Kazmierczak B I, Jou T S, Mostov K, Engel J N
Department of Medicine, University of California, San Francisco 94143-0654, USA.
Cell Microbiol. 2001 Feb;3(2):85-98. doi: 10.1046/j.1462-5822.2001.00091.x.
The Gram-negative pathogen Pseudomonas aeruginosa invades epithelial cells in vivo and in vitro. We have examined the pathway(s) by which epithelial cells internalize P. aeruginosa strain PA103 using Madin-Darby canine kidney (MDCK) cells. We have recently demonstrated that P. aeruginosa internalization occurs by an actin-dependent Toxin B-inhibited pathway which becomes downregulated as epithelial cells become polarized, suggesting that one or more of the Rho family GTPases is involved in bacterial internalization. Here, we demonstrate that activation of the Rho family GTPases by cytotoxic necrotizing factor 1 (CNF-1) stimulates P. aeruginosa internalization. Examination of the roles of the individual Rho family GTPases in internalization shows that expression of a constitutively active allele of RhoA (RhoAV14), but not of constitutively active Rac1 (Rac1V12) or Cdc42 (Cdc42V12), is sufficient to increase uptake of PA103pscJ. This relative increase persists when bacterial infection is established at the basolateral surface of polarized cells, suggesting that the effect of RhoAV14 is not simply due to its known ability to disrupt tight junction integrity in polarized cells. RhoAV14-mediated stimulation of bacterial uptake is actin dependent as it is abrogated by exposure to latrunculin A. We also find that endogenous Rho GTP levels in epithelial cells are increased by infection with an internalized strain of P. aeruginosa; conversely, a poorly internalized isogenic strain expressing the bacterial anti-internalization protein ExoT causes decreased Rho GTP levels. Experimental inhibition of Rho, either by expressing dominant negative RhoAN19 or by inhibiting native Rho using a membrane permeable fusion construct of a Rho-specific inhibitor, C3 ADP-ribosyltransferase, does not inhibit PA103pscJ internalization in MDCK or HeLa cells. Models consistent with these data are presented.
革兰氏阴性病原体铜绿假单胞菌可在体内和体外侵袭上皮细胞。我们利用犬肾上皮细胞（MDCK）研究了上皮细胞内化铜绿假单胞菌PA103菌株的途径。我们最近证明，铜绿假单胞菌的内化是通过一种肌动蛋白依赖性且受毒素B抑制的途径发生的，随着上皮细胞极化，该途径会下调，这表明一种或多种Rho家族GTP酶参与了细菌内化过程。在此，我们证明细胞毒性坏死因子1（CNF-1）激活Rho家族GTP酶可刺激铜绿假单胞菌的内化。对单个Rho家族GTP酶在内化中的作用进行研究表明，组成型活性RhoA等位基因（RhoAV14）的表达足以增加PA103pscJ的摄取，但组成型活性Rac1（Rac1V12）或Cdc42（Cdc42V12）的表达则不然。当在极化细胞的基底外侧表面建立细菌感染时，这种相对增加仍然存在，这表明RhoAV14的作用不仅仅是由于其已知的破坏极化细胞紧密连接完整性的能力。RhoAV14介导的细菌摄取刺激是肌动蛋白依赖性的，因为暴露于拉春库林A可消除这种刺激。我们还发现，用内化的铜绿假单胞菌菌株感染可增加上皮细胞内源性Rho GTP水平；相反，表达细菌抗内化蛋白ExoT的内化能力较差的同基因菌株会导致Rho GTP水平降低。通过表达显性负性RhoAN19或使用Rho特异性抑制剂C3 ADP-核糖基转移酶的膜通透性融合构建体抑制天然Rho来进行Rho的实验性抑制，并不会抑制MDCK或HeLa细胞中PA103pscJ的内化。文中给出了与这些数据一致的模型。
