Abstract:

Objective: To investigate the effects of acetyltransferase fadA3 in Mycobacterium tuberculosis (MTB) on acetylation modification of host protein, gene expression, and MTB survival in vivo. Methods: MTB standard strain (H37Rv) was selected from the Molecular Biology Laboratory of Institute of Tuberculosis and Thoracic Tumor/Beijing Chest Hospital, and the H37Rv-fadA3 knockout gene strain (ΔfadA3) was constructed by CRISPR-cas system-assisted non-homologous terminal conjugation (CRISPR-NHEJ). The absorbance (A value) and bacterial activity of H37Rv and ΔfadA3 were detected by microplate method in 7H9 liquid medium, and the growth curve and minimum inhibitory concentration (MIC) table were drawn. The changes in protein acetylation and gene expression of macrophages infected with H37Rv and ΔfadA3 was analyzed by Western blotting and transcriptome sequencing. The survival and pathological modifications of H37Rv and ΔfadA3 in lung tissue and macrophages of C57BL/6J mice was analyzed by colony count and hematoxylin-eosin staining. Results: The 1116 bp fragment of fadA3 was successfully knocked out to obtain the ΔfadA3 knockout strain. There was no significant difference in the A₆₀₀ values between H37Rv and ΔfadA3 on days 3, 6, 9, and 12 of cultivation (0.245±0.005 and 0.232±0.013, 0.403±0.122 and 0.385±0.009, 0.444±0.010 and 0.442±0.005, 0.675±0.027 and 0.662±0.026, respectively)(t values were 1.623, 2.351, 0.178, 0.848, respectively, all Ps>0.05). The MIC₉₀ of isoniazid, ethambutol, streptomycin, levofloxacin, PA-824, linezolid, clofazimine, rifampicin, bedaquiline, delamanid and other first and second-line antituberculosis drugs on ΔfadA3 was the same as that on H37Rv (0.006, 2.000, 0.313, 0.156, 0.250, 0.625, 1.250, 0.003, 0.125, 0.320 μg/ml, respectively). The grey value of acetylation modification in macrophages whole protein infected with H37Rv (243.100±7.125) was significantly different from that in the uninfected group (204.800±9.348) and ΔfadA3 infected group (154.500±14.890)(t=5.294, P=0.013; t=9.350, P=0.003). In ΔfadA3 infection group, 94 differential genes were up-regulated, and 7 were down-regulated compared to H37Rv infection. Moreover, there were significant differences in intracellular CFU count ((41.000±4.583)×10⁴ and (18.670±1.155)×10⁴, log₁₀ (5.531±0.203) and log₁₀ (4.541±0.276)) after H37Rv and ΔfadA3 infection in the macrophage model (72h) and mouse lung tissue (28d)(t=8.815, P=0.001; t=6.466, P<0.001). The pathological inflammatory infiltration of lung tissue in mice infected with ΔfadA3 was significantly weaker than in H37Rv-infected mice. Conclusion: The germicidal efficacy of anti-tuberculosis drugs on ΔfadA3 knockout strain was equivalent to H37Rv. The acetylation modification of host protein and gene expression could be significantly regulated by fadA3, which may act as an essential virulence factor for maintaining the survival of H37Rv in macrophages and mouse lung tissue, leading to the inflammatory infiltration of lung tissue. These finding provides the theoretical data for host-directed therapy targeting fadA3 and host protein acetylation.

Key words: Mycobacterium tuberculosis, Acetyltransferase, Mononuclear phagocyte system, Acetylation, Gene expression, Cell survival