Genistein is effective in inhibiting Orf virus infection in vitro by targeting viral RNA polymerase subunit RPO30 protein.

Orf virus (ORFV), a typical member of the genus Parapoxvirus, Poxvirus family, causes a contagious pustular dermatitis in sheep, goats, and humans. Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm, which is a viral factor essential to poxvirus replication. Due to its vital role in viral life, vRNAP has emerged as one of the potential drug targets. In the present study, we investigated the antiviral effect of genistein against ORFV infection. We provided evidence that genistein exerted antiviral effect through blocking viral genome DNA transcription/replication and viral protein synthesis and reducing viral progeny, which were dosedependently decreased in genistein-treated cells. Furthermore, we identified that genistein interacted with the vRNAP RPO30 protein by CETSA, molecular modeling and Fluorescence quenching, a novel antiviral target for ORFV. By blocking vRNAP RPO30 protein using antibody against RPO30, we confirmed that the inhibitory effect exerted by genistein against ORFV infection is mediated through the interaction with RPO30. In conclusion, we demonstrate that genistein effectively inhibits ORFV transcription in host cells by targeting vRNAP RPO30, which might be a promising drug candidate against poxvirus infection.

The role of lysosomes as intermediates in betacoronavirus PHEV egress from nerve cells.

IMPORTANCE: Betacoronaviruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. However, whether all betacoronaviruses members use the same pathway to exit cells remains unknown. Here, we demonstrated that porcine hemagglutinating encephalomyelitis virus (PHEV) egress occurs by Arl8b-dependent lysosomal exocytosis, a cellular egress mechanism shared by SARS-CoV-2 and MHV. Notably, PHEV acidifies lysosomes and activates lysosomal degradative enzymes, while SARS-CoV-2 and MHV deacidify lysosomes and limit the activation of lysosomal degradative enzymes. In addition, PHEV release depends on V-ATPase-mediated lysosomal pH. Furthermore, this is the first study to evaluate ßCoV using lysosome for spreading through the body, and we have found that lysosome played a critical role in PHEV neural transmission and brain damage caused by virus infection in the central nervous system. Taken together, different betacoronaviruses could disrupt lysosomal function differently to exit cells.

Isovanillic acid protects mice against Staphylococcus aureus by targeting vWbp and Coa.

Aim: Our primary objective was to investigate the protective effects and mechanisms of isovanillic acid in mice infected with Staphylococcus aureus Newman. Methods: In vitro coagulation assays were used to validate vWbp and Coa as inhibitory targets of isovanillic acid. The binding mechanism of isovanillic acid to vWbp and Coa was investigated using molecular docking and point mutagenesis. Importantly, a lethal pneumonia mouse model was used to assess the effect of isovanillic acid on survival and pathological injury in mice. Results & Conclusion: Isovanillic acid reduced the virulence of S. aureus by directly binding to inhibit the clotting activity of vWbp and Coa, thereby reducing lung histopathological damage and improving the survival rate in mice with pneumonia.

The natural product, echinatin, protects mice from methicillin-resistant Staphylococcus aureus pneumonia by inhibition of alpha-hemolysin expression.

Antimicrobial resistance (AMR) is a global, multifaceted crisis that poses significant challenges to the successful eradication of devastating pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), a persistent superbug that causes devastating infections. The scarcity of new antibacterial drugs is obvious, and antivirulence strategies that reduce the pathogenicity of bacteria by weakening their virulence have become the subject of intense investigation. Alpha-hemolysin (Hla), a cytolytic pore-forming toxin, has a pivotal role in S. aureus pathogenesis. Here, we demonstrated that echinatin, a natural compound isolated from licorice, effectively inhibited the hemolytic activity of MRSA at 32 µg/mL. In addition, echinatin did not interfere with bacterial growth and had no significant cytotoxicity at the inhibitory concentration of S. aureus hemolysis. Heptamer formation tightly correlated with Hla-mediated cell invasion, whereas echinatin did not affect deoxycholic acid-induced oligomerization of Hla. Echinatin affected hemolytic activity through indirect binding to Hla as confirmed by the neutralization assay and cellular thermal shift assay (CETSA). Furthermore, qRT-PCR and western blot analyses revealed that echinatin suppressed Hla expression at both the mRNA and protein levels as well as the transcript levels of Agr quorum-sensing system-related genes. Additionally, when echinatin was added to a coculture system of A549 cells and S. aureus, it significantly reduced cell damage. Importantly, echinatin exhibited a significant therapeutic effect in an MRSA-induced mouse pneumonia model. In conclusion, the present findings demonstrated that echinatin significantly inhibits the hemolysin effect and may be a potential candidate compound for combating drug-resistant MRSA infections.

Oroxylin a glucuronide as a novel class of reversible inhibitors of Sortase a, combats MRSA-induced infections.

AIMS: The main purpose of this study was to study the therapeutical effect of oroxylin A glucuronide (OAG) on methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: By substrate peptide reaction-based fluorescence resonance energy transfer (FRET) screening, we identified that OAG was an efficient inhibitor of Sortase A (SrtA) with an IC50 of 45.61 µg mL-1, and achieved efficacy in the treatment of Staphylococcus aureus (S. aureus) infections. We further demonstrated that OAG inhibited the adhesion of the S. aureus to fibrinogen, the surface protein A anchoring and diminished biofilm formation. Results obtained from fluorescence quenching assay elucidated a direct interaction between OAG and SrtA. Employing molecular dynamics simulations, we proved that OAG binds to the binding sites of R197, G192, E105, and V168 in the SrtA. Notably, OAG exhibited a robust therapeutic effect in a MRSA-induced pneumonia model. CONCLUSIONS: We identified that OAG as a novel class of reversible inhibitors of SrtA, combats MRSA-induced Infections.

Orf virus DNA prime-protein boost strategy is superior to adenovirus-based vaccination in mice and sheep.

Contagious ecthyma (Orf), an acute and highly contagious zoonosis, is prevalent worldwide. Orf is caused by Orf virus (ORFV), which mainly infects sheep/goats and humans. Therefore, effective and safe vaccination strategies for Orf prevention are needed. Although immunization with single-type Orf vaccines has been tested, heterologous prime-boost strategies still need to be studied. In the present study, ORFV B2L and F1L were selected as immunogens, based on which DNA, subunit and adenovirus vaccine candidates were generated. Of note, heterologous immunization strategies using DNA prime-protein boost and DNA prime-adenovirus boost in mice were performed, with single-type vaccines as controls. We have found that the DNA prime-protein boost strategy induces stronger humoral and cellular immune responses than DNA prime-adenovirus boost strategy in mice, which was confirmed by the changes in specific antibodies, lymphocyte proliferation and cytokine expression. Importantly, this observation was also confirmed when these heterologous immunization strategies were performed in sheep. In summary, by comparing the two immune strategies, we found that DNA prime-protein boost strategy can induce a better immune response, which provides a new attempt for exploring Orf immunization strategy.

Orf virus induces complete autophagy to promote viral replication via inhibition of AKT/mTOR and activation of the ERK1/2/mTOR signalling pathway in OFTu cells.

Orf virus (ORFV) is the causative agent of contagious ecthyma, which is an important zoonotic pathogen with a widespread distribution affecting sheep, goats and humans. Our previous research showed that autophagy can be induced in host cells by ORFV infection. However, the exact mechanism of ORFV-induced autophagy remains unknown. In this study, we investigated the underlying mechanisms of autophagy induced by ORFV in OFTu cells and the impact of autophagy on ORFV replication. By using specific autophagy inhibitors and activators, Western blotting, immunofluorescence and transmission electron microscopy imaging, we confirmed that ORFV infection triggered intracellular autophagosome accumulation and the activation of autophagic flux. Moreover, ORFV-induced autophagic activity was found to rely on an increase in the phosphorylation of tuberous sclerosis complex 2 (TSC2) and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR), which is mediated by the suppression of the PI3K/AKT/mTOR signalling pathway and activation of the ERK1/2/mTOR signalling pathway. Furthermore, we investigated the role of mTOR-mediated autophagy during ORFV replication using pharmacological agents and demonstrated that ORFV-induced autophagy correlated positively with viral replication. Taken together, our data reveal the pathways of ORFV-induced autophagy and the impact of autophagy on ORFV replication, providing new insights into ORFV pathogenesis.

Rapid Onsite Visual Detection of Orf Virus Using a Recombinase-Aided Amplification Assay.

Orf is an important zoonotic disease caused by the Orf virus (ORFV) which can cause contagious pustular dermatitis in goats and sheep. Orf is widespread in most sheep-raising countries in the world, causing huge economic losses. Although diagnostic methods for ORFV infection already exist, it is still necessary to develop a time-saving, labor-saving, specific, low-cost and visual diagnostic method for rapid detection of ORFV in the field and application in grassroots laboratories. This study establishes a DNA extraction-free, real-time, visual recombinase-aided amplification (RAA) method for the rapid detection of ORFV. This method is specific to ORFV and does not cross-react with other common DNA viruses. The detection limits of the real-time RAA and visual judgment of the RAA assay at 95% probability were 13 and 21 copies per reaction for ORFV, respectively. Compared with qPCR, the sensitivity and specificity of the real-time RAA assay were 100%, and those of the visual RAA assay were 92.31% and 100.0%, respectively. The DNA extraction-free visual detection method of RAA established in this study can meet the needs of rapid onsite detection and grassroots laboratories and has important reference value and significance for the early diagnosis of diseased animals.

Oncolytic Parapoxvirus induces Gasdermin E-mediated pyroptosis and activates antitumor immunity.

The advantage of oncolytic viruses (OV) in cancer therapy is their dual effect of directly killing tumours while prompting anti-tumour immune response. Oncolytic parapoxvirus ovis (ORFV) and other OVs are thought to induce apoptosis, but apoptosis, being the immunogenically inert compared to other types of cell death, does not explain the highly inflamed microenvironment in OV-challenged tumors. Here we show that ORFV and its recombinant therapeutic derivatives are able to trigger tumor cell pyroptosis via Gasdermin E (GSDME). This effect is especially prominent in GSDME-low tumor cells, in which ORFV-challenge pre-stabilizes GSDME by decreasing its ubiquitination and subsequently initiates pyroptosis. Consistently, GSDME depletion reduces the proportion of intratumoral cytotoxic T lymphocytes, pyroptotic cell death and the success of tumor ORFV virotherapy. In vivo, the OV preferentially accumulates in the tumour upon systemic delivery and elicits pyroptotic tumor killing. Consequentially, ORFV sensitizes immunologically 'cold' tumors to checkpoint blockade. This study thus highlights the critical role of GSDME-mediated pyroptosis in oncolytic ORFV-based antitumor immunity and identifies combinatorial cancer therapy strategies.

Natural flavonoid sinensetin inhibits cisplatin-induced pyroptosis and attenuates intestinal injury.

The demand of exploring strategies to enhance chemotherapy drug efficacy and alleviate adverse effects by using natural compounds is increasing. Sinensetin (SIN) is a kind of natural flavonoids with anti-inflammatory activities. However, its protective impact on chemotherapy-induced adverse effects has not been well demonstrated. Here, we found that SIN could inhibit Cisplatin-induced release of proinflammatory cellular contents and inflammatory cell death-pyroptosis. In addition, Cisplatin-induced activation of gasdermin E (GSDME), a critical mediator of chemotherapy-induced tissue injury, could also be reversed by SIN. Furthermore, SIN impaired Cisplatin-induced intracellular damages, including ROS release and DNA damages. Importantly, SIN was able to alleviate intestinal injury in Cisplatin-challenged mice, which was accompanied by the decrease of lytic cell death and immune cell infiltration. Of note, SIN administration did not reverse Cisplatin-caused tumor suppression in vivo. In conclusion, our result provides a potential application of SIN to reduce Cisplatin-caused adverse effects, without impairing its anti-tumor capacity.

Porcine Hemagglutinating Encephalomyelitis Virus Co-Opts Multivesicular-Derived Exosomes for Transmission.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the family Coronaviridae, genus Betacoronavirus, and subgenus Embecovirus that causes neurological disorders, vomiting and wasting disease (VWD), or influenza-like illness (ILI) in pigs. Exosomes regulate nearby or distant cells as a means of intercellular communication; however, whether they are involved in the transmission of viral reference materials during PHEV infection is unknown. Here, we collected exosomes derived from PHEV-infected neural cells (PHEV-exos) and validated their morphological, structural, and content characteristics. High-resolution mass spectrometry indicated that PHEV-exos carry a variety of cargoes, including host innate immunity sensors and viral ingredients. Furthermore, transwell analysis revealed that viral ingredients, such as proteins and RNA fragments, could be encapsulated in the exosomes of multivesicular bodies (MVBs) to nonpermissive microglia. Inhibition of exosome secretion could suppress PHEV infection. Therefore, we concluded that the mode of infectious transmission of PHEV is likely through a mixture of virus-modified exosomes and virions and that exosomal export acts as a host strategy to induce an innate response in replicating nonpermissive bystander cells free of immune system recognition. IMPORTANCE The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a large number of deaths worldwide. Clinical neurological complications have occurred in some cases; however, knowledge of the natural history of coronavirus in the central nervous system (CNS) is thus far limited. PHEV is a typical neurotropic betacoronavirus (ß-CoV) that propagates via neural circuits in the host CNS after peripheral incubation rather than through the bloodstream. It is therefore a good prototype pathogen to investigate the neuropathological pathogenesis of acute human coronavirus infection. In this study, we demonstrate a new association between host vesicle-based secretion and PHEV infection, showing that multivesicular-derived exosomes are one of the modes of infectious transmission and that they mediate the transfer of immunostimulatory cargo to uninfected neuroimmune cells. These findings provide novel insights into the treatment and monitoring of neurological consequences associated with ß-CoV, similar to those associated with SARS-CoV-2.

Parapoxvirus-based therapy eliminates SARS-CoV-2-loaded fine aerosol and blocks viral transmission in hamster models.

Currently, it is believed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an airborne virus, and virus-containing aerosol particles have been found concurrent with the onset of COVID-19, which may contribute to the noncontact transmission of SARS-CoV-2. Exploring agents to block SARS-CoV-2 transmission is of great importance to prevent the COVID-19 pandemic. In this study, we found that inactivated Parapoxvirus ovis (iORFV), a kind of immunomodulator, could compress the proportion of small particle aerosols exhaled by Syrian golden hamsters. Notably, the concentration of SARS-CoV-2 RNA-containing aerosol particles was significantly reduced by iORFV in the early stages after viral inoculation. Importantly, smaller aerosol particles (<4.7 µm) that carry infectious viruses were completely cleared by iORFV. Consistently, iORFV treatment completely blocked viral noncontact (aerosol) transmission. In summary, iORFV may become a repurposed agent for the prevention and control of COVID-19 by affecting viral aerosol exhalation and subsequent viral transmission.

The protection effect of rhodionin against methicillin-resistant Staphylococcus aureus-induced pneumonia through sortase A inhibition.

Methicillin-resistant Staphylococcus aureus (MRSA) is a zoonotic antibiotic-resistant pathogen that negatively impacts society from medical, veterinary, and societal standpoints. The search for alternative therapeutic strategies and innovative anti-infective agents is urgently needed. Among the pathogenic mechanisms of Staphylococcus aureus (S. aureus), sortase A is a virulence factor of great concern because it is highly linked with the ability of MRSA to invade the host. In this study, we identified that rhodionin, a natural compound of flavonoid glucosides, effectively inhibited the activity of SrtA without affecting the survival and growth of bacteria, and its half maximal inhibitory concentration (IC50) value was 22.85 µg/mL. In vitro, rhodionin prominently attenuated the virulence-related phenotype of SrtA by reducing the adhesion of S. aureus to fibrinogen, reducing the capacity of protein A (SpA) on the bacterial surface and biofilm formation. Subsequently, fluorescence quenching and molecular docking were performed to verify that rhodionin directly bonded to SrtA molecule with KA value of 6.22 × 105 L/mol. More importantly, rhodionin showed a significant protective effect on mice pneumonia model and improved the survival rate of mice. According to the above findings, rhodionin achieved efficacy in the treatment of MRSA-induced infections, which holds promising potential to be developed into a candidate used for MRSA-related infections.

Antiviral effect of lysosomotropic disaccharide trehalose on porcine hemagglutinating encephalomyelitis virus, a highly neurotropic betacoronavirus.

Many members of the genus Betacoronavirus are neurotropic viruses that frequently cause serious harm to humans or animals, including highly neurotropic porcine hemagglutinating encephalomyelitis virus (PHEV). Nevertheless, very few approved treatments exist to combat these viruses. Lysosomotropic trehalose, a widely used, nontoxic, natural disaccharide that can traverse the blood-brain barrier, has been proposed as a potential antiviral agent for use in prevention or treatment of betacoronavirus-associated infections. The purpose of this study was to determine if trehalose could inhibit PHEV infection of cells of a mouse central nervous system-derived neuroblastoma cell line in vitro or brain cells in vivo. Our results demonstrated that treatment of PHEV-infected mouse neuroblastoma cells and mice with trehalose reduced viral replication and that these trehalose antiviral effects were dependent on expression of lysosomal protein progranulin. Collectively, these results indicated that trehalose holds promise as a new antiviral agent for use in controlling neurotropic betacoronavirus infections.

Bavachin Suppresses Alpha-Hemolysin Expression and Protects Mice from Pneumonia Infection by Staphylococcus aureus.

Staphylococcus aureus (S. aureus) infection causes dramatic harm to human health as well as to livestock development. As an important virulence factor, alpha-hemolysin (hla) is critical in the process of S. aureus infection. In this report, we found that bavachin, a natural flavonoid, not only efficiently inhibited the hemolytic activity of hla, but was also capable of inhibiting it on transcriptional and translational levels. Moreover, further data revealed that bavachin had no neutralizing activity on hla, which did not affect the formation of hla heptamers and exhibited no effects on the hla thermal stability. In vitro assays showed that bavachin was able to reduce the S. aureus-induced damage of A549 cells. Thus, bavachin repressed the lethality of pneumonia infection, lung bacterial load and lung tissue inflammation in mice, providing potent protection to mice models in vivo. Our results indicated that bavachin has the potential for development as a candidate hla inhibitor against S. aureus.

Vesicular stomatitis virus sensitizes immunologically cold tumors to checkpoint blockade by inducing pyroptosis.

BACKGROUND: Traditionally, vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) are thought to kill tumors by inducing apoptosis. However, cell apoptosis leads to immune quiescence, which is incompatible with the ability of OVs to activate the antitumor immune microenvironment. Thus, studying OVs-mediated oncolytic mechanisms is of great importance for the clinical application of OVs. METHODS: We examined the pyroptosis in tumor cells and tissues by morphological observation, Lactate Dehydrogenase (LDH) assay, frozen section observation, and western-blotting techniques. The critical role of GSDME in VSV-induced pyroptosis was confirmed by CRISPR/Cas9 technique. VSV virotherapy-recruited cytotoxic lymphocytes in the tumors were examined by flow cytometry assay. VSV-activated antitumor immunity was further enhanced by the co-administration with anti-PD-1 antibody. RESULTS: Here, we observed that VSV was able to trigger tumor pyroptosis through Gasdermin E (GSDME) in tumor cells, human tumor samples, and tumor-bearing mouse models. Importantly, the effectiveness of VSV-based virotherapy is highly dependent on GSDME, as depletion of GSDME not only reverses VSV-induced tumor-suppressive effects but also diminishes the ability of VSV to activate antitumor immunity. Notably, VSV treatment makes immunologically 'cold' tumors more sensitive to checkpoint blockade. CONCLUSIONS: Oncolytic VSV induces tumor cell pyroptosis by activating GSDME. GSDME is critical in recruiting cytotoxic T lymphocytes in the context of VSV therapy, which can switch immunologically 'cold' tumors into 'hot' and enhance immune checkpoint therapy efficacy.

Epigenetic regulation of cutaneous T-cell lymphoma is mediated by dysregulated lncRNA MALAT1 through modulation of tumor microenvironment.

Cutaneous T-Cell Lymphoma (CTCL) is a rare non-Hodgkin lymphoma marked by migration of T-lymphocytes to the skin. It has many subtypes some of which are aggressive with documented metastasis. We investigated a possible role of lncRNA MALAT1 in CTCL cells because of its documented involvement in cancer metastasis. A screening of MALAT1 in CTCL patients revealed its elevated levels in the patients, compared to healthy individuals. For our investigation, we employed HH and H9 CTCL cells and silenced MALAT1 to understand the MALAT1 mediated functions. Such silencing of MALAT1 resulted in reversal of EMT and inhibition of cancer stem cell phenotype, along with reduced cell growth and proliferation. EMT reversal was established through increased E-cadherin and reduced N-cadherin while inhibition of cancer stem cell phenotype was evident through reduced Sox2 and Nanog. CTCL patients had higher circulating levels of IL-6, IL-8, IL-10, TGFß, PGE2 and MMP7 which are factors released by tumor-associated macrophages in tumor microenvironment. MALAT1 sponged miR-124 as this tumor suppressive miRNA was de-repressed upon MALAT1 silencing. Moreover, downregulation of miR-124 attenuated MALAT1 silencing effects. Our study provides a rationale for further studies focused on an evaluation of MALAT1-miR-124 in CTCL progression.

Evaluation of the Immune Response Afforded by Combined Immunization with Orf Virus DNA and Subunit Vaccine in Mice.

Contagious ecthyma (Orf) is a highly contagious disease caused by Orf virus (ORFV) infection. Orf is prevalent all over the world and, not only affects the healthy development of sheep husbandry, but also threatens human health. However, there are no safe and effective vaccines or drugs for the prevention and treatment of Orf at present. In this study, we constructed a DNA plasmid expressing ORFV B2L and F1L genes as a DNA vaccine candidate, with purified B2L full-length protein and F1L truncated protein as subunit vaccine candidates. BALB/c mice were immunized with the DNA vaccine, subunit vaccine, as well as DNA prime-protein boost strategies. The results showed that compared with the DNA vaccine and subunit vaccine alone, the DNA prime-protein boost immunization group had a higher level of specific antibodies, stronger lymphocyte proliferation, and higher expression of cytokines such as IL-2, IL-4, IL-6, IFN-γ, and TNF-α, which are considered to cause a Th1/Th2 mixed cytokine response. Our results demonstrated that the DNA prime-protein boost immunization strategy induced stronger humoral and cellular immune responses, which have potential advantages in preventing ORFV 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.

Hibifolin, a Natural Sortase A Inhibitor, Attenuates the Pathogenicity of Staphylococcus aureus and Enhances the Antibacterial Activity of Cefotaxime.

This study aimed to identify hibifolin as a sortase A (SrtA) inhibitor and to determine whether it could attenuate the virulence of methicillin-resistant Staphylococcus aureus (MRSA). We employed a fluorescence resonance energy transfer (FRET) assay to screen a library of natural molecules to identify compounds that inhibit SrtA activity. Fluorescence quenching assay and molecular docking were performed to verify the direct binding interaction between SrtA and hibifolin. The pneumonia model was established using C57BL/6J mice by MRAS nasal administration for evaluating the effect of hibifolin on the pathogenicity of MRSA. Herein, we found that hibifolin was able to inhibit SrtA activity with an IC50 of 31.20 µg/mL. Further assays showed that the capacity of adhesion of bacteria to the host cells and biofilm formation was decreased in hibifolin-treated USA300. Results obtained from fluorescence quenching assay and molecular docking indicated that hibifolin was capable of targeting SrtA protein directly. This interaction was further confirmed by the finding that the inhibition activities of hibifolin on mutant SrtA were substantially reduced after mutating the binding sites (TRP-194, ALA-104, THR-180, ARG-197, ASN-114). The in vivo study showed that hibifolin in combination with cefotaxime protected mice from USA300 infection-induced pneumonia, which was more potent than cefotaxime alone, and no significant cytotoxicity of hibifolin was observed. Taken together, we identified that hibifolin attenuated the pathogenicity of S. aureus by directly targeting SrtA, which may be utilized in the future as adjuvant therapy for S. aureus infections. IMPORTANCE We identified hibifolin as a sortase A (SrtA) inhibitor by screening the natural compounds library, which effectively inhibited the activity of SrtA with an IC50 value of 31.20 µg/mL. Hibifolin attenuated the pathogenic behavior of Staphylococcus aureus, including adhesion, invasion, and biofilm formation. Binding assays showed that hibifolin bound to SrtA protein directly. Hibifolin improved the survival of pneumonia induced by S. aureus USA300 in mice and alleviated the pathological damage. Moreover, hibifolin showed a synergistic antibacterial effect with cefotaxime in USA300-infected mice.