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Structure of CFA/I Fimbriae from Enterotoxigenic Escherichia Coli

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Structure of CFA/I Fimbriae from Enterotoxigenic Escherichia Coli Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli Yong-Fu Lia, Steven Pooleb, Kazuya Nishioa, Ken Jangc, Fatima Rasulovab, Annette McVeighb, Stephen J. Savarinob,d,1, Di Xiaa,1, and Esther Bullittc,1 aLaboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4256; bEnteric Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910-7500; cDepartment of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118-2526; and dDepartment of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799 Edited by Roy Curtiss III, Arizona State University, Tempe, AZ, and approved April 22, 2009 (received for review December 16, 2008) Adhesion pili (fimbriae) play a critical role in initiating the events of inducing protective immune responses against bacterial dis- that lead to intestinal colonization and diarrheal disease by ente- ease (15), structural insights gained into fimbrial assembly and rotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts adhesion will lead to new strategies to prevent and treat such an enormous global disease burden. We elucidate atomic struc- diseases. tures of an ETEC major pilin subunit, CfaB, from colonization factor An atomic-resolution structure for a class 5 fimbrial compo- antigen I (CFA/I) fimbriae. These data are used to construct models nent was recently reported for the minor adhesive subunit CfaE for 2 morphological forms of CFA/I fimbriae that are both observed (16). This advance notwithstanding, a detailed elucidation of the in vivo: the helical filament into which it is typically assembled, and structure and assembly of the CFA/I fimbria has posed a an extended, unwound conformation. Modeling and corroborative challenge. We have shown that gold-labeled anti-CfaE poly- mutational data indicate that proline isomerization is involved in clonal antibodies decorate only the tip region of a mature CFA/I the conversion between these helical and extended forms. Our fimbria (17). In addition, EM imaging of CFA/I filaments findings affirm the strong structural similarities seen between class showed that although most fimbriae are helical filaments, re- 5 fimbriae (from bacteria primarily causing gastrointestinal dis- gions of CFA/I fimbriae can undergo unwinding to a thin ease) and class 1 pili (from bacteria that cause urinary, respiratory, fibrillum (18). The structural basis for this morphologic heter- MICROBIOLOGY and other infections) in the absence of significant primary se- ogeneity has not been determined. quence similarity. They also suggest that morphological and bio- To better understand the structure and assembly of CFA/I chemical differences between fimbrial types, regardless of class, fimbria, we have determined the atomic structure of the major provide structural specialization that facilitates survival of each pilin subunit. The geometry of adjacent CfaB subunits covalently bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic linked by genetic engineering was examined in the crystal variation to evade host immune responses, in that residues occu- structures, and compared with fibrillar and helical filament pying the predicted surface-exposed face of CfaB and related class models assembled using constraints provided by EM data. We 13 5 pilins show much higher genetic sequence variability than the found evidence that isomerization of Pro provides a switch for the remainder of the pilin protein. transition from CFA/I pilins arranged in an extended fibrillar form to the helical filamentous form. Last, we provide a spatial frame- crystal structure ͉ pili ͉ diarrheal disease ͉ adhesion ͉ proline isomerization work for understanding class 5 pilus antigenic variation (19). Results nterotoxigenic Escherichia coli (ETEC) use surface fimbriae Structure of CfaEB. Because previous characterizations showed E(also called pili) to attach to host intestinal epithelia, an early, vital step in diarrhea pathogenesis (1). Colonization factor that CfaB subunits follow right after the initiating minor adhe- antigen I (CFA/I) fimbriae are prevalent among ETEC strains sive subunit CfaE in a mature pilus (7) and the structure of and represent the archetype of class 5 fimbriae, the largest class dscCfaE is known (PDB ID 2HB0) (16), we extended dscCfaE of human-specific ETEC colonization factors (2, 3). A 4-gene at its C terminus to include CfaB, with the resulting fusion operon for fimbria-related proteins is shared by all class 5 protein designated CfaEB (Fig. 1A). Resulting crystals dif- fimbriae, which are assembled via the ‘‘alternate’’ chaperone fracted X-rays to 2.1-Å resolution (Table S1), yielding data for pathway, as distinguished from the classic chaperone-usher atomic models of CfaE and CfaB, and insights into intersubunit pathway that guides assembly of class 1 fimbriae, such as P- and orientations (Fig. 2 A–C). type I pili (4). Mature CFA/I fimbriae are a polymer typically The CfaB structure has an anticipated 7-stranded ␤-sandwich consisting of Ͼ1,000 copies of the major pilin subunit CfaB, and fold, belonging to the Ig-like family (Fig. 2B). As in CfaE, CfaB 1 or a few copies of the tip-residing adhesive minor subunit has a barrel-like shape, with the engineered donor strand filling CfaE (5–7). the hydrophobic groove that would be exposed in a native Structures of fimbriae-associated components and pilin com- plexes from bacterial fimbriae have been defined by using X-ray crystallography, electron microscopy (EM), and NMR (8–12). Author contributions: S.J.S., D.X., and E.B. designed research; Y-F.L., S.P., K.N., K.J., F.R., A.M., D.X., and E.B. performed research; Y-F.L., K.N., D.X., and E.B. analyzed data; and Despite low sequence similarity among Gram-negative bacterial Y-F.L., S.J.S., D.X., and E.B. wrote the paper. fimbriae, all pilin subunit structures determined to date, except The authors declare no conflict of interest. those of type IV, share an immunoglobulin (Ig)-like fold with an This article is a PNAS Direct Submission. exposed hydrophobic groove (10, 13). A ␤-strand contributed by Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, the adjacent subunit fills this groove via ‘‘donor strand ex- www.pdb.org (PDB ID code 3F83 for CfaEB, 3F84 for CfaBB, and 3F85 for CfaBBB). change’’ (9, 10), visualized in Fig. 1B. By engineering recombi- 1To whom correspondence may be addressed. E-mail: [email protected], [email protected], nant proteins in which a subunit is created that fills its own or [email protected]. groove, researchers have successfully expressed subunits suitable This article contains supporting information online at www.pnas.org/cgi/content/full/ for structure determination (8, 14). Because fimbriae are capable 0812843106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812843106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 1, 2021 The CfaEB structure elucidates the structural relationship between CfaE and the adjoining CfaB subunit. Chemical inter- actions between CfaEpd and CfaB in CfaEB are very limited, featuring only 4 hydrogen bonds and no salt bridges. The total buried surface area at the CfaEpd–CfaB interface is 505 Å2 (Table S3), indicative of minimal interactions between the 2 domains. Last, there is a significant bend (joint angle, 138°; twisting angle, 138.4°) between CfaEpd and CfaB, such that the entire CfaEB fusion is J-shaped (Fig. 2C and Table S2). Structures of Multiple CfaB Subunit Fusions. As characterized bio- chemically, a CFA/I fimbria is composed of about 1,000 copies of the major fimbrial subunit arranged in an ordered assembly governed by the donor-strand exchange mechanism. To eluci- date the geometric relationship between 2 neighboring CfaB subunits, we followed the same strategy as that used to design the CfaEB fusion, genetically engineering a CfaBB fusion protein that emulates 2 adjacent, noncovalently linked CfaB subunits (see Methods and Fig. 1B). Crystals of CfaBB diffracted X-rays to 2.3-Å resolution. Molecular replacement methods were used to identify 2 CfaBB molecules, called ‘‘A’’ and ‘‘B,’’ in a crystallographic asymmetric unit cell, corresponding to 4 pilin subunits for which atomic models were refined (Table S1). The 2 CfaB subunits in each CfaBB are identically folded (Fig. 2D). Although both subunits in molecule A are tightly packed, 1 subunit of molecule B is flexible, giving rise to poor density. These differences between molecules A and B suggest a degree of flexibility in the CfaB intersubunit spatial orientation. This significant degree of Fig. 1. Schematic depiction of recombinant fusion proteins containing CfaB. freedom is corroborated by the relatively large variations in joint (A) CfaEB comprises (in sequence from N to C terminus) native full-length CfaE, (8°) and twisting (18.4°) angles between adjacent major subunits including the N-terminal signal peptide, adhesin domain, and pilin domain; a (Table S2). In addition, contacts between 2 CfaB subunits are tetrapeptide linker (designated hereafter as linker); mature CfaB (i.e., without signal peptide); linker; the first 19 aa of a mature CfaB (containing the donor weak, with only 3 hydrogen bonds (2 water-mediated), and no strand, ds); and a hexahistidine affinity tag (His)6.(B) CfaBB comprises mature salt bridges (Table S3). CfaB with an N-terminal deletion (ntd) of its first 15 residues; linker; a copy of We genetically engineered a trimeric, donor-strand-exchanged mature CfaB; linker; the first 15 residues from the mature CfaB N terminus CfaB fusion, designated CfaBBB (Fig. 1C), to determine (containing the ds); (His)6. The diagram illustrates how this genetic construct whether the relationship derived from the CfaBB fusion is replicates the donor-strand exchange mechanism of assembled CFA/I fim- generally applicable to all polymerized CfaB subunits. Crystals briae.
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