Natural flavone hispidulin protects mice from Staphylococcus aureus pneumonia by inhibition of α-hemolysin production via targeting AgrAC.

The emergence of drug-resistant Staphylococcus aureus (S. aureus) has limited drug options for the clinical treatment of S. aureus infections. Considering recent reports, therapeutic strategies targeting bacterial virulence hold great promise, and alpha-hemolysin (encoded by hla), a critical virulence factor of S. aureus, plays a vital role during bacterial infection. Herein, we demonstrated that hispidulin effectively inhibited the hemolytic activity of S. aureus USA300 without suppressing bacterial growth, along with inhibiting hla transcription and expression in a dose-dependent manner. As heptamer formation is essential for hla-mediated invasion of cells, nevertheless, hispidulin did not affect the deoxycholate-induced oligomerization of hla, suggesting that hispidulin did not affect the protein activity of hla. In vitro assays illustrated that hispidulin bound to agrAC, a crucial protein in quorum sensing. Meanwhile, hispidulin alleviated A549 cell damage caused by S. aureus USA300 and reduced lactate dehydrogenase release. In vivo studies showed that hispidulin had a protective effect against pneumonia caused by S. aureus USA300 in mice. S. aureus did not develop resistance to hispidulin in the short term. Interestingly, our research indicated that hispidulin synergized with the antibacterial activity of cefoxitin. These results showed that hispidulin effectively inhibited α-hemolysin expression by inhibiting the agr quorum sensing of S. aureus. It has promise as an agent to treat S. aureus infection.

Nepetin reduces virulence factors expression by targeting ClpP against MRSA-induced pneumonia infection.

The resistance of Staphylococcus aureus (S. aureus) to various antibiotics has increased dramatically due to the misuse of antibiotics, and thus the development of new anti-infective drugs with new targets is urgently needed to combat resistance. Caseinolytic peptidase P is a case in hydrolase that regulates the virulence level of S. aureus. Here, we found that nepetin, a small-molecule compound from traditional Chinese herbal flavonoids, effectively inhibits ClpP activity. Nepetin suppressed the virulence of S. aureus and effectively combated the lethal pneumonia caused by MRSA. The results of cellular thermal shift assay showed that nepetin could bind to ClpP and reduce the thermal stability of ClpP, and the KD value of 602 nM between them was determined using localized surface plasmon resonance. The binding mode of nepetin and ClpP was further investigated by molecular docking, and it was found that Ser-22 and Gln-47 of ClpP residues were found to be involved in the binding of nepetin to ClpP. In conclusion, we determined that nepetin is a ClpP inhibitor and an effective lead compound for the development of a virulence factor-based treatment for MRSA infection.

Scutellarin potentiates vancomycin against lethal pneumonia caused by methicillin-resistant Staphylococcus aureus through dual inhibition of sortase A and caseinolytic peptidase P.

The strategy of targeting virulence factor has received great attention as it barely develops bacterial resistance. Sortase A (SrtA) and caseinolytic peptidase P (ClpP), as important virulence factors, are considered to be ideal pharmacological targets for methicillin-resistant Staphylococcus aureus (MRSA) infection. Through screening hundreds of compounds, we found scutellarin, a natural flavonoid, markedly inhibited SrtA and ClpP activities of MRSA strain USA300 with an IC50 of 53.64 µg/mL and 107.00 µg/mL, respectively. Subsequently, we observed that scutellarin could inhibit the SrtA-related virulence of MRSA. To demonstrate whether scutellarin directly binding to SrtA, fluorescence quenching assay and molecular docking were performed and the results indicated that scutellarin directly bonded to SrtA molecule with a KA value of 7.58 × 104 L/mol. In addition to direct SrtA inhibition, scutellarin could also inhibit hemolytic activity of S. aureus by inhibiting the expression of Hla in a SrtA-independent manner. Further assays confirmed that scutellarin inhibited hemolysis by inhibiting ClpP. The combination of scutellarin and vancomycin showed enhancing inhibition of USA300 in vitro and in vivo, evidenced by decreased MIC from 3 µg/mL to 0.5 µg/mL and increased survival and improvement of lung pathology in pneumonia mice. Taken together, these results suggest that scutellarin exhibited di-inhibitory effects on SrtA and ClpP of USA300. The di-inhibition of virulence factors by scutellarin combined with vancomycin to prevent MRSA invasion of A549 cells and pneumonia in mice, indicating that scutellarin is expected to be a potential adjuvant against MRSA in the future.

Galangin as a direct inhibitor of vWbp protects mice from Staphylococcus aureus-induced pneumonia.

The surge in multidrug resistance in Staphylococcus aureus (S. aureus) and the lag in antibiotic discovery necessitate the development of new anti-infective strategies to reduce S. aureus infections. In S. aureus, von Willebrand factor-binding protein (vWbp) is not only the main coagulase that triggers host prothrombin activation and formation of fibrin cables but also bridges the bacterial cell wall and von Willebrand factor, thereby allowing S. aureus to bind to platelets and endothelial cells, playing a vital role in pathogenesis of S. aureus infections. Here, we have identified that galangin, a bioactive compound found in honey and Alpinia officinarum Hance, is a potent and direct inhibitor of vWbp by coagulation activity inhibition assay, thermal shift assay and biolayer interferometry assay. Molecular dynamic simulations and verification experiments revealed that the Trp-64 and Leu-69 residues are necessary for the binding of galangin to vWbp. Significantly, galangin attenuated S. aureus virulence in a mouse S. aureus-induced pneumonia model. In addition, we also identified that galangin can enhance the therapeutic effect of latamoxef on S. aureus-induced pneumonia. Taken together, the results suggest that galangin may be used for the development of therapeutic drugs or utilized as adjuvants to combine with antibiotics to combat S. aureus-related infections.

Myricetin protects mice against MRSA-related lethal pneumonia by targeting ClpP.

Methicillin-resistant Staphylococcus aureus is one of the leading causes of community and nosocomial infections, which has created the urgent need for innovative anti-infective agents to control MRSA-associated infections. A conserved serine protease, caseinolytic peptidase P (ClpP) in Staphylococcus aureus is highly associated with pathogenicity and has been claimed to be a novel antimicrobial target. We aim to search suitable inhibitors of ClpP to attenuate the virulence of MRSA and combat their infections in vivo. Over 500 natural compounds were pre-screened via fluorescence resonance energy transfer using the Suc-LY-AMC substrate. The binding of myricetin to ClpP was determined and the mechanism of action was elucidated by thermal shift assay, surface plasmon resonance, and molecular dynamics simulations. The therapeutic effects of myricetin on S. aureus infection were further investigated using a S. aureus-induced pneumonia model. We revealed that myricetin could effectively block the activity of ClpP without disturbing the growth of the bacteria and the Gln-47 and Met-31 residues were necessary for myricetin binding to ClpP. Importantly, myricetin attenuated the pathogenicity of S. aureus in vivo, while improving the efficacy of the traditional antibiotic oxacillin against MRSA infection and protecting mice from fatal lung infections caused by MRSA. These findings indicate that myricetin has the potential to be applied in the pharmaceutical industry as a promising therapeutic agent.

Isovitexin Is a Direct Inhibitor of Staphylococcus aureus Coagulase.

Staphylococcus aureus (S. aureus) is a major pathogen that causes human pneumonia, leading to significant morbidity and mortality. S. aureus coagulase (Coa) triggers the polymerization of fibrin by activating host prothrombin, which then converts fibrinogen to fibrin and contributes to S. aureus pathogenesis and persistent infection. In our research, we demonstrate that isovitexin, an active traditional Chinese medicine component, can inhibit the coagulase activity of Coa but does not interfere with the growth of S. aureus. Furthermore, we show through thermal shift and fluorescence quenching assays that isovitexin directly binds to Coa. Dynamic simulation and structure-activity relationship analyses suggest that V191 and P268 are key amino acid residues responsible for the binding of isovitexin to Coa. Taken together, these data indicate that isovitexin is a direct Coa inhibitor and a promising candidate for drug development against S. aureus infection.

Analysis of Long Noncoding RNAs in Aila-Induced Non-Small Cell Lung Cancer Inhibition.

Non-small cell lung cancer (NSCLC) has the highest morbidity and mortality among all carcinomas. However, it is difficult to diagnose in the early stage, and current therapeutic efficacy is not ideal. Although numerous studies have revealed that Ailanthone (Aila), a natural product, can inhibit multiple cancers by reducing cell proliferation and invasion and inducing apoptosis, the mechanism by which Aila represses NSCLC progression in a time-dependent manner remains unclear. In this study, we observed that most long noncoding RNAs (lncRNAs) were either notably up- or downregulated in NSCLC cells after treatment with Aila. Moreover, alterations in lncRNA expression induced by Aila were crucial for the initiation and metastasis of NSCLC. Furthermore, in our research, expression of DUXAP8 was significantly downregulated in NSCLC cells after treatment with Aila and regulated expression levels of EGR1. In conclusion, our findings demonstrate that Aila is a potent natural suppressor of NSCLC by modulating expression of DUXAP8 and EGR1.

Chrysin Induced Cell Apoptosis Through H19/let-7a/COPB2 Axis in Gastric Cancer Cells and Inhibited Tumor Growth.

BACKGROUND: Chrysin is a natural flavone that is present in honey and has exhibited anti-tumor properties. It has been widely studied as a therapeutic agent for the treatment of various types of cancers. The objectives of this present study were to elucidate how chrysin regulates non-coding RNA expression to exert anti-tumor effects in gastric cancer cells. METHODS: Through the use of RNA sequencing, we investigated the differential expression of mRNAs in gastric cancer cells treated with chrysin. Furthermore, COPB2, H19 and let-7a overexpression and knockdown were conducted. Other features, including cell growth, apoptosis, migration and invasion, were also analyzed. Knockout of the COPB2 gene was generated using the CRISPR/Cas9 system for tumor growth analysis in vivo. RESULTS: Our results identified COPB2 as a differentially expressed mRNA that is down-regulated following treatment with chrysin. Moreover, the results showed that chrysin can induce cellular apoptosis and inhibit cell migration and invasion. To further determine the underlying mechanism of COPB2 expression, we investigated the expression of the long non-coding RNA (lncRNA) H19 and microRNA let-7a. Our results showed that treatment with chrysin significantly increased let-7a expression and reduced the expression of H19 and COPB2. In addition, our results demonstrated that reduced expression of COPB2 markedly promotes cell apoptosis. Finally, in vivo data suggested that COPB2 expression is related to tumor growth. CONCLUSIONS: This study suggests that chrysin exhibited anti-tumor effects through a H19/let-7a/COPB2 axis.

Improved Antiviral Activity of Classical Swine Fever Virus-Targeted siRNA by Tetrahedral Framework Nucleic Acid-Enhanced Delivery.

DNA self-assembled nanostructures have been considered as effective vehicles for biomolecule delivery because of their excellent biocompatibility, cellular permeability, noncytotoxicity, and small size. Here, we report an efficient antiviral strategy with self-assembled tetrahedral framework nucleic acids (tFNAs) delivering small interfering RNA (t-siRNA) to silence classical swine fever virus (CSFV) gene in porcine host cells. In this study, two previously reported siRNAs, C3 and C6, specifically targeting the CSFV genome were selected and modified on tFNAs, respectively, and termed t-C3 and t-C6. Results indicate that t-C3 and t-C6 can inhibit the viral proliferation of CSFV in kidney derived porcine cells, PK-15, effectively and that inhibition was markedly stronger than free siRNA-C3 or siRNA-C6 only. In addition, the DNA nanostructure also has high cargo-carrying capacity, allowing to deliver multiple functional groups. To improve the antiviral ability of tFNAs, a dual-targeting DNA nanostructure t-C3-C6 was constructed and used to silence the CSFV gene in porcine host cells. This study found that t-C3-C6 can inhibit the viral release and replication, exhibiting outstanding anti-CSFV capabilities. Therefore, these dual-targeting tFNAs have great potential in virus therapy. This strategy not only provides a novel method to inhibit CSFV replication in porcine cells but also verifies that tFNAs are effective tools for delivery of antiviral elements, which have great application potential.

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.

Baicalein mediates protection against Staphylococcus aureus-induced pneumonia by inhibiting the coagulase activity of vWbp.

The emergence and spread of multidrug-resistant Staphylococcus aureus (S. aureus) necessitate the research on therapeutic tactics which are different from classical antibiotics in overcoming resistance andtreatinginfections. In S. aureus, von Willebrand factor-binding protein (vWbp) is one of the key virulence determinants because it mediates not only the activation of thrombin to convert fibrinogen to fibrin, thereby enabling S. aureus to escape from the host immune clearance, but also the adhesion of S. aureus to host cells. Thus, vWbp is regarded as a promising druggable target to treat S. aureus-associated infections. Here we identify that baicalein, a natural compound isolated from the Chinese herb Scutellaria baicalensis, can effectively block the coagulase activity of vWbp without inhibiting the growth of the bacteria. Through thermal shift and fluorescence quenching assays, we demonstrated that baicalein directly binds to vWbp. Molecular dynamics simulations and mutagenesis assays revealed that the Asp-75 and Lys-80 residues are necessary for baicalein binding to vWbp. Importantly, we demonstrated that baicalein treatment attenuates the virulence of S. aureus and protects mice from S. aureus-induced lethal pneumonia. In addition, baicalein can improve the therapeutic effect of penicillin G by 75% in vivo. These findings indicate that baicalein might be developed as a promising therapeutic agent against drug-resistant S. aureus infections.

Targeting staphylocoagulase with isoquercitrin protects mice from Staphylococcus aureus-induced pneumonia.

Staphylocoagulase (Coa) is a virulence factor of Staphylococcus aureus (S. aureus) that promotes blood coagulation by activating prothrombin to convert fibrinogen to fibrin. Coa plays a crucial role in disease pathogenesis and is a promising target for the treatment of S. aureus infections. Here, we identified that isoquercitrin, a natural flavonol compound, can markedly reduce the activity of Coa at concentrations that have no effect on bacterial growth. Mechanistic studies employing molecular dynamics simulation revealed that isoquercitrin binds to Coa by interacting with Asp-181 and Tyr-188, thereby affecting the binding of Coa to prothrombin. Importantly, in vivo studies showed that isoquercitrin treatment significantly reduced the bacterial burden, pathological damage, and inflammation of lung tissue and improved the percentage of survival of mice infected with S. aureus Newman strain. These data suggest that isoquercitrin is a promising inhibitor of Coa that can be used for the development of therapeutic drugs to combat S. aureus infections.Key Points• Staphylocoagulase plays a key role in the pathogenesis of S. aureus infection.• We identified that isoquercitrin is a direct inhibitor of staphylocoagulase.• Isoquercitrin treatment can significantly attenuate S. aureus virulence in vivo.

The combination of salvianolic acid A with latamoxef completely protects mice against lethal pneumonia caused by methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus), especially methicillin-resistant Staphylococcus aureus (MRSA), is a major cause of pneumonia, resulting in severe morbidity and mortality in adults and children. Sortase A (SrtA), which mediates the anchoring of cell surface proteins in the cell wall, is an important virulence factor of S. aureus. Here, we found that salvianolic acid A (Sal A), which is a natural product that does not affect the growth of S. aureus, could inhibit SrtA activity (IC50 = 5.75 µg/ml) and repress the adhesion of bacteria to fibrinogen, the anchoring of protein A to cell wall, the biofilm formation, and the ability of S. aureus to invade A549 cells. Furthermore, in vivo studies demonstrated that Sal A treatment reduced inflammation and protected mice against lethal pneumonia caused by MRSA. More significantly, full protection (a survival rate of 100%) was achieved when Sal A was administered in combination with latamoxef. Together, these results indicate that Sal A could be developed into a promising therapeutic drug to combat MRSA infections while limiting resistance development.

An Inhibitory Effect of Dryocrassin ABBA on Staphylococcus aureus vWbp That Protects Mice From Pneumonia.

Von Willebrand factor-binding protein (vWbp), secreted by Staphylococcus aureus (S. aureus), can activate host prothrombin, convert fibrinogen to fibrin clots, induce blood clotting, and contribute to pathophysiology of S. aureus-related diseases, including infective endocarditis, staphylococcal sepsis and pneumonia. Therefore, vWbp is an promising drug target in the treatment of S. aureus-related infections. Here, we report that dryocrassin ABBA (ABBA), a natural compound derived from Dryopteris crassirhizoma, can significantly inhibit the coagulase activity of vWbp in vitro by directly interacting with vWbp without killing the bacteria or inhibiting the expression of the vWbp. Using molecular dynamics simulations, we demonstrate that ABBA binds to the "central cavity" in the elbow of vWbp by interacting with Arg-70, His-71, Ala-72, Gly-73, Tyr-74, Glu-75, Tyr-83, and Gln-87 in vWbp, thus interfering with the binding of vWbp to prothrombin. Furthermore, in vivo studies demonstrated that ABBA can attenuate injury and inflammation of mouse lung tissues caused by S. aureus and increase survival of mice. Together these findings indicate that ABBA is a promising lead drug for the treatment of S. aureus-related infections. This is the first report of potential inhibitor which inhibit the coagulase activity of vWbp by directly interacting with vWbp.

Astilbin Inhibits the Activity of Sortase A from Streptococcus mutans.

Streptococcus mutans (S. mutans) is the primary etiological agent of dental caries. The S. mutans enzyme sortase A (SrtA) is responsible for anchoring bacterial cell wall surface proteins involved in host cell attachment and biofilm formation. Thus, SrtA is an attractive target for inhibiting dental caries caused by S. mutans-associated acid fermentation. In this study, we observed that astilbin, a flavanone compound extracted from Rhizoma Smilacis Glabrae, has potent inhibitory activity against the S. mutans SrtA, with an IC50 of 7.5 µg/mL. In addition, astilbin was proven to reduce the formation of biofilm while without affecting the growth of S. mutans. The results of a molecular dynamics simulation and a mutation analysis revealed that the Arg213, Leu111, and Leu116 of SrtA are important for the interaction between SrtA and astilbin. The results of this study demonstrate the potential of using astilbin as a nonbactericidal agent to modulate pathogenicity of S. mutans by inhibiting the activity of SrtA.

Large-scale prediction of ADAR-mediated effective human A-to-I RNA editing.

Adenosine-to-inosine (A-to-I) editing by adenosine deaminase acting on the RNA (ADAR) proteins is one of the most frequent modifications during post- and co-transcription. To facilitate the assignment of biological functions to specific editing sites, we designed an automatic online platform to annotate A-to-I RNA editing sites in pre-mRNA splicing signals, microRNAs (miRNAs) and miRNA target untranslated regions (3' UTRs) from human (Homo sapiens) high-throughput sequencing data and predict their effects based on large-scale bioinformatic analysis. After analysing plenty of previously reported RNA editing events and human normal tissues RNA high-seq data, >60 000 potentially effective RNA editing events on functional genes were found. The RNA Editing Plus platform is available for free at https://www.rnaeditplus.org/, and we believe our platform governing multiple optimized methods will improve further studies of A-to-I-induced editing post-transcriptional regulation.

Exploiting a conjugative CRISPR/Cas9 system to eliminate plasmid harbouring the mcr-1 gene from Escherichia coli.

The transfer of multi-drug-resistance plasmids by bacterial conjugation is largely responsible for the development of drug resistance in bacteria, and causes serious problems in the treatment of infectious diseases. Since the first discovery of plasmid-borne colistin resistance gene mcr-1 was reported in late 2016, this gene has been found in a great number of Escherichia coli and other Gram-negative pathogens separated from different types of sources worldwide. The elimination of plasmids carrying mcr-1 and restoration of polymyxin sensitivity has very important clinical significance because polymyxins are frequently used as last-resort antibiotics to treat extensively drug-resistant Gram-negative bacterial infections. A host-independent conjugative plasmid was constructed in this study, and an engineered CRISPR/Cas9 system was used to remove plasmid harbouring mcr-1 from bacteria. This study found that this conjugative plasmid can not only be used as a new tool to remove resistance plasmids and sensitize the recipient bacteria to antibiotics, but can also make the recipient cell acquire immunity against mcr-1. This strategy provides a novel method to counteract the ever-worsening spread of mcr-1 among bacterial pathogens.

Genetically modified pigs are protected from classical swine fever virus.

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.

Isovitexin, a Potential Candidate Inhibitor of Sortase A of Staphylococcus aureus USA300.

Staphylococcus aureus (S. aureus) causes a broad variety of diseases. The spread of multidrugresistant S. aureus highlights the need to develop new ways to combat S. aureus infections. Sortase A (SrtA) can anchor proteins containing LPXTG binding motifs to the bacteria surface and plays a key role in S. aureus infections, making it a promising antivirulence target. In the present study, we used aSrtA activity inhibition assay to discover that isovitexin, a Chinese herbal product, can inhibit SrtA activity with an IC50 of 28.98 µg/ml. Using a fibrinogenbinding assay and a biofilm formation assay, we indirectly proved the SrtA inhibitory activity of isovitexin. Additionally, isovitexin treatment decreased the amount of staphylococcal protein A (SpA) on the surface of the cells. These data suggest that isovitexin has the potential to be an anti-infective drug against S. aureus via the inhibition of sortase activity.