Effect And Mechanism Of Engineered Phage On Chlamydia Trachomatis

BackgroundBacterial sexually transmitted disease caused by Chlamydia trachomatis is an important public health problem in the world today.The incidence of urogenital Chlamydia trachomatis infection continues to rise as other sexually transmitted diseases are gradually controlled.In view of the increasing number of cases of treatment failure due to Chlamydia trachomatis resistance,antibiotics have been unable to control its development,and we urgently need a strong treatment to improve the status of Chlamydia trachomatis infection and prevalence.Chlamydia phage is recognized as the best choice for solving this problem.Objective1.To obtain recombinant bacteriophages with high inhibitory or destructive effects on Chlamydia trachomatis,which are specific and safe.It lays a foundation for providing a new precise treatment method for chlamydia trachomatis infection in clinical practice.2.To preliminarily explore the possible mechanism of action of recombinant phage on Chlamydia trachomatis.Methods1.The effect of recombinant M13-RGD-IN5 phage on Chlamydia trachomatis and its mechanism:?1?Recombination of phage M13-RGD-IN5,M13-RGD,M13-IN5 by genetic engineering,ie,integrin RGD and Chlamydia phage PhiCPG1 IN5 was inserted into the major coat protein pVIII and the minor coat protein pIII of M13 phage,respectively;?2?The recombinant M13 phage was purified and the titer of each recombinant M13 phage was tested by plaque assay;?3?CCK-8 assay was used to detect the cytotoxicity of recombinant M13 phage against Hela cells and to screen out the optimal concentration of recombinant phage;?4?Chlamydia trachomatis?CT?and chlamydia conjunctivitis in guinea pigs?GPIC?were treated with different recombinant M13 phage.The effects of recombinant phage on Hela cells,Chlamydia trachomatis and chlamydia conjunctivitis in guinea pigs were observed by immunofluorescence assay;?5?Observation of the localization of recombinant M13-RGD-IN5 phage in Hela cells infected with Chlamydia trachomatis by confocal microscopy;?6?The ultrastructural changes of Chlamydia trachomatis infected by recombinant M13-RGD-IN5 phage for 36 hours were observed by transmission electron microscopy;?7?Polyclonal antibody against IN5 was used to detect the specificity of recombinant M13-RGD-IN5 on Chlamydia trachomatis;?8?Verification of whether the recombinant phage can proliferate in Chlamydia trachomatis by QPCR.2.Effect and mechanism of recombinant pGFP-PhiCPG1 bacteriophage on Chlamydia trachomatis:?1?Construction of recombinant pGFP-PhiCPG1 plasmid by genetic engineering;?2?CaCl2 method was used to transform the recombinant plasmid into the elementary body?EB?of chlamydia conjunctivitis in guinea pigs;?3?The recombinant pGFP-PhiCPG1 phage was amplified by passage,and the copy number of each generation of recombinant pGFP-PhiCPG1 phage was quantified by QPCR;?4?The recombinant pGFP-PhiCPG1 phage was purified and quantified by QPCR;?5?CCK-8 assay was used to detect the cytotoxicity of recombinant pGFP-PhiCPG1 phage to Hela cells and screen out the optimal concentration of intervention;?6?The effect of different concentrations of recombinant pGFP-PhiCPG1 on Chlamydia trachomatis and chlamydia conjunctivitis in guinea pigs was observed by immunofluorescence assay;?7?The effect of recombinant pGFP-PhiCPG1 phage at the optimal intervention concentration on Chlamydia trachomatis and chlamydia conjunctivitis in guinea pigs was observed by immunofluorescence at different time periods after infection;?8?The ultrastructural changes of Chlamydia trachomatis and chlamydia conjunctivitis in guinea pigs infected by recombinant pGFP-PhiCPG1 phage for 36 hours were observed by transmission electron microscopy.Results1.Recombinant M13-RGD-IN5 phage:?1?The recombinant phages M13-RGD-IN5,M13-RGD and M13-IN5 were successfully constructed;?2?According to CCK-8test results,it was proved that the titer of recombinant M13 phage was less than or equal to 10¹⁰ pfu/mL without obvious cytotoxicity to Hela cells;?3?The insertion of RGD enhances the endocytosis of cells in a dose-dependent manner;the IN5 insertion did not affect the uptake of recombinant M13-RGD-IN5 phage by Hela cells;?4?The inhibitory effect of recombinant M13-RGD-IN5 phage on Chlamydia trachomatis was dose-dependent,and 10⁹ pfu/ml was selected as the optimal intervention concentration;?5?The recombinant phage significantly reduced the infection of Chlamydia trachomatis and chlamydia conjunctivitis in guinea pigs,with inhibition rates of 69.05%and 77.06%,respectively;?6?Recombinant M13-RGD-IN5 phage can penetrate the cell mucosal barrier and inclusion body membrane into the inclusion bodies formed by Chlamydia trachomatis in host cells,and this phenomenon does not occur in the recombinant M13-RGD phage;?7?If the recombinant M13-RGD-IN5 phage is incubated with the polyclonal antibody against IN5 before infection with Chlamydia trachomatis,it will not change the infection process of Chlamydia trachomatis;?8?Transmission electron microscopy showed that recombinant M13-RGD-IN5 phage affects the normal transformation of elementary body?EB?to reticulate body?RB?,resulting in abnormally enlarged reticulate body?mRB?to inhibit EB production;?9?QPCR results showed that the recombinant M13-RGD-IN5 phage did not proliferate in Chlamydia trachomatis and Hela cells,and observed strict host restriction.2.Recombinant pGFP-PhiCPG1 phage:?1?The recombinant pGFP-PhiCPG1phage was successfully constructed;?2?pGFP insertion did not alter the packaging or infectivity of PhiCPG1;?3?Recombinant pGFP-PhiCPG1 phage was readily able to infect Chlamydia trachomatis and chlamydia conjunctivitis in guinea pigs;?4?According to CCK-8 test results,it was proved that the titer of recombinant pGFP-PhiCPG1 phage was less than 10¹⁶v.g/mL without obvious cytotoxicity to Hela cells;?5?The recombinant pGFP-PhiCPG1 phage could inhibit CT and GPIC in a dose-dependent fashion;?6?This inhibition was most pronounced during the mid and late stages of the CT infection,disrupting the reticular body?RB?to EB transition,leading to the formation of enlarged RBs.conclusion1.The recombinant M13 phage that can stably express the integrin-binding protein RGD and the capsid protein IN5 of chlamydiaphages can overcome the barrier of the cell membrane and the mucous membrane,and it has a significant inhibitory effect on the growth of CT without affecting its own protein folding.2.The growth and infectious characteristics of the recombinant pGFP-PhiCPG1phage were comparable to those of the Chlamydia bacteriophage PhiCPG1,indicating that this recombinant phage is suitable for use in experimental studies in place of PhiCPG1;The pGFP-PhiCPG1 phage was able to readily infect CT cells and significantly inhibit their growth and proliferation.3.The mechanism of recombinant phage M13-RGD-IN5 inhibiting chlamydia trachomatis infection may be the destruction triggered by signal pathway.The mechanism of recombinant phage pGFP-phiCPG1 inhibiting the infection of Chlamydia trachomatis is not only the mechanical damage caused by the proliferation of phages,but also the destruction triggered by signal pathways.4.The overall results suggest that Chlamydia trachomatis maybe suitable host for chlamydiaphage PhiCPG1 or that chlamydia trachomatis may have its own phage and is closely related to chlamydia phage PhiCPG1.As such our study provides a theoretical foundation for the identification and characterization of phages specific for Chlamydia trachomatis.