Isovitexin Protects Mice from Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia by Targeting Sortase A.

The rise of methicillin-resistant Staphylococcus aureus (MRSA) has resulted in significant morbidity and mortality, and clinical treatment of MRSA infections has become extremely difficult. Sortase A (SrtA), a virulence determinant that anchors numerous virulence-related proteins to the cell wall, is a prime druggable target against S. aureus infection due to its crucial role in the pathogenicity of S. aureus. Here, we demonstrate that isovitexin, an active ingredient derived from a variety of traditional Chinese medicines, can reversibly inhibit SrtA activity in vitro with a low dose (IC50=24.72 µg/ml). Fluorescence quenching and molecular simulations proved the interaction between isovitexin and SrtA. Subsequent point mutation experiments further confirmed that the critical amino acid positions for SrtA binding to isovitexin were Ala-92, Ile-182, and Trp-197. In addition, isovitexin treatment dramatically reduced S. aureus invasion of A549 cells. This study shows that treatment with isovitexin could alleviate pathological injury and prolong the life span of mice in an S. aureus pneumonia model. According to our research, isovitexin represents a promising lead molecule for the creation of anti-S. aureus medicines or adjuncts.

WYBQ-4: a New Bactericidal Agent against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that currently poses a serious threat to global health. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bactericidal activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. In addition, WYBQ-4 exhibited low cytotoxicity without hemolytic activity according to a safety evaluation. Importantly, WYBQ-4 showed potent in vivo efficacy in an MRSA-induced mouse pneumonia model, systemic infection model, and intramuscular infection model. The efficacy of this new cephalosporin against MRSA was associated with a high affinity for penicillin-binding proteins (PBP1, PBP2, PBP3, PBP4, PBP2a) evaluated in a competition assay using bocillin as a reporter. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection. IMPORTANCE Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bacteriostatic activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. WYBQ-4 showed potent in vivo efficacy in MRSA-induced mouse models. Subsequently, we further revealed its antibacterial mechanism. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection.

Molecular docking and proteomics reveals the synergistic antibacterial mechanism of theaflavin with ß-lactam antibiotics against MRSA.

Recurrent epidemics of methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) have illustrated that the effectiveness of antibiotics in clinical application is rapidly fading. A feasible approach is to combine natural products with existing antibiotics to achieve an antibacterial effect. In this molecular docking study, we found that theaflavin (TF) preferentially binds the allosteric site of penicillin-binding protein 2a (PBP2a), inducing the PBP2a active site to open, which is convenient for ß-lactam antibiotics to treat MRSA infection, instead of directly exerting antibacterial activity at the active site. Subsequent TMT-labeled proteomics analysis showed that TF treatment did not significantly change the landscape of the S. aureus USA300 proteome. Checkerboard dilution tests and kill curve assays were performed to validate the synergistic effect of TF and ceftiofur, and the fractional inhibitory concentration index (FICI) was 0.1875. The antibacterial effect of TF combined with ceftiofur was better than that of single-drug treatment in vitro. In addition, TF effectively enhanced the activity of ceftiofur in a mouse model of MRSA-induced pneumonia. Our findings provide a potential therapeutic strategy to combine existing antibiotics with natural products to resolve the prevalent infections of multidrug-resistant pathogens.

2,3-Dehydrokievitone combats methicillin-resistant Staphylococcus aureus infection by reducing alpha-hemolysin expression.

Due to powerful drug resistance and fatal toxicity of methicillin-resistant Staphylococcus aureus (MRSA), therapeutic strategies against virulence factors present obvious advantages since no evolutionary pressure will induce bacterial resistance. Alpha-hemolysin (Hla) is an extracellular toxin secreted by Staphylococcus aureus and contributes to bacterial pathogenicity. Herein, we identified a natural product 2,3-dehydrokievitone (2,3-DHKV) for inhibiting Hla activity of MRSA strain USA300 but not affecting bacteria growth. 2,3-DHKV significantly decreased hemolysin expression in a dose-dependent manner, but it did not potently neutralize hemolysin activity. Subsequently, cellular thermal shift and heptamer formation assays confirmed that 2,3-DHK affects hemolytic activity through indirect binding to Hla. RT-qPCR and western blot revealed that 2,3-DHKV suppressed Hla expression at the mRNA and protein levels, and further decreased accessory gene regulator A (agrA) transcription levels. We also observed that 2,3-DHK significantly attenuated the damage of A549 cells by S. aureus and reduced the release of lactate dehydrogenase (LDH). Moreover, in the MRSA-induced pneumonia mouse model, 2,3-DHK treatment prolonged the life span of mice and reduced the bacterial load in the lungs, which significantly alleviated the damage to the lungs. In summary, this study proved that 2,3-DHK as a Hla inhibitor is a potential antivirulence agent against MRSA infection.

Cyanidin chloride protects mice from methicillin-resistant Staphylococcus aureus-induced pneumonia by targeting Sortase A.

Methicillin-resistant Staphylococcus aureus (MRSA) has been developing rapidly in recent years. It poses a severe peril to global health care, and the new strategies to against the MRSA is urgently needed. Sortase A (SrtA) regulates the anchoring of many surface proteins. Compounds repress Staphylococcus aureus (S. aureus) cysteine transpeptidase SrtA are considered adequate potent virulence inhibitors. Then, we describe the identification of an effective SrtA inhibitor, cyanidin chloride, a bioflavonoid compound isolated from various plants. It has a reversible inhibitory effect on SrtA activity at an IC50 of 21.91 µg/mL. As a SrtA inhibitor, cyanidin chloride antagonizes SrtA-related virulence phenotypes due to its breadth and specificity, including fibrinogen adhesion, A549 cell invasion, biofilm formation, and surface protein (SpA) anchoring. Subsequently, molecular docking and fluorescence quenching revealed that SrtA and cyanidin chloride had robust mutual affinity. Further mechanistic studies revealed that Arg-197, Gly-167, and Sep-116 were the key-binding sites mediating the interaction between SrtA and cyanidin chloride. Notably, a significant therapeutic effect of cyanidin chloride in vivo was also observed on the mouse pneumonia model induced by MRSA. In conclusion, our study indicates that cyanidin chloride potentially represents a new candidate SrtA inhibitor for S. aureus and potentially be developed as a new antivirulence agent.

Orientin mediates protection against MRSA-induced pneumonia by inhibiting Sortase A.

Drug-resistant pathogenic Staphylococcus aureus (S. aureus) has severely threatened human health and arouses widespread concern. Sortase A (SrtA) is an essential virulence factor of S. aureus, which is responsible for the covalent anchoring of a variety of virulence-related proteins to the cell wall. SrtA has always been regarded as an ideal pharmacological target against S. aureus infections. In this research, we have determined that orientin, a natural compound isolated from various medicinal plants, can effectively inhibit the activity of SrtA with an IC50 of 50.44 ± 0.51 µM. We further demonstrated that orientin inhibited the binding of S. aureus to fibrinogen and diminished biofilm formation and the attaching of Staphylococcal protein A (SpA) to the cell wall in vitro. Using the fluorescence quenching assay, we demonstrated a direct interaction between orientin and SrtA. Further mechanistic studies revealed that the residues Glu-105, Thr-93, and Cys-184 were the key sites for the binding of SrtA to orientin. Importantly, we demonstrated that treatment with orientin attenuated S. aureus virulence of in vivo and protected mice against S. aureus-induced lethal pneumonia. These findings indicate that orientin is a potential drug to counter S. aureus infections and limit the development of drug resistance.

Eriodictyol as a Potential Candidate Inhibitor of Sortase A Protects Mice From Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia.

New anti-infective approaches are urgently needed to control multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Sortase A (SrtA) is a membrane-bound cysteine transpeptidase that plays an essential role in the catalysis of covalent anchoring of surface proteins to the cell wall of Staphylococcus aureus (S. aureus). The present study reports identification of a flavonoid, eriodictyol, as a reversible inhibitor of SrtA with an IC50 of 2.229 ± 0.014 µg/mL that can be used as an innovative means to counter both resistance and virulence. The data indicated that eriodictyol inhibited the adhesion of the bacteria to fibrinogen and reduced the formation of biofilms and anchoring of staphylococcal protein A (SpA) on the cell wall. The results of fluorescence quenching experiments demonstrated a strong interaction between eriodictyol and SrtA. Subsequent mechanistic studies revealed that eriodictyol binds to SrtA by interacting with R197 amino acid residue. Importantly, eriodictyol reduced the adhesion-dependent invasion of A549 cells by S. aureus and showed a good therapeutic effect in a model of mouse pneumonia induced by S. aureus. Overall, the results indicated that eriodictyol can attenuate MRSA virulence and prevent the development of resistance by inhibiting SrtA, suggesting that eriodictyol may be a promising lead compound for the control of MRSA infections.