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Gad Frankel

Publications and source records attributed to Gad Frankel.

At least 37 records · Page 2Linked to original sources
PubMed2006

EspF of enteropathogenic Escherichia coli binds sorting nexin 9.

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.

Amino Acid Motifs↗
PubMed2005

Structure of the bundle-forming pilus from enteropathogenic Escherichia coli.

Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization.

Amino Acid Sequence↗
PubMed2005

Molecular basis of antigenic polymorphism of EspA filaments: development of a peptide display technology.

Like many Gram-negative pathogens, enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) use a macromolecular type III secretion system (TTSS) to inject effector proteins into eukaryotic host cells. The membrane-associated needle complex (NC) of the TTSS, which shows broad similarity to the flagellar basal body, is conserved amongst bacterial pathogens. However, the extracellular part of the TTSS of EPEC and EHEC is unique, in that it has a hollow, approximately 12 nm in diameter, filamentous extension to the NC. EspA filaments are homo-polymers made of the translocator protein EspA. The three-dimensional structure of EspA filaments is comparable to that of flagella; the helical symmetry and packing of the subunits forming both filamentous structures are very similar. Like flagella, EspA filaments show antigenic polymorphism as EspA from different EPEC and EHEC clones show no immunological cross-reactivity. In this study, we determined the molecular basis of the antigenic polymorphism of EspA filaments and identified a surface-exposed hypervariable domain that contains the immunodominant EspA epitope. By exchanging the hypervariable domains of EspA(EPEC) and EspA(EHEC) we swapped the antigenic specificity of the EspA filaments. As for the flagellin D3 domain, which is known to tolerate insertions of natural and artificial amino acid sequences, we have inserted short peptides into the surface-exposed, hypervariable domain of EspA. We demonstrated that the inserted peptides are presented on the surface of the recombinant EspA filaments forming a new immunodominant epitope. Accordingly, EspA filaments have a potential to be developed into a novel epitope display system.

Actins↗
PubMed2005

Operon structure and gene expression of the espJ--tccP locus of enterohaemorrhagic Escherichia coli O157:H7.

In this study, we investigated the genetic organisation and expression of the espJ--tccP locus of enterohaemorrhagic Escherichia coli (EHEC O157:H7), which encodes the type III secretion system effector proteins EspJ and TccP. Using flow cytometry and fluorescence microscopy, we found that espJ and tccP constitute an operon, whose expression in vitro is affected by the composition of the medium and environmental signals such as temperature, pH, osmolarity and O(2) pressure. Moreover, expression of the espJ--tccP operon is not regulated by Ler, a LEE-encoded transcriptional regulator, but by an as yet unidentified regulatory system(s).

Escherichia coli O157↗
PubMed2005

Enteropathogenic Escherichia coli: unravelling pathogenesis.

Enteropathogenic Escherichia coli (EPEC) is a gram-negative bacterial pathogen that adheres to intestinal epithelial cells, causing diarrhoea. It constitutes a significant risk to human health and remains an important cause of infant mortality in developing countries. Although EPEC was the first E. coli strain to be implicated in human disease in the 1940s and 1950s, the mechanisms by which this pathogen induced diarrhoea remained a complete mystery throughout most of the 40 years since its description. It was only during the late 1980s that major advances were made in unravelling the mechanisms behind EPEC pathogenesis. Ever since, progress has been made at a stunning pace and there have been major breakthroughs in identifying the bacterial factors involved in attaching and effacing (A/E) lesion formation, host signal transduction pathways in response to EPEC infection and the genetic basis of EPEC pathogenesis. The rapid pace of discovery is a result of intensive research by investigators in this field and portends that EPEC will soon be among one of the most understood diarrhoea-causing infectious agents. This review aims to trace the progress of EPEC research since its existence was first reported by John Bray in 1945, highlighting the major findings that have revolutionised our understanding of EPEC pathogenesis.

Enteritis↗
PubMed2005

Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ.

The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 - amino acid residues 1-55 in the mature protein, and D2 - amino acid residues 90-170), each comprising a three-stranded beta-sheet flanked by two alpha-helices. A flexible region (residues 60-85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ(D1) and EscJ(D2) in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ(D1) and EscJ(D2) in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.

Amino Acid Sequence↗
PubMed2005

Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract.

Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens exploit attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. However, both EHEC and EPEC are poorly pathogenic in mice. In contrast, C. rodentium, which is genetically highly related to E. coli, relies on A/E lesion formation as an essential step in both colonization and infection of the murine mucosa, providing an excellent in vivo model. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity and dynamics of colonization of mice by LB-grown and mouse-passaged C. rodentium in situ and in real time. We have demonstrated the appearance of a 'hyperinfectious' state after passage of C. rodentium through the murine gastrointestinal tract. The 'hyperinfectious' state was found to dramatically reduce the dose required to infect secondary individuals, and also influenced the tissue distribution of colonizing bacteria, removing the requirement for primary colonization of the caecal patch. In addition, the 'hyperinfectious' phenotype was found to be transient with one overnight passage in rich medium sufficient to return C. rodentium to 'culture' infectivity.

Administration, Oral↗
PubMed2005

Citrobacter rodentium of mice and man.

The major classes of enteric bacteria harbour a conserved core genomic structure, common to both commensal and pathogenic strains, that is most likely optimized to a life style involving colonization of the host intestine and transmission via the environment. In pathogenic bacteria this core genome framework is decorated with novel genetic islands that are often associated with adaptive phenotypes such as virulence. This classical genome organization is well illustrated by a group of extracellular enteric pathogens, which includes enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium, all of which use attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. Both EHEC and EPEC are poorly pathogenic in mice but infect humans and domestic animals. In contrast, C. rodentium is a natural mouse pathogen that is related to E. coli, hence providing an excellent in vivo model for A/E lesion forming pathogens. C. rodentium also provides a model of infections that are mainly restricted to the lumen of the intestine. The mechanism's by which the immune system deals with such infections has become a topic of great interest in recent years. Here we review the literature of C. rodentium from its emergence in the mid-1960s to the most contemporary reports of colonization, pathogenesis, transmission and immunity.

Adhesins, Bacterial↗
PubMed2005

Characterization of two non-locus of enterocyte effacement-encoded type III-translocated effectors, NleC and NleD, in attaching and effacing pathogens.

Intestinal colonization by enteropathogenic and enterohemorrhagic Escherichia coli requires the locus of enterocyte effacement-encoded type III secretion system. We report that NleC and NleD are translocated into host cells via this system. Deletion mutants induced attaching and effacing lesions in vitro, while infection of calves or lambs showed that neither gene was required for colonization.

Amino Acid Sequence↗
PubMed2005

Interaction of enteropathogenic Escherichia coli with human intestinal mucosa: role of effector proteins in brush border remodeling and formation of attaching and effacing lesions.

Enteropathogenic Escherichia coli (EPEC) strains deliver effector proteins Tir, EspB, Map, EspF, EspH, and EspG into host cells to induce brush border remodeling and produce attaching and effacing (A/E) lesions on small intestinal enterocytes. In this study, the role of individual EPEC effectors in brush border remodeling and A/E lesion formation was investigated with an in vitro human small intestinal organ culture model of EPEC infection and specific effector mutants. tir, map, espB, and espH mutants produced "footprint" phenotypes due to close bacterial adhesion but subsequent loss of bacteria; an espB mutant and other type III secretion system mutants induced a "noneffacing footprint" associated with intact brush border microvilli, whereas a tir mutant was able to efface microvilli resulting in an "effacing footprint"; map and espH mutants produced A/E lesions, but loss of bacteria resulted in a "pedestal footprint." An espF mutant produced typical A/E lesions without associated microvillous elongation. An espG mutant was indistinguishable from the wild type. These observations indicate that Tir, Map, EspF, and EspH effectors play a role in brush border remodeling and production of mature A/E lesions.

Adhesins, Bacterial↗
PubMed2005

EspJ is a prophage-carried type III effector protein of attaching and effacing pathogens that modulates infection dynamics.

Enterohemorrhagic Escherichia coli, enteropathogenic E. coli, and Citrobacter rodentium are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions. These pathogens utilize a type III secretion system (TTSS) apparatus, encoded by the locus of enterocyte effacement, to translocate bacterial effector proteins into epithelial cells. Here, we report the identification of EspJ (E. coli-secreted protein J), a translocated TTSS effector that is carried on the 5' end of the cryptic prophage CP-933U. Infection of epithelial cells in culture revealed that EspJ is not required for A/E lesion activity in vivo and ex vivo. However, in vivo studies performed with mice demonstrated that EspJ possesses properties that influence the dynamics of clearance of the pathogen from the host's intestinal tract, suggesting a role in host survival and pathogen transmission.

Animals↗
PubMed2005

Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells.

Enteropathogenic Escherichia coli infection of intestinal epithelial cells leads to localized depletion of the microtubule cytoskeleton, an effect that is dependent on delivery of type III translocated effector proteins EspG and Orf3 (designated EspG2) to the site of depletion. Microtubule depletion involved disruption rather than displacement of microtubules.

Actins↗
PubMed2005

Polarity of enteropathogenic Escherichia coli EspA filament assembly and protein secretion.

Type III secretion systems (TTSS) are sophisticated macromolecular structures that play an imperative role in bacterial infections and human disease. The TTSS needle complex is conserved among bacterial pathogens and shows broad similarity to the flagellar basal body. However, the TTSS of enteropathogenic and enterohemorrhagic Escherichia coli, two important human enteric pathogens, is unique in that it has an approximately 12-nm-diameter filamentous extension to the needle that is composed of the secreted translocator protein EspA. EspA filaments and flagellar structures have very similar helical symmetry parameters. In this study we investigated EspA filament assembly and the delivery of effector proteins across the bacterial cell wall. We show that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament. Moreover, EspA filament length is modulated by the availability of intracellular EspA subunits. Finally, we provide direct evidence that EspA filaments are hollow conduits through which effector proteins are delivered to the extremity of the bacterial cell (and subsequently into the host cell).

Cell Line↗
PubMed2005

Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.

Animals↗
PubMed2004

Impaired immunity to intestinal bacterial infection in stromelysin-1 (matrix metalloproteinase-3)-deficient mice.

Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine. We have previously shown enhanced pathology in infected TNFRp55-/-, IL-12p40-/-, and IFN-gamma-/- mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3-/- mice. In MMP3-/- mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3-/- mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3-/- mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3-/- mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa.

Animals↗