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Integrative Reverse Genetic Analysis Identifies Polymorphisms Contributing to Decreased Antimicrobial Agent Susceptibility in Streptococcus pyogenes.

ABSTRACT: Identification of genetic polymorphisms causing increased antibiotic resistance in bacterial pathogens traditionally has proceeded from observed phenotype to defined mutant genotype. The availability of large collections of microbial genome sequences that lack antibiotic susceptibility metadata provides an important resource and opportunity to obtain new information about increased antimicrobial resistance by a reverse genotype-to-phenotype bioinformatic and experimental workflow. We analyzed 26,465 genome sequences of Streptococcus pyogenes, a human pathogen causing 700 million infections annually. The population genomic data identified amino acid changes in penicillin-binding proteins 1A, 1B, 2A, and 2X with signatures of evolution under positive selection as potential candidates for causing decreased susceptibility to β-lactam antibiotics. Construction and analysis of isogenic mutant strains containing individual amino acid replacements in penicillin-binding protein 2X (PBP2X) confirmed that the identified residues produced decreased susceptibility to penicillin. We also discovered the first chimeric PBP2X in S. pyogenes and show that strains containing it have significantly decreased β-lactam susceptibility. The novel integrative reverse genotype-to-phenotype strategy presented is broadly applicable to other pathogens and likely will lead to new knowledge about antimicrobial agent resistance, a massive public health problem worldwide. IMPORTANCE The recent demonstration that naturally occurring amino acid substitutions in Streptococcus pyogenes PBP2X are sufficient to cause severalfold reduced susceptibility to multiple β-lactam antibiotics in vitro raises the concern that these therapeutic agents may become compromised. Substitutions in PBP2X are common first-step mutations that, with the incremental accumulation of additional adaptive mutations within the PBPs, can result in high-level resistance. Because β-lactam susceptibility testing is not routinely performed, the nature and extent of such substitutions within the PBPs of S. pyogenes are poorly characterized. To address this knowledge deficit, polymorphisms in the PBPs were identified among the most comprehensive cohort of S. pyogenes genome sequences investigated to date. The mutational processes and selective forces acting on the PBPs were assessed to identify specific substitutions likely to influence β-lactam susceptibility and to evaluate factors posited to be impediments to resistance emergence.

SUBMITTER: Beres SB

PROVIDER: S-EPMC8764543 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledTwo-component systems (TCSs) are regulatory systems in bacteria that play important roles in sensing and adapting to the environment. In this study, we systematically evaluated the roles of TCSs in the susceptibility of the group A Streptococcus (GAS; Streptococcus pyogenes) SF370 strain to several types of lantibiotics. Using individual TCS deletion mutants, we found that the deletion of srtRK (spy_1081-spy_1082) in SF370 increased the susceptibility to nisin A, which is produced by Lactococcus lactis ATCC 11454, but susceptibility to other types of lantibiotics (nukacin ISK-1, produced by Staphylococcus warneri, and staphylococcin C55, produced by Staphylococcus aureus) was not altered in the TCS mutants tested. The expression of srtFEG (spy_1085 to spy_1087), which is located downstream of srtRK and is homologous to ABC transporters, was increased in response to nisin A. However, srtEFG expression was not induced by nisin A in the srtRK mutant. The inactivation of srtFEG increased the susceptibility to nisin A. These results suggest that SrtRK controls SrtFEG expression to alter the susceptibility to nisin A. Further experiments showed that SrtRK is required for coexistence with L. lactis ATCC 11454, which produces nisin A. Our results elucidate the important roles of S. pyogenes TCSs in the interactions between different bacterial species, including bacteriocin-producing bacteria.ImportanceIn this study, we focused on the association of TCSs with susceptibility to bacteriocins in S. pyogenes SF370, which has no ability to produce bacteriocins, and reported two major new findings. We demonstrated that the SrtRK TCS is related to susceptibility to nisin A by controlling the ABC transporter SrtFEG. We also showed that S. pyogenes SrtRK is important for survival when the bacteria are cocultured with nisin A-producing Lactococcus lactis This report highlights the roles of TCSs in the colocalization of bacteriocin-producing bacteria and non-bacteriocin-producing bacteria. Our findings provide new insights into the function of TCSs in S. pyogenes.

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Integrative Reverse Genetic Analysis Identifies Polymorphisms Contributing to Decreased Antimicrobial Agent Susceptibility in Streptococcus pyogenes.

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