Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA.
Infect Immun. 2010 Nov;78(11):4500-10. doi: 10.1128/IAI.00417-10. Epub 2010 Aug 23.
Pseudomonas aeruginosa can establish a niche within the plasma membrane of epithelial cells (bleb niches) within which bacteria can survive, replicate, and swim at speeds detectable by real-time phase-contrast imaging. This novel virulence strategy is dependent on the bacterial type three secretion system (T3SS), since mutants lacking the T3SS needle or known T3SS effectors localize to perinuclear vacuoles and fail to replicate. Here, we determined which of the three effectors (ExoS, ExoT, or ExoY) were required for bleb niche formation and intracellular replication. PAO1 strains with mutations in exoS, exoT, exoY, or combinations thereof were compared to wild-type and complemented strains. P. aeruginosa exoS mutants, but not exoT or exoY mutants, lost the capacity for bleb niche formation and intracellular replication. Complementation with exoS rescued both phenotypes, either in the background of an exoS mutant or in a mutant lacking all three known effectors. Complementation with activity domain mutants of exoS revealed that the ADP-ribosyltransferase (ADP-r) activity of ExoS, but not the Rho-GAP activity nor the membrane localization domain (MLD) of ExoS, was required to elicit this phenotype. Membrane bleb niches that contained P. aeruginosa also bound annexin V-enhanced green fluorescent protein (EGFP), a marker of early apoptosis. These data show that P. aeruginosa bleb niches and intracellular survival involve ExoS ADP-r activity and implicate a connection between bleb niche formation and the known role(s) of ExoS-mediated apoptosis and/or Rab GTPase inactivation.
铜绿假单胞菌可以在细胞上皮细胞膜的小泡（小泡生态位）内建立一个小生境，细菌可以在其中生存、复制并以实时相差成像可检测的速度游动。这种新的毒力策略依赖于细菌的 III 型分泌系统（T3SS），因为缺乏 T3SS 针或已知 T3SS 效应器的突变体定位于核周空泡中并不能复制。在这里，我们确定了三个效应器（ExoS、ExoT 或 ExoY）中的哪一个对于小泡生态位形成和细胞内复制是必需的。与野生型和互补菌株相比，PAO1 菌株中的 exoS、exoT、exoY 或它们的组合突变被比较。铜绿假单胞菌 exoS 突变体，但不是 exoT 或 exoY 突变体，丧失了形成小泡生态位和细胞内复制的能力。用 exoS 进行互补，无论是在 exoS 突变体的背景下还是在缺乏所有三种已知效应器的突变体中，都挽救了这两种表型。用 exoS 的活性结构域突变体进行互补表明，ExoS 的 ADP-核糖基转移酶（ADP-r）活性，而不是 Rho-GAP 活性或 ExoS 的膜定位结构域（MLD），是引发这种表型所必需的。含有铜绿假单胞菌的膜小泡也结合了膜联蛋白 V-增强型绿色荧光蛋白（EGFP），这是早期细胞凋亡的标志物。这些数据表明，铜绿假单胞菌的膜小泡和细胞内存活涉及 ExoS ADP-r 活性，并暗示了膜小泡生态位的形成与 ExoS 介导的细胞凋亡和/或 Rab GTPase 失活的已知作用之间的联系。
