Construction of A Mycobacterium tuberculosis H37RaΔcfp10 Gene Knockout Strain and Study on Its Role in Virulence and Immune Response

FAN Chao; XUE Yuqin; HUANG Yun; JIN Ying; ZHENG Yijing; SHI Yi

doi:10.3639/j.issn.2095-3097.2025.12.024

Abstract：Objective To investigate the function of the culture filtrate protein 10(CFP-10),a core effector protein of the Mycobacterium tuberculosis early secretory antigen 6(ESX-1)secretion system,construct its gene knockout strain,and systematically analyze the effect and mechanism of the cfp10 gene on virulence regulation of the H37Ra strain and host immune responses.Methods Primers were designed according to the cfp10 gene sequence in GenBank.Using H37Ra genomic DNA as the template,the H37RaΔcfp10 knockout strain was constructed by phage transduction-mediated gene knockout technology.The expression of ESX-1-related genes and virulence-associated genes was detected by quantitative real-time polymerase chain reaction(qRT-PCR).THP-1 macrophage infection experiments were conducted to measure intracellular bacterial survival,cell viability,inflammatory cytokine secretion,and the expression of autophagy-and apoptosis-related proteins.C57BL/6 mice were infected via aerosol to determine the bacterial load in organs 8 weeks post-infection.Results A genetically stable H37RaΔcfp10 significantly lower than that in the wild-type H37Ra(P<0.05).In macrophage infection experiments,compared with the wild-type H37Ra group,the intracellular survival of the H37RaΔcfp10 knockout group was significantly reduced at 6 h,24 h,48 h,and 72 h post-infection(P<0.05).At 72 h post-infection,macrophage viability was significantly increased(P<0.05),the levels of tumor necrosis factor-α and interleukin-6 were significantly decreased(P<0.05),the ratio of microtubule-associated protein 1 light chain 3(LC3)-Ⅱ to LC3-Ⅰ was significantly increased(P<0.05),the expression level of cleaved caspase-3 was significantly decreased(P<0.05),and the expression level of B-cell lymphoma 2 was significantly increased(P<0.05).The bacterial loads in the lung,spleen,and liver tissues of mice infected with the H37RaΔcfp10 knockout strain were significantly lower than those in the wild-type H37Ra infection group(P<0.05).Conclusion The cfp10 gene is a key factor regulating the virulence of the H37Ra strain and the function of the ESX-1 system.Its deletion attenuates bacterial virulence by downregulating the expression of related genes,enhancing host cell autophagy,and inhibiting apoptosis.