CEACAMs serve as toxin-stimulated receptors for enterotoxigenic Escherichia coli Alaullah Sheikha, Brunda Tumalaa, Tim J. Vickersa, David Alvaradob, Matthew A. Ciorbab, Taufiqur Rahman Bhuiyanc, Firdausi Qadric, Bernhard B. Singerd, and James M. Fleckensteina,e,f,1 aDepartment of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO 63110; bDepartment of Medicine, Division of Gastroenterology, Washington University School of Medicine, Saint Louis, MO 63110; cInternational Centre for Diarrhoeal Disease Research, Bangladesh (ICDDRB), Dhaka, Bangladesh; dInstitute of Anatomy, Medical Faculty, University Duisburg-Essen, 45147 Essen, Germany; eMolecular Microbiology and Microbial Pathogenesis Program, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO 63110; and fMedicine Service, Veterans Affairs Medical Center, St. Louis, MO 63106 Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved October 7, 2020 (received for review June 17, 2020) The enterotoxigenic Escherichia coli (ETEC) are among the most com- in enterocyte apical membranes, resulting in a net loss of salt and mon causes of diarrheal illness and death due to diarrhea among water into the lumen of the small intestine and ensuing watery young children in low-/middle-income countries (LMICs). ETEC have diarrheal illness (7). also been associated with important sequelae including malnutrition However, more recent studies have suggested that the molec- and stunting, placing children at further risk of death from diarrhea ular pathogenesis of these organisms is considerably more com- and other infections. Our understanding of the molecular pathogen- plex than had been appreciated (8, 9). One aspect of ETEC esis of acute diarrheal disease as well as the sequelae linked to ETEC virulence which has not been completely explored is the role are still evolving. It has long been known that ETEC heat-labile toxin played by ETEC toxins in mediating pathogen–host interactions. (LT) activates production of cAMP in the cell, signaling the modulation Interestingly, heat-labile toxin has been shown to promote colo- of cellular ion channels that results in a net efflux of salt and water nization of the small intestine in animal models of ETEC infection into the intestinal lumen, culminating in watery diarrhea. However, (10, 11) and to enhance bacterial adhesion in vitro (12). While the as LT also promotes ETEC adhesion to intestinal epithelial cells, we intracellular signaling pathways involved in LT-stimulated adhe- postulated that increases in cAMP, a critical cellular “second messen- sion have been explored (13), the precise mechanism by which LT ” ger, may be linked to changes in cellular architecture that favor promotes pathogen–host interactions remains undefined. MICROBIOLOGY pathogen–host interactions. Indeed, here we show that ETEC use LT Interestingly, E. coli pathovars other than ETEC (14, 15) in- to up-regulate carcinoembryonic antigenrelated cell adhesion mole- teract with certain carcinoembryonic antigen-related cell adhesion cules (CEACAMs) on the surface of small intestinal epithelia, where molecules (CEACAMs), surface glycoproteins belonging to a su- they serve as critical bacterial receptors. Moreover, we show that perfamily of immunoglobulin-like cell adhesion molecules enco- bacteria are specifically recruited to areas of CEACAM expression, in ded on chromosome 19 (16). Each CEACAM possess an amino particular CEACAM6, and that deletion of this CEACAM abrogates terminal Ig-like variable domain and varying numbers of domains both bacterial adhesion and toxin delivery. Collectively, these results homologous to Ig constant region (SI Appendix,Fig.S1A). Data to provide a paradigm for the molecular pathogenesis of ETEC in which date suggest that chromosomally encoded type 1 fimbriae (pili), the bacteria use toxin to drive up-regulation of cellular targets that common to virtually all E. coli, engage these glycoprotein mole- enhances subsequent pathogen–host interactions. cules, including CEA (CD66e, CEACAM5), as well as NCA enterotoxin | CEACAM | diarrhea Significance Escherichia coli he burden of illness caused by enterotoxigenic Enterotoxigenic Escherichia coli (ETEC), a leading cause of acute T(ETEC) is remarkable, with yearly estimates of hundreds of watery diarrheal illness in children, have also been linked to millions of infections worldwide (1). Although the death rate from nondiarrheal sequelae including malnutrition. ETEC produce acute diarrheal illness has declined appreciably in the past few heat-labile toxin (LT) to stimulate the production of cellular decades (2), ETEC and other enteric pathogens have recently cAMP, ultimately modulating ion channels to release salt and been recognized as a cause of substantial morbidity in children water into the intestinal lumen, resulting in diarrhea. Given the who survive the acute infection. Increasingly, these organisms have importance of cAMP as a “second messenger,” we reasoned that been implicated in postdiarrheal sequelae including growth cellular features could be altered by LT to benefit the bacteria. stunting, malnutrition, and cognitive deficiencies, thereby exacting Interestingly, we found that LT-mediated increases in cAMP a tremendous global health burden (3). stimulate production of CEACAMs, a family of cell surface gly- ETEC were discovered as a cause of severe cholera-like di- – coproteins, which then serve as receptors for ETEC. Collectively, arrhea more than five decades ago (4 6), and the ETEC path- our findings may have implications for our understanding of ovar came to be defined as organisms capable of producing heat-labile toxin-mediated illness as well as sequelae associated with these (LT) and/or heat-stable (ST) enterotoxins. LT is a heterohexameric important pathogens. protein, similar to cholera toxin (CT), that is composed of a pentameric B subunit that engages host cell receptors to pro- Author contributions: A.S., M.A.C., B.B.S., and J.M.F. designed research; A.S., B.T., T.J.V., mote uptake of the enzymatically active A subunit. Heat-stable T.R.B., F.Q., and J.M.F. performed research; D.A., M.A.C., T.R.B., F.Q., and B.B.S. contrib- toxins are small peptides of 18 or 19 amino acids that bind to uted new reagents/analytic tools; A.S., B.T., T.J.V., D.A., B.B.S., and J.M.F. analyzed data; guanylate cyclase C. In the canonical paradigm for ETEC patho- and A.S., M.A.C., B.B.S., and J.M.F. wrote the paper. genesis, these organisms adhere to the small intestinal mucosa via The authors declare no competing interest. plasmid-encoded colonization factors, where they deliver their toxin This article is a PNAS Direct Submission. payloads to cognate receptors on intestinal epithelia (7). Toxin- Published under the PNAS license. mediated activation of adenylate cyclase by LT and guanylate cy- 1To whom correspondence may be addressed. Email: [email protected]. clase by ST result in respective production of host cAMP and This article contains supporting information online at https://www.pnas.org/lookup/suppl/ cGMP. Intracellular accumulation of these cyclic nucleotides results doi:10.1073/pnas.2012480117/-/DCSupplemental. in activation of protein kinases that in turn modulate ion channels First published November 2, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2012480117 PNAS | November 17, 2020 | vol. 117 | no. 46 | 29055–29062 Downloaded by guest on September 24, 2021 − − (CD66c, CEACAM6) (17–19), through specific lectin-mediated CEACAM6 / cells in trans by lentiviral transduction with CEA- interactions with mannosylated residues (20, 21). We have re- CAM6, restoring the adhesion phenotype (Fig. 2 E and F). Sim- cently demonstrated that, in addition to plasmid-encoded patho- ilarly, heterologous expression of CEACAM6 in HeLa cells var specific pili, known as colonization factors, the majority of promoted ETEC adhesion (Fig. 2 G–I), and, among the individual ETEC express chromosomally encoded type 1 fimbriae, and that CEACAMs, CEACAM6 expression was most efficient in pro- these structures play an essential role in virulence (22). We moting bacterial adhesion (SI Appendix,Fig.S3A and B). therefore questioned whether CEACAMs expressed in the intes- Earlier studies demonstrated that E. coli can interact not only tine contribute to the pathogenesis of ETEC. Here we demon- with CEACAM6, but also with CEA (CEACAM5) (17–19). While strate that heat-labile toxin stimulates production of CEACAMs we found that treatment of Caco-2 cells with CEACAM5 siRNA that then serve as critical receptors for ETEC type 1 fimbriae, resulted in significant decreases in adhesion, this was not true of − − accelerating bacterial adhesion and toxin delivery to host tissues. CEACAM6 / cells treated with the same siRNA (SI Appendix, Fig. S4A). Moreover, we found that this siRNA treatment resulted Results in decreased production of both CEACAM5 and CEACAM6 (SI Heat-Labile Toxin Induces CEACAM Expression in Intestinal Epithelial Appendix,Fig.S4B). In contrast to distinct localization of bacteria Cells. Of the different CEACAMs, CEACAM1, CEACAM5, and within areas of CEACAM6 expression, we found that ETEC were CEACAM6 are normally expressed predominantly in the colon, not preferentially distributed to areas of CEACAM5 production but also by small intestinal epithelia, while CEACAM7 is largely (SI Appendix,Fig.S4C). Nevertheless, we observed clear areas of restricted to the colon (SI Appendix, Table S1 and Fig. S1B).