Screening And Validation Of Mycobacterium Tuberculosis Effector Proteins That Regulating Host Autophagy Process

Mycobacterium tuberculosis(Mtb)is a facultative intracellular pathogen and the causative agent of tuberculosis(TB).During the long-term confrontation and struggle with the host immune system,Mtb has evolved a number of strategies to escape the host immune system,ultimately leading to its long-term survival in the infected host cells.According to the Global Tuberculosis Report released by the World Health Organization in 2020,there were about 10 million new cases of TB worldwide in 2019 about 1.4 million people died from this disease in 2019.Once again,tuberculosis was the deadliest of infectious disease last year.Xenophagy is a selective autophagy process in cells that can target pathogens to promote bacterial clearance.During infection,Mtb-containing phagosomes fuses with early and late endosomes,respectively,resulting in phagosomes maturation.It has been reported that Mtb could damage the phagosomal membrane and gain access to the cytosol,and then the escaped Mtb could be recaptured into double-membraned vesicles called autophagosomes through a type of selective autophagy called xenopahgy,which represents a conserved host defense response against intracellular pathogens through innate immune clearance.However,Mtb has evolved a series of strategies to interfere with host autophagy to achieve its intracellular survival in the host cells.Therefore,an improved understanding of the regulatory roles of Mtb effector proteins during host autophagy process will lead to the development of new therapeutic strategies for TB treatment basing on pathogen-host interaction.In this study,we sought to screen Mtb effector proteins that modulate host autophagy process during infection.LC3 is a classical autophagy marker protein,and we can analyze host autophagy levels by detecting the colors(red,green,yellow)and proportion of LC3 spots by high-throughput screening.Thus,we first constructed an RFP-GFP-LC3 stablely expressing macrophage cell line using p LL3.7 lentiviruses system.We first performed flow cytometry to detect the autophagy flux in cells under EBSS starvation induction and rapamycin stimulation,and we determined that the RFPGFP-LC3 stablely expressing macrophages could well indicate the occurrence of autophagy.In addition,we constructed a BCG mutagenesis library,and we then used BCG trains in this library to infect the macrophages stably expressing RFP-GFP-LC3.By flow cytometry,we screened a series of candidate Mtb effector proteins involved in host autophagy flux regulation.Through further experimental verification,we identified that the BCG mutant,(in which Rv1375 gene was inactivated)-infected macrophage cells exhibited lower levels of autophagosome formation as compared to the cells infected by the wild-type BCG strain.This result indicates that Rv1375 effector protein of Mtb may promote autophagy induction in host cells during infection.