Insights Into Persistence And Clerance Mechanisem Of Mycobacterium Tuberculosis

Tuberculosis is an infectious zoonosis caused by M.tuberculosis with high morbidity and mortality.Tuberculosis causes great threat to the public health and development of livestock.M.tuberculosis is an intracellular parasitic pathogen that infects a variety of cells(macrophages,epithelial cells,endothelial cells,etc.)and could persists for long periods of time.However,there is a difference in the survival of M.tuberculosis in these cells,and the mechanism which makes these differences remains unclear.In this study,M.tuberculosis was used to infect five cell lines of human and murine macrophages,epithelial cells and endothelial cells,respectively.The distinction in survival of M.tuberculosis in different cells and their differences were analyzed at different time points after infection.The following results were obtained by intracellular survival assay,immunofluorescence assay,transcriptome sequencing analysis,etc.1.Distinct survival capability of M.tuberculosis in different cells:The number of CFU/Cell in M.tuberculosis in h BMEC and b End.3 cells decreased slowly,but increased in THP-1 and A549 cells.The amount of M.tuberculosis in Raw 264.7 cells reached the lowest level on day 4,but increased slightly afterwards.These data show that M.tuberculosis persists and replicates in THP-1 cells and A549 cells,but survives only for a long time in h BMEC,b End.3 and Raw 264.7 cells with weaker viability.2.Distinct trafficking of M.tuberculosis in different cells:In Raw 264.7 and THP-1 cells,M.tuberculosis mainly arrests phagosome maturation in early stage,and in b End.3,h BMEC and A549 cells,M.tuberculosis mainly blocks the maturation of phagosomes in late stage.In THP-1,h BMEC,A549 and Raw 264.7 cells,M.tuberculosis significantly inhibited the acidification of lysosomes to ensure long-term survival in cells,whereas in b End.3 cells,M.tuberculosis was transported to phagolysosomes.,and degraded in autolysosomes.In THP-1 and h BMEC cells,M.tuberculosis inhibits autophagosome formation and avoids its own clearance in both cell lines.In A549,Raw 264.7 and b End.3 cells,M.tuberculosis is delivered to autolysosomes for degradation.3.Distinct basal gene expression profile in different cells:In different type of cells,the baseline expression levels of phagosome and autophagy-related pathway genes are different.For example,b End.3 cells highly express ATG3 and ITGB3.M.tuberculosis has stronger intracellular viability in ATG3 and ITGB3 gene expression interference b End.3 cell line compared with wild-type b End.3 cell line.The phagocytosis pathway signaling of M.tuberculosis infected with gene interference expression cell lines and wild-type b End.3 was found to be weaker after ITGB3 interference expression.After the mice were treated with ITGB3 agonist aspirin,the degree of lesions and bacterial load in the lung and spleen of the mice were weakened compared with the untreated group.Our result showed that ITGB3 may regulate intracellular survival of M.tuberculosis in a phagocytosis modulation manner.4.M.tuberculosis cause up-regulation of expression level of type-Ⅰ interferon related genes in THP-1 cells:After infection with M.tuberculosis,the transcription levels of a large number of type I interferon signaling pathway-related genes were increased in macrophages,including OASL,IFNB1 and ISG15.Among which OASL was the most up-regulated gene in THP-1 cells.The expression of OASL homologues gene was also upregulated in the lung and spleen of M.tuberculosis infected mice.Meanwhile,the human OASL-interacting protein-encoding genes and the murine OASL1-interacting protein-encoding genes annotated through the STRING database were up-regulated after THP-1 and Raw 264.7 infection with M.tuberculosis,respectively.Our result indicated that OASL may play a key role in the regulation of intracellular survival of M.tuberculosis by macrophages.5.Knockdown of OASL in THP-1 cells caused increased intracellular viability of M.tuberculosis:In order to explore the specific regulatory effect of OASL on the intracellular survival of M.tuberculosis in macrophages,we constructed a OASL expression interference THP-1 cell line.Compared with wild-type THP-1,the intracellular viability of M.tuberculosis was reduced in OASL expression interference cell lines.In addition,compared with wild-type THP-1 cells,M.tuberculosis infection of the OASL interference-expressing cell line caused a lower degree of up-regulation of IFIT1,IFIT2,IFIT3 and MX1 transcriptional levels.Our result indicated that OASL may facilitate the intracellular survival of M.tuberculosis by promoting the type I interferon response.6.M.tuberculosis genome transfection could induce OASL related signaling:Since OASL is closely related to the c GAS-STING pathway which related to intracellular DNA sensing.M.tuberculosis genome was used to transfect THP-1 cells,and the results showed that M.tuberculosis genome transfection could induce upregulation of OASL,IFN-α,,IFN-β and IL-10 transcription levels similar to infection with M.tuberculosis.Indicating that M.tuberculosis may release DNA,or use the degradation of macrophages to release part of bacterial DNA,causing type I interferon response,thereby promoting the intracellular survival of other bacteria.Taken together,our study first explored M.tuberculosis’ s distinct intracellular viability within different cells and some of the mechanisms that contribute to this divergence.This study will benefit further acknowledgement of M.tuberculosis invasion mechanism,escape from host cell immune clearance,maintain long term persistence and proliferation.This study could also facilitates exploration of host antiM.tuberculosis genes,and provide theoretical basis for the construction of anti-TB animal breeds and discovery the target for the development and screening of anti-TB drugs.