海分枝杆菌ppk基因的生物学功能研究

Objective To construct the ppk mutant in Mycobacterium marinum ( M. marinum) and to investigate its role in the physiology. Methods The ppk mutant in M.marinum was constructed by homologous recombination. Intracellular poly inorganic phosphate (poly-Pi) concentration was measured in cell suspensions by using a DAPI-based fluorescence approach. Then in vitro bacterial viability of ppk mutant type and the wild type strain was compared under different stress conditions. Furthermore, in vivo mycobacterial virulence was checked by infecting the adult zebrafish with mutant and wild type strain. And the intracellular growth ability was investigated by infection of the murine macrophage cell line RAW267.4. Results The intracellular poly-Pi concentration of M. marinum was significantly decreased after the deletion of ppk gene. In addition, the ppk mutant displayed attenuated phenotype in the zebrafish model. In mice-derived macrophage cell line, the cell proliferation of ppk mutant type declined significantly compared with wild type. Moreover, the ppk mutant type was more sensitive to nutrient starvation and antibiotic treatment, including rifampicin and levo-floxacin. Conclusions These results indicate that ppk is involved in stress response and required for mycobacterial virulence.