Localization of uroplakin Ia, the urothelial receptor for bacterial adhesin FimH, on the six inner domains of the 16 nm urothelial plaque particle

The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense.     

Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified beta1 and alpha3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.     

Surfactant Protein D Inhibits Adherence of Uropathogenic Escherichia coli to the Bladder Epithelial Cells and the Bacterium-induced Cytotoxicity: A POSSIBLE FUNCTION IN URINARY TRACT*

The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca²⁺-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.     

Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-A resolution

Tetraspanin uroplakins (UPs) Ia and Ib, together with their single-spanning transmembrane protein partners UP II and IIIa, form a unique crystalline 2D array of 16-nm particles covering almost the entire urothelial surface. A 6 A-resolution cryo-EM structure of the UP particle revealed that the UP tetraspanins have a rod-shaped structure consisting of four closely packed transmembrane helices that extend into the extracellular loops, capped by a disulfide-stabilized head domain. The UP tetraspanins form the primary complexes with their partners through tight interactions of the transmembrane domains as well as the extracellular domains, so that the head domains of their tall partners can bridge each other at the top of the heterotetramer. The secondary interactions between the primary complexes and the tertiary interaction between the 16-nm particles contribute to the formation of the UP tetraspanin network. The rod-shaped tetraspanin structure allows it to serve as stable pilings in the lipid sea, ideal for docking partner proteins to form structural/signaling networks.     

Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization

The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex.     

Type 1 pilus-mediated bacterial invasion of bladder epithelial cells

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.     

Distinct glycan structures of uroplakins Ia and Ib: structural basis for the selective binding of FimH adhesin to uroplakin Ia

Although it has been shown that mouse uroplakin (UP) Ia, a major glycoprotein of urothelial apical surface, can serve as the receptor for the FimH lectin adhesin of type 1-fimbriated Escherichia coli, the organism that causes a great majority of urinary tract infections, the glycan structure of this native receptor was unknown. Using a sensitive approach that combines in-gel glycosidase and protease digestions, permethylation of released glycans, and mass spectrometry, we have elucidated for the first time the native glycoform structures of the mouse UPIa receptor and those of its non-binding homolog, UPIb, and have determined the glycosylation site occupancy. UPIa presents a high level of terminally exposed mannose residues (located on Man(6)GlcNAc(2) to Man(9)GlcNAc(2)) that are capable of specifically interacting with FimH. We have shown that this property is conserved not only in the mouse uroplakins but also in cattle and, even more importantly, in human UPIa, thus establishing the concept that UPIa is a major urothelial receptor in humans and other mammals for the mannose-specific FimH variant. In contrast, our results indicate that most terminally exposed glycans of mouse UPIb are non-mannose residues, thus explaining the failure of FimH to bind to this UPIb. In cattle, on the other hand, complex carbohydrates constituted only about 20% of the UPIb N-linked glycans. Human UPIa contained exclusively high mannose glycans, and human UPIb contained only complex glycans. The drastically different carbohydrate processing of the UPIa and UPIb proteins, two closely related members of the tetraspanin family, may reflect differences in their folding and masking due to their interactions with their associated proteins, UPII and UPIIIa, respectively. Results from this study shed light on the molecular pathogenesis of urinary tract infections and may aid in the design of glyco-mimetic inhibitors for preventing and treating this disease.     

Expression of the carbohydrate recognition domain of FimH and development of a competitive binding assay

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). In the first step of this infective process, the virulence factor FimH located on type 1 pili allows UPEC to specifically adhere to oligosaccharides, which are part of glycoproteins on the urinary bladder mucosa. This initial step prevents the clearance of E. coli from the urinary tract and enables the invasion of the host cells. Because FimH antagonists can block this interaction, they exhibit a promising therapeutic potential as anti-infectives. For the evaluation of their binding properties, a reliable, target-based affinity assay is required. Here, we describe the expression and purification of the carbohydrate recognition domain of FimH (FimH-CRD) as well as the development of a competitive binding assay. FimH-CRD linked with a thrombin cleavage site to a 6His-tag is recombinantly expressed and purified by affinity chromatography. For the evaluation of FimH antagonists, a cell-free binding assay based on the interaction of a biotinylated polyacrylamide glycopolymer with the FimH-CRD was developed. Complexation of the biotinylated glycopolymer with streptavidin coupled to horseradish peroxidase allows the quantification of the binding properties of FimH antagonists. The assay format was optimized and validated by a comparison with affinity data from reported assays.     

Synthesis and evaluation of thiomannosides,potent and orally active FimH inhibitors

FimH is a type I fimbrial lectin located at the tip of type-1 pili of Gram-negative uropathogenic Escherichia coli (UPEC) guiding its ability to adhere and infect urothelial cells. Accordingly, blocking FimH with small molecule inhibitor is considered as a promising new therapeutic alternative to treat urinary tract infections caused by UPEC. Herein, we report that compounds having the S-glycosidic bond (thiomannosides) had improved metabolic stability and plasma exposures when dosed orally. Especially compound 5h showed the potential to inhibit biofilm formation and also to disrupt the preformed biofilm. And compound 5h showed prophylactic effect in UTI model in mice.     

Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like alpha3beta1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol- and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of beta1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.     

ATG16L1 and pathogenesis of urinary tract infections

Autophagy is generally considered to be antipathogenic. The autophagy gene ATG16L1 has a commonly occurring mutation associated with Crohn disease (CD) and intestinal cell abnormalities. Mice hypomorphic for ATG16L1 (ATG16L1^HM) recreate specific features of CD. Our recent study shows that the same ATG16L1^HM mice that are susceptible to intestinal inflammatory disease are protected from urinary tract infections (UTI), a common and important human disease primarily caused by uropathogenic E. coli (UPEC). UPEC colonize the bladder and exhibit both luminal and intra-epithelial stages. The host responds by recruiting innate immune cells and shedding infected epithelial cells to clear infection. Despite these countermeasures, UPEC can persist within the bladder epithelium as membrane-enclosed quiescent intracellular reservoirs (QIRs) that can seed recurrent UTI. The mechanisms of persistence remain unknown. In this study, we show that ATG16L1 deficiency protects the host against acute UTI and UPEC latency. ATG16L1^HM mice clear urinary bacterial loads more rapidly and thoroughly due to ATG16L1-deficient innate immune components. Furthermore, ATG16L1^HM mice exhibit superficial urothelial cell-autonomous architectural aberrations that also result in significantly reduced QIR numbers. Our findings reveal a host-protective effect of ATG16L1 deficiency in vivo against a common pathogen.     

FimH-mediated Escherichia coli K1 invasion of human brain microvascular endothelial cells

Adhesion to brain microvascular endothelial cells, which constitute the blood-brain barrier is considered important in Escherichia coli K1 bacterial penetration into the central nervous system. Type 1 fimbriae are known to mediate bacterial interactions with human brain microvascular endothelial cells (HBMEC). Here, we demonstrate that type 1 fimbriae, specifically FimH adhesin is not only an adhesive organelle that provides bacteria with a foothold on brain endothelial cells but also triggers signalling events that promote E. coli K1 invasion in HBMEC. This is shown by our demonstrations that exogenous FimH increases cytosolic-free-calcium levels as well as activates RhoA. Using purified recombinant mannose-recognition domain of FimH, we identified a glycosylphosphatidylinositol-anchored receptor, CD48, as a putative HBMEC receptor for FimH. Furthermore, E. coli K1 binding to and invasion of HBMEC were blocked by CD48 antibody. Taken together, these findings indicate that FimH induces host cell signalling cascades that are involved in E. coli K1 invasion of HBMEC and CD48 is a putative HBMEC receptor for FimH.     

Design, synthesis and biological evaluation of mannosyl triazoles as FimH antagonists

Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is one of the most prevalent infectious diseases. Particularly affected are women, who have a 40-50% risk to experience at least one symptomatic UTI episode at some time during their life. In the initial step of the infection, the lectin FimH, located at the tip of bacterial pili, interacts with the high-mannosylated uroplakin Ia glycoprotein on the urinary bladder mucosa. This interaction is critical for the ability of UPEC to colonize and invade the bladder epithelium. X-ray structures of FimH co-crystallized with two different ligands, the physiological binding epitope oligomannose-3 and the antagonist biphenyl α-D-mannoside 4a revealed different binding modes, an in-docking-mode and an out-docking-mode, respectively. To accomplish the in-docking-mode, that is the docking mode where the ligand is hosted by the so-called tyrosine gate, FimH antagonists with increased flexibility were designed and synthesized. All derivatives 5-8 showed nanomolar affinities, but only one representative, the 4-pyridiyl derivative 5j, was as potent as the reference compound n-heptyl α-D-mannoside (1b). Furthermore, a loss of affinity was observed for C-glycosides and derivatives where the triazole aglycone is directly N-linked to the anomeric center. A conformational analysis by NMR revealed that the triazolyl-methyl-C-mannosides 8 adopt an unusual (1)C(4) chair conformation, explaining the comparably lower affinity of these compounds. Furthermore, to address the druglikeness of this new class of FimH antagonists, selected pharmacokinetic parameters, which are critical for oral bioavailability (lipophilicity, solubility, and membrane permeation), were determined.     

Matrix stiffness regulates endosomal escape of uropathogenic E. coli

Uropathogenic E. coli (UPEC) infection in vivo is characterized by invasion of bladder umbrella epithelial cells followed by endosomal escape and proliferation in the cytoplasm to form intracellular bacterial communities. By contrast, UPEC infection in tissue culture models results in bacteria being trapped within Lamp1‐positive endosomes where proliferation is limited. Pharmacological disruption of the actin cytoskeleton has been shown to facilitate UPEC endosomal escape in vitro and extracellular matrix stiffness is a well‐characterized physiological regulator of actin dynamics; therefore, we hypothesized that substrate stiffness may play a role in UPEC endosomal escape. Using functionalized polyacrylamide substrates, we found that at physiological stiffness, UPEC escaped the endosome and proliferated rapidly in the cytoplasm of bladder epithelial cells. Dissection of the cytoskeletal signaling pathway demonstrated that inhibition of the Rho GTPase RhoB or its effector PRK1 was sufficient to increase cytoplasmic bacterial growth and that RhoB protein level was significantly reduced at physiological stiffness. Our data suggest that tissue stiffness is a critical regulator of intracellular bacterial growth. Due to the ease of doing genetic and pharmacological manipulations in cell culture, this model system may provide a useful tool for performing mechanistic studies on the intracellular life cycle of uropathogens.     

FimH Adhesin of Type 1 Fimbriae Is a Potent Inducer of Innate Antimicrobial Responses Which Requires TLR4 and Type 1 Interferon Signalling

Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs). We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-β production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88^−/−, Trif^−/−, IFN−α/βR^−/− or IRF3^−/− mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88^−/−, IFN-α/βR^−/−, IRF3^−/− or TLR4^−/− mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-β, but not IFN-α or IFN-γ. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC) and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6) mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4^−/− mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens.     

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.     

Syndecan transmembrane domain modulates intracellular signaling by regulating the oligomeric status of the cytoplasmic domain

Cell surface receptors must specifically recognize an extracellular ligand and then trigger an appropriate response within the cell. Their general structure enables this, as it comprises an extracellular domain that can bind an extracellular ligand, a cytoplasmic domain that can transduce a signal inside the cell to produce an appropriate response, and a transmembrane domain that links the two and is responsible for accurately delivering specific information on a binding event from the extracellular domain to the cytoplasmic domain, to trigger the proper response. A vast body of research has focused on elucidating the specific mechanisms responsible for regulating extracellular binding events and the subsequent interactions of the cytoplasmic domain with intracellular signaling. In contrast, far less work has focused on examining how the transmembrane domain links these domains and delivers the necessary information. In this review, we propose the importance of the transmembrane domain as a signal regulator. We highlight the cell adhesion receptor, syndecan, as a special case, and propose that the transmembrane domain-mediated oligomerization of the syndecan cytoplasmic domain is a unique regulatory mechanism in syndecan signaling.     

Molecular and cellular mechanisms of invasion of the intestinal barrier by enteric pathogens the paradigm ofShigella

The pathogenesis of bacillary dysentery can be studied at different levels of integration of the cellular components that constitute the colonic mucosal barrier. We considered the interaction ofShigella flexneri in three experimental systems that provide complementary information and a scheme of events occurring in human colorectal mucosa asShigella invasion proceeds. Interaction ofS. flexneri with individual epithelial cells shows a series of events in which the bacterium, upon contact with the cell surface, releases a set of Ipa proteins (i.e. invasins) through a specialized, activable, type-III secretory apparatus (i.e. Mxi/Spa).Via a complex signaling process, these invasins cause major rearrangements of the subcortical cytoskeletal network which allow bacterial entry by a macropinocytotic event. Then the bacterium lyses its phagocytotic vacuole and initiates intracytoplasmic movement, due to polar assembly of actin filaments caused by a bacterial surface protein, IcsA. This allows very efficient colonization of the host cell cytoplasm and passage to adjacent cellsvia protrusions which are engulfed by a cadherin-dependent process. However, when invasiveShigella are deposited on the apical side of polarized monolayers of human colonic cells, they appear unable to invade, indicating that bacteria need to reach the subepithelial area to invade the epithelium. In this system, it has been shown that transepithelial signaling caused by apical bacteria induces adherence and transmigration of basal polymorphonuclears (PMN), thus disrupting the monolayer permeability and facilitating bacterial invasion. LPS accounts for a large part of this transepithelial signalization to PMN. Such a process could account for invasion in intestinal crypts. Finally, models of infection, such as the rabbit ligated intestinal loop show that initial bacterial entry occurs essentiallyvia M cells of the follicular associated epithelium. It then causes apoptosis of macrophages located in the follicular dome, inducing release of IL-1β which, in turn, initiates inflammation, leading to destabilization of the epithelial structures as modeled above. These data can now be used to understand the mechanisms of mucosal protection against bacillary dysentery. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXφ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls^AEGL was rescued by the insertion of a classical YXXφ motif in the cytoplasmic tail. Significantly, a Drosophila Wls^AEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXφ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.