The role of the epidermal growth factor receptor in microbial infections of the gastrointestinal tract

Microbes and Infection, 1, 1999, 1139−1144 © 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

Review

The role of the epidermal growth factor receptor in microbial infections of the gastrointestinal tract Andre Bureta*, D. Grant Gallb, Merle E. Olsonb, James A. Hardinb a

Department of Biological Sciences, University of Calgary, Alberta T2N 1N4, Canada Gastrointestinal Research Group, University of Calgary, Alberta T2N 1N4, Canada

b

ABSTRACT – The epidermal growth factor receptor (EGFr) is a transmembrane glycoprotein with an intrinsic tyrosine kinase. Ligand-binding to the EGFr activates cell signaling, phosphorylates protein kinases, and rearranges cytoskeletal proteins – responses that resemble those induced by microbial attachment to cell surfaces, a process known to be mediated by host cell receptors in a number of cases. This article critically reviews the possible role played by the EGFr in microbial colonization, and discusses how modulation of the EGF-EGFr axis may affect infection of the gastrointestinal tract. © 1999 Éditions scientifiques et médicales Elsevier SAS epidermal growth factor / gastrointestinal / infection / epithelial

The scientific literature of the past three decades has underscored the pivotal role played by gastrointestinal peptides in the regulation of epithelial proliferation, differentiation, maturation, and function [1, 2]. Peptide growth factors modulate these processes by binding to specific receptors. The epidermal growth factor (EGF)-epidermal growth factor receptor (EGFr) axis belongs to one of the families of gastrointestinal growth peptides [1–3]. In addition to its broad mitogenic and trophic bioactivities, it has recently been suggested that the EGFr (erbB) may be implicated in the initial attachment and the cellular invasion by some microbial organisms. Moreover, work from our laboratory suggests that EGF may act as an antiinfective agent, further implying a role for the EGF-EGFr axis in infectious processes [4]. Finally, activation of the EGFr induces cytoskeletal alterations which closely resemble the rearrangements of cytoskeletal proteins associated with microbial colonization and subsequent invasion of host cells. Following a brief overview of the properties of the EGFr, the aim of this review is to critically discuss the EGFr in the context of gastrointestinal infections and to suggest directions for future research.

1. EGFr ligands

include transforming growth factor alpha (TGF-α), amphiregulin, heparin-binding EGF, and betacellulin [3–9]. While these ligands can mimic the mitogenic effects of EGF, some of the bioactivities induced by EGFr activation, like the upregulation of microvillar membrane constituents on enterocytes, appear to be ligand-specific [10]. As discussed in the following paragraphs, poxvirus growth factors from vaccinia and Shope viruses, as well as other viral constituents can also bind the EGFr. All mature EGF-related peptides contain a conserved region of six cysteines bound by three disulfide bridges which, together, confer a distinctive three-dimensional structure to the peptide. This structure, and the spacing of the cysteine, residues constitute the essential requirements for ligandbinding to the EGFr. Although newly described receptors such as EGFr-2 (erbB-2), EGFr-3 (erbB-3), and EGFr-4 (erbB-4) are structurally similar to EGFr, they appear to bind EGFr ligands, including EGF itself, with a lower degree of affinity than other peptides, which were recently shown to belong to the heregulin protein family [11]. Therefore, this review will focus on EGFr, the best-known and potentially most physiologically relevant EGF receptor.

In addition to EGF itself, a growing number of other peptides are known to bind to the EGFr. To date, these

2. Molecular and biochemical properties of the EGFr

* Correspondence and reprints

The molecular characteristics of the EGFr are summarized in table I. The cellular location of the EGFr in the

Microbes and Infection 1999, 1139-1144

1139

Review

Buret et al.

Table I. The molecular characteristics of the EGFr. Feature

Characteristic

Molecular weight Structure

170 kDa Single N-glycosylated chain of 1186 amino acid residues Extracellular ligand-binding domain Single hydrophobic transmembrane spanning domain Intrinsic cytoplasmic tyrosine kinase domain

Conformation

intestinal epithelium remains a subject of controversy. In some species, including humans, reports have suggested that the EGFr resides on the basolateral membrane [12]. In other species, including pigs, EGFr has been demonstrated on the apical membrane [13]. Regardless, studies in vivo have demonstrated that luminal EGF can bind to the apical brush border membrane of villus enterocytes within minutes after exposure [14]. After binding, the EGF-EGFr complexes enter the cell via clathrin-coated pits, accumulate in endosomes and then in lysosomes, where the complexes are degraded [15]. Ligand-binding to the EGFr causes dimerization of the receptor and activation of the EGFr intrinsic tyrosine kinase, which in turn phosphorylates residues in the receptor itself as well as intracellular substrates such as other phosphokinases, protein kinase C, GTP-dependent processes, and phospholipases. Upon phosphorylation of cellular substrates, signal transduction is initiated via pathways implicating phosphatidylinositol, Ras, and G proteins. As a result, the cellular responses to EGFr activation include induction of genes required for cell proliferation, changes in cytoskeletal proteins, regulation of ion channels, increased cytosolic calcium, and inhibition of arachidonic acid metabolites [1, 3, 16, 17]. Moreover, recent findings have shown that EGF rapidly upregulates the length and absorptive function of brush border microvilli, possibly by redistributing existing intracellular pools of preformed apical membrane constituents via actin polymerization-dependent mechanisms [18, 19]. Studies have shown that within a few minutes of exposure to the enterocyte, EGF also induces the formation of membrane ruffles, via the phosphorylation and reorganization of cytoskeletal proteins, including actin and ezrin, in a mechanism that is reminiscent of the actin-dependent cellular phenomena that accompany enterocyte invasion by microbial pathogens [20–23].

3. Microbial attachment to host cells The vast majority of microbial pathogens require binding to the host cell surface in order to develop an efficient infection. Following adhesion, microorganisms multiply on the colonized surface, and/or invade the host cell. While not necessarily sufficient to cause disease on its own, this interplay between the pathogen and the host is a determining factor in microbial pathogenicity. Mucosal epithelia, including those in the gastrointestinal tract, are 1140

particularly prone to such modes of colonization. In most instances, binding of a pathogen to intestinal epithelia leads to, via multifactorial processes, the fluid transport abnormalities which ultimately are responsible for diarrheal disease. In addition to microbial adhesion factors, extracellular attachment may involve host-encoded, secreted polysaccharides and proteins, and/or specific receptors located on the epithelial surface [24, 25]. EGFr is one such host-encoded epithelial receptor used by some microorganisms to colonize a host. As EGFr is found along the entire length of the gastrointestinal tract [26], it is tempting to speculate that EGFr may act as a port of colonization for enteric pathogens.

4. Interactions of viral pathogens with the EGFr Attachment to, and subsequent entry by viral particles into, mammalian cells is a crucial step in the pathogenesis of viral infections, as well as a topic of great relevance to the development of successful strategies for the targeted delivery of vectors used in gene therapy. While there is evidence implicating the EGFr in viral colonization, little information is available on the role of EGFr in gastrointestinal viral infections. A wide range of host cell components have been implicated in interactions leading to viral attachment. Differential sensitivity of various cell lines to infection by the same virus suggests that efficient surface binding to a single receptor site, like that mediated by sialic acid for reovirus, does not necessarily result in productive infection [27]. Molecular studies of viral gene sequences have since demonstrated that polypeptides homologous to EGF and TGF-α are encoded by several viruses [25]. Some poxviruses encode EGF homologs capable of binding the EGFr. Shope fibroma virus contains a gene with a high degree of sequence homology with the genes encoding EGF and TGF-α [28]. Infection with this virus is tumorigenic. Similarly, vaccinia virus encodes a polypeptide homologous to EGF and TGF-α [29]. This polypeptide resembles EGF not only in its amino acid sequence, but also in its pattern of disulfide bridges, and, as a result, in its three-dimensional structure [29]. Other studies have shown that occupancy of the EGFr on murine L-cells with either EGF or with synthetic decapeptide antagonists corresponding to the third disulfide loop of EGF inhibited vaccinia infection in a dose-dependent fashion [30]. Binding of viral components to the EGFr stimulates its intrinsic tyrosine kinase and activates cell signal transduction pathways [31]. Together, these observations suggest that vaccinia virus can utilize the EGFr as a port of entry into eukaryotic cells. More recently, it has been shown that successful reovirus infection depends on the tyrosine kinase activity of the EGFr, and, although not an absolute requirement to establish infection, reovirus-binding to the EGFr significantly facilitates invasion [32]. Overall, viral activation of the EGFr signaling cascade induces mitogenic activation in immature infected and uninfected neighboring cells. One can postulate that the resulting cell proliferation at the site of infection may facilitate perpetuation of viral multiplication in target cells. Microbes and Infection 1999, 1139-1144

EGF receptor and infection

The viral infections most detrimental to the gastrointestinal tract are those caused by rotavirus. There is no evidence available to date suggesting that rotavirus uses the EGFr in the infectious process. It was recently suggested that oral EGF administration may be protective in neonatal piglets infected with rotavirus, although this study did not assess the effects of EGF on viral colonization [33]. Furthermore, recent findings suggest that the pathophysiology of simian rotavirus infection of enterocytes in vitro is associated with disruption of cytoskeletal F-actin [34]. Whether these changes implicate the EGFrdependent signaling cascade is unknown. More research is warranted in order to further clarify the interactions between rotavirus and the EGF-EGFr axis.

5. Interactions of bacteria with the EGFr Initial bacterial attachment to eukaryotic cells initiated by bacterial pili, fimbriae, or afimbrial adhesins may be followed by 1) local replication and extracellular surface colonization, or 2) internalization and/or bacterial invasion. The latter has been particularly well studied for Listeria monocytogenes and for enteropathogenic bacteria including species of Salmonella, Yersinia, Shigella, and Escherichia [22, 23, 35]. These pathogens use or otherwise modulate host cell receptor expression and exploit host cell signal transduction cascades, which aid in adherence and invasive processes [23]. Bacteria-host cell interactions following colonization by these latter enteropathogens share a number of similarities. Despite these similarities, salmonella has the capability to activate the EGFr signaling cascade, while yersinia, shigella, or escherichia do not. These similarities and differences are discussed in the following paragraphs. Members of the β1 integrin receptor family mediate cell invasion by Yersinia pseudotuberculosis and Y. enterocolitica by serving as targets for bacterial outer membrane invasins [35, 36]. As β1 integrin receptors are linked to cytoskeletal proteins, microbial attachment leads to cytoplasmic structural rearrangements which implicate actin and facilitate internalization. This mechanism clearly exploits cell signaling events, as tyrosine kinase inhibitors block bacterial entry [36]. Binding of invasin or host fibronectin to the integrin receptor is mutually exclusive, which allows the bacteria to compete effectively with fibronectin for host cell attachment. These invasindependent events are known to be mediated by several β1 integrins [37], but not by the EGFr. Like Yersinia spp., enteropathogenic Escherichia coli (EPEC) causes local reorganization of cellular actin in the apical half of epithelial cells. While secretory proteins of EPEC, including EspA and EspB, can stimulate cell signaling and alter cellular actin, contact of the intact microorganisms with the cell is required to induce signal transduction responses [38]. In addition to actin, myosin, α-actinin, talin, and ezrin accumulate at sites of bacterial attachment [38–40]. These events can be activated by tyrosine kinase and induce the formation of characteristic pedestals on which EPEC adheres [22, 23, 41]. Intriguingly, recent observations suggest that some enterohemorMicrobes and Infection 1999, 1139-1144

Review

rhagic E. coli O157:H7 strains may induce cytoskeletal reorganization and attaching-effacing lesions independently of phosphorylation of host cell proteins at tyrosine residues [40]. The signaling cascades induced by various E. coli strains causing attaching-effacing lesions, as well as epithelial cell responses to EGFr activation, remain the topic of intense ongoing research. The EPEC strain RDEC-1 causes reproducible intestinal pathology in rabbits and induces attaching-effacing lesions on the apical surface of enterocytes. E. coli RDEC-1 binds, primarily via its plasmid-encoded AF/R1 pilus, to an enterocyte membrane-glycoprotein receptor complex linked to the cell cytoskeleton, which is distinct from the EGF receptor [42]. Binding of RDEC-1 to this receptor contributes both to initial mucosal adherence and to direct cytoskeletal alterations which may result in the attaching-effacing lesions of the microvillous border. Human EPEC isolates also use a plasmid-encoded adhesive factor, which binds human and porcine enterocytes [41, 43]. The identity of the eukaryotic receptor for this factor remains to be determined. Interestingly, administration of oligosaccharides found in human colostrum and milk inhibits the adhesive factor-dependent EPEC adhesion to HEp-2 cells in vitro [44]. EGF, another important component of milk, was recently shown to protect the gastrointestinal tract against colonization by RDEC-1 in rabbits [4]. While the exact nature of the interaction between EPEC and the EGFr-EGF axis needs to be further characterized, these observations suggest possible mechanisms for preventing or modifying EPEC infection by inhibiting adhesion. Prior to invasion, shigella adheres to epithelial cells on their basolateral membrane. Cell invasion by shigella is initiated by the bacterial secretory proteins Ipa-B, C, and D, which are released upon contact with target epithelial cells [23, 45]. The mechanism of entry involves binding of these invasins to the integrin α5β1 [46, 47]. This triggers host cell signaling pathways and facilitates entry [23, 35]. Cellular invasion is associated with actin reorganization in the cell, much like the response to yersinia and EPEC, which causes focal cytoskeletal alterations and results in the formation of membrane ruffles. A similar strategy is used by Salmonella species. However, in contrast with host cell binding by shigella, attachment occurs on the apical surface of the enterocyte. Activation of the EGFr has been implicated in host cell invasion by salmonella. In a classic study, Galan et al. demonstrated that invasion of epithelial cells by Salmonella typhimurium was associated with stimulation of the EGFr and subsequent induction of its tyrosine kinase, and that InvA gene-deficient mutants unable to invade did not induce such alterations in host cells [48]. Apical delivery of EGF enabled internalization of the noninvasive strain, but not of a noninvasive E. coli strain, further supporting a role for the EGFr in cell invasion by S. typhimurium. More recently, findings demonstrated that S. typhimurium substrates of the protein-secretion apparatus type III, including SopE, directly activate Rho GTPases, inducing cytoskeletal changes and membrane ruffling, and stimulate nuclear responses in host cells [49]. Thus, instead of inducing signaling events by stimulating a host cell receptor, this pathogen may initiate cell signaling cascades involv1141

Review

ing cytoskeletal rearrangement through direct delivery of a bacterial effector protein. Moreover, tyrosine kinase inhibitors do not inhibit Salmonella invasion, further implying that ports of entry other than the EGFr must be involved [36]. Studies using waved-2 mice, which express an EGFr with reduced kinase activity, have demonstrated that S. typhimurium invades EGFr-deficient cells as effectively as cells from control animals, further questioning the role played by the EGFr in cell invasion by Salmonella sp. [50]. Furthermore, genistein, an EGFr inhibitor, and anti-EGFr antibodies do not affect Salmonella invasion [51]. Finally, S. typhimurium can invade cells that are devoid of the EGFr [52]. While these observations imply that the EGFr does not act as a port of entry for Salmonella sp. in host cells, it remains possible that EGFr activation may be associated with this process. Clearly, further research is needed to define the role played by the EGFr and possibly other enterocyte surface receptors in the pathogenesis of Salmonella infection. Findings from such studies will help establish a rational basis for the development of novel therapeutic approaches to control this disease.

6. Interactions of enteric protozoa with EGF Enteric protozoan parasites, including species of Cryptosporidium and Giardia, can cause diarrhea. Cryptosporidiosis and giardiasis are acquired from ingestion of water or food contaminated with sporulated oocysts or cysts. The apical membrane of small intestinal enterocytes is the preferential site of attachment for sporozoites or trophozoites, respectively, and subsequent infection. As was observed in the viral and bacterial infections discussed above, epithelial colonization with species of Cryptosporidium and Giardia results in the rearrangement of cytoskeletal proteins, including F-actin, villin, and ezrin [53–55]. We recently demonstrated that administration of EGF to the apical surface of enterocytes significantly reduces epithelial colonization by these protozoa [56, 57]. The mechanisms involved in attachment to host cells as well as the factors that may regulate the preferential sites of colonization for these parasites require further investigation. Also, the possible interactions of metazoan parasites with the EGF-EGFr axis have yet to be investigated.

7. Conclusion Rather than attempting to fully review the current state of knowledge on microbial adherence to host cells, this article discusses how selected binding processes may relate to EGFr-dependent attachment and subsequent cell signaling events, and whether the EGFr may provide a port of colonization for enteric microbes. Clearly, the EGFr is involved in numerous biological events in the gastrointestinal tract. Whether these include an important role in colonization and infection with enteropathogens requires further investigation. Indeed, although activation of the 1142

Buret et al.

EGFr by ligand-binding and microbial colonization share a number of similarities in the resulting cell signaling cascade, including phosphorylation of protein kinases and the rearrangement of cytoskeletal proteins, the microbes for which a role for EGFr in invasion is well established are limited to nonenteric viral pathogens. For bacterial enteropathogens, the evidence is less supportive of a role for EGFr. Salmonella, which was first thought to use the EGFr for invasion, seems now to use infective strategies that are independent of the EGFr, albeit resembling the response induced by EGFr activation. Intriguingly, recent observations have suggested that administration of the EGF peptide significantly reduces microbial attachment on gastrointestinal epithelial cells in vivo and in vitro [4, 56, 57]. The mechanisms responsible for this antiinfective benefit of EGF have yet to be identified. To date, studies have shown that the protective benefits of EGF against gastrointestinal colonization are not due to a microbiocidal effect of EGF and are independent of competitive binding to the EGFr. Regardless, pharmacological manipulations of the EGFr-EGF axis may prove valuable in a variety of gastrointestinal disorders [58] and include the control of infectious diseases. We speculate that such manipulations may include administration of EGF to inhibit gastrointestinal colonization by enteric pathogens.

Acknowledgments This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Medical Research Council of Canada (MRC), the Alberta Agricultural Research Institute (AARI), and the Canada-Alberta Hog Industry Development fund.

References [1] Podolsky D.K.,in: Johnson L.R. (Ed.), Physiology of the Gastrointestinal Tract, Raven Press, New York, 3rd Ed., Vol. 1, 1994, pp. 129–167. [2] Cohen S., Isolation of a submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal, J. Biol. Chem. 237 (1962) 1555–1562. [3] Barnard J.A., Beauchamp R.D., Russell W.E., Dubois R.N., Coffey R.J., Epidermal Growth Factor – related peptides and their relevance to gastrointestinal pathophysiology, Gastroenterology 108 (1995) 564–580. [4] Buret A., Olson M.E., Gall, D.G., Hardin J.A., Effects of orally administered epidermal growth factor on enteropathogenic Escherichia coli infection in rabbits, Infect. Immun. 66 (1998) 4917–4923. [5] Marquardt H., Hunkapiller M.W., Hood L.E., Todaro G.J., Rat Transforming Growth Factor type 1: Structure and relationship to EGF, Science 213 (1984) 1079–1082. [6] Shoyab M., McDonald V.L., Bradley J.F., Todaro G.J., Structure and function of amphiregulin: a member of the epidermal growth factor family, Science 243 (1989) 1074–1076. Microbes and Infection 1999, 1139-1144

EGF receptor and infection

[7] Higashiyama S., Abraham J.A., Miller J., Fiddes J.C., Klagsburn M., A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF, Science 251 (1991) 936–939. [8] Sasada R., Ono Y., Taniyama Y., Sing Y., Folkman J., Igarishi K., Cloning and expression of cDNA encoding human betacellulin, a new member of the EGF family, Biochem. Biophys. Res. Commun. 190 (1993) 1173–1179. [9] Prigent S.A., Lemoine N.R., Type 1 (EGFr-related) family of growth factor receptors and their ligands, Prog. Growth Factor Res. 4 (1992) 1–24. [10] Hardin J.A., Gall D.G., The effect of TGFα on intestinal solute transport, Regul. Pept. 39 (1992) 169–176. [11] Plowman G.D., Green, J.M., Culouscou J.M., Carlton G.W., Rothwell V.M., Buckley S., Heregulin induces tyrosine phosphorylation of HERA/p180erbB4, Nature 366 (1993) 473–475. [12] Playford R.J., Hanby A.M., Gschmeissner S., Pfeiffer L.P., Wright N.A., McGarrity T., The epidermal growth factor receptor is present on the basolateral but not apical surface of enterocytes in the human gastrointestinal tract, Gut 39 (1996) 262–266. [13] Kelly D., McFayden M., King T.P., Morgan P.J., Characterization and localization of the epidermal growth factor receptor in the jejunumn of the neonatal and weaning pig, Reprod. Fertil. Dev. 4 (1992) 183–189. [14] Gonnella P.A., Siminoski K., Murphy R., Neutra M., Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum, J. Clin. Invest. 80 (1987) 22–32. [15] Willingham, M.C., Haigler H.T., Fitzgerald J.P., Gallo M.G., Rutherford A.V., Pastan I.H., The morphologic pathway of binding and internalization of epidermal growth factor in cultured cells, Exp. Cell Res. 146 (1983) 163–175. [16] Carpenter G., Cohen S., Epidermal Growth Factor, J. Biol. Chem. 265 (1990) 7709–7712. [17] Mitchell M.D., Epidermal Growth Factor actions on arachidonic acid metabolism in human amnion cells, Biochem. Biophys. Acta 928 (1987) 240–242. [18] Hardin J.A., Buret A., Meddings J.B., Gall D.G., Effect of epidermal growth factor on enterocyte brush-border surface area, Am. J. Physiol. 264 (1993) G312–G318. [19] Chung B.M., Wong J.K., Hardin J.A., Gall D.G., Role of actin in EGF-induced alterations in enterocytes SGLT1 expression, Am. J. Physiol. 276 (1999) 39 G463–G469. [20] Bretscher A., Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor, J. Cell Biol. 108 (1989) 921–930. [21] Higley S., Way M., Actin and cell pathogenesis, Curr. Op. Cell Biol. 9 (1997) 62–69. [22] Bliska J.B., Galan J.E., Falkow ., Signal transduction in the mammalian cell during bacterial attachment and entry, Cell 73 (1993) 903–920. [23] Finlay B.B., Cossart P., Exploitation of mammalian host cell function by bacterial pathogens, Science 276 (1997) 718–725. Microbes and Infection 1999, 1139-1144

Review

[24] Isberg R.R., Discrimination between intracellular uptake and surface adhesion of bacterial pathogens, Science 252 (1991) 934–938. [25] Todaro G.J., Rose T.M., Spooner C.E., Shoyab M., Plowman G.D., Cellular and viral ligands that interact with the EGF receptor, Canc. Biol. 1 (1990) 257–263. [26] Thompson J.F., Specific receptors for Epidermal Growth Factor in rat intestinal microvillous membranes, Am. J. Physiol. 254 (1988) G429–G435. [27] Duncan M.R., Stanish S.M., Cox D.C., Differential sensitivity of normal and transformed human cells to reovirus infection, J. Virol. 28 (1978) 444–449. [28] Chang W., Upton C., Hu S.L., Purchio A.F., McFadden G., The genome of Shope fibroma virus, a tumorigenic virus, containing a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha, Mol. Cell. Biol. 7 (1987) 535–540. [29] Brown J.P., Twardzik D.R., Marquardt H., Todaro G., Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor alpha, Nature 313 (1985) 491–492. [30] Eppstein D.A., Marsh Y.V., Schreiber A.B., Newman S.R., Todaro G.J., Nestor J.J., Epidermal Growth Factor receptor occupancy inhibits vaccinia virus infection, Nature 318 (1985) 663–664. [31] King C.S., Cooper J.A., Moss B., Twardzik D.R., Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors, Mol. Cell. Biol. 6 (1986) 332–336. [32] Strong J.E., Tang D., Lee P.W.K., Evidence that the epidermal growth factor receptor on host cells confers reovirus infection efficiency, Virology 197 (1993) 405–411. [33] Zjilstra R.T., Odle J., Hall W.F., Petschow B.W., Gelberg H.B., Litov R.E., Effect of orally administered epidermal growth factor on intestinal recovery of neonatal pigs infected with rotavirus, J. Pediatr. Gastroenterol. 19 (1994) 382–390. [34] Jourdan N., Brunet J.P., Sapin C., Blais A., Cotte-Lafitte J., Forestier F., Quero A., Trugnan G., Servin A.L., Rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton, J. Virol. 72 (1998) 7228–7236. [35] Falkow S., Isberg R.R., Portnoy D.A., The interaction of bacteria with mammalian cells, Annu. Rev. Cell. Biol. 8 (1992) 333–363. [36] Pace J., Hayman M.J., Galan J.E., Signal transduction and invasion of epithelial cells by S. typhimurium, Cell 72 (1993) 505–514. [37] Isberg R.R., Leong J.M., Multiple β1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells, Cell 60 (1990) 861–871. [38] Kenny B., Finlay B.B., Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells, Proc. Natl. Acad. Sci. USA 92 (1995) 7991–7995. 1143

Review

[39] Rosenshine I., Donnenberg M.S., Kaper J.B., Finlay B.B., Signal transduction between enteropathogenic Escherichia coli (EPEC) and epithelial cells: EPEC induces tyrosine phosphorylation of host cell proteins to initiate cytoskeletal rearrangement and bacterial uptake, EMBO J. 11 (1992) 3551–3560. [40] Ismaili A., McWhirter E., Handelsman M.Y.C., Brunton J.L., Sherman P.M., Divergent signal transduction responses to infection with attaching and effacing Escherichia coli, Infect. Immun. 66 (1998) 1688–1696. [41] Law D., Adhesion and its role in the virulence of enteropathogenic E. coli, Clin. Microbiol. Rev. 7 (1994) 152–173. [42] Rafiee P., Leffler H., Byrd J.C., Cassels F.J., Boedecker E.C., Kim Y.S., A sialoglycoprotein complex linked to the microvillous cytoskeleton acts as a receptor for pilus (AF/R1) mediated adhesion of enteropathogenic Escherichia coli (RDEC-1) in rabbit small intestine, J. Cell Biol. 115 (1991) 1021–1029. [43] Giron J.A., Ho A.S.Y., Schoolnick C.K., An inducible bundle-forming pilus of enteropathogenic Escherichia coli, Science 254 (1991) 710–713. [44] Cravioto A., Tello A., Villafan H., Ruiz J., DelVedovo S., Neeser J.R., Inhibition of localized adhesion of enteropathogenic Escherichia coli to Hep-2 cells by immunoglobulin and oligosaccharide fractions of human colostrum and breast milk, J. Infect. Dis. 163 (1991) 1247–1255. [45] Watari M., Tobe T., Yoshikawa M., Sasakawa C., Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness, Eur. Mol. Biol. Organ. J. 14 (1995) 2461–2470. [46] Dehio C., Prevost M.C., Sansonetti P.J., Embo J. 14 (1995) 2471. [47] Watarai M., Funato S., Sasakawa C., Interaction of Ipa proteins of Shigella flexeneri with α5β1 integrin promotes entry of the bacteria into mammalian cells, J. Exp. Med. 183 (1996) 991–999. [48] Galan J.E., Pace J., Hayman M.J., Involvement of the epidermal growth factor receptor in the invasion of cultured mammalian cells by S. typhimurium, Nature 357 (1992) 588–589.

1144

Buret et al.

[49] Hardt W.D., Chen L.M., Schuebel K.E., Bustelo X.R., Galan J.E., S. typhimurium encodes an activator of the Rho GTPases that induces membrane ruffling and nuclear responses in host cells, Cell 93 (1998) 815–826. [50] McNeil A., Dunstan S.J., Clark S., Strugnell A., Salmonella typhimurium displays normal invasion of mice with defective epidermal growth factor receptors, Infect. Immun. 63 (1995) 2770–2772. [51] Rosenshine I., Ruschkowski S., Foubister V., Finlay B.B., S. typhimurium invasion of epithelial cells: role of induced host cell tyrosine protein phosphorylation, Infect. Immun. 62 (1994) 4969–4974. [52] Francis C.L., Ryan T.A., Jones B.D., Smith S.J., Falkow, S., Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria, Nature 364 (1993) 639–642. [53] Forney, J.R., Dewald D.B., Yang S., Speer C.A., Healey M.C., A role for host phosphoinositide 3-kinase and cytoskeletal remodeling during Cryptosporidium parvum infection, Infect. Immun. 67 (1999) 844–852. [54] Teoh, D.A., Kamieniecki D., Pang G., Buret, A., Decreased electrical resistance in human colonic and duodenal monolayers exposed to Giardia lamblia is associated with rearrangement of F-actin in the terminal web. Gastroenterology 114 (1998) A1098. [55] Teoh D., Buret A., Decreased electrical resistance in duodenal monolayers exposed to Giardia lamblia is associated with relocalization of villin and ezrin in vitro, Gastroenterology 116 (1999) A831. [56] Buret A., Kamieniecky D., Olson M.E., Gall D.G., Hardin J.A., Epithelial colonization by Cryptosporidium and transepithelial electrical resistance: Effects of epidermal growth factor, Gastroenterology 116 (1999) A865. [57] Hardin, J.A., Olson M.E., Buret A., Gall D.G., The effect of oral EGF on giardiasis in gerbils, Gastroenterology 112 (1997) A992. [58] Guglietta A., Sullivan B., Clinical applications of epidermal growth factor, Eur. J. Gastroenterol. 7 (1995) 945–950.

Microbes and Infection 1999, 1139-1144