Research On The Correlation Of Genital Chlamydia Trachomatis Infection And Autophagy In Hela Cells

BackgroundC.trachomatis(CT)infection in the genital tract is one of the most common sexually transmitted diseases.CT is a pathogen that is strictly parasitic in cells while Autophagy is one of the common mechanisms for cells to resist pathogen infection.Previous studies have shown that different chlamydia infection macrophages or non-phagocytic cells cause different effects of autophagy.Reproductive tract CT(D-K type)is susceptible to human reproductive tract epithelial cells,and the relationship between its infection and autophagy has not been reported.This article aimed to explore the characteristics of autophagy caused by CT infection of human cervical cancer epithelial cells(Hela cells)in order to lay a foundation for in-depth researches.Methods and Materials1.Hela cells were cultured and infected with mRFP-GFP-LC3 lentivirus to construct dual-fluorescent Hela cells.They were infected with CT and then placed in a living cell workstation for continuous culturing and fluorescence shooting.2.Infected Hela cells with Chlamydia trachomatis D type,uninfected cells as a control,and collected the cells in the early(6h),middle(12h),and late(24-48h)stages of infection.The autophagy proteins LC3 ?/? and p62 were detected by Western Blot.Immunofluorescence staining was used to observe CT growth and intracellular LC3 marked autophagosomes and Lamp2 marked lysosomes.3.Data analysisImage J software was used to analyze integrated density of the protein bands,and so were the fluorescence indensity of the immunofluorescence stained cells.SPSS 20.0 statistical software was used to compare the differences.Prism GraphPad 8.0 was used for graphing.Results1.We successfully constructed the infection model of CT,and so were the lentiviral Hela cells that expressing mRFP-GFP-LC3 double-fluorescence.2.During the early and middle infecting stages(6h and 12h),there was no significant difference of LC3B ?/? and p62 protein between the CT infected group and the negative control group.Also,there was no significant difference between the two groups when comparing the intracellular LC3 punctas both in immunofluorescence and double-fluorescence Hela cells.However,there were differences of the CT infection in each cell.The average number of infected CT in high autophagy cells(intracellular LC3 is higher than average)was 4.83 per cell,and 8.23 per cell in the low autophagy cells(intracellular LC3 is lower than average),with significant differences(t-test,p<0.05).LC3 was negatively correlated with the number of CT infected in each cell(r=-0.3342,p<0.01).The cells were pre-treated with autophagy drugs and then infected with CT.The results were:the infection rate of the control group(DMSO)was 66.8%,the Rapamycin group was 57.8%,and the BafA1 group was 72.2%.One-way ANOVA,p<0.05.3.In the late stage(24h and 48h),LC3 ?/? and p62 of the CT group was significantly higher than those of the Control group.Both the results of immunofluorescence and dual-fluorescence Hela cells showed increasing autophagosomes in the CT group,(t-test,p<0.05).Results showed that co-localization of LC3 labeled autophagosomes and lysosomal protein Lamp2 were less in the CT group.Conclusions1.During the early and middle stages of infection,Chlamydia trachomatis did not cause an overall increasingly autophagy of the host cells.But there are differences of CT infection and autophagy in each cell.Cells with stronger autophagy were significantly less likely to be infected than those cells with weaker autophagy.Intracellular LC3 marked autophagosomes are negatively correlated with CT infection in each cell.2.During the late stage of CT infection,autophagy of Hela cells increased,but CT may inhibited the formations of autophagolysosomes.