Repurposing cinacalcet suppresses multidrug-resistant Staphylococcus aureus by disruption of cell membrane and inhibits biofilm by targeting IcaR.

BACKGROUND: MDR Staphylococcus aureus infections, along with the severity of biofilm-associated infections, continue to threaten human health to a great extent. It necessitates the urgent development of novel antimicrobial and antibiofilm agents. OBJECTIVES: To reveal the mechanism and target of cinacalcet as an antibacterial and antimicrobial agent for S. aureus. METHODS: Screening of non-antibiotic drugs for antibacterial and antibiofilm properties was conducted using a small-molecule drug library. In vivo efficacy was assessed through animal models, and the antibacterial mechanism was studied using quantitative proteomics, biochemical assays, LiP-SMap, BLI detection and gene knockout techniques. RESULTS: Cinacalcet, an FDA-approved drug, demonstrated antibacterial and antibiofilm activity against S. aureus, with less observed development of bacterial resistance. Importantly, cinacalcet significantly improved survival in a pneumonia model and bacterial clearance in a biofilm infection model. Moreover, the antibacterial mechanism of cinacalcet mainly involves the destruction of membrane-targeted structures, alteration of energy metabolism, and production of reactive oxygen species (ROS). Cinacalcet was found to target IcaR, inhibiting biofilm formation through the negative regulation of IcaADBC. CONCLUSIONS: The findings suggest that cinacalcet has potential for repurposing as a therapeutic agent for MDR S. aureus infections and associated biofilms, warranting further investigation.

YjgM is a crotonyltransferase critical for polymyxin resistance of Escherichia coli.

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.

Unveiling a novel mechanism for competitive advantage of ciprofloxacin-resistant bacteria in the environment through bacterial membrane vesicles.

Ciprofloxacin (CIP) is a prevalent environmental contaminant that poses a high risk of antibiotic resistance. High concentrations of antibiotics can lead to the development of resistant bacteria with high fitness costs, which often face a competitive disadvantage. However, it is unclear whether low-cost resistant bacteria formed by exposure to sub-MIC CIP in the environment can evolve competitive mechanisms against sensitive Escherichia coli (SEN) other than stronger resistance to CIP. Our study exposed E. coli to sub-MIC CIP levels, resulting in the development of CIP-resistant E. coli (CIPr). In antibiotic-free co-culture assays, CIPr outcompeted SEN. This indicates that CIPr is very likely to continue to develop and spread in antibiotic-free environments such as drinking water and affect human health. Further mechanism investigation revealed that bacterial membrane vesicles (BMVs) in CIPr, functioning as substance delivery couriers, mediated a cleavage effect on SEN. Proteomic analysis identified Entericidin B (EcnB) within CIPr-BMVs as a key factor in this competitive interaction. RT-qPCR analysis showed that the transcription of its negative regulator ompR/envZ was down-regulated. Moreover, EcnB plays a crucial role in the development of CIP resistance, and some resistance-related proteins and pathways have also been discovered. Metabolomics analysis highlighted the ability of CIPr-BMVs to acidify SEN, increasing the lytic efficiency of EcnB through cationization. Overall, our study reveals the importance of BMVs in mediating bacterial resistance and competition, suggesting that regulating BMVs production may be a new strategy for controlling the spread of drug-resistant bacteria.

Crizotinib Shows Antibacterial Activity against Gram-Positive Bacteria by Reducing ATP Production and Targeting the CTP Synthase PyrG.

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.

Potential Role of Lysine Acetylation in Antibiotic Resistance of Escherichia coli.

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.