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

PLOS ONE, Dec 2019

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.

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

Lau GW (2012) Saturated Alanine Scanning Mutagenesis of the Pneumococcus Competence Stimulating Peptide Identifies Analogs That Inhibit Genetic Transformation. PLoS ONE 7(9): e44710. doi:10.1371/journal.pone.0044710 Saturated Alanine Scanning Mutagenesis of the Pneumococcus Competence Stimulating Peptide Identifies Analogs That Inhibit Genetic Transformation Chaohui Duan 0 Luchang Zhu 0 Ying Xu 0 Gee W. Lau 0 Patrick M. Schlievert, University of Iowa Carver College of Medicine, United States of America 0 1 Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 2 Laboratory of Clinical Immunology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University , Guangzhou, Guangdong , People's Republic of China 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. - Funding: This work was partially supported by the National Institutes of Health (HL090699) and a University of Illinois Research Board Arnold O. Beckman research award to G.W. Lau. This investigation was conducted in a facility constructed with the support from Research Facilities Improvement Program Grant Number C06 RR 16515-01 from the National Center for Research Resources, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Streptococcus pneumoniae is a major cause of numerous diseases worldwide, including pneumonia, otitis media, meningitis, bacteremia and sepsis [1]. In recent decades, antibiotic resistant S. pneumoniae have been increasingly isolated in clinical settings [28]. Genetic transformation, which occurs when S. pneumoniae enters the competent state, contributes to the transfer and acquisition of antibiotic resistance genes [911]. The competence regulon is activated when CSP binds to its membrane-associated histidine kinase receptor ComD, which phosphorylates the response regulator ComE [1215]. In turn, ComE activates the transcription of genes in the competence regulon, including the transcription of ComX, a competence specific sigma factor. ComX positively regulates the transcription of genes encoding effectors for DNA uptake and recombination [16]. Interestingly, we and others have shown that the competence regulon is also important for virulence during acute pneumonia and bacteremia models of mouse infection [1722]. DNA sequence analysis predicts the existence of six distinct CSP subtypes, with an overwhelming majority of S. pneumoniae strains producing CSP1 or CSP2 [23,24], corresponding to two variants of ComD, namely ComD1 and ComD2 [25]. Competence in ComD1 strains could be induced more efficiently with the compatible CSP1. In contrast, ComD2 strains are more sensitive to induction by the compatible CSP2 [26,27]. Alanine scanning was previously used to identify amino acid residues that are important for the activity of CSP1 [28]. However, the author did not test the ability of these analogs to competitively inhibit CSP1. Most recently, by using amino acid substitution and deletion, we have identified synthetic analogs of CSP1 and CSP2 that competitively inhibit CSPs ability to induce the competence regulon, to control pneumococcal pneumonia and to reduce horizontal gene transfer of an (...truncated)


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Chaohui Duan, Luchang Zhu, Ying Xu, Gee W. Lau. Saturated Alanine Scanning Mutagenesis of the Pneumococcus Competence Stimulating Peptide Identifies Analogs That Inhibit Genetic Transformation, PLOS ONE, 2012, Volume 7, Issue 9, DOI: 10.1371/journal.pone.0044710