1.B.54 The Intimin/Invasin (Int/Inv) or Autotransporter-3 (AT-3) Family

The Intimin/Invasin (Int/Inv) or Autotransporter-3 (AT-3) family of adhesins are outer membrane (OM) proteins found in strains of Yersinia spp. (Inv), pathogenic E. coli (Int), and Citrobacter spp. (Int). These homologous proteins mediate bacterial attachment and invasion of these pathogens into their host cells. Intimins/Invasins are translocated from the cytoplasm across the IM via the Sec-translocase and are related to each other both in terms of sequence and structure (Gal-Mor et al., 2008; Adams et al., 2005; Wentzel et al., 2001; Tsai et al., 2010).

Both intimins and invasins expose structurally similar domains on the bacterial cell surface resembling an extended rigid rod made of domains similar to eukaryotic members of the immunoglobulin superfamily. The carboxy-termini have a folding topology related to C-type lectin-like receptor-binding domains, which are separated from a membrane-embedded N-terminal domain by several tandem Ig-like repeats, four in invasins and three in intimins. With >36% identity existing within the first 500 amino acids, the conserved N-terminal domains are believed to form porin-like β-barrels in the OM. These domains are probably used to export the C-terminal passenger domains across the outer bacterial cell membrane (Adams et al., 2005; Batchelor et al., 2000; Gal-Mor et al., 2008). Leo et al. (2012) review these and other (putative) autotransporters.

The extracellular C-terminus of an Int/Inv is responsible for receptor binding to Tir (translocated intimin receptor) and b1 integrin, respectively. Intimins are surface proteins of enteropathogenic and enterohemorrhagic E. coli that promote intimate bacterial adhesion associated with attaching and effacing lesion formation ( Adams et al., 2005; Gal-Mor et al., 2008 ). The Tir binding sites of intimins are located at the opposite side of the C-terminal lectin-like domain. Invasins lack the short α-helix (residues 904-909 of intimin) involved in Tir binding (Luo et al., 2000). Invasins bind to high-affinity members of the b1 family of integrins to mediate bacterial entry into eukaryotic cells (Adams et al., 2005).

Intimin-mediated adhesion of bacterial cells to eukaryotic target cells can be mimicked by surface display of a short fibrinogen receptor binding peptide. Intimate bacterial adhesion associated with attaching and effacing lesion formation is promoted by intimin. Intimin targets the translocated intimin receptor (Tir) which is exported by the bacterium and integrated into the host cell membrane. Tir is introduced into the host cell membrane via a type III protein secretion/translocation system. For both Int/Inv, a C-terminal fragment of ~190 aa is sufficient for function although no significant sequence similarity is observed. At least five different subtypes of intimins have been described. They are integrated into the E. coli outer membrane by their amino-terminal regions, while the carboxy-terminal 280 amino acids are surface exposed (Batchelor et al., 2000; Wentzel et al., 2001).

Outer membrane intimin directs attachment of enteropathogenic Escherichia coli (EPEC) via its Tir receptor in mammalian target cell membranes. Phosphorylation of Tir triggers local actin polymerization and the formation of 'pedestal-like' pseudopods. Touzé et al., 2004 demonstrated that the intimin protein contains three domains, a flexible N-terminus (residues 40-188), a central membrane-integrated β-barrel (189-549), and a tightly folded Tir-binding domain (550-939). Intimin was shown by electron microscopy to form ring-like structures with an approximately 7 nm external diameter and an electron dense core, and to form channels of 50picoSiemens conductance in planar lipid bilayers. Gel filtration, multiangle light scattering and cross-linking showed that this central β-barrel membrane-anchoring domain directs intimin dimerization. A high affinity, with a 2 : 1 stoichiometry between dimeric intimin and Tir was shown (Touzé et al., 2004). This interaction determines a reticular array-like superstructure underlying receptor clustering.

The EaeA intimin from EHEC O157:H7 is 939 amino acids long. It contains an N-terminal transporter domain, which resides in the bacterial OM and promotes translocation of 4 C-terminally attached passenger domains across the bacterial cell outer membrane. The cell binding activity of EaeA has been localized to its C-terminal 280 residues. It is assumed that the amino-terminal 550 residues of intimin form a porin-like structure and are folded into an antiparallel β-barrel. The entire extracellular segment forms an elongated and relatively rigid rod made up of three immunoglobulin-like domains and a C-terminal lectin-like domain which interacts with the receptors. This domain resides on a rigid extracellular arm, which is most likely anchored to the amino-terminal transmembrane domain through a flexible hinge that includes two noncontiguous conserved glycine residues. They allow mechanical movement between the extracellular rod and the bacterial outer membrane (Wentzel et al., 2001; Luo et al., 2000 ). Intimin forms a ring shaped structure with a 7nm diameter and a channel (with conductance of 50 pS) which can accommodate peptide chains, but not fully folded passenger domains (Adams et al., 2005).

Int/Inv systems are primarily involved in virulence of Gram-negative pathogens. Pathogenic gram-negative bacteria have developed many distinct secretion mechanisms for the efficient surface display of binding domains which specifically interact with their complementary receptors on host cell surfaces (Saier, 2006; Wentzel et al., 2001). While not much is known about the secretion mechanisms of Int/Inv, the passenger domains may be secreted by an Autotransporter-like mechanism ( Gal-Mor et al., 2008).

The transport reaction catalyzed by the Int/Inv family is:

Passenger domain (periplasm) --> passenger domain (extracellular medium)