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Summary: Lysine crotonylation has attracted widespread attention in recent years. However, little is known about bacterial crotonylation, particularly crotonyltransferase and decrotonylase, and its effects on antibiotic resistance. Our study demonstrates the ubiquitous presence of crotonylation in E. coli, which promotes bacterial resistance to polymyxin. We identify the crotonyltransferase YjgM and its regulatory pathways in E. coli with a focus on crotonylation. Further studies show that YjgM upregulates the crotonylation of the substrate protein PmrA, thereby boosting PmrA’s affinity for binding to the promoter of eptA, which, in turn, promotes EptA expression and confers polymyxin resistance in E. coli. Additionally, we discover that PmrA’s crucial crotonylation site and functional site is Lys 164. These significant discoveries highlight the role of crotonylation in bacterial drug resistance and offer a fresh perspective on creating antibacterial compounds.

ABSTRACT 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.

ABSTRACT Infections caused by drug-resistant bacteria are a serious threat to public health worldwide, and the discovery of novel antibacterial compounds is urgently needed. Here, we screened an FDA-approved small-molecule library and found that crizotinib possesses good antimicrobial efficacy against Gram-positive bacteria. Crizotinib was found to increase the survival rate of mice infected with bacteria and decrease pulmonary inflammation activity in an animal model. Furthermore, it showed synergy with clindamycin and gentamicin. Importantly, the Gram-positive bacteria showed a low tendency to develop resistance to crizotinib. Mechanistically, quantitative proteomics and biochemical validation experiments indicated that crizotinib exerted its antibacterial effects by reducing ATP production and pyrimidine metabolism. A drug affinity responsive target stability study suggested crizotinib targets the CTP synthase PyrG, which subsequently disturbs pyrimidine metabolism and eventually reduces DNA synthesis. Subsequent molecular dynamics analysis showed that crizotinib binding occurs in close proximity to the ATP binding pocket of PyrG and causes loss of function of this CTP synthase. Crizotinib is a promising antimicrobial agent and provides a novel choice for the development of treatment for Gram-positive infections. IMPORTANCE Infections caused by drug-resistant bacteria are a serious problem worldwide. Therefore, there is an urgent need to find novel drugs with good antibacterial activity against multidrug-resistant bacteria. In this study, we found that a repurposed drug, crizotinib, exhibits excellent antibacterial activity against drug-resistant bacteria both in vivo and in vitro via suppressing ATP production and pyrimidine metabolism. Crizotinib was found to disturb pyrimidine metabolism by targeting the CTP synthase PyrG, thus reducing DNA synthesis. This unique mechanism of action may explain the decreased development of resistance by Staphylococcus aureus to crizotinib. This study provides a potential option for the treatment of drug-resistant bacterial infections in the future.