Immunopathology and Infectious Diseases The Bacterial Virulence Factor Lymphostatin Compromises Intestinal Epithelial Barrier Function by Modulating Rho GTPases

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 -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. (Am J Pathol 2009, 174:1347–1357; DOI: 10.2353/ajpath.2009.080640) Enteric Gram-negative bacteria are a significant cause of intestinal inflammation and diarrhea worldwide. Pathogens such as enteropathogenic Escherichia coli (EPEC) or enterohemorrhagic E. coli (EHEC) are associated with significant morbidity and mortality in humans, leading to dehydration and occasionally extraintestinal manifestations, including renal failure. Citrobacter rodentium, a related mouse pathogen, functions as a model for human Gram-negative infection. Like EPEC and EHEC, C. rodentium harbors a large pathogenicity island, termed locus for enterocyte effacement, encoding for numerous effector proteins. After oral infection and adhesion to epithelial cells, Gram-negative enteric pathogens induce dense actin accumulation underneath the site of bacterial attachment and loss of microvilli, termed attaching and effacing lesions (A/E). Furthermore, EPEC activates sophisticated mechanisms to breach the intestinal epithelial barrier. Previously, we have described a large immunomodulatory virulence factor in EPEC, termed lymphostatin, which suppresses cytokine expression in vitro. Lymphostatin is encoded for by the lifA gene, which is present in Chlamydia psittaci, Chlamydia muridarum, EPEC, EHEC, and C. rodentium. Lymphostatin expression has been associated with high virulence, and has the strongest statistical association with diarrhea caused by atypical EPEC strains. lifA consists of 9669 bp in EPEC and 9627 bp in C. rodentium, and encodes for three enzymatic motifs: a glucosyltransferase, a protease, and an aminotransferase motif. Infection with C. rodentium identified lymphostatin as an important bacterial effector protein regulating large bowel colonization and development of transmissible murine colonic hyperplasia.