Babbin Brian A, Sasaki Maiko, Gerner-Schmidt Kirsten W, Nusrat Asma, Klapproth Jan-Michael A
Department of Pathology and Laboratory Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322, USA.
Am J Pathol. 2009 Apr;174(4):1347-57. doi: 10.2353/ajpath.2009.080640. Epub 2009 Mar 12.
Lymphocyte inhibitory factor A (lifA) in Citrobacter rodentium encodes the large toxin lymphostatin, which contains two enzymatic motifs associated with bacterial pathogenesis, a glucosyltransferase and a protease. Our aim was to determine the effects of each lymphostatin motif on intestinal epithelial-barrier function. In-frame mutations of C. rodentium lifA glucosyltransferase (CrGlM21) and protease (CrPrM5) were generated by homologous recombination. Infection of both model intestinal epithelial monolayers and mice with C. rodentium wild type resulted in compromised epithelial barrier function and mislocalization of key intercellular junction proteins in the tight junction and adherens junction. In contrast, CrGlM21 was impaired in its ability to reduce barrier function and influenced the tight junction proteins ZO-1 and occludin. CrPrM5 demonstrated decreased effects on the adherens junction proteins beta-catenin and E-cadherin. Analysis of the mechanisms revealed that C. rodentium wild type differentially influenced Rho GTPase activation, suppressed Cdc42 activation, and induced Rho GTPase activation. CrGlM21 lost its suppressive effects on Cdc42 activation, whereas CrPrM5 was unable to activate Rho signaling. Rescue experiments using constitutively active Cdc42 or C3 exotoxin to inhibit Rho GTPase supported a role of Rho GTPases in the epithelial barrier compromise induced by C. rodentium. Taken together, our results suggest that lymphostatin is a bacterial virulence factor that contributes to the disruption of intestinal epithelial-barrier function via the modulation of Rho GTPase activities.
鼠柠檬酸杆菌中的淋巴细胞抑制因子A（lifA）编码大毒素淋巴抑素，其包含与细菌致病机制相关的两个酶基序，一个葡糖基转移酶和一个蛋白酶。我们的目的是确定每个淋巴抑素基序对肠上皮屏障功能的影响。通过同源重组产生了鼠柠檬酸杆菌lifA葡糖基转移酶（CrGlM21）和蛋白酶（CrPrM5）的读码框内突变。用鼠柠檬酸杆菌野生型感染模型肠上皮单层细胞和小鼠均导致上皮屏障功能受损以及紧密连接和黏附连接中关键细胞间连接蛋白的定位错误。相比之下，CrGlM21降低屏障功能的能力受损，并影响紧密连接蛋白ZO-1和闭合蛋白。CrPrM5对黏附连接蛋白β-连环蛋白和E-钙黏蛋白显示出降低的影响。机制分析表明，鼠柠檬酸杆菌野生型对Rho GTP酶激活有不同影响，抑制Cdc42激活，并诱导Rho GTP酶激活。CrGlM21失去了对Cdc42激活的抑制作用，而CrPrM5无法激活Rho信号。使用组成型活性Cdc42或C3外毒素抑制Rho GTP酶的拯救实验支持了Rho GTP酶在鼠柠檬酸杆菌诱导的上皮屏障损伤中的作用。综上所述，我们的结果表明淋巴抑素是一种细菌毒力因子，通过调节Rho GTP酶活性导致肠上皮屏障功能破坏。
