ASAP1 Involved In Host Resistance To Mycobacterium Tuberculosis By Regulating Macrophages And Neutrophils Migration

Mycobacterium tuberculosis(Mtb)is a single pathogen that causes major chronic infectious diseases in humans and animals-tuberculosis(TB).The genetic traits of the host itself affect susceptibility to TB.Many large-scale genome-wide association studies(GWAS)have shown that ASAP1(Arf GAP with SH3 domain,ankyrin repeat and PH domain 1)polymorphisms are significantly associated with TB susceptibility in Russian and Chinese populations.However,how ASAP1 affects susceptibility to tuberculosis has not been reported.This article analyzes the susceptibility of the host to TB after knocking out or knocking down ASAP1 in zebrafish models and macrophage cell lines THP1 and Raw264.7 from the perspective of trans-genetics,and further explores the immune physiological function of phagocytes which may be the mechanisms of ASAP1 for the host susceptibility to TB.The main findings are as follows: 1.Establishment of asap1 knockout zebrafish modelZebrafish has now become a common model organism for studying TB.Firstly,this article analyzed the zebrafish asap1 a and asap1 b genes from a bioinformatics perspective.The zebrafish Asap1 a and Asap1 b proteins were compared with human,mouse and rat ASAP1 for multiple sequence comparison and gene chromosome linkage analysis.Phylogenetic tree analysis identified the evolutionary positions of the zebrafish Asap1 a and Asap1 b proteins.It was found by q RT-PCR that the asap1 a and asap1 b genes of zebrafish were under maternally control,and in situ hybridization shows that these two genes ubiquitously distributed throughout the body in zebrafish.CRISPR/Cas9 gene editing technology was used to establish asap1 a and asap1 b knockout zebrafish,resulting in a total of 3 lines of asap1 a gene knockout mutants,and a total of 3 lines of asap1 b gene knockout mutants,and 1 line of asap1 a and asap1 b co-knockout mutants,and 1 line of asap1 a and asap1 b co-knockout and neutrophil fluorescent markers mutants.Survival analysis revealed that asap1 a and asap1 b co-knockout mutant zebrafish are susceptible to lethality in 0-3 dpf,while asap1 a and asap1 b single-knockout mutant zebrafish are not significantly susceptible to early childhood lethality.According to the results of q RT-PCR,the expression level of asap1 b gene in asap1 a single knockout mutants is increased,while the expression level of asap1 a gene in asap1 b single knockout mutants is not compensated.In addition,we tried to knock down the zebrafish asap1 a and asap1 b genes by morpholino,and the knockdown efficient was verified by q PCR and western blotting.The first part of the results showed that asap1 a and asap1 b knockout mutants or knockdown morphants were successfully established in zebrafish by CRISPR/Cas9 gene editing and morpholinos.2.Application of zebrafish model to explore the relationship between migration of macrophages,neutrophils and Asap1-mediated susceptibility to tuberculosisMycobacterium marinum(Mm)shares a high degree of homology with Mycobacterium tuberculosis,and is the pathogenic bacterium that causes tuberculosis in zebrafish.Zebrafish is transparent throughout the early stages of development,so it has a unique visualization advantage in studying innate immune cells such as macrophage(M?)and neutrophil.In this chapter,we first systematically study the effect of Asap1 on susceptibility to tuberculosis at the zebrafish level.We found that asap1 a and asap1 b single gene knockout mutants had no significant difference in susceptibility to Mm compared with wild type,but asap1 a and asap1 b gene co-knockout zebrafish had a higher mycobacterial load than wild-type,which was consistent with the results of studies on asap1 a and asap1 b knockdown zebrafish with morpholino.In asap1 a and asap1 b co-knockout mutants,we also found that the migration ability of macrophages and neutrophils were obviously weakened than that of wild-type zebrafish no matter whether by tail wounds or directed chemotaxis after mycobacterial infection in zebrafish.3.To study the relationship between ASAP1 and tuberculosis susceptibility by using human mononuclear macrophage cell line THP1 and mouse macrophage cell line Raw264.7.ASAP1 knockdown model was established in human mononuclear macrophage cell line THP1 and mouse macrophage cell line Raw264.7,and the knockdown efficiency was verified by q RT-PCR and western blotting.Then,we explored the intracellular bacterial load was higher in ASAP1 knockdown cells compared with the control cells infected with H37 Ra.THP1 and Raw264.7 cells of knockdown ASAP1 displayed weak migration ability to Mycobacterium by Transwell experiments,which validates the previous results in zebrafish.In the cell model,we further explored the cytoskeleton remodeling after knocking down ASAP1,and the number of actin pucta was increased significantly,and the aggregation state of the adhesion proteins Vinculin and Paxillin were also changed.The studies above found that the reduced expression level of ASAP1 enhanced the infection degree of cell to Mycobacterium tuberculosis,and affected the migration ability of macrophages and neutrophils which may be the biological mechanism of ASAP1 affecting TB susceptibility.The implementation and completion of this project will enrich the understanding of host genetic genes affecting TB susceptibility,and have certain scientific significance and application value for TB prevention,early screening or breeding of TB-resistant livestock.