Molecular Mechanism Of Induction Of Endoplasmic Reticulum Stress In Host Cells By Mycobacterium Tuberculosis Effector Protein CdhM

According to the Global Tuberculosis Report 2021,about 25% of the world’s population was infected withMycobacterium tuberculosis(Mtb).Until the COVID-19 pandemic,tuberculosis(TB)was the leading cause of death from a single infectious agent,ranking above AIDS.TB not only seriously endangers human health and life but also heavily increases the social burden,especially in poverty-stricken areas.It is therefore urgent to end TB.Unveiling the molecular mechanisms of the interaction of humans and Mtb is an indispensable theory basis for TB prevention and treatment.For purpose of survival and proliferation,pathogens command numerous effector proteins to perturb or modulate the immune system or organelle function of the host during infection.For example,pathogens can induce endoplasmic reticulum(ER)stress to disrupt the homeostasis of the ER,which may cause cell death eventually.According to research in 2010,most of the genes involved in ER stress were found to be upregulated in TB granulomas of humans and mice,implicating that infection withMtb can also induce ER stress.However,limited studies have been performed on Mtb induction of ER stress,and little is known about the molecular mechanism of them,the physiological significance has not been addressed until now.Hence we explored the scientific issue about Mtb induction of ER stress in host cells,and obtained results at three levels,including phenotypes,molecular mechanisms,and physiological significance,as presented below.Phenotypes: Through live-cell imaging with fluorescence protein label we found that a novel Mtb effector protein,CdhM,could target the ER and cause abnormal ER morphology.Further time-lapse live imaging revealed that CdhM could eventually cause cell death with condensation morphology,which resembled apoptosis.In experiments with a single conserved amino acid mutant of CdhM we found that the mutants,including L44 A,G96A,H150 A and W175 A,could not cause those phenotypes anymore,which proved that the phenotypes caused by CdhM are specific and functional.Molecular mechanisms: First,through transcriptome analysis of the host cells transfected with CdhM or H150 A,we found that CdhM,but not H150 A,could significantly affect the ER-related pathways,and most of the genes involved in ER stress were regulated by CdhM.Biochemical experiments,like q RT-PCR and western blot,showed that the transcriptional and protein levels of ER stress marker,BiP and CHOP,were significantly upregulated by CdhM,but not its mutants,which confirmed that CdhM could induce ER stress alone.Then,we constructed CdhM-related recombinant strains and investigated the function of CdhM under the infection model of macrophage in vitro.The transcriptome analysis of macrophages infected with CdhM-related strains showed that:(1)The profile of ER stress-related genes was significantly changed by Mtb during infection,which verified the findings in 2010 about ER stress occurring in TB granulomas at the level of cellular model;(2)CdhM could promote ER stress during Mtb infection of host macrophage.These conclusions were also confirmed by the measure of the transcriptional and protein levels of ER stress marker BiP and CHOP during infection.In addition,further assessments of the levels of XBP1 splicing and eIF2α phosphorylation under both transfection and infection were performed,and we found that the levels of XBP1 splicing and eIF2α phosphorylation were significantly increased by CdhM,but not its mutants,which proved that CdhM could activate the UPR alone or during Mtb infection of macrophage through at least two pathways,including IRE1 and PERK signaling pathway.Physiology significance: We found that the ER stress induced by CdhM could mediate apoptosis of macrophages during Mtb infection.Measure of the CFU during infection of macrophages in vitro revealed that the apoptosis mediated by CdhM could promote the release of Mtb to the extracellular environment.Given the ER stress-mediated apoptosis of macrophage has been observed in the TB caseous granulomas,the apoptosis mediated by CdhM-induced ER stress may facilitate dissemination and proliferation of Mtb at the late stages of TB granulomas.Finally,in the infection model of mouse,we observed the pathological sections of lung tissue of the mice infected with CdhM-related strains and preliminarily found that CdhM could increase the number of lung lesions,indicating that CdhM may also play a role in vivo.Taken together,our study identified a novel Mtb effector protein CdhM which may promote Mtb dissemination and proliferation by induction of ER stress and mediating apoptosis of host cells.This study is the first to address the physiological significance of the ER stress induced by Mtb during infection.It also provides a new theoretical basis for understanding the pathogenesis and clinical therapeutic of TB.