Analysis Of Correlation Between Mobiluncus Curtisii And Bacterial Vaginosis And The Functional And Structural Study Of Mobiluncus Curtisii CAMP Factor

Bacterial Vaginosis(BV)is a common gynecological disease with a variety of clinical symptoms caused by abnormal proliferation of anaerobic bacteria which replaces Lactobacillus vaginalis.BV not only discomforts patients,but also increases the risk of infections such as HPV,HIV and other sexually transmitted diseases.It also causes pelvic inflammatory disease,vaginal bleeding and premature delivery during pregnancy.The pH of the normal vagina is between 3.8and 4.2,while the vaginal pH of patients with BV rises to 4.5 to 6.At present,in order to kill abnormal proliferating bacteria in the vagina,the main treatment strategy of BV is oral and intravaginal use of antibiotics such as metronidazole.However,approximately 10%to 15%of patients did not receive a cure after the first course of antibiotic treatment,and the recurrence rate of BV was 80%.Therefore,studying the pathogenesis of BV provides a basis for clinical development of better drugs,which has high application value.Mobiluncus curtisii(M.curtisii)is a Gram-staining variable anaerobic bacterium that is resistant to the most commonly used therapeutic drug metronidazole.It has been reported abroad that M.curtisii was found in the vagina of 65%-85%of BV patients,and they confirmed that the presence of M.curtisii was highly correlated with recurrence after BV treatment.Until now,no data about the relationship between M.curtisii and BV was reported in China.A research declared that M.curtisii medium supernatant can destroy vaginal epithelial cells,which means that M.curtisii may participate in the development of BV by secreting cytotoxin,but the specific ingredient is not clear yet.CAMP factor was first discovered in Group B streptococcus(GBS).It is a cytotoxin which can form pore in the cell membrane then cause cell death.According to homology analysis,M.curtisii can also secret CAMP factor,but several questions are still not clear.Can M.curtisii CAMP cause cell death as GBS CAMP?What's the relationship between BV and M.curtisii CAMP factor?Furthermore,what's the molecular mechanism of the function of M.curtisii CAMP factor?In order to figure out the above questions,this subject has carried out the following research and obtained the following main results:(1)To analyze the infection status of M.curtisii in BV patients,we collected 257 vaginal swabs from the hospital,genomic DNA of the bacteria on the swabs were extracted and used as template,PCR was performed using M.curtisii specific primers.The positive ratio of M.curtisii of healthy woman was 5.7%,which in mild BV patients was 12.3%,and in patients with severe BV was 81.2%.These results is consistent with the foreign researchs,and indicate that M.curtisii infection in Chinese BV patients is also positively correlated the degree of disease.(2)In order to explore the molecular mechanism of M.curtisii,we found that M.curtisii can also express a kind of cytotoxin which called CAMP factor.We expressed and purified the recombinant protein of M.curtisii CAMP factor,and found that the oligomerization of this protein is pH dependent.(3)In order to explore the effect of pH on M.curtisii CAMP factor,size exclusion chromatography was performed,M.curtisii CAMP factor was confirmed to be monomer at pH 4.4,and it gradually formed polymer when increasing pH.To further explore the effect of pH conditions on the function of M.curtisii CAMP factor,the hemolysis assay was carried out,results showed that the M.curtisii CAMP factor has the activity to lysis sheep red blood cells at pH 5.7?7.4,which indicates that CAMP factor can function in BV vagina with relative high pH.(4)To reveal the mechanism of M.curtisii CAMP factor,980of crystallization conditions were screened,and monomeric CAMP factor crystal was finally obtained by optimization.The M.curtisii CAMP factor crystal diffraction data was obtained by X-ray diffraction,and a 1.85(?)crystal structure which is a pattern of 8?-helix composed of 5+3 mode was successfully refined.(5)The result of circular dichroism(CD)showed that CAMP factor has two energy barriers.Then we purified the N terminal domain(NTD)and C terminal domain(CTD)respectively,the CD results reflected that the NTD is more stable than CTD.(6)To further verifing the results above,we use engineered disulfide bonds which introduced two cystein residue into NTD or CTD of CAMP WT(called N_mut or C_mut,respectively).The CD results showed that the structure of N_mut and C_mut are stable.Further than that,hemolysis experiments showed that the introduction of disulfide bonds at the NTD would destroy its hemolysis function.Conversely,the hemolysis function would restored when the disulfide is reduced.Interestingly,when introducing disulfide bonds at CTD would increased the hemolysis ability.These results disclosed that NTD plays an important role in its function and stabilized CTD is helpful for CAMP function.In summary,our results show that the severity of Chinese BV patients is positively associated with M.curtisii infection.In addition,we found that the M.curtisii CAMP factor can destroy the cell membrane.We successfully solved the first crystal structure of monomer M.curtisii CAMP factor,which laid the foundation for further analysis of its mechanism of cytotoxicity.So we hypothesize that M.curtisii CAMP factor is able to exert its biological functions in vaginal environment of BV,then destroy vaginal epithelial cells,thereby promoting the the development of BV.This study may provide theoretical guidance for clinical treatment of BV and related gynecological diseases in the future.