Biochanin A as an α-hemolysin inhibitor for combating methicillin-resistant Staphylococcus aureus infection

World Journal of Microbiology and Biotechnology https://doi.org/10.1007/s11274-021-03182-4 (2022) 38:6 ORIGINAL PAPER Biochanin A as an α‑hemolysin inhibitor for combating methicillin‑resistant Staphylococcus aureus infection Jiaxuan Feng1 · Dazhong Sun3 · Li Wang1 · Xueting Li1 · Jiyu Guan4 · Lin Wei1 · Donghui Yue1 · Xingye Wang1 · Yicheng Zhao1 · Haimiao Yang2 · Wu Song1 · Bingmei Wang1 Received: 9 September 2021 / Accepted: 28 October 2021 © The Author(s), under exclusive licence to Springer Nature B.V. 2021 Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. In S. aureus, α-hemolysin is an important virulence factor as it contributes to the capacity of the bacteria to infect the host. Here, we showed that biochanin A (bioA), an isoflavone present in red clover, cabbage and alfalfa, effectively inhibited hemolytic activity at a dose as low as 32 μg/mL. Further, western blot and RT-qPCR data showed that bioA reduced the production and expression of MRSA hemolysin in a dose-dependent manner. In addition, when different concentrations of bioA were added to a coculture system of A549 cells and S. aureus, it could significantly decrease cell injury. Importantly, the in vivo study showed that bioA could protect mice from pneumonia caused by a lethal dose of MRSA, as evidenced by improving their survival and reducing the number of bacterial colonies in lung tissues, the secretion of hemolysin into alveolar lavage fluid and the degree of pulmonary edema. In conclusion, biochanin A protected the host from MRSA infection by inhibiting the expression of the hemolysin of MRSA, which may provide experimental evidence for its development to a potential anti-MRSA drug. Keywords Methicillin-resistant Staphylococcus aureus · Biochanin A · Antivirulence · α-Hemolysin Introduction Staphylococcus aureus (S. aureus) is a seriously dangerous human and animal pathogen that cause endocarditis, skin and soft tissue infections, bacteremia and pneumonia (Kyaw Jiaxuan Feng and Dazhong Sun have contributed equally to this work. * Wu Song * Bingmei Wang 1 Changchun University of Chinese Medicine, Changchun 130117, China 2 Affiliated Hospital to Changchun University of Chinese Medicine, Changchun 130021, China 3 First School of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China 4 Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, China et al. 2015; McColl et al. 1998; Ni et al. 2018). The difficulty of treating S. aureus infection is due to the emergence of antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to most antibiotics currently used (Fridman et al. 2014; Levin-Reisman et al. 2017; Mechler et al. 2015). The World Health Organization (WHO) identified MRSA as a high-priority pathogen (Shrivastava et al. 2018). To cope with the increasingly problematic emergence of antibiotic resistance and the postantibiotic era (Dickey et al. 2017), novel strategies against MRSA are urgently needed. Pathogens express a variety of virulence factors, which affect the host by destroying the relationship between the host immune system and pathogens. In addition, inhibiting the virulence of pathogens will not place selection pressure on the host, which dramatically reduces the probability of drug resistance. Therefore, screening of antivirulence inhibitors is an ideal strategy to control multidrug resistant pathogens (Casadevall and Pirofski 1999). S. aureus can produce a large number of cytotoxic molecules, including four hemolysins (α, β, δ and γ). Among them, alpha-hemolysin (Hla) is a pore-forming exotoxin 13 Vol.:(0123456789) 6 Page 2 of 10 that has been widely studied. Hla, a 33 kDa polypeptide, is secreted by 95% of S. aureus (2019; Grumann et al. 2014). One of the remarkable characteristics of hemolysin is its ability to dissolve red blood cells. Hla has been classified as a phenol soluble regulatory protein (PSM), and its hemolytic activity is not dependent on any receptor (Fritz et al. 2013; Kolata et al. 2011). As a water-soluble monomer, it can integrate into the cell membrane to form a transmembrane oligomer β-barrel heptamer channel, leading to host cell lysis or death (Dinges et al. 2000; Wu et al. 2019b). Previous studies have shown that the Hla mutant of S. aureus has significantly reduced virulence compared with the wildtype strain (Tran et al. 2020). Hla has hemolytic, cytotoxic, dermonecrotic, and other lethal properties and is considered an essential virulence target against S. aureus infection. Natural products from plants and microorganisms are the main sources of antivirulence inhibitors because of their extensive physiological activities and environmental friendliness. A number of natural products have been reported to protect the host against S. aureus by inhibiting the production (Jiang et al. 2016; Teng et al. 2017) and self-assembly of Hla (Dong et al. 2013; Marathe et al. 2012). Biochanin A (4′-methoxy-5,7-dihydroxy isoflavone, bioA) (Fig. 1A) is a bioactive isoflavone found in diverse plants (Sarkar et al. 2006). It has been proven to produce marked anti-inflammatory (Kole et al. 2011), antioxidant (Derangula et al. 2020) and antitumor effects (Jain et al. 2015). In addition, bioA has been reported to inhibit the efflux pump of MRSA (Dan et al. 2014) and can be used as an adjuvant in combination with ciprofloxacin against clinical isolates of S. aureus (Liu et al. 2011), while its effect on S. aureus-related virulence factors has not been reported. In our study, a concentration of bioA at 32 μg/mL almost completely inhibited hemolysin activity. In addition, bioA also significantly inhibited the hemolytic World Journal of Microbiology and Biotechnology (2022) 38:6 activity of MRSA clinical isolates. Subsequently, the protective effect of bioA on S. aureus pulmonary infection was confirmed by establishing a mice pneumonia model. In conclusion, our results showed that bioA effectively inhibit the virulence of S. aureus Hla in vivo and in vitro, indicating it should be further explored as a drug against MRSA infection. Materials and methods Bacterial strains, reagents and growth conditions S. aureus Newman and S. aureus BAA-1717 (USA 300) were purchased from American Type Culture Collection (ATCC, Manassas, V A, United States). Clinically isolated MRSA strain SA28 was obtained within the past 3 years and was identified by 16S RNA and quality control. BioA was obtained from the Rui Fensi Biological Company (Chengdu, China). BioA was dissolved in dimethyl sulfoxide (DMSO, Sigma, United States) to obtain a master stock (20 mg/mL) for standby application. Other chemical reagents were provided by Sangon Biotech (Shanghai, China). S. aureus was grown in trypticase soy broth (TSB, Hopebio, Qingdao, China) in an (...truncated)