Proteomics Analysis Of Mycobacterium Bovis-infected THP-1 Cells And Function Exploration Of Neutrophils In Host Response Infection

Tuberculosis(TB)is a zoonotic infectious disease mainly caused by Mycobacterium tuberculosis(M.tb)and Mycobacterium bovis(M.bovis)through the inhalation of aerosolized droplets.M.bovis is virulent to human and cattle,and transmission between cattle and human warrants reconsideration concerning food safety and public health.An estimated one-third of the world’s population are infected with M.tb,and develop latent TB infection(LTBI)with no clinically pathological features.About 5% to 10% subjects with LTBI develop clinical active TB later in life,but the prevalence increases up to 21 to 34 times after coinfection with HIV.Mycobacterium bovis bacillus Calmette–Guérin(BCG)is a live-attenuated strain of M.bovis originally developed for its potential to prevent TB,which has been the one and only vaccine approved for the prevention of TB so far.However,its efficacy against pulmonary TB(PTB)in different human populations is discrepant,the protection wanes to 60% by adolescence and is likely to develop disseminated BCG in the immunodeficient inoculated population.The triggering receptor expressed on myeloid cells(TREM)regulates the innate immune response by amplifying or inhibiting TLR-induced signals,and thus plays a key role in fine-tuning the inflammatory response.TREM1 has diagnostic potential in infectious acute inflammatory diseases,including tuberculous pleuropneumonia;as well as a promising therapeutic target in non-infectious inflammatory diseases and cancers,such as atherosclerosis.At present,the challenges faced by TB include the lack of understanding of the pathogenic mechanisms of M.bovis infected with human beings,as well as the mechanism of BCGpoor protection for PTB remains unclear,and lack of effective diagnostic methods and treatments for patients with LTBI.For the present study,we utilized a global and comparative labeling strategy of isobaric tag for relative and absolute quantitation(i TRAQ)to assess proteomic changes in the human monocyte cell line(THP-1)using a vaccine strain and two virulent strains H37 Rv and M.bovis.The potential immune response and transmission mechanism of the virulent strain M.bovis in infected human cells were explored.In addition,mice were infected with the attenuated vaccine strain BCG to analyze the host immune response process and explore the mechanism of host cell killing of BCG.Meanwhile,TREM1 gene-deleted mice were used as animal model to investigate the differential response of TREM1-mediated BCG infection in the host.Our results uncovered the BCG-induced host immune response and the molecular mechanism of active tuberculosis development,it provides a new target for clinical tuberculosis treatment.We have constructed mycobacteriophage elements to obtain mutant strains and successfully achieved trigenic deletion in M.tuberculosis,which mainly involved in pho P,Rv1759 c and Lpr G.Futhermore,the differential responses in the interaction between related genes and host were explored.Researches have been conducted on the three aspects of genetic modification,immunomodulation and drug regulation to explore the pathogen and host interaction,eventually provide new ideas for the prevention and treatment of tuberculosis.The main contents are as following:1.Comparative proteomics analysis of human macrophages infected with different virulence MTBCWe successfully obtained differential protein expression profiles of THP-1 cells infected with the MTBC strains,especially the M.bovis induced human macrophage proteins.We measured 2,032 proteins,of which 61 were significantly differentially regulated.Ingenuity Pathway Analysis was employed to investigate the canonical pathways and functional networks involved in the infection.Several pathways,most notably the phagosome maturation pathway and TNF signaling pathway,were differentially affected by virulent strains’ treatment,including the key proteins IFIT1,IFIT3,CCL20 and ICAM1.Of the 61 differential proteins,31 were involved in vesicle trafficking.Five common differential proteins induced by virulent strains include NCF1 and ICAM1,which interacted with each other and involved in immune-related signaling pathways,such as the leukocyte transendothelial migration pathway.BCG and H37 Rv induced different phagosome maturation pathway.Different virulence mycobacterial strain infections could significantly up-regulate the expression of inflammatory cytokines,but M.bovis induced a significant down-regulation of IL-4 m RNA expression.String analysis also suggested that the vacuolar protein VPS26 A interacted with TBC1D9 B uniquely induced by M.bovis.The key enzyme MTHFD2,which was mainly involved in metabolism pathways,as well as LAMTOR2 might be effective upon M.bovis infection.2.Function exploration of PMN in host response to BCG infectionAfter C57BL/6 mice were infected with BCG by respiratory tract using nasal drip,the host killed the BCG through neutrophils(PMN)by flow cytometric analysis and different degrees of histopathological damage in organs were observed.With the time of infection,the BCG in the lung of mice was gradually cleared.At 28 days post infection,no BCG was isolated and cultured in the lungs.Through different infection routes,the degree of histopathological damage produced by TREM1-/-mice was different,as well as the process of host response.Compared with the tail vein injection,TREM1-/--infected BCG by the respiratory tract could produce more severe lung injury and body weight loss,and the mass of lung and spleen significantly increased.BCG were also isolated from the lung tissue after infection for 7 days.Different infection routes,TREM1-/-mice could produce high levels of PMN by immunohistochemical analysis,but the ability of killing the pathogen in lung was different.At 50 days post infection with the tail vein injection,TREM1-/-mice survived longer within 2 weeks of immunosuppressive agent dexamethasone treatment.And infected with virulent Mycobacterium tuberculosis H37 Rv by tail vein infection,TREM1-/-mice were able to produce significantly different IL-1β and IL-12.In summary,the host could effectively kill BCG through PMN,but the lack of TREM1 impaired the ability to kill BCG in lung.TREM1 and PMN may be the bridges linking innate immunity and specific immunity in the process of mycobacterium tuberculosis infection.The inhibitors of TREM1 and dexamethasone provide a novel approach for the treatment of patients with clinically active tuberculosis in the future.3.Construction of H37RvΔpho PΔRv1759cΔLpr G mutant strainThe allelic exchange substrates(AES)were constructed and cloned into shuttle vector ph AE159 which contains temperature sensitive mycobacteriophage element.Gene deletion mutants H37RvΔpho PΔRv1759cΔLpr G and M.bovisΔpho PΔLpr G were successfully constructed.Moreover,the colony morphology of wild type and mutants grown on solid medium were compared and found that H37RvΔpho P and H37RvΔpho PΔRv175cΔLpr G formed receded cording.The intracellular invasion of wild type and mutants in the process of infection to THP-1 and were compared,as well as mice infection by tail intravenous injection.The results showed that the H37RvΔpho PΔRv1759cΔLpr G showed significantly reduced ability in invasion into macrophages,inducing high level of IL-1β.H37RvΔpho PΔRv1759cΔLpr G could produce minor lung injury and induce significantly IL-1β and TNF-α in mice,indicating the attenuated virulence of mutants.These provided the further understand the relationship between pathogen-genetic modification and host immune response.