Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, UK.
J Invertebr Pathol. 2010 Nov;105(3):352-62. doi: 10.1016/j.jip.2010.09.020. Epub 2010 Oct 8.
Many bacteria persist within phagocytes, deploying complex sets of tightly regulated virulence factors to manipulate and survive within host cells. So far, no single factor has been identified that is sufficient to allow intracellular persistence of an otherwise non-pathogenic bacterium. Here we report that the two-component KdpD/KdpE sensor kinase/response regulator of the insect and human pathogen Photorhabdus asymbiotica (Pa) is sufficient to allow a harmless laboratory strain of E. coli to resist phagocytic killing and persist within insect hemocytes, ultimately killing the insect. Screening of a cosmid library of Pa in E. coli by injection into the moth Manduca sexta, previously identified three overlapping clones which caused the insect to cease feeding and subsequently die. Transposon mutagenesis revealed a cosmid encoded kdp high affinity potassium pump regulon was responsible for this phenotype. Gentamycin protection assays and confocal microscopy revealed the cosmid clones were persisting inside insect hemocytes far longer than control bacteria. Cloning and expression of PakdpD/kdpE alone into E. coli recapitulated the phenotype. Bioassay results and transcriptional analysis of various E. coli kdp mutants harboring the Pa kdp genes confirmed that Pa KdpD/KdpE was able to induce the E. coli kdp pump structural genes in response to exposure to insect hemocytes but not blood plasma alone. The finding that Pa KdpD/KdpE can facilitate resistance of E. coli to phagocytic killing suggests a central role for potassium in this process, supporting previous work implicating potassium sensing in virulence of other bacteria and also in the normal process of protease killing of engulfed bacteria by neutrophils.
许多细菌存在于吞噬细胞内，通过复杂的、严格调控的毒力因子来操纵和在宿主细胞内生存。到目前为止，还没有发现一种单一的因素足以使原本非致病性的细菌在细胞内持续存在。在这里，我们报告昆虫和人类病原体 Photorhabdus asymbiotica (Pa) 的双组分 KdpD/KdpE 传感器激酶/反应调节剂足以使无害的实验室大肠杆菌菌株抵抗吞噬作用并在昆虫血细胞内持续存在，最终杀死昆虫。通过向鳞翅目昆虫烟夜蛾 Manduca sexta 注射 Pa 的 cosmid 文库进行筛选，先前确定了三个重叠克隆，这些克隆导致昆虫停止进食并随后死亡。转座子诱变显示，一个 cosmid 编码的 kdp 高亲和力钾泵调控子是导致这种表型的原因。庆大霉素保护试验和共聚焦显微镜显示，cosmid 克隆在昆虫血细胞内的存活时间远远长于对照细菌。将 PakdpD/kdpE 单独克隆到大肠杆菌中并表达，重现了这一表型。含有 Pa kdp 基因的各种大肠杆菌 kdp 突变体的生物测定结果和转录分析证实，Pa KdpD/KdpE 能够诱导大肠杆菌 kdp 泵结构基因对昆虫血细胞而非单独的血浆作出反应。发现 Pa KdpD/KdpE 能够促进大肠杆菌抵抗吞噬作用的杀伤作用，这表明钾在这个过程中起着核心作用，支持了先前的工作，即钾感应在其他细菌的毒力以及中性粒细胞吞噬细菌后蛋白酶杀伤细菌的正常过程中起作用。
