INSERM, U1036, Biology of Cancer and Infection, Grenoble, France.
PLoS One. 2012;7(1):e30488. doi: 10.1371/journal.pone.0030488. Epub 2012 Jan 27.
Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation.
铜绿假单胞菌 III 型分泌装置将四种外毒素输出并转运到宿主细胞质中。这种转运需要两种疏水性细菌蛋白，PopB 和 PopD，它们在感染后与宿主细胞膜结合。在这项工作中，我们研究了宿主细胞成分对毒素转运效率的影响。我们开发了一种基于定量流式细胞术的外毒素转运检测方法，该方法使用 ExoS 或 ExoY 与β-内酰胺酶报告酶之间的蛋白融合。同时，通过蔗糖密度梯度分离膜后免疫检测 PopB/D，评估了转位蛋白与宿主质膜的结合情况。虽然 Pro-myelocytic 细胞系 (HL-60) 和 Pro-monocytic 细胞系 (U937) 中的 PopB/D 与宿主质膜结合，但发现它们对毒素注射具有抗性。将这些细胞分化为巨噬细胞样或中性粒细胞样细胞系会导致注射敏感表型，而不会显著改变插入质膜的转位蛋白的水平。由于之前的体外研究表明，PopB 和 PopD 裂解脂质体需要胆固醇和磷脂酰丝氨酸，我们首先研究了胆固醇在转运效率中的作用。用甲基-β-环糊精处理敏感的 HL-60 细胞，这是一种消耗胆固醇的试剂，导致 ExoS-Bla 的注射减少。此外，PopB 转位器存在于蔗糖梯度纯化获得的膜部分中，其中含有脂质筏标记蛋白 flotillin。通过药理学方法进一步检测了影响毒素注射的信号通路成分。系统检测宿主细胞膜中的转位蛋白表明，除了膜组成外，一些参与肌动蛋白聚合的一般信号通路可能对形成功能孔至关重要。总之，我们提供了有关转运过程调控的新见解，并提出了真核细胞和病原体在毒素转运水平上可能存在的交叉对话。
