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.

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.

Ciprofloxacin-Resistant Staphylococcus aureus Displays Enhanced Resistance and Virulence in Iron-Restricted Conditions.

The unreasonable misuse of antibiotics has led to the emergence of large-scale drug-resistant bacteria, seriously threatening human health. Compared with drug-sensitive bacteria, resistant bacteria are difficult to clear by host immunity. To fully explore the adaptive mechanism of resistant bacteria to the iron-restricted environment, we performed data-independent acquisition-based quantitative proteomics on ciprofloxacin (CIP)-resistant (CIP-R) Staphylococcus aureus in the presence or absence of iron. On bioinformatics analysis, CIP-R bacteria showed stronger amino acid synthesis and energy storage ability. Notably, CIP-R bacteria increased virulence by upregulating the expression of many virulence-related proteins and enhancing the synthesis of virulence-related amino acids under iron-restricted stress. This study will help us to further explain the adaptive mechanisms that lead to bacterial resistance to antibiotics depending on the host environment and provide insights into the development of novel drugs for the treatment of drug-resistant bacterial infections.

Quantitative secretome analysis of polymyxin B resistance in Escherichia coli.

Bacterial resistance has become a serious threat to human health. In particular, the gradual development of resistance to polymyxins, the last line of defense for human infections, is a major issue. Secreted proteins contribute to the interactions between bacteria and the environment. In this study, we compared the secretomes of polymyxin B-sensitive and -resistant Escherichia coli strains by data-independent acquisition mass spectrometry. In total, 87 differentially expressed secreted proteins were identified in polymyxin B-resistant E. coli compared to the sensitive strain. A GO enrichment analysis indicated that the differentially expressed proteins were involved in biological processes, including bacterial-type flagellum-dependent cell motility, ion transport, carbohydrate derivative biosynthetic process, cellular response to stimulus, organelle organization, and cell wall organization or biogenesis. The differentially expressed secreted proteins in polymyxin B-resistant bacteria were enriched for multiple pathways, suggesting that the resistance phenotype depends on complex regulatory mechanisms. A potential biomarker or drug target (YebV) was found in polymyxin B-resistant E. coli. This work clarifies the secretome changes associated with the acquisition of polymyxin resistance and may contribute to drug development.

SPD_1495 Contributes to Capsular Polysaccharide Synthesis and Virulence in Streptococcus pneumoniae.

Streptococcus pneumoniae, a Gram-positive human pathogen, causes a series of serious diseases in humans. SPD_1495 from S. pneumoniae is annotated as a hypothetical ABC sugar-binding protein in the NCBI database, but there are few reports on detailed biological functions of SPD_1495. To fully study the influence of SPD_1495 on bacterial virulence in S. pneumoniae, we constructed a deletion mutant (D39Δspd1495) and an overexpressing strain (D39spd1495+). Comparative analysis of iTRAQ-based quantitative proteomic data of the wild-type D39 strain (D39-WT) and D39Δspd1495 showed that several differentially expressed proteins that participate in capsular polysaccharide synthesis, such as Cps2M, Cps2C, Cps2L, Cps2T, Cps2E, and Cps2D, were markedly upregulated in D39Δspd1495 Subsequent transmission electron microscopy and uronic acid detection assay confirmed that capsular polysaccharide synthesis was enhanced in D39Δspd1495 compared to that in D39-WT. Moreover, knockout of spd1495 resulted in increased capsular polysaccharide synthesis, as well as increased bacterial virulence, as confirmed by the animal study. Through a coimmunoprecipitation assay, surface plasmon resonance, and electrophoretic mobility shift assay, we found that SPD_1495 negatively regulated cps promoter expression by interacting with phosphorylated ComE, a negative transcriptional regulator for capsular polysaccharide formation. Overall, this study suggested that SPD_1495 negatively regulates capsular polysaccharide formation and thereby enhances bacterial virulence in the host. These findings also provide valuable insights into understanding the biology of this clinically important bacterium.IMPORTANCE Capsular polysaccharide is a key factor underlying the virulence of Streptococcus pneumoniae in human diseases. Thus, a deep understanding of capsular polysaccharide synthesis is essential for uncovering the pathogenesis of S. pneumoniae infection. In this study, we show that protein SPD_1495 interacts with phosphorylated ComE to negatively regulate the formation of capsular polysaccharide. Deletion of spd1495 increased capsular polysaccharide synthesis and thereby enhanced bacterial virulence. These findings further reveal the synthesis mechanism of capsular polysaccharide and provide new insight into the biology of this clinically important bacterium.

Novel Mechanistic Insights into Bacterial Fluoroquinolone Resistance.

Advancements in studies on the evolutionary mechanisms underlying bacterial antibiotic resistance are unclear. This study aimed to investigate the evolutionary mechanism underlying bacterial antibiotic resistance using isobaric tags for relative and absolute quantitation-based quantitative proteomics along with functional validation. Quantitative analysis revealed 101, 325, and 428 differentially expressed proteins (DEPs) at three drug resistance levels (low-R, 0.2 µg/mL; medium-R, 5 µg/mL; high-R, 15 µg/mL). Continuous adjustment of metabolic patterns to enhance nucleotide synthesis and energy generation may underlie evolution. Indeed, nucleotide levels were elevated and strengthened ciprofloxacin resistance. Quorum sensing (QS) genes were upregulated in the early growth phase, thus potentially improving survival. Further, a thicker cell wall potentially serves as a stronger barrier and reduces drug permeation. The aforementioned three drug resistance levels displayed continuity and differences; the low-resistant level displayed no prominent mechanism; medium, a more focused change in nucleotide metabolism; and high, a thorough evolution to a complete systematic mechanism with higher adenosine 5'-triphosphate levels, serving as a defense mechanism for reducing drug-induced stress. Thus, gradual increments in nucleotide synthesis, energy synthesis, cell wall synthesis, QS, and biofilm formation may direct the evolution of bacterial resistance.