Biomedical subjects

Stuart Knutton

Publications and source records attributed to Stuart Knutton.

At least 19 recordsLinked to original sources

PubMed2006

Function and distribution of EspG2, a type III secretion system effector of enteropathogenic Escherichia coli.

The enteropathogenic Escherichia coli (EPEC) effector protein EspG, like the Shigella effector VirA, functions through disruption of the host cell microtubule network. Reports have differed as to whether the EspG homologue, EspG2, is also responsible for microtubule disruption. In this study we show that following translocation, EspG2 and VirA are localised under adherent bacteria and able to restore the microtubule disruption phenotype to an espG/espG2 double EPEC mutant. The espG/espG2 double mutant produced A/E lesions similar to wild-type EPEC on human intestinal in vitro organ cultures. Determining the distribution of espG and espG2 among clinical EPEC isolates revealed two different types of espG (espG alpha and espG beta) and espG2 (intact and pseudo genes), which were associated with specific EPEC serotypes and closely followed the EPEC lineage. This investigation has established a role for EspG2 in the disruption of the microtubule network and associated different espG and espG2 types with different groups of EPEC.

Caco-2 Cells↗

PubMed2006

Subversion of actin dynamics by EPEC and EHEC.

During the course of infection, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) subvert the host cell signalling machinery and hijack the actin cytoskeleton to tighten their interaction with the gut epithelium, while avoiding phagocytosis by professional phagocytes. Much progress has been made recently in our understanding of how EPEC and EHEC regulate the pathways leading to local activation of two regulators of actin cytoskeleton dynamics, the Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex. A recent highlight is the unravelling of functions for effector proteins (particularly Tir, TccP, Map and EspG/EspG2) that are injected into the host cell by a type III secretion system.

Actin-Related Protein 2-3 Complex↗

PubMed2006

The enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: implication for cell signalling and diarrhoea.

Enteropathogenic Escherichia coli (EPEC) is the single most important contributor to child diarrhoea in developing countries. Nevertheless, the mechanism responsible for EPEC diarrhoea remains elusive. Using the yeast two-hybrid system to determine the target host cell protein of the EPEC type III secretion system effector Map led to identification of ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+/H+ exchanger regulatory factor 1 (NHERF1). Protein interaction is mediated by the carboxy-terminal Thr-Arg-Leu (TRL) motif of Map and the PSD-95/Disk-large/ZO-1 domain 1 (PDZ1) of EBP50/NHERF1. Although EBP50/NHERF1 is recruited to site of EPEC adhesion in a Map-independent mechanism, co-immunoprecipitation and immunostaining revealed that Map binds to, induces proteolysis of, and colocalizes with EBP50/NHERF1 during infection of cultured epithelial cells. The TRL motif of Map was involved in Map-induced filopodia formation and brush border elongation on infected HeLa and Caco-2 cells respectively. As EBP50/NHERF1 regulates ion channels in the intestine we assessed the involvement of Map in diarrhoea using the Citrobacter rodentium mouse model of EPEC. We report significantly greater diarrhoea following infections with wild-type C. rodentium compared with C. rodentiumDeltamap. These results provide new insights into the mechanisms of EPEC diarrhoea.

Actins↗

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

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

Celiac disease with mild to moderate histologic changes is a common cause of chronic diarrhea in Indian children.

OBJECTIVES: In developed countries, small bowel histology in coeliac disease is a spectrum, ranging from normal with increased intraepithelial lymphocytes to the classic flat mucosa. In developing countries, mild to moderate enteropathies in children with chronic diarrhea and growth failure are assumed to be caused by tropical sprue, persistent infections, or malnutrition with bacterial overgrowth. We report the prevalence and histology of coeliac disease in children with chronic diarrhea at a tertiary referral hospital in North India. METHODS: Two hundred fifty-nine children with symptoms indicating coeliac disease attended the All India Institute of Medical Sciences. Histology was graded after a modified Marsh classification. Serum immunoglobulin A anti-endomysial antibodies (AEA) were assayed using indirect immunofluorescence. Subjects with abnormal histology and positive AEA were put on a gluten free diet (GFD). Coeliac disease was diagnosed on small intestinal biopsy changes and a clinical response to a GFD. RESULTS: Severe enteropathies were present in 63 (24%) subjects, and 58 (92%) responded to a GFD. Sixty-six (25%) had moderate histologic changes, 61 responding to a GFD. AEA was positive in 56 of 63 patients with severe and 65 of 66 with moderate enteropathies. Fifty-seven children had mild enteropathies, and 19 of 20 with positive AEA responded clinically to a GFD. CONCLUSIONS: Coeliac disease is more common than previously believed. It presents a variable histology, and diagnoses may be missed or delayed if based only on severe enteropathies. Serology is a useful adjunct to diagnosis, and diagnostic criteria need to be developed appropriately for coeliac disease in developing countries despite limited facilities.

Adolescent↗

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

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

Identification of an Escherichia coli operon required for formation of the O-antigen capsule.

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.

Bacterial Capsules↗

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↗

PubMed2004

Arylamine N-acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target.

Mycolic acids represent a major component of the unique cell wall of mycobacteria. Mycolic acid biosynthesis is inhibited by isoniazid, a key frontline antitubercular drug that is inactivated by mycobacterial and human arylamine N-acetyltransferase (NAT). We show that an in-frame deletion of Mycobacterium bovis BCG nat results in delayed entry into log phase, altered morphology, altered cell wall lipid composition, and increased intracellular killing by macrophages. In particular, deletion of nat perturbs biosynthesis of mycolic acids and their derivatives and increases susceptibility of M. bovis BCG to antibiotics that permeate the cell wall. Phenotypic traits are fully complemented by introduction of Mycobacterium tuberculosis nat. We infer from our findings that NAT is critical to normal mycolic acid synthesis and hence other derivative cell wall components and represents a novel target for antituberculosis therapy. In addition, this is the first report of an endogenous role for NAT in mycobacteria.

Anti-Bacterial Agents↗

PubMed2004

Involvement of the intermediate filament protein cytokeratin-18 in actin pedestal formation during EPEC infection.

While remaining extracellular, enteropathogenic Escherichia coli (EPEC) establish direct links with the cytoskeleton of the target epithelial cell leading to the formation of actin-rich pedestals underneath attached bacteria. The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins; and recruitment of some is dependent on phosphorylation of Tyr 474. Using Tir as bait and HeLa cell cDNA library as prey in a yeast two-hybrid screen, we identified cytokeratin 18 as a novel Tir partner protein. Cytokeratin 18 is recruited to the EPEC-induced pedestal and has a direct role in actin accretion and cytoskeleton reorganization. This study is the first to implicate intermediate filaments in microfilament reorganization following EPEC infection.

Actins↗

PubMed2004

Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin.

Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic 'attaching and effacing' (A/E) lesions on small intestinal enterocytes. EPEC adhesins that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and intimin. BFP are involved in bacteria-bacteria interaction and microcolony formation but their role in cell adhesion remains unclear; EspA filaments are components of the EPEC type III secretion system but since they interact directly with host cells they may also function as adhesins; intimin is the well characterized intimate EPEC adhesin which binds the translocated intimin receptor, Tir. However, other uncharacterized host cell receptors have been implicated in intimin-mediated adhesion. In this study, the role of BFP, EspA filaments and intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in bfpA, espA and eae (intimin gene) to differentiated human intestinal Caco-2 cells. E2348/69 (bfpA(+) espA(+) eae(+)) adhered rapidly (<10 min) to the brush border of Caco-2 cells and subsequently produced microcolonies and typical A/E lesions. Non-intimate brush border adhesion of double mutant strain UMD880 (bfpA(+) espA(-) eae(-)) also occurred rapidly, whereas adhesion of strain UMD886 (bfpA(-) espA(+) eae(-)) occurred later in the infection (>1 h) and with much lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively. Strain UMD883 (bfpA(-) espA(-) eae(+)), which is unable to translocate Tir, was non-adherent although this strain was able to form intimate attachment and A/E lesions when co-cultured with strain CVD206 (bfpA(+) espA(+) eae(-)) which supplied Tir to the membrane. Single mutant strains CVD206 (bfpA(+) espA(+) eae(-)) and UMD872 (bfpA(+) espA(-) eae(+)) showed adherence characteristics of strain UMD880 (bfpA(+) espA(-) eae(-)), whilst triple mutant strain UMD888 (bfpA(-) espA(-) eae(-)) was totally non-adherent. These results support an adhesive role for BFP and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as initial attachment factors in atypical EPEC which lacks BFP. The study found no evidence for an independent host cell intimin receptor or for other adhesive factors able to support bacterial adherence.

Adhesins, Bacterial↗

PubMed2004

SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD.

Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.

Bacterial Adhesion↗

PubMed2004

TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton.

Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion. When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo. Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro. These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.

Actins↗

PubMed2004

Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 subverts host cells through a type III secretion system encoded by the locus for enterocyte effacement (LEE). Genome sequencing of this pathotype revealed the existence of a gene cluster encoding components of a second cryptic type III secretion system, E. coli type III secretion system 2 (ETT2). Recently, we showed that the ETT2 gene cluster is present in whole or in part in the majority of E. coli strains but is unable to encode a functional secretion system in most strains, including EHEC O157:H7. However, here we show that mutational inhibition of two regulatory genes (ECs3720 or etrA and ECs3734 or eivF) from the ETT2 cluster in EHEC O157:H7 leads to greatly increased secretion of proteins encoded by the LEE and to increased adhesion to human intestinal cells. Studies in which transcriptional fusions and microarrays were used indicated that EtrA and EivF exert profound negative effects on gene transcription within the LEE. Consistent with these observations, expression of these regulators in an EHEC O26:H- strain led to suppression of protein secretion under LEE-inducing conditions. These findings provide fresh examples of the influence of mobile genetic elements on regulation of the LEE and of cross talk between type III secretion system gene clusters. In addition, they provide a cautionary tale because they show that the effects of regulatory genes can outlive widespread decay of other genes in a functionally coherent gene cluster, a phenomenon that we have named the "Cheshire cat effect." It also seems likely that variations in the ETT2 regulator repertoire might account for strain-to-strain variation in secretion of LEE-encoded proteins.

Bacterial Adhesion↗