Robin M. Delahay

Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells

Enteropathogenic 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 confirmed 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 lectin‐like 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 lectin‐like manner with a host cell intimin receptor.

Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler.

Enteropathogenic 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 −10 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 −247 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 −300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the −300 to +1 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 −246 bp of the LEE2 operon. A lacZ fusion from the −300 to +1 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.

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA–EspB protein interactions

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for ‘attaching and effacing’ (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co‐immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp‐2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.

Coiled‐coil proteins associated with type III secretion systems: a versatile domain revisited

Summary The 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 coiled‐coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

ABSTRACT Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagicEscherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenicE. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

A Small Fibronectin-mimicking Protein from Bacteria Induces Cell Spreading and Focal Adhesion Formation

Fibronectin, a 250-kDa eukaryotic extracellular matrix protein containing an RGD motif plays crucial roles in cell-cell communication, development, tissue homeostasis, and disease development. The highly complex fibrillar fibronectin meshwork orchestrates the functions of other extracellular matrix proteins, promoting cell adhesion, migration, and intracellular signaling. Here, we demonstrate that CagL, a 26-kDa protein of the gastric pathogen and type I carcinogen Helicobacter pylori, mimics fibronectin in various cellular functions. Like fibronectin, CagL contains a RGD motif and is located on the surface of the bacterial type IV secretion pili as previously shown. CagL binds to the integrin receptor α5β1 and mediates the injection of virulence factors into host target cells. We show that purified CagL alone can directly trigger intracellular signaling pathways upon contact with mammalian cells and can complement the spreading defect of fibronectin−/− knock-out cells in vitro. During interaction with various human and mouse cell lines, CagL mimics fibronectin in triggering cell spreading, focal adhesion formation, and activation of several tyrosine kinases in an RGD-dependent manner. Among the activated factors are the nonreceptor tyrosine kinases focal adhesion kinase and Src but also the epidermal growth factor receptor and epidermal growth factor receptor family member Her3/ErbB3. Interestingly, fibronectin activates a similar range of tyrosine kinases but not Her3/ErbB3. These findings suggest that the bacterial protein CagL not only exhibits functional mimicry with fibronectin but is also capable of activating fibronectin-independent signaling events. We thus postulate that CagL may contribute directly to H. pylori pathogenesis by promoting aberrant signaling cross-talk within host cells.

The Coiled-coil Domain of EspA Is Essential for the Assembly of the Type III Secretion Translocon on the Surface of EnteropathogenicEscherichia coli

Enteropathogenic E. coli (EPEC) utilize a type III secretion system to deliver virulence-associated effector proteins to the host cell. Four proteins, EspA, EspB, EspD, and Tir, which are integral to the formation of characteristic “attaching and effacing” (A/E) intestinal lesions, are known to be exported via the EPEC type III secretion system. Recent work demonstrated that EspA is a major component of a filamentous structure, elaborated on the surface of EPEC, which is required for translocation of EspB and Tir. The carboxyl terminus of EspA is predicted to comprise an α-helical region, which demonstrates heptad periodicity whereby positions a and d in the heptad repeat unitabcdefg are occupied by hydrophobic residues, indicating a propensity for coiled-coil interactions. Here we demonstrate multimeric EspA isoforms in EPEC culture supernatants and EspA:EspA interaction on solid phase. Non-conservative amino acid substitution of specific EspA heptad residues generated EPEC mutants defective in filament assembly but which retained the ability to induce A/E lesions; additional mutation totally abolished EspA filament assembly and A/E lesion formation. These results demonstrate a similarity to flagellar biosynthesis and indicate that the coiled-coil domain of EspA is required for assembly of the EspA filament-associated type III secretion translocon.

CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map

Map is an enteropathogenic Escherichia coli (EPEC) protein that is translocated into eukaryotic cells by a type III secretion system. Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death. Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD. In this study, we present evidence that the Tir‐specific chaperone, CesT, also performs a chaperone function for Map. Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map. Similar to other chaperone–effector pairings, binding is apparent at the amino‐terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction. Map secretion from a cesT mutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp‐2 cells is almost totally abrogated. Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.

Involvement of the gonococcal MtrE protein in the resistance of Neisseria gonorrhoeae to toxic hydrophobic agents

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 o 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 alpha-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.

Analysis of post-translational modification of mycobacterial proteins using a cassette expression system

A recombinant expression system was developed to analyse sequence determinants involved in O‐glycosylation of proteins in mycobacteria. By expressing peptide sequences corresponding to known glycosylation sites within a chimeric lipoprotein construct, amino acids flanking modified threonine residues were found to have an important influence on glycosylation. The expression system was used to screen mycobacterial sequences selected using a neural network (NetOglyc) trained on eukaryotic O‐glycoproteins. Evidence of glycosylation was obtained for eight of 11 proteins tested. The results suggest that sites involved in O‐glycosylation of mycobacterial and eukaryotic proteins share similar structural features.