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Urinary tract infection (UTI) is the second most common infectious disease, and is caused predominantly by type 1-fimbriated uropathogenic E. coli (UPEC). UPEC initiates infection by attaching to uroplakin Ia, its urothelial surface receptor, via the FimH adhesins capping the distal end of its fimbriae. Uroplakin Ia, together with uroplakins Ib, II and IIIa, forms a 16 nm receptor complex that is assembled into hexagonally packed two-dimensional crystals (urothelial plaques) covering >90% of the urothelial apical surface. Recent studies indicate that FimH is the invasin of UPEC as its attachment to the urothelial surface can induce cellular signaling events including calcium elevation and the phosphorylation of the uroplakin IIIa cytoplasmic tail, leading to cytoskeletal rearrangements and bacterial invasion. However, it remains unknown how the binding of FimH to the uroplakin receptor triggers a signal that can be transmitted through the highly impermeable urothelial apical membrane. We show here by cryo-electron microscopy that FimH-binding to the extracellular domain of UPIa induces global conformational changes in the entire uroplakin receptor complex, including a coordinated movement of the tightly bundled transmembrane helices. This movement of the transmembrane helix bundles can cause a corresponding lateral translocation of the uroplakin cytoplasmic tails, which can be sufficient to trigger downstream signaling events. Our results suggest a novel pathogen-induced transmembrane signal transduction mechanism that plays a key role in the initial stages of UPEC invasion and receptor-mediated bacterial invasion in general.
Bacterial infections of the urinary tract, caused predominantly by type 1-fimbriated uropathogenic E. coli (UPEC), are the second most common human bacterial infection.1-3 Initial binding of UPEC to the urothelium is mediated by the FimH adhesin, a lectin that caps the tip of the fimbria and binds with high affinity to mannose glycans exposed on the apical surface of the bladder.4 Over 90% of the luminal surface of the superficial umbrella cell layer of the bladder urothelium is covered by rigid-looking plaques (“urothelial plaques”), which are 0.3-1 μm in diameter and consist of two-dimensional crystals of hexagonally packed 16-nm particles5-8 composed of four integral membrane proteins, the uroplakins (UPs; reviewd in 9). We and others have shown that the 16-nm uroplakin particles have 6 inner and 6 outer structural subdomains,10-13 and that each of these subdomains contains a cylindrical 4-helix-bundled tetraspanin uroplakin (UP Ia for the inner subdomains and UP Ib for the outer ones) in association with a single-pass transmembrane uroplakin (UP II and UP IIIa, respectively).14,15 These 16-nm particles are packed hexagonally into 2D crystalline arrays that form the urothelial plaques, and contribute to the extraordinary permeability barrier function of the mammalian urothelium.15-19 The uroplakin complex is also a unique example of tetraspanin webs – membrane protein networks that play important signaling roles in a wide range of cells.20,21 Importantly, uroplakin Ia has been identified as the urothelial receptor for the FimH protein.22,23
FimH binding to the uroplakin receptor complex enables the bacterium to adhere to the urothelial surface, thus allowing it to establish a foothold for infection.24 Moreover, FimH plays a key role as the “invasin” that enables the UPEC to invade into the host cells, i.e., it is necessary and sufficient for triggering host cell signaling cascades leading to bacterial internalization by the so-called “zipper”-type invasion mechanism.25-27 UPEC invasion subsequently leads to the formation of intracellular bacterial communities in the superficial umbrella cells.28,29 Despite the critical roles of the uroplakin receptor complex in the establishment of UTI, the mechanism by which the bacterial binding-induced signal is transmitted through the lipid bilayer to initiate the downstream signaling cascade is unclear.
We speculated earlier that the binding of FimH to the uroplakin particle might induce the receptor complex to undergo conformational changes that can mediate transmembrane signal transduction.14,15 We report here a three-dimensional (3D) cryo-EM structure, at 8 Å resolution, of the mouse uroplakin particle in complex with the FimH adhesin. Comparison of this 3D structure with that of the native particle14 shows that FimH binding to UP Ia induces large conformational changes in the extracellular domains of the two single-pass transmembrane uroplakins, UPs II and IIIa, as well as a twisting movement of the transmembrane helices of all the uroplakin molecules of the 16-nm particle. The movements of these transmembrane helices can cause conformational changes in the cytoplasmic tail of UP IIIa, thus triggering the downstream signaling pathways and enabling bacterial invasion and host cell response.
The structure of the 16-nm uroplakin particles bound to recombinant FimH (Fig. 1), in the form of a FimH/C complex, was determined by cryo-EM at 8 Å resolution (Table 1 and Fig. S1). The FimH/C complex was used as a ligand because FimC, a periplasmic chaperone, is required to stabilize the FimH adhesin.27,30-32 The FimH/C-bound 16-nm uroplakin particle retained the stellate-shape of the native particle,14,15 with six easily recognizable subunits, each consisting of an inner subdomain made of UPs Ia and II and an outer subdomain made of UPs Ib and IIIa (Figs. 2A and 2C). To compare the electron density maps of the FimH-bound particle with the previously-determined native one, we contoured their density maps to enclose similar volumes and segmented the maps into individual uroplakins.14
Major FimH-induced conformational changes could be readily detected in the top regions of the UPs II and IIIa extracellular domains – the so-called “joint” domains (“JT” in Fig. 2C) which bridge the inner and outer subdomains at the top of the uroplakin particle.14,15 In the native particle, the joint domain of UP II has an elongated globular shape connected by a long helical electron density anchored to the single transmembrane helix (Figs. 3A and 3C). After FimH binding to UP Ia, however, the globular joint domain of UP II opens up to form two smaller domains linked at their lower parts to form an approximately “Y” shaped structure (Fig. 3B), in which one of the arms swings ~120° towards the opposite side (Fig. 3G, curved arrow). Thus, the joint domain of UP II undergoes a drastic change in both its shape and orientation (Figs. 3A-3G). The rotation of the UP II head resulting from FimH/UP Ia binding can be appreciated from Figures Figures2D2D and and3F.3F. In addition to the changes in the joint domain, FimH binding also affected the conformation of the trunk domains of UP II (Fig. 3). In the unliganded native particle there is a discontinuity of density in the trunk regions that link the joint domains to the transmembrane helices14 of both uroplakins II and IIIa (Figs. 3A and 3H, dashed lines), indicating a greater degree of flexibility in these regions. However, in the FimH-bound structure of UP II, the joint and transmembrane regions are connected by an uninterrupted trunk region density (Figs. 3B and 3D), suggesting that this helical region has been stabilized by FimH binding.
Although less drastic than those observed for UP II, FimH binding also induced major conformational changes in UP III (Figs. 3H-3N). In its native structure, a density tunnel in the joint region of UP III runs approximately perpendicular to the plane of the membrane and is thus visible only from the top (Figs. 3J and 3L, asterisks). In the FimH-bound structure, however, this tunnel is re-oriented to run horizontally, thus becoming clearly visible from the side (Fig. 3K, asterisk). In addition, FimH binding forces the head of UP IIIa, i.e., its joint region, to lean away from its UP Ib partner, thus creating a separation between these two uroplakins (Fig. 3K, arrow). Interestingly, the large conformational changes of the joint domains of both UPs II and IIIa appeared to be centered on the FimH-binding site, which lies in a crevice between the two domains (Fig. 2D, asterisk). This suggests that the interactions between FimH and the uroplakins cause the receptor complex to open up its joint regions thus facilitating FimH binding to the mannose moiety in the shorter UP Ia receptor.
Although the affinity between FimH and uroplakins is sufficiently strong (Kd ~ 100 nM)23 to allow a stable binding (Fig. 1), we did not observe densities that directly correspond to the whole FimH/C complex itself, an elongated mass with a total molecular weight of about 50-kDa.30 However, this is anticipated for the following reasons: (i) the high mannose moiety of UP Ia to which FimH binds is likely flexible, (ii) the sugars of the high mannose moiety of UP Ia do not have a unique number of branches and length35 and FimH only binds the terminal mannose, and (iii) the opening of the crevice that FimH has to reach in for binding the mannose is larger than the diameter of FimH. Hence, FimH may not occupy a crystallographically unique position when complexed with the 16-nm particle. In our previous molecular model of the native tetraspanin UP Ia,14 we predicted the location of its single glycosylation site. We now observed in the FimH-bound receptor complex some density at that site (Figs. (Figs.2D2D and 3F, asterisks; see also Fig. S2), possibly a residual density of the FimH/C complex after crystallographic average.33
To compare the transmembrane helices of the 4 uroplakins in native and FimH-bound 16-nm particles,14,15 we segmented the transmembrane helices by marking the center of the transmembrane helices in cross-sections of density maps parallel to the plane of the membrane (Fig. 4A). A similar method was used by Unwin and Ennis to detect conformational changes in the transmembrane helices of gap junctions induced by calcium ions.34 Superimposition of the segmented helices of the native and FimH-bound 16-nm particle showed that FimH-binding induced all the uroplakin transmembrane helices to move in a concerted fashion (Figs. 4B-4D). The top ends (near the luminal side) of the 5 helices of each subdomain underwent a clockwise rotation around the center of the subdomain (Fig. 4C). Since the swing of the UPII head was also clockwise (Fig. 3G, curved arrow), the rotation of the transmembrane helices most likely resulted from the conformational changes in the joint regions induced by FimH binding. Notably, the movement of the cytoplasmic ends of the helices differed from that of the top ends, in both direction and magnitude (Fig. 4D), indicating that a degree of twisting between helices takes place upon ligand binding (see also Fig. S3 in Supplementary Data). For example, the cytoplasmic end of transmembrane helix 3 of UP Ib tilted ~10° more in the FimH-bound structure than that in the native structure (Figs. 3J and 3K, black dashed lines). A comparison of the cytoplasmic ends of the helices in the native and the FimH-bound uroplakins showed that FimH-binding causes displacements of up to ~6 Å (Table 2).
Our data showed that FimH binding to its UP Ia receptor induces major conformational changes in the extracellular domains as well as concerted movements of the transmembrane helices of all the uroplakins in the 16-nm uroplakin receptor complex (Figs. (Figs.22--4).4). The fact that FimH, which binds to the mannose moiety of UP Ia, can induce widespread conformational changes throughout the uroplakin complex is consistent with the unique architecture of the 16-nm particle. First, FimH has to reach into a crevice on the top of the uroplakin particle, because UP Ia, the only uroplakin harboring the high mannose moiety bound by FimH,23,35 is shorter than the single-pass UPs II and IIIa.14 The crevice formed by the two joints (connecting UPs II and IIIa between the inner and outer subdomains at the top) of the neighboring subunits of the 16 nm particle is ~3 nm wide, large enough to accommodate the lectin domain (~2.5 nm in diameter and 5 nm long) of the FimH molecule.14 Therefore the binding of FimH to UP Ia, located at the bottom of the crevice formed with the joint regions of UPs II and IIIa, favors the formation of protein-protein contacts between FimH with the UP II and IIIa joints. These contacts can then induce large conformational changes in the joint region. Second, we showed previously that the joint region is more flexible, indicated by the weaker electron density, than the rest of the 16-nm particle.14 Hence FimH binding can relatively easily induce conformational changes in this region (Fig. 3). Third, all uroplakins in the 16-nm particle are closely connected, thus enabling conformational changes in one region to be propagated to other regions.14 This high degree of connection occurs on 4 levels: (i) in each subdomain, the single-pass uroplakin is closely packed against the extracellular loop as well as the transmembrane helical bundle of its tetraspanin uroplakin partner; (ii) within each subunit, the inner domain is connected to the outer domain through the top joint; (iii) in the 16-nm particle, the 6 inner domains are interconnected through linkages at the juxtamembrane region;14 (iv) the 5 transmembrane helices of each subdomain form a tightly packed helix bundle;14 thus the helices within a subdomain have to move in a concerted manner. Collectively, these considerations suggest that FimH establishes its initial binding to the UP Ia receptor via its specific interaction with the mannose moiety, and then interacts with a broader area of the uroplakin protein complex. Such a secondary protein-protein interaction then induces global conformational changes in the entire 16-nm particle. Interestingly, we did not observe electron densities in the 3D construction that can be interpreted as the FimH/C complex used in our experiments, even though we have used enough FimH/C to saturate the binding sites on the urothelial plaques and the binding appears stoichiometric (Fig. 1). This suggests that FimH in the complex may be somewhat mobile and its density was averaged out.33
Recent data indicate that the uroplakin complex can transmit signals across the cell membrane. Klumpp and coworkers showed that FimH binding to the uroplakin receptor complex can induce the phosphorylation of a specific threonine residue located in the UP IIIa cytoplasmic tail, and that this binding is followed by an elevation of the intracellular calcium concentration.36 Pharmacological inhibition of these signaling events can block the invasion of the UPEC into the host urothelial cells. These data support the idea that the uroplakin receptor complex is a mediator of urothelial responses to UPEC attachment. In addition, uroplakins are known to play signaling roles in the fertilization of Xenopus eggs.37-39 Fertilization of Xenopus eggs requires the phosphorylation of a cytoplasmic tyrosine residue of UP IIIa by the egg tyrosine kinase Src, and can be blocked by antibodies to the extracellular domain of UPIIIa. These data indicate that uroplakins have multiple transmembrane signaling roles and regulate cellular functions in both mammals and amphibians.
Although the FimH-induced movements of the transmembrane helices of uroplakins are relatively small in comparison with the conformational changes of the extracellular domains, these movements may be sufficient to transmit signals recognizable by downstream molecules that interact with the cytoplasmic domains of the uroplakins. First, an excellent example is provided by G protein-coupled receptors that are known to be able to transmit signals via conformational changes of their transmembrane helices.40 Recent X-ray crystallographic studies have shown that ligand-induced conformational changes of the human β2-adrenergic receptor and A2A adenosine receptor include a movement of the transmembrane helices in the range of 2-7 Å.41,42 This is comparable with the magnitude of the FimH-induced movements of the uroplakin transmembrane helices, which amount to ~6 Å at the cytoplasmic ends of these helices. Such a movement of the helices was shown to be sufficient to expose the G protein binding site on the cytoplasmic surface, thus allowing binding and the initiation of downstream signaling events. Second, the binding of FimH to the high-mannose glycans can exert a mechanical force of up to a hundred pico-Newtons,43,44 and it has been suggested that a force of this magnitude is strong enough to modify the interactions between the cytoplasmic tails of the transmembrane receptors complexes and the cytoskeleton.45 Finally, since the FimH/FimC complex alone, in the absence of other bacterial virulence factors, is sufficient to mediate bacterial uptake by the umbrella cells,27,46 the FimH-induced conformational changes in the uroplakin receptor complex most likely occur in vivo. In addition, EM studies have revealed that, after the bacteria attach to the host urothelial surface, their type 1 fimbriae undergo retraction.25,47 This retraction may further enhance the conformational changes of the uroplakin receptor complex beyond what we have observed in our cryo-EM study performed under static conditions.
The tetraspanin network nature of the urothelial plaques may further enhance the FimH binding-induced signal sensed by the host urothelial umbrella cells. UP Ia and UP Ib belong to the tetraspanin family of proteins that often form ‘tetraspanin-enriched microdomains’ (TEMs), or tetraspanin networks.48 TEMs are membrane domains similar to but distinct from lipid rafts; they serve as a signaling platform and play important roles in regulating cell migration, fusion and other signaling events.20,21,49 The urothelial plaque may be regarded as a unique TEM as thousands of 16-nm particles in each plaque aggregate into 2D membrane crystalline arrays.14,21 This crystalline network may enhance the propagation of the bacterial binding-signal to the cytoplasmic side through multiple cytoskeleton connections within each plaque. The almost complete coverage of the urothelial apical surface by the crystalline plaques may be particularly advantageous to UPEC, allowing this pathogen, which previously thought to be extracellular only, to become invasive in the urinary tract.50 UPEC is able to initiate invasion into the urothelial umbrella cells by the FimH-binding-induced conformational changes in its receptor complex because the structural features of the complex are particularly suitable to transmit this signal.
This is the first report to show that bacterial adhesin-binding can induce conformational changes in transmembrane domains of the host receptor leading to bacterial invasion using the zipper-mechanism. Bacterial pathogens invade the non-phagocytic host cells by two general mechanisms.25,26 In the much better understood trigger mechanism, bacteria such as Shigella and Salmonella use a specialized type III secretion system to inject bacterial proteins into the host cells in order to modulate the activity of the host cell's Rho GTPases, leading to the formation of actin-rich membrane ruffles promoting bacterial invasion. In the zipper mechanism, bacteria such as Listeria and Yersinia as well as UPEC invade the host cells by binding to specific receptors using adhesin molecules.25,51 This binding eventually leads to cytoskeletal rearrangements and bacterial internalization. However, there is a gap in our understanding of how the initial transmembrane signal in this receptor-mediated mechanism sets off the downstream signaling cascade leading to the cytoskeleton rearrangement.51 We showed here that the UPEC adhesin can induce conformational changes in the transmembrane domains of its uroplakin receptor complex, hence transmitting a signal to the cytoplasmic side. The urothelial plaque is known to be connected to the cytoskeleton,52 and the bacterial binding signal may propagate through these linkers to activate Rho-family GTPase pathways and cause cytoskeletal rearrangements and down stream events similar to those of the trigger mechanism.27,53 Hence, results from this study provide an improved understanding of how transmembrane signaling facilitates the early stages of the zipper mechanism of bacterial invasion.
Mouse urothelial plaques (AUM) were isolated by sucrose density gradient centrifugation and differential detergent wash;54,55 the quality of the purified urothelial plaques was assessed by negative staining and EM. FimH/C complex was expressed in E. coli HM 125 56 harboring pFimH-FimC and purified by ion-exchange chromatography as described.57 FimC, which does not affect the function of FimH, is the periplasmic chaperone required to stabilize FimH in its active mannose binding state.23,30-32,58 To form the FimH-bound 16-nm uroplakin particle, two times excess FimH/C was mixed with purified mouse AUM (0.1 mg/ml) and incubated on ice for 45 minutes to 1 hour. The formation of the FimH-uroplakin complexes was verified by pull-down assays and gel electrophoresis (Fig. 1). Judging from the intensities of the protein bands on the gels, FimH bound UP Ia in an approximately 1:1 stoichiometry. All experiments on animals received prior approval from the institutional animal care and use committee of New York University School of Medicine and conformed to the standards set by the appropriate regulatory agencies.
Preparation of the frozen-hydrated samples for cryo-EM has been described previously.14 collecting un-tilted data, we used the back-injection method for grid preparation.59 Drops of ~4.5μl of FimH-bound mouse urothelial plaques were absorbed onto newly prepared carbon films on a molybdenum grid (300 mesh) and transferred to a tannic acid solution (0.75%, pH 6.0). After the excess liquid was blotted with filter paper, the sample was quickly immersed into liquid nitrogen. For collecting tilted data, the carbon sandwich technique,60 for which the urothelial plaques were sandwiched between two sheets of carbon film, was adopted to prepare cryo-EM samples. The frozen samples were then loaded onto an Oxford cryo-holder and transferred to a Philips CM200 FEG electron microscope operated at 200 kV.
Data recorded from a tilted series of images of large urothelial plaques with a diameter up to ~1 μm enabled us to reconstruct a 3D structure of the FimH-liganded particle at 8 Å resolution in the membrane plane and 19.2 Å in the vertical direction (Figs. 2, Table 1 and Fig. S1 in Supplementary Data). Electron Micrographs were taken at a magnification of 50,000× under low-dose mode and with 0.5-1.5 μm underfocus, at up to 55° tilt angles. Only one image was acquired from each plaque. The micrographs were selected using an optical diffractometer and digitized using a ZEISS SCAI scanner at a step of 14 μm, which corresponds to 2.8 Å in the crystal. A total of 65 images was selected from more than 1000 micrographs and processed using the MRC software suite.61 The 3D density maps were visualized and segmented using AMIRA (Mercury Computer System, Inc.). To compare the 3D map of the FimH-liganded 16-nm particle with that of the native one, we first contoured the two density maps, both calculated at 8 Å resolution, at compatible levels (1.2σ for the native and 1.47σ for the FimH bound maps, respectively) so that the electron density map-enclosed volumes are about the same for the two structures. We then segmented the two maps into individual uroplakins to compare the conformational changes in the two structures.14
We thank Drs. George Georgiou (University of Texas at Austin) for the bacterial strain for overexpress FimH/C, Anchi Cheng for the program to calculate the resolution in the vertical direction of the 3D reconstruction. We thank Dr. David Klumpp for sharing a manuscript before publication, and Drs. Milton Adesnik, Gert Krebich and David Sabatini for critically reading the manuscript. This work was supported by National Institutes of Health grant DK52206 and the Schweizerische Nationalfonds.
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