Dynamin is required for F-actin assembly and pedestal formation by enteropathogenic Escherichia coli (EPEC)

Unsworth, Kate E.; Mazurkiewicz, Piotr; Senf, Freya; Zettl, Markus; McNiven, Mark A.; Way, Michael; Holden, David W.

doi:10.1111/j.1462-5822.2006.00801.x

Cited by 41 publications

(46 citation statements)

References 49 publications

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“…For instance, internalized occludin colocalizes with caveolin-1 and dynamin II, which is blocked by dominant negative dynamin II (K44A) and inhibition of caveola-mediated endocytosis by cholesterol extraction prevented both latrunculin A-induced TER loss and occludin internalization (41). Conversely, for EPEC infections, other authors have found that dynamin is required for F-actin assembly and pedestal formation by EPEC E2348/69 (47) and that EPEC E2348/69 Tir translocation and pedestal formation require membrane cholesterol (1). Recently, Alto et al (2) also reported that that the type III effector EspF coordinates membrane remodeling and F-actin polymerization during EPEC pathogenesis, since EspF activated both SNX9 and N-WASP in a coordinated spatiotemporal pattern at clathrin-coated pits, again indicating a relationship between endocytosis and actin polymerization.…”

Section: Infect Immunmentioning

confidence: 99%

EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

Peralta-Ramirez¹,

Hernández²,

Manning‐Cela

et al. 2008

Infect Immun

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Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability.

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Section: Infect Immunmentioning

confidence: 99%

EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

Peralta-Ramirez¹,

Hernández²,

Manning‐Cela

et al. 2008

Infect Immun

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“…We attempted to assess this but were unable to accurately localize dynamin at pedestals. This was likely due to the different antibodies that were used by Unsworth and coworkers (18) and those that we could acquire commercially. How does clathrin and the endocytic machinery mediate pedestal formation and not bacterial internalization?…”

Section: Discussionmentioning

confidence: 92%

“…Dynamin-2 was also recently identified at EPEC pedestals (18). During endocytosis, dynamin-2 has a dual function in both formation of the endocytic vesicle and pinching off from the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…It was demonstrated that dynamin recruitment required Tir, Nck, and N-WASP (18). However, cells with dynamin knockdown prevented the recruitment of N-WASP, Arp3, and cortactin (18). The precise arrangement of dynamin-2 either upstream or downstream of N-WASP requires further clarification.…”

Section: Discussionmentioning

confidence: 99%

“…Although the role of clathrin during enteropathogenic E. coli infections was not investigated until recently (20), the finding of clathrin at the tips of EPEC pedestals, coupled with the discovery of dynamin-2, another protein known to be involved in endocytosis, associated within the actin stalk of EPEC pedestals (18), suggests a possible role for additional endocytosis-associated proteins and indicates that a unique mechanism is employed by EPEC to remain extracellular despite the presence of these endocytic components. Other proteins, including the actin-associated protein cortactin, are also prominent at these structures.…”

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confidence: 99%

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Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation

et al. 2010

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Enteropathogenic Escherichia coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.The extracellular bacterial pathogen enteropathogenic Escherichia coli (EPEC) causes serious diarrheal disease in humans and is a prevalent microbe involved in childhood mortality in the developing world. This microbe is part of a larger family of bacteria called the attaching and effacing (A/E) pathogens that also includes the human-specific pathogen enterohemorrhagic E. coli (EHEC) and the murine disease-causing bacterium Citrobacter rodentium. These bacteria attach to intestinal epithelial cells and use a type III secretion system to directly deliver effector proteins from the bacterial cytosol into the cytoplasm of host cells. Among other functions, these effectors harness the host cell's cytoskeleton (4) to generate actin-rich pedestals that are hallmarks of virulence for this class of pathogens (14). One of these effectors, the translocated intimin receptor (Tir), is key to pedestal formation. Following translocation into the host cell, Tir becomes embedded in the host cell plasma membrane, where its extracellular domain acts to firmly anchor the pathogen to the epithelial cell. In cultured cells, the intracellular cytoplasmic domain of EPEC Tir can become phosphorylated at tyrosine 474 (Y474) (6), where it recruits the adaptor protein Nck (7). These events all occur prior to actin filament polymerization beneath the attached bacteria via an N-WASP-and Arp2/3-based mechanism (7, 11). Although this is the prominent strategy used by EPEC to recruit actin to pedestals, a Y474-independent strategy also exists, but it occurs at a much lower frequency. During such instances, EPEC Tir becomes phosphorylated at Y454 and actin recruitment is independent of Nck (1).Previous work highlighted a role for clathrin during some bacterial infections (19,20). A...

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Desmosomes are unaltered during infections by attaching and effacing pathogens

Guttman

Kazemi

Lin

et al. 2007

The Anatomical Record

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The human attaching and effacing (A/E) intestinal pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and the murine A/E pathogen Citrobacter rodentium cause serious diarrhea in their hosts. These bacteria alter numerous host cell components, including organelles, the host cell cytoskeleton, and tight junctions during the infectious process. One of the proteins that contribute to the intermediate filament network in host cells, cytokeratin-18, is extensively altered during EPEC infections.Based on this, we tested the hypothesis that desmosomes, the only intercellular junctions that interact with intermediate filaments, are also influenced by A/E pathogen infections. We found that the desmosomal transmembrane proteins desmoglein and desmocollin, as well as the desmosome plaque protein desmoplakin, all remain unchanged during EPEC infection in vitro. This evidence is corroborated by the unaltered localization of desmoglein and desmoplakin in vivo in mice infected with C. rodentium for 7 days. Electron microscopic analysis of 7-day C. rodentium-infected murine colonocytes also show no observable differences in the desmosomes when compared to uninfected controls. Our data suggest that, unlike tight junctions, the desmosome protein levels and localization, as well as desmosome morphology, are unaltered during A/E pathogenesis.

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Dynamin is required for F-actin assembly and pedestal formation by enteropathogenic Escherichia coli (EPEC)

Cited by 41 publications

References 49 publications

EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation

Desmosomes are unaltered during infections by attaching and effacing pathogens

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