Structural And Functional Studies On Glycoside Hydrolase From Gardnerella Vaginalis

Bacterial vaginosis(BV)is a disease that seriously affects globe women's reproductive health.It can increase the risk of many diseases,including HIV infection and transmission,pelvic inflammation,cervical cancer,preterm and urethral infection.Gardnerella vaginalis plays an important role in this process.Studies related have shown that Gardnerella vaginalis not only possesses specific virulence factors(such as vaginal hemolysin,adhesion factor and drug efflux system),but also is likely to be the "culprit" of BV.As a conditional pathogen,Gardnerella vaginalis probably firstly colonized vaginal epithelial cells and formed biofilm,providing invasion for other related anaerobic pathogens.This function is similar to scaffolding,thus forming a biofilm system coexisting with other bacteria.Because of the biofilm,BV is famous for its stubborn,recurrent and difficult to cure.On the other hand,bacteria in biofilm are often facing the problem of nutrition deficiency.Carbonhydrate,as the main energy source,is not only the "food" for most organisms to survive,but also the important component of biofilm(extracellular polysaccharides).However,there is almost no study on glycoside hydrolase from Gardnerella vaginalis.ADP38981.1 is a member of glycoside hydrolase 20 family(GH20)from Gardnerella vaginalis spp.It has the property of beta-N-acetylhexosaminase and is probably an important virulence factor in animal pathogens.ADP38499.1 is another glycoside hydrolase from Gardnerella vaginalis spp.belonging to the G5 family.Domain prediction indicates that it is probably a protein that plays adhesive role in the formation of biofilm.In addition,it has a lysozyme-like domain,indicating that it may have the function of degrading extracellular polysaccharides to provide carbon source.In addition,by aligning in PDB,we found these two glycoside hydrolases not only have no structure,but also have low homology with the known structure proteins in the database.More importantly,there is no relevant research on the glycoside hydrolase of Gardnerella.Therefore,it is very meaningf?l to select them as the research object.With the methods of molecular biology and structural biology,the above two glycoside hydrolases were analyzed by expression and purification.crystallizaiton,X-ray diffraction.enzymatic assay and biofilm degradation in vitro to reveal the properties and pathogenic mechanism of related proteins from molecular level and function,and find the relevant drug targets for the theoretical basis.In this paper,we have obtained the following preliminary results:1.By constructing prokaryotic expression system.we have obtained the related proteins with high expression and purity;2.Through the screening and optimization of protein crystals,we have obtained the native protein and SeMet protein crystals of ADP38981.1.and obtained high-resolution crystal diffraction data at Shanghai Synchrotron Radiation Facililty(SSRF).The highest resolution of the native protein crystal is about 3.2A.3.Through enzymatic activity test and biofilm degradation in vitro,we preliminarily know the related characteristics of ADP38981.1 and ADP38499.1,including some enzymatic parameters and biotilm degradation effect.The shortcoming of this paper is that no ADP38499.1 protein crystal has been obtained,and because the diffraction quality of ADP38981.1 protein crystal is poor,we cannot obtain high resolution protein structure finaly.