Functional Genomic Studies of the Intestinal Response to a Foodborne Enteropathogen in a Humanized Gnotobiotic

Members of the genus Listeria provide a model for defining host responses to invasive foodborne enteropathogens. Active translocation of Listeriamonocytogenes across the gut epithelial barrier is mediated by interaction of bacterial internalin (InlA) and its species-specific host receptor, E-cadherin, whereas translocation across Peyer’s patches through M-cells is InlAindependent. To define microbial determinants and molecular correlates of the host response to translocation via these two routes, we colonized germ-free transgenic mice expressing the human enterocyte-associated E-cadherin receptor with wildtype (WT) or mutant L. monocytogenes strains, or its nonpathogenic noninvasive relative Listeria innocua, or with Bacteroides thetaiotaomicron, a prominent gut symbiont. Mouse GeneChips, combinedwith Ingenuity Pathway software, were used to identify canonical signaling pathways that comprise the response to WT L. monocytogenes versus the other species. Gainand loss-of-function experiments with L. innocua and L. monocytogenes, respectively, demonstrated that the 773-member transcriptional signature of the response to WT L. monocytogenes is largely conserved in the inlAmutant. Internalin-dependent responses include down-regulation of gene networks involved in various aspects of lipid, amino acid, and energymetabolism and up-regulation of immunoinflammatory responses. The host response is markedly attenuated in a listeriolysin-deficient ( hly) mutant despite its ability to be translocated to the lamina propria. Together, these studies establish that hly, rather than bacterial invasion of the lamina propria mediated by InlA, is a dominant determinant of the intensity of the host response to L. monocytogenes infection via the oral route. The human gut is inhabited by a complex community of trillions of microorganisms representing all three known domains of life: Bacteria, Archaea, and Eukarya (1–3). Our microbiota is dominated by members of Bacteria, with components of two divisions, the Firmicutes and the Bacteroidetes, comprising 90% of all phylogenetic types in those few individuals where comprehensive 16S rRNA gene sequence-based enumerations have been performed (1, 2). Although most of the estimated 500–1000 bacterial species in the gut microbiota appear to enjoy a mutually beneficial relationship with their host, potential pathogens are also present or may be introduced through the consumption of food or water. Members of the genus Listeria provide a model for comparing host responses to invasive versus noninvasive foodborne bacteria. Fully sequenced genomes are available from two species: Listeria monocytogenes, an enteroinvasive human pathogen that can cross the intestinal as well as blood-brain and placental barriers; and Listeria innocua, a nonpathogenic and noninvasive relative that shares 84% of its genes with L. monocytogenes (4). L. monocytogenes is estimated to be present in the small intestines of up to 5% of individuals yet only a few, typically those who are immunocompromised, develop invasive symptomatic disease (5). L. monocytogenes and L. innocua can cross the follicle-associated epithelium (FAE)5 that overlies the lymphoid follicles of Peyer’s patches with equal efficiency (6). L. monocytogenes, unlike L. innocua, expresses internalin (InlA), a surface protein that is sufficient to promote bacterial internalization into enterocytes that express its receptor, human E-cadherin (hEcad). Epidemiological (7) and histopathological data (8, 9), as well as experiments using human primary cells and tissue explants (8), indicate that InlA is an important virulence factor in humans, mediating targeting and crossing of both intestinal and placental barriers. A single amino acid difference (Pro16 in human versus Glu16 in mouse) enables human but not mouse E-cadherin to function as a receptor for InlA (10). In conventionally raised, adult transgenic mice expressing hEcad under the control of an enterocyte-specific promoter (Fabpi-hEcad), L. monocytogenes is able to invade enterocytes that cover small intestinal villi and enter the underlying lamina propria. This * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and Tables S1–S8. 1 These authors contributed equally to this work. 2 Supported by a European Molecular Biology Organization fellowship and the Philippe Foundation. Present address: Avenir INSERM U604, Bacteria Cell Interactions Unit, Institut Pasteur, and Dept. of Infectious Diseases and Tropical Medicine, Necker-Enfants Malades Hospital, René Descartes Paris-5 University, Paris 75015, France. 3 A Howard Hughes Medical Institute international research scholar. 4 Supported by National Institutes of Health Grant DK30292 and the Ellison Foundation. To whom correspondence should be addressed: Center for Genome Sciences, Washington University School of Medicine, 4444 Forest Park Blvd., Campus Box 8510, St. Louis, MO 63108. Tel.: 314-362-7243; Fax: 314-362-7047; E-mail: [email protected]. 5 The abbreviations used are: FAE, follicle-associated epithelium; InlA, internalin; hEcad, human E-cadherin; WT, wild type; LLO, listeriolysin; CFU, colony-forming unit; SAM, significance analysis of microarrays; IPA, Ingenuity pathway analysis; GF, germ-free; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 20, pp. 15065–15072, May 18, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.