The Inhibitory Effect Of Isorhamnetin On Staphylococcus Aureus ?-Hemolysin And Sortase A

Staphylococcus aureus,(S. aureus), a clinically common gram positive bacterium, is widely distributed in nature due to its low requirements for nutrients and high environmental adaptability. This bacterium could be detected by more than50% of the healthy population, mainly in the groin, perineal region, neonatal umbilical cord, the mouth pharynx, intestinal and other parts of the body. Although most of the S. aureus is not pathogenic, some nubers of S. aureus can cause a variety of deadly diseases in human and animal, such as endocarditis, pneumonia and meningitis. S. aureus has become a leading cause for hospital infection and the isolation of this bacteira is increasing year by year, which increased the burden for healthacre and also caused a potential threat to the public health. Antibiotics are still the mainly agents for the infection cuased by S. aureus. However, the inceasing emergence of drug resistance for S. aureus(including methicillin-resistant S. aureus(MRSA) and vancomycin-resistant S. aureus) due to the abuse or long-term use of antibiotics further complicated the treatment of S. aureus infection. Additionally, the formation of biofilm increases the resistance to harsh environment, furhter worsening the treatment of S. aureus infection. Thus, there is an urgent need for new agents or anti-infective strategies against S. aureus infection.Recently researches have showed that the virulence factors, such as hemolysin,Sortase A, enterotoxin and TSST-1, synthesized by S. aureus have played an important role in the pahthogenecity of S. aureus. The surface proteins anchored by Sortase A facilitate the adhesion, invasion and the formation of biofilm of S. aureus.Following these steps, the secreted ?-hemolysin, a pore-forming toxin, could lyses most host cells and is indispensable in many animal models of S. aureus infection.Therefore, identification of inhibitors of virulence factors according to the functionof protein from traditional Chinese medicine has certain social significance and potential economic value. Since the anti-virulence strategy would put less selection pressure on targeted bacteria and not easy to induce the occurrence and development of drug resistance. Furtermore, anti-virulence drugs can be combined with existing antibiotics would reduce the use of antibiotics and prolong the service life of antibiotics.In this study, the hemolytic activity of bacterial culture supernatant was significantly decreased after co-cultured with isorhamnetin(2-16 ?g/m L) using hemolysis assay. The expression of ?-hemolysin was remarkably attenuated after treated with isorhamnetin. However, the MIC values of isorhamnetin for S. aureus strains tested in this study were all >1024 ?g/m L. Furthermore, the growth of S.aureus was not visibly affected by the addition of increasing sub-inhibitory concentrations of isorhamnetin, indicating that isorhamnetin, as an agent, would put less pressure on S. aureus. Treatment with sub-inhibitory concentrations of isorhamnetin led to inhibition, also in a dose-dependent manner, of the transcriptional levels for both the hla, the gene encoding ?-hemolysin, and RNAIII genes, the effective molecule of the agr two-component system positively regulating the transcription of hla. Treatment with 16 ?g/m L isorhamnetin provided robust protection against S. aureus-induced cell injury. In addition, the catalytic activity of Sortase A was also significantly decreased in the presence of isorhamnetin by sortase activity inhibition assay. Significantly, treatment of S. aureus with the oligopeptide resulted in decreased adherence to the fibronectin coating on the well bottom,biofilm formation and invasion of S. aureus into J774 cells. Consistent with the in vitro results, treatment with isorhamnetin provided a robust protection against both mouse model of S. aureus pneumonia or systemic infection.Taken together, our results establisehd that isorhamnetin, a natrual compound without anti-S. aureus acitivitiy, could simultaneously inhibiting ?-hemolysin production and directly neutralizing Sortase A activity. The virulence of S. aureus was both decreased in the presence of isorhamnetin. Additionally, Srt A is highlyconserved among the members of its familiy. Thus, anti-infective therapy with a Srt A inhibitor, isorhamnetin, may be broadly used to combat infections with gram-positive bacteria. Thus, it is potential that isorhamnetin to become a new anti-infective agent against S. aureus or other gram-positive bacteria with novel mechanism.