Research On Function Of TRIM Protein Family In Tuberculosis And Screening Diagostic Biomarkers For Tuberculosis

Upon Mycobacterium tuberculosis(Mtb)infection,host innate immunity is critical for the progression or control of Mtb.Recently,accumulating evidence demonstrated that tripartite motif(TRIM)family proteins play important regulatory roles in innate immune responses,the dysregulation of which causes several diseases,such as virus infective diseases,cancers,immunological diseases and neuropsychiatric disorders.However,there is little known about the role and function of TRIM protein family in tuberculosis(TB).In this study,we first employed realtime PCR to profile the transcript levels of more than 70 TRIM genes in active TB patients,latent TB infection(LTBI)subjects and health controls.Results showed that 20 TRIM genes were decreased in active TB patients compared to LTBI and healthy controls.Subsequently,we further evaluated the expression level of TRIM4,TRIM16,TRIM27,TRIM32,TRIM35,TRIM46,TRIM47,TRIM65 and TRIM68 by in vitro cell infection experiment,and found that the expression of these TRIM genes were correlated with initial bacteria loads and infection time.Next,we selected the TRIM65 to construct the over expression eukaryotic recombinant plasmid,the purpose is to analyze the regulatory function of TRIM65 in macrophages against Mtb infection.The results showed that over expression TRIM65 can increase the level of IL-6 and decrease the level of IL-12 in RAW264.7 cells after Mycobacterium smegmatis(M.sm)infection;while the over expression TRIM65 can increase the level of IL-6 and decrease the level of iNOS2 in RAW264.7 cells after Mtb H37 Rv infection.Therefore,the regulatory function of TRIM65 in macrophages against Mtb infection is complex and may correlated with virulence and growth rate,but the detailed mechanism needs further study.The source of TB transmission is active pulmonary TB patients,Mtb is mainly transmitted by the respiratory route of infection,and the transmission is highly efficient as demonstrated by the fact that nearly one-third of the world’s human population is infected with Mtb.Thus,acurrate and early diagnosis of active TB cases,and as early as possible to give treatment,is the most effective method for reducing TB transmission.The current diagnostic methods for TB have several limitations.Therefore,screening new biomarkers for active TB are urgently needed.This paper screened the diagnostic biomarkers from two aspects.One is related with TRIM potein,we collected more clinical samples,then dectected the mRNA levels of 9 TRIM genes(TRIM4,TRIM16,TRIM27,TRIM32,TRIM35,TRIM46,TRIM47,TRIM65 and TRIM68)in peripheral blood cells by using real-time PCR.The receiver operating characteristic curve(ROC)analysis showed that 9 TRIM genes were all exhibited the better diagnostic power to distinguish active TB from LTBI or healthy controls,among which the TRIM4 and TRIM27 showed the strongest diagnostic power to discriminate active TB from healthy controls with both sensitivity and specificity of 100%,while TRIM4 and TRIM65 had the best ability reaching both 100% of sensitivity and specificity to discriminate active TB from LTBI.However,only the moderate ability of some TRIM genes singly achieved to differentiate LTBI from healthy controls,but the combination of TRIM genes showed improved diagnostic power.The other perspective for screening biomarkers is correlated with serum antibodies;we screened serum biomarkers for active TB at a system level by using Mtb proteome microarray.The results showed that antibody responses(IgG and IgM)against multiple antigens were significantly different between the active TB patients and healthy controls.Next,Rv2026 c and Rv2421 c recognized simultaneously by IgG and IgM antibodies were further validated by ELISA with larger samples,we found that IgG levels against Rv2026 c and Rv2421 c and IgM level against Rv2421 c were higher in active TB patients than healthy controls.ROC analysis showed that they had moderate ability in identifying active TB,while logistic regression model established by antigen combination had improved diagnostic power,with sensitivity and specificity of 82.5% and 88.12%,respectively.Therefore,these results provide a reference value to establish a new method for diagnosing active TB.