Crystal Structures of the Outer Membrane Domain of Intimin and Invasin from Enterohemorrhagic E. coli and Enteropathogenic Y. pseudotuberculosis

National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Structure (Impact Factor: 5.62). 05/2012; 20(7):1233-43. DOI: 10.1016/j.str.2012.04.011
Source: PubMed


Intimins and invasins are virulence factors produced by pathogenic Gram-negative bacteria. They contain C-terminal extracellular passenger domains that are involved in adhesion to host cells and N-terminal β domains that are embedded in the outer membrane. Here, we identify the domain boundaries of an E. coli intimin β domain and use this information to solve its structure and the β domain structure of a Y. pseudotuberculosis invasin. Both β domain structures crystallized as monomers and reveal that the previous range of residues assigned to the β domain also includes a protease-resistant domain that is part of the passenger. Additionally, we identify 146 nonredundant representative members of the intimin/invasin family based on the boundaries of the highly conserved intimin and invasin β domains. We then use this set of sequences along with our structural data to find and map the evolutionarily constrained residues within the β domain.

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Available from: Teresa Przytycka, Oct 22, 2015
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    • "The shortest TAA we have encountered, BN80 143 of the Yersinia phage phiR1 RT, consists entirely of the anchor domain, with the central coiled-coil stalk barely extended by 15 N-terminal residues. The anchor domain is most similar to the translocation domain of canonical single-chain autotransporters (Oomen et al., 2004), and to that of intimins and invasins (Fairman et al., 2012), both of which are 12-stranded -barrels as well; in the latter, however, located at the N-, not the C-terminus. A recent study, which analyzed the similarity of outer membrane proteins in sequence and structure, concluded on the common origin of all OMPs by amplification and divergence of an ancestral -hairpin (Remmert et al., 2010). "
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    ABSTRACT: Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive outer membrane proteins that mediate adhesion to external surfaces in many Gram-negative bacteria. In recent years, several TAAs have been investigated in considerable detail, also at the structural level. However, in their vast majority, putative TAAs in prokaryotic genomes remain poorly annotated, due to their sequence diversity and changeable domain architecture. In order to achieve an automated annotation of these proteins that is both detailed and accurate we have taken a domain dictionary approach, in which we identify recurrent domains by sequence comparisons, produce bioinformatic descriptors for each domain type, and connect these to structural information where available. We implemented this approach in a web-based platform, daTAA, in 2008 and demonstrated its applicability by reconstructing the complete fiber structure of a TAA conserved in enterobacteria. Here we review current knowledge on the domain structure of TAAs.
    International Journal of Medical Microbiology 12/2014; 305(2). DOI:10.1016/j.ijmm.2014.12.010 · 3.61 Impact Factor
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    • "In the case of ATs, the β-barrel is preceded by α-helix linker that fills the lumen and connects its N-terminus with the passenger region [27,28]. In contrast, the β-barrel of Int/Inv proteins is followed by a peptide linker that runs through the lumen connecting its C-terminus to the passenger region [29]. In addition, the passengers of AT and Int/Inv proteins also have distinct structures, namely β-helical rods in most ATs and tandems of Ig-like domains in Int/Inv proteins [30,31]. "
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    ABSTRACT: Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.
    PLoS ONE 09/2013; 8(9):e75126. DOI:10.1371/journal.pone.0075126 · 3.23 Impact Factor
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    • "The same effect is observed when mutating an equivalent glycine residue in a trimeric autotransporter [28]. In both cases, the most conserved residue is a glycine in the middle of the third β-strand counting from the C-terminus, and this is confirmed by the crystal structures [39]. The effects are somewhat less pronounced in invasin compared to YadA. "
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    ABSTRACT: Invasin and intimin are major virulence factors of enteropathogenic Yersiniae and Escherichia coli, mediating invasion into and intimate adherence to host cells, respectively. Several studies have hinted that extracellular portion of these homologous proteins might be exported via an autotransport mechanism, but rigorous experimental proof has been lacking. Here, we present a topology model for invasin and intimin, consistent with the hypothesis that the N-terminal β-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. We confirmed this topology model by inserting epitope tags into the loops of the β-barrel. We further show that obstructing the pore of β-barrel hinders the export of the passenger domain. As for classical autotransport, the biogenesis of invasin and intimin is dependent on the Bam complex and the periplasmic chaperone SurA, whereas the chaperone/protease DegP is involved in quality control. However, compared to classical autotransporters (Type Va secretion), the domain structure of intimin and invasin is inverted. We conclude that proteins of the intimin and invasin family constitute a novel group of autotransported proteins, and propose that this class of autotransporters be termed Type Ve secretion.
    PLoS ONE 11/2012; 7(10):e47069. DOI:10.1371/journal.pone.0047069 · 3.23 Impact Factor
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