Source: http://www.asmscience.org/content/book/10.1128/9781555818395.chap4
Timestamp: 2019-04-24 06:02:36+00:00

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Proposed steps in A/E lesion formation by EPEC. (a) The first step of EPEC infection involves localized adherence to the host intestinal epithelium, which does not require the LEE pathogenicity island and is mediated by the plasmid-encoded bundle forming pilus (BFP). (b) The second step involves assembly of the LEE-encoded TTSS and its translocon. Type III effectors including Tir are delivered into the host. Translocated Tir is phosphorylated and inserts into the host cell membrane by an unknown process. (c) The last step involves intimin binding to Tir. The clustering of Tir by intimin results in very intimate attachment. This binding also transmits a signal via the transmembrane domains and cytoplasmic tails of Tir, which results in recruitment of cytoskeletal proteins, massive reorganization of the host cytoskeleton, and, ultimately, formation of the actin-rich pedestal.
Genetic organization of the LEE pathogenicity island of EPEC. The EPEC LEE contains 41 open reading frames (ORFs) organized into at least five operons, designated LEE1, LEE2, LEE3, LEE4, and LEE5/Tir. The esc genes encode structural components of the TTSS, whereas the esp genes encode type III secreted/translocated proteins. The LEEencoded regulator Ler is located in the LEE1 operon.
EPEC type III secretion and proposed membrane topology of EPEC Tir. Upregulation of LEE gene expression results in the assembly of a TTSS across the bacterial membrane. Three type III secreted proteins, EspA, EspB, and EspD, then assemble a translocon structure which mediates protein translocation into the host cell. EspA polymerizes into a filament which is thought to be the translocation tube, and EspB and EspD form a hetero-oligomeric pore on the surface of host cells. Type III effectors including Tir are translocated into the host on proper assembly of the TTSS and its translocon. Once translocated into the host cell, Tir is thought to insert into the host cell membrane and adopt a hairpin-like conformation. Tir is predicted to contain two transmembrane segments (TM1, residues 259 to 234; TM2, residues 363 to 382). The N-terminal (residues 1 to 233) and C-terminal (residues 383 to 549) tails of Tir localize to the host cytoplasm. The intervening region between the two putative transmembrane domains makes up the intiminbinding domain.
Structural similarities among type III secretion chaperones. Shown are ribbon representations of three type III secretion chaperones: EHEC CesT, Salmonella SigE, and Yersinia SycE. Despite the lack of sequence identities among these three proteins, their three-dimensional structures are very similar. All contain hydrophobic surfaces. Diagrams were generated with Molscript ( Kraulis, 1991 ) and Raster3D ( Merritt and Bacon, 1997 ), using PDB accession codes 1K3E (CesT), 1K3S (SigE), and 1JYA (SycE). Due to domain swapping, two possible homodimers can be chosen for the CesT structure ( Luo et al., 2001 ), only one of which is shown here.
Isothermal titration calorimetric analysis of Tir-intimin in the presence of the Tir chaperone CesT. CesT-bound Tir was titrated against the extracellular fragment of intimin in solution ( Luo et al., 2001 ). Tir retains its ability to bind intimin with high affinity (K a , 2.2 × 107 M-1) even in the presence of CesT, suggesting that the CesT chaperone does not act to globally unfold Tir.
The EPEC-host cell adhesion interface based on the structural model of Tir IBD-intimin. Intimin contains an eight-residue linker with a glycine near each end that serves as a flexible hinge, mediating mechanical movement between the rigid extracellular domains (D0 to D3) and the bacterial outer membrane to which it is anchored. Dimeric Tir IBD, which forms a four-helix bundle, is stabilized by multiple hydrophobic and hydrogenbonded interactions with intimin. Finally, EPEC is anchored to the host actin cytoskeleton via the N-terminal tails of Tir, which binds focal adhesion proteins.
Structural similarities between the extracellular fragment of Yersinia invasin and EPEC intimin. (a) Despite low sequence identity (22%), the overall architecture between invasin and intimin is conserved, with 241 of 282 backbone Cα of the intact intimin structure matching invasin, with a 2.9-Å root mean squared deviation. Both molecules are shaped as rigid rods composed of tandemly repeated Ig domains (three for intimin and four for invasin) and terminate with a receptor-binding domain which adopts a fold similar to that of C-type lectins (D3 for intimin and D5 for invasin) and which directly mediates the specific and unique interactions with the cognate receptor ligand (Tir for intimin and β1-integrins for invasin). In both systems, the two most C-terminal domains are intimately associated, with ∼1,500 Å2 of buried surface between them. Ribbon representations were generated using data in PDB accession codes 1CWV (invasin) and 1F00 (intimin). (b) Comparison of the C-type lectin-like domains. The D3 domain of intimin and the D5 domain of invasin structurally resemble the C-type lectin family, a well-characterized example of which is MBP. However, unlike MBP, which is shown with bound calcium (spheres) and carbohydrate (ball and stick), intimin D3 and invasin D5 lack the necessary structural features required for such interactions with calcium and carbohydrate. The Tir-binding face of intimin and the putative integrin- binding surface of invasin are shown in grey. The ribbon representation of the MBP-carbohydrate complex was generated using PDB accession code 2MSB.
Tir is a multifunctional protein. The three different domains of Tir perform unique biological functions. Tir acts as a receptor of intimin, and the central extracellular intimin-binding domain mediates its interaction with intimin, resulting in intimate attachment. The N-terminal region of Tir recruits focal adhesion proteins, including talin and α-actinin, which anchors EPEC to the host actin cytoskeleton. The C-terminal region of Tir functions as a signaling platform. Phosphorylation of tyrosine 474 by an unknown host kinase results in recruitment of the host adaptor protein Nck and subsequent downstream activation of WASP and the Arp2/3 complex. These events ultimately lead to formation of actin-rich pedestals.
Dimerization of Tir and structural comparison with ColE1 Rop. Each Tir IBD monomer consists of two long α-helices that mediate dimerization by forming an antiparallel four-helix bundle similar to ColE1 Rop (the root mean squared deviation on 80 common backbone Cα atoms is 3.2 Å). As shown, the dimerization interface between the two Tir IBDs is relatively hydrophobic. Data used for this representation were taken from PDB accession codes 1ROP (ColE1 Rop) and 1F02 (Tir IBD).
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