敲除PPK基因对幽门螺杆菌逃避巨噬细胞免疫清除的影响

PPK knockout attenuates evasion of immune elimination of Helicobacter pylori by macrophages

目的:研究敲除多聚磷酸激酶(polyphosphate kinase,PPK)基因后对幽门螺旋杆菌(Helicobacterpylori,H.pylori)逃避巨噬细胞免疫清除的影响.方法:通过同源重组的原理构建H.pylori PPK敲除菌株.提取PPK敲除菌株体内多聚磷酸盐,转化为ATP进行定量,并与野生型菌株内多聚磷酸盐水平进行比较.将PPK敲除菌株及野生型菌株分别与小鼠巨噬细胞系RAW264.7共同培养,比较存活的H.pylori数量.结果:成功构建敲除PPK的H.pylori菌种.Western blot显示该菌无PPK蛋白表达.对敲除PPK的H.pylori菌株中的多聚磷酸盐定量结果显示,其内PP含量为0.46±0.25nmol Pi/mg Protein,显著低于G27野生菌种(175.33±21.22nmol Pi/mg Protein,P<0.01).将该菌株与小鼠巨噬细胞系RAW264.7共同培养,2h时间点,G27及G27ΔPPK菌种在巨噬细胞内的存活无显著差异;而在24h时间点,G27ΔPPK菌种在巨噬细胞内的存活率显著低于野生型G27菌种,巨噬细胞内的BacLightkit染色结果显示,G27ΔPPK菌种在巨噬细胞内获得染色的活菌显著低于野生型G27菌种.结论:PPK是H.pylori合成多聚磷酸盐的关键酶.敲除该基因后,H.pylori合成多聚磷酸盐的能力显著下降,其逃避巨噬细胞清除的能力明显减弱.

AIM： To evaluate the impact of knockout of the polyphosphate kinase gene in Helicobacter pylori （H. pylori） on bacterial evasion of immune elimi- nation by macrophages. METHODS： A PPK null mutant of H. pylori was constructed by gene homologous recombination. The polyphosphate was extracted from the PPK null mutant and wild type bacteria to compare the amount of polyphosphate by conversion intoATP. PPK null mutant H. pylori or wild type bac- teria were co-cultured with murine macrophage cell line Raw 264.1 to compare the bacterial sur- vival in macrophages at 24 h. RESULTS： A PPK null mutant H. pylori strain was successfully constructed. The amount of polyphosphate in PPK null mutant bacteria was significantly lower than that in wild type bac- teria （0.46 nmol Pi/mg Protein ＋ 0.25 nmol Pi/ mg Protein vs 175.33 nmol Pi/mg Protein ± 21.22 nmol Pi/mg Protein, P 〈 0.01）. Compared to wild type H. pylori, the survival rate of PPK null mutant bacteria in macrophages was similar at 2 h but was significantly reduced at 24 h. CONCLUSION： PPK plays a critical role in synthesizing polyphosphate in H. pylori. PPK knockout in H. pylori significantly impaired their ability to synthesize polyphosphate and to evade immune elimination by macrophages.