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Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli.

ABSTRACT: The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.

SUBMITTER: Suarez SA

PROVIDER: S-EPMC8579933 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Intraspecific genomic diversity brings the potential for an unreported and diverse reservoir of cryptic antibiotic resistance genes in pathogens, as cryptic resistance can occur without major mutations and horizontal transmission. Here, we predicted the differences in the types of antibiotics and genes that induce cryptic and latent resistance between micro-diverse Escherichia coli strains. For example, we hypothesize that known resistance genes will be the culprit of latent resistance within clinical strains. We used a modified functional metagenomics method to induce expression in eight E. coli strains. We found a total of 66 individual genes conferring phenotypic resistance to 11 out of 16 antibiotics. A total of 14 known antibiotic resistance genes comprised 21% of total identified genes, whereas the majority (52 genes) were unclassified cryptic resistance genes. Between the eight strains, 1.2% of core orthologous genes were positive (conferred resistance in at least one strain). Sixty-four percent of positive orthologous genes conferred resistance to only one strain, demonstrating high intraspecific variability of latent resistance genes. Cryptic resistance genes comprised most resistance genes among laboratory and clinical strains as well as natural, semisynthetic, and synthetic antibiotics. Known antibiotic resistance genes primarily conferred resistance to multiple antibiotics from varying origins and within multiple strains. Hence, it is uncommon for E. coli to develop cross-cryptic resistance to antibiotics from multiple origins or within multiple strains. We have uncovered prospective and previously unknown resistance genes as well as antibiotics that have the potential to trigger latent antibiotic resistance in E. coli strains from varying origins.IMPORTANCEIntraspecific genomic diversity may be a driving force in the emergence of adaptive antibiotic resistance. Adaptive antibiotic resistance enables sensitive bacterial cells to acquire temporary antibiotic resistance, creating an optimal window for the development of permanent mutational resistance. In this study, we investigate cryptic resistance, an adaptive resistance mechanism, and unveil novel (cryptic) antibiotic resistance genes that confer resistance when amplified within eight E. coli strains derived from clinical and laboratory origins. We identify the potential of cryptic resistance genes to confer cross-resistance to antibiotics from varying origins and within multiple strains. We discern antibiotic characteristics that promote latent resistance in multiple strains, considering intraspecific diversity. This study may help detect novel resistance genes and functional genes that could become responsible for cryptic resistance among diverse strains and antibiotics, thus also identifying potential novel antibiotic targets and mechanisms.

Project description:Antibiotic resistance is increasingly becoming a challenge to public health. The regulation of bacterial metabolism by post-translational modifications (PTMs) has been widely studied. However, the mechanism underlying the regulation of acetylation in bacterial resistance to antibiotics is still unknown. Here, we performed a quantitative analysis of the acetylated proteome of a wild-type (WT) Escherichia coli (E. coli) sensitive strain and ampicillin- (Re-Amp), kanamycin- (Re-Kan), and polymyxin B-resistant (Re-Pol) strains. Based on bioinformatics analysis combined with biochemical validations, we found a common regulatory mechanism between the different resistant strains. Our results showed that protein acetylation negatively regulates bacterial metabolism to regulate antibiotic resistance and positively regulates bacterial motility. Further analyses revealed that key enzymes in various metabolic pathways were differentially acetylated. In particular, pyruvate kinase (PykF), a glycolytic enzyme that regulates bacterial metabolism, and its acetylated form were highly expressed in the three resistant strains and were identified as reversibly acetylated by the deacetylase CobB and the acetyl-transferase PatZ (peptidyl-lysine N-acetyltransferase). Results showed that PykF also could be acetylated by nonenzymatic acetyl phosphatase (AcP) in vitro. Furthermore, the deacetylation of Lys413 in PykF increased PykF enzymatic activity by changing the conformation of its ATP binding site, resulting in an increase in energy production which, in turn, increased the sensitivity of drug-resistant strains to antibiotics. This study provides novel insights for understanding bacterial resistance and lays the foundation for future research on the regulation of acetylation in antibiotic-resistant strains. IMPORTANCE The misuse of antibiotics has resulted in the emergence of many antibiotic-resistant strains which seriously threaten human health. Protein post-translational modifications, especially acetylation, tightly control bacterial metabolism. However, the comprehensive mechanism underlying the regulation of acetylation in bacterial resistance remains unexplored. Here, acetylation was found to positively regulate bacterial motility and negatively regulate energy metabolism, which was common in all antibiotic-resistant strains. Moreover, the acetylation and deacetylation process of PykF was uncovered, and deacetylation of the Lys 413 in PykF was found to contribute to bacterial sensitivity to antibiotics. This study provides a new direction for research on the development of bacterial resistance through post-translational modifications and a theoretical basis for developing antibacterial drugs.

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Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli.

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