| Tuberculosis(TB)is an infectious disease caused by Mycobacterium tuberculosis(MTB)infection,which is the second most deadly infectious disease.According to the statistics of the Global tuberculosis Report 2022,there will be 10.6 million new TB patients worldwide in 2021.During 2020-2021,the incidence rate of TB will increase by 3.6%,and the number of deaths will increase.Currently,the pathogenesis of TB is not very clear,and researchers generally believe that it is related to cell dysfunction caused by membrane components and secreted antigens of MTB cells.Therefore,the study of MTB virulence proteins and their effects on host cell function has become a focus and hotspot.Rv3874 and Rv3875 are the main virulent proteins encoded by the RD1 region of Mycobacterium tuberculosis,which play an important role in the infection process and immune escape process of MTB.Some studies have reported that sphingolipids and intermediate products of sphingolipids metabolism are associated with changes in macrophage function,but no reports have been reported on the effects of MTB virulent proteins Rv3874 and Rv3875 on macrophage metabolic function.Therefore,this study aims to explore the effects of MTB virulent proteins Rv3874 and Rv3875 on the immune function and sphingolipids metabolism of macrophages after infection.In this study,recombinant BCG vaccines r BCG-Rv3874 and r BCG-Rv3875 expressing virulent proteins Rv3874 and Rv3875 were first used to infect macrophages.Transcriptomic,non targeted,and targeted liquid chromatography mass spectrometry(UPLC-MS/MS)metabolomic studies were conducted on infected macrophages,and the changes in transcriptional and lipid metabolism levels of infected macrophages were extensively analyzed.In addition,we have preliminarily verified the relevant genes and signal pathways targeted by the analysis by means of fluorescence quantitative PCR and flow cytometry.The main research results are as follows:(1)Transcriptome studies showed that at the RNA-seq level,7100 genes in the r BCGRv3874 group changed compared to the BCG control group,and 1263 genes in the r BCG-Rv3875 group changed.(2)The metabolomic results showed that 612 and 485 substances were detected in the nontargeted positive and negative ion modes of lipid metabolism in r BCG-Rv3874 and r BCG-Rv3875 group samples,respectively,and 1240 and 210 metabolites were detected to change in the targeted positive and negative ions of lipid metabolism.After systematic analysis,it was finally found that the lipid changes related to sphingomyelin metabolism pathway were the most significant.Finally,a total of 220 types of sphingomyelin metabolites were detected by positive ion detection,among which the main metabolites involved were sphingomyelin(SM),ceramide(Cer),and ceramide 1phosphate(C1P).r BCG-Rv3874 enriched more Cer and C1 P than r BCG-Rv3875 sample group.(3)The expression of genes related to macrophage phagocytosis lysosomal acidification,TOLL like receptor inflammatory pathway,cell apoptosis,and key enzymes of sphingomyelin metabolism pathway all showed an upward trend(p<0.05),providing support for transcriptome and metabolomic analysis results.(4)During the process of MTB infection of macrophages,the way in which Rv3874 and Rv3875 affect the immune function of macrophages and cause MTB immune escape may be by altering the metabolism and reprogramming of sphingolipids in macrophages.In summary,our research results demonstrate the important role of sphingolipids in regulating the immune function of macrophages,and reveal the possibility of searching for new targets in sphingolipids metabolism pathways for anti TB treatment. |
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