Division of Infection Medicine, Department of Clinical Sciences, BMC, B14, Lund University, SE-221 84 Lund, Sweden.
Microbes Infect. 2012 Nov;14(14):1319-29. doi: 10.1016/j.micinf.2012.08.005. Epub 2012 Aug 30.
Streptococcus pyogenes, a significant bacterial pathogen in humans, interferes with the membrane traffic of human neutrophils and survives following phagocytosis. The mechanism(s) behind this property is not known, but in contrast to wild-type bacteria, mutant bacteria lacking virulence factors regulated by the transcriptional regulator Mga, are phagocytosed and killed. In the present work we investigated whether differences in phagosomal acidification may contribute to this difference. Phagosomal pH in neutrophil-differentiated HL-60 cells was studied by fluorescence ratio imaging, and phagosomes containing wild-type S. pyogenes bacteria of the M1 serotype exhibited little or no acidification, whereas Mga mutant bacteria were found in more acidic phagosomes. With phagosomes containing these bacteria, proton delivery was inhibited by adding folimycin, a vacuolar-type adenosine triphosphatase (V-ATPase) inhibitor. This inhibitor had no effect on phagosomes containing wild-type bacteria, indicating either inactivation or removal of V-ATPases by the bacteria. Analysis of isolated bacteria-containing phagosomes confirmed the latter scenario and showed a more efficient delivery of V-ATPases to phagosomes containing Mga mutant bacteria. The results demonstrate that V-ATPase-mediated phagosomal proton delivery is reduced during phagocytosis of wild-type S. pyogenes, leading to impaired acidification, and that surface proteins of the mga regulon are responsible for this effect.
化脓性链球菌是一种重要的人类细菌病原体，它能干扰人中性粒细胞的膜转运，并在吞噬后存活。这种特性的机制尚不清楚，但与野生型细菌不同的是，缺乏转录调节因子 Mga 调控的毒力因子的突变细菌在被吞噬后被杀死。在本工作中，我们研究了吞噬体酸化的差异是否可能导致这种差异。通过荧光比成像研究了中性粒细胞分化的 HL-60 细胞中的吞噬体 pH 值，M1 血清型野生型化脓性链球菌的吞噬体几乎没有或没有酸化，而 Mga 突变细菌则存在于更酸性的吞噬体中。用含有这些细菌的吞噬体，通过添加folimycin（一种液泡型三磷酸腺苷酶（V-ATPase）抑制剂）来抑制质子传递。该抑制剂对含有野生型细菌的吞噬体没有影响，这表明细菌要么失活要么去除了 V-ATPases。对分离的含有细菌的吞噬体的分析证实了后一种情况，并显示出更有效地将 V-ATPase 递送至含有 Mga 突变细菌的吞噬体。结果表明，在吞噬野生型化脓性链球菌的过程中，V-ATPase 介导的吞噬体质子传递减少，导致酸化受损，而 mga 调控子的表面蛋白是导致这种效应的原因。
