SummaryEnteropathogenic Escherichia coli (EPEC) induce characteristic attaching and effacing (A/E) lesions on epithelial cells. This event is mediated, in part, by binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study, we have localized the intimin-binding domain of Tir to a central 107-amino-acid region, designated Tir-M. We provide evidence that both the amino-and carboxy-termini of Tir are located within the host cell. In addition, using immunogold labelling electron microscopy, we have con®rmed that intimin can bind independently to host cells even in the absence of Tir. This Tir-independent interaction and the ability of EPEC to induce A/E lesions requires an intact lectinlike module residing at the carboxy-terminus of the intimin polypeptide. Using the yeast two-hybrid system and gel overlays, we show that intimin can bind both Tir and Tir-M even when the lectin-like domain is disrupted. These data provide strong evidence that intimin interacts not only with Tir but also in a lectinlike manner with a host cell intimin receptor.

SummaryEnteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping 210 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than 2247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the 2300 to 11 region of LEE2; however, it failed to bind to a DNA fragment containing the 2300 to 11 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3::lacZ fusions extended to what would be 2246 bp of the LEE2 operon. A lacZ fusion from the 2300 to 11 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.

Low-level resistance of Neisseria gonorrhoeae to toxic hydrophobic agents (HAS), including some antibiotics, is chromosomally mediated via the multiple transferable resistance (mtr) efflux system. The gene encoding the 48.3 kDa outer-membrane protein MtrE, which is associated with the mtr phenotype, was identified and is homologous to export-associated outer-membrane proteins, including the OprM (formerly OprK) lipoprotein of Pseudomonas aeruginosa. Insertional inactivation of the mtrE gene in N. gonorrhoeae strain FA19 resulted in the loss of the outer-membrane protein, with concomitant hypersusceptibility of the mutant strain to a range of HAS. The properties of this mutant confirmed the role of MtrE in multidrug resistance mediated by an active efflux mechanism. Secondary structure predictions for MtrE indicated a largely hydrophilic protein with a single a-helical transmembrane region. A transposon-like element, similar to that found downstream of the region containing the promoters for mtrR and mtrC in Neisseria meningitidis, was identified 63 bp downstream of the mtrE gene.I I

Eukaryotic cell adhesion is a fundamental process in tissue development, homeostasis, and disease and is mediated by specific interactions of cell surface receptors with extracellular matrix (ECM) 2 proteins (1-5). The ECM is a meshwork of fibrillar and nonfibrillar components assembled into complex structures such as basement membranes. The latter provide a scaffold for cell adhesion, spreading, and migration. ECM regulates numerous cell functions by activating multiple signaling pathways at the adhesion sites. ECMs, composed of collagens, laminins, and other glycoproteins such as fibronectin (FN), serve as substrates for different adhesion molecules including the integrin family of transmembrane receptors. The assembly of ECM components into functional supramolecular modules is highly regulated (3-7). FN matrix assembly alone is a dynamic cell-driven process in which the soluble FN molecules assemble into insoluble fibrillar polymeric ECM structures (8).FN and integrin receptors play crucial roles in a variety of morphogenetic processes, which are regulated by processes termed outside-in and inside-out signaling cascades (3-5). Deregulation of integrin and FN functions associates with disease development including chronic inflammation, heart failure, cancer, and metastasis (7, 9 -11). The outside-in signaling triggered by ligation of integrin receptors with FN and other ECM components results in the reorganization of cytoskeletal and signaling molecules into complexes of more than 90 proteins (9 -13). This occurs by synergistic processes dependent on integrin aggregation and occupancy, as well as tyrosine phosphorylation. Integrins also cooperate with growth factor receptors such as epidermal growth factor receptor (EGFR) to enhance signaling (14).FN consists of multiple domains (classified types I-III) that show binding specificities for specific cell membrane receptors, collagen, fibrin, and heparin. FN alone is sufficient to induce highly efficient spreading of many mammalian cell types including fibroblast and epithelial cells in vitro. An important functional unit of FN is its RGD tripeptide motif, which acts in

SummaryThe pathogenic potential of many Gram-negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host. Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step. Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen. Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled-coil structural feature. Coiled-coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins. The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled-coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins. The known functional flexibility that coiledcoil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.