at the first position created analogs that could competitively inhibit CSP1-mediated competence development in a concentration-dependent manner. Additional substitutions of the negatively-charged glutamate residue with amino acids of different charge, acidity and hydrophobicity, as well as enantiomeric D-glutamate, generated analogs that efficiently outcompeted CSP1, suggesting the importance of negative charge and enantiomericity of the first glutamate residue for the function of CSP1. Collectively, these results indicate that glutamate residue at the first position is important for the ability of CSP1 to induce ComD, but is dispensable for the peptide to bind the receptor. Furthermore, these results demonstrate the potential applicability of competitive CSP analogs to control horizontal transfer of antibiotic resistance genes in  S. pneumoniae .

Saturated Alanine Scanning Mutagenesis of the Pneumococcus Competence Stimulating Peptide Identifies Analogs That Inhibit Genetic Transformation