The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection.
Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of Tir(EPEC/CR) Y474/1 leads to recruitment of Nck and neural Wiskott-Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. Tir(EPEC/CR) also contains an Asn-Pro-Tyr (NPY(454/1)) motif, which triggers weak actin polymerization. In EHEC the NPY(458) actin polymerization pathway is amplified by TccP/EspF(U), which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in Tir(CR) abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.
Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway.
ABSTRACT The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH , which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC Δ nleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium , which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.
Feruloyl esterases (E.C. 3.1.1.73), a subclass of the carboxylic acid esterases (E.C. 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell walls, and have been classified as Types A or B based on their substrate specificity for aromatic moieties. They constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the production of feruloyl esterases by the filamentous fungi Talaromyces slipitalus and Neurospora crassa.
Neurospora crassa has been shown to produce multiple feruloyl esterase activities depending upon the time of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and over-expressed in Pichia pasloris. The gene encodes a single domain feruloyl esterase (NcFae-l), which represents the first report of a nonmodular Type-B enzyme and the purified recombinant protein has been shown to exhibit concentration dependent substrate inhibition. The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilisation of plant cell wall materials and their respective modes of action.
A novel feruloyl esterase (TsF AEC, Type-C feruloyl esterase) that exhibits broad substrate specificity in culture supernatants of Talaromyces slipitalus when grown on sugar beet pulp, has been cloned and over-expressed in Pichiapasloris. Various gene fusions have been constructed to investigate the use of alternative signal peptides by P. pastoris and to produce an authentic feruloyl esterase featuring the N-terminal sequence determined for the native enzyme. It has been demonstrated that additional amino acids at the N-terminus of the FAEC sequence do not influence the catalytic capacity of the enzyme, and that the nature of the signal sequence does not appreciably alter the yield of the secreted enzyme.
NcFae-l and TsF AEC contain internal peptide sequences that correspond with the consensus motif G-X-S-X-G that contains the catalytic serine nucleophile conserved in the esterase enzyme supcrfamily. The serine residues at the centre of these peptide motifs have been independently mutated and the corresponding enzymes overexpressed in P. pastoris to identify essential serine residues as candidate nucleophiles responsible for catalysing the enzymatic reaction.
Based on activity profile data and supported by the characterisation of a recombinant Type-D feruloyl esterase from N. crassa, a feruloyl esterase sub-classification is proposed and discussed in terms of the evolutionary relationships existing between carbohydrate esterases.
Summary 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 β‐sheet flanked by two α‐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.
Enteropathogenic Escherichia coli (EPEC) strains are defined as extracellular pathogens which nucleate actin rich pedestal-like membrane extensions on intestinal enterocytes to which they intimately adhere. EPEC infection is mediated by type III secretion system effectors, which modulate host cell signaling. Recently we have shown that the WxxxE effector EspT activates Rac1 and Cdc42 leading to formation of membrane ruffles and lamellipodia. Here we report that EspT-induced membrane ruffles facilitate EPEC invasion into non-phagocytic cells in a process involving Rac1 and Wave2. Internalized EPEC resides within a vacuole and Tir is localized to the vacuolar membrane, resulting in actin polymerization and formation of intracellular pedestals. To the best of our knowledge this is the first time a pathogen has been shown to induce formation of actin comets across a vacuole membrane. Moreover, our data breaks the dogma of EPEC as an extracellular pathogen and defines a new category of invasive EPEC.
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