| Bacterial vaginosis(BV)is a common disease in obstetrics and gynecology,which is prevalent among women of childbearing age worldwide,seriously threatening women’s reproductive health.BV usually manifests as a sharp decline in probiotics such as Lactobacillus vaginalis,while anaerobic bacteria such as Gardnerella vaginalis(GV)overmultiply and dominate.G.vaginalis can not only secrete a variety of virulence factor to damage germ cell and tissues,but also adhere to vaginal epithelial cells to form a biofilm,increasing the ability to resist the body’s immune system.The clinical treatment of BV mainly uses antibiotics,However,the widespread occurrence of postoperative recurrence has shown that the effectiveness of antibiotic treatment is not ideal.At present,it is believed that the presence of biofilms is the main reason for the recurrence and prolonged treatment of BV.This study found that extracellular protease LasA derived from Pseudomonas aeruginosa can effectively disrupt the biofilm of G.vaginalis and lyse its cell wall,which has good prospects for the treatment of recurrent BV.The main achievements are as follows:1.LasA can effectively disintegrate the G.vaginalis biofilmUnder atomic force microscope,it was observed that the proportion of protein in the extracellular polymer of G.vaginalis was higher than that of polysaccharide,and it was found that protease LasA had a destructive effect on its biofilm,and 0.02 mg/mL LasA could reach 85%of the disintegration rate of G.vaginalis biofilm.In contrast,lysozyme and antibiotics have the highest biofilm clearance effect on G.vaginalis,which is only about 10%.LasA acts on the biofilm,and its substrate may be a structural protein in the biofilm.Several possible structural protein components were screened from extracellular proteins of G.vaginalis and digested for validation.It was found that some proteins,such as the CHAP domain containing protein,can indeed be digested by LasA and can be the substrate of LasA biofilm.2.LasA can lyse the cell wall of G.vaginalisThe cell lysis rate of LasA above 0.01 mg/mL can reach 80%within 180 min.In contrast,lysozymes that can cleave cell walls have no effect on G.vaginalis.The combination of lysozyme and LasA can actually affect the effectiveness of LasA.Lactobacillus crispatus is a common probiotic in the reproductive tract of Asian women,and LasA does not have a killing effect on L.crispatus isolated from clinical BV patients.3.LasA has ideal therapeutic effects in animal modelsMice were infected with clinical isolates of G.vaginalis JNFY14 and model strain ATCC1401 9,and treated with LasA and antibiotics,respectively.Compared to antibiotics,the number of G.vaginalis decreased significantly after LasA treatment,while the number of probiotics decreased slightly.The natural susceptible host fox of G.vaginalis was screened and treated.The results of secretion smear and qPCR showed that LasA had a better therapeutic effect than antibiotics,indicating that LasA had good prospects for treating BV.4.Determination of LasA protein properties and optimization of purification methodThe heterologous expression and purification of LasA through E.coli obtained a protein with high purity and good stability.The determination of its optimal pH,Tm value and other enzymatic properties provides a reference for future applications.We purified LasA in the endotoxin deficient E.coli host ClearColi BL21(DE3)and tried to directly purify its mature catalytic domain.The protein yield purified in ClearColi BL21 was twice that in E.coli BL21,and the enzyme activity was stable.The yield of the mature catalytic domain directly expressed LasA was halved and the enzyme activity was lost,providing guidance for future large-scale fermentation.In summary,this article screened a protease LasA that can damage the biofilm of G.vaginalis.If it can be applied,it is of great significance for improving the treatment effect of BV. |
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