The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment

Mallick, Emily M.; Garber, John J.; Vanguri, Vijay K.; Balasubramanian, Sowmya; Blood, Timothy; Clark, Stacie; Vingadassalom, Didier; Louissaint, Christopher; McCormick, Beth A.; Snapper, Scott B.; Leong, John M.

doi:10.1111/cmi.12302

Cited by 25 publications

(31 citation statements)

References 96 publications

(160 reference statements)

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“…High levels of colonization were also observed in colonic luminal washes and colonic homogenates following sacrifice. These titers of CR(ΦStx 2dact ) were roughly equal to the peak fecal shedding, colonic colonization, and luminal washes previously reported (Mallick et al, 2012, 2014) by intra-gastric inoculation. These data indicate that ingestion of food inoculated with mid-log phase bacteria grown without shaking results in efficient intestinal colonization.…”

Section: Resultssupporting

confidence: 77%

“…The production of Stx by C. rodentium has been associated with slightly higher levels of intestinal colonization following inoculation by oral gavage (Mallick et al, 2012, 2014). Therefore, we also infected mice with CR(ΦStx 2dact :: kan R ), a CR(ΦStx 2dact ) mutant that does not produce Stx.…”

Section: Resultsmentioning

confidence: 99%

“…Two days prior to infection, CR(ΦStx 2dact ) and CR(ΦStx 2dact :: kan R ), described previously (Mallick et al, 2012, 2014), were streaked from frozen glycerol stock onto LB agar containing the appropriate antibiotic (i.e., 10 μg/ml chloramphenicol for CR(ΦStx 2dact ), and 10 μg/ml chloramphenicol and 25 μg/ml kanamycin CR(ΦStx 2dact :: kan R ) for single colonies. The following day, a single colony was inoculated into 40 ml LB broth (without antibiotic) in a 50 ml conical tube (VWR, catalog number 89039-662) and incubated at 37°C with 5% CO 2 , without shaking but with the cap loosened to allow for gas exchange.…”

Section: Methodsmentioning

confidence: 99%

“…To address these limitations, an alternative model, using conventional mice, was developed. The native murine AE pathogen C. rodentium (CR), which like EHEC forms attaching and effacing lesions, was lysogenized with phage Φ1720a-02, isolated from an Stx-producing E. coli strain (Mallick et al, 2012, 2014). This phage encodes and produces Stx variant Stx2dact, which is activated ~18-fold by intestinal mucus (Mallick et al, 2012; Bunger et al, 2013), resulting in high potency in mice (Teel et al, 2002; Bunger et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation

Flowers

Ghanem

Leong

2016

Front. Cell. Infect. Microbiol.

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Upon colonization of the intestinal epithelium, the attaching and effacing (AE) pathogen Enterohemorrhagic Escherichia coli (EHEC) effaces microvilli and forms pedestal-like structures beneath the adherent bacterium. The production of one of its virulence factors, the phage-encoded Shiga toxin (Stx) results in systemic disease, including the development of renal failure. Although EHEC does not productively infect conventional mice, EHEC infection can be modeled in mice utilizing a derivative of the natural murine AE pathogen Citrobacter rodentium (CR). Gavage of mice with CR(ΦStx2dact), a C. rodentium lysogenized by a phage encoding an Stx variant with high potency in mice, features AE lesion formation on intestinal epithelium and Stx-mediated systemic disease, including renal damage. This model is somewhat limited by mouse-to-mouse variation in the course of disease, with the time to severe morbidity (and required euthanasia) varying by as many as 5 days, a feature that limits pathological analysis at defined stages of disease. In the current study, we altered and optimized the preparation, dose, and mode of delivery of CR(ΦStx2dact), using food-borne route of infection to generate highly synchronous disease model. We found that food-borne inoculation of as few as 3 × 104 CR(ΦStx2dact) resulted in productive colonization and severe systemic disease. Upon inoculation of 1 × 108 bacteria, the majority of infected animals suffered weight loss beginning 5 days post-infection and all required euthanasia on day 6 or 7. This enhanced murine model for EHEC infection should facilitate characterization of the pathology associated with specific phases of Stx-mediated disease.

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Section: Resultssupporting

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation

Flowers

Ghanem

Leong

2016

Front. Cell. Infect. Microbiol.

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“…Similarly, a C. rodentium strain deficient for pedestal formation was outcompeted by a wild-type strain in mixed infections (34). Finally, the ability of A/E pathogens to induce pedestals in cultured cells is correlated with their ability to induce disease in humans and in animal models (31,35).…”

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confidence: 99%

Actin Pedestal Formation by Enterohemorrhagic Escherichia coli Enhances Bacterial Host Cell Attachment and Concomitant Type III Translocation

et al. 2014

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c Attachment of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelial cells is critical for colonization and is associated with localized actin assembly beneath bound bacteria. The formation of these actin "pedestals" is dependent on the translocation of effectors into mammalian cells via a type III secretion system (T3SS). Tir, an effector required for pedestal formation, localizes in the host cell plasma membrane and promotes attachment of bacteria to mammalian cells by binding to the EHEC outer surface protein Intimin. Actin pedestal formation has been shown to foster intestinal colonization by EHEC in some animal models, but the mechanisms responsible for this remain undefined. Investigation of the role of Tir-mediated actin assembly promoting host cell binding is complicated by other, potentially redundant EHEC-encoded binding pathways, so we utilized cell binding assays that specifically detect binding mediated by Tir-Intimin interaction. We also assessed the role of Tir-mediated actin assembly in two-step assays that temporally segregated initial translocation of Tir from subsequent Tir-Intimin interaction, thereby permitting the distinction of effects on translocation from effects on cell attachment. In these experimental systems, we compromised Tir-mediated actin assembly by chemically inhibiting actin assembly or by infecting mammalian cells with EHEC mutants that translocate Tir but are specifically defective in Tir-mediated pedestal formation. We found that an inability of Tir to promote actin assembly resulted in a significant and striking decrease in bacterial binding mediated by Tir and Intimin. Bacterial mutants defective for pedestal formation translocated type III effectors to mammalian cells with reduced efficiency, but the decrease in translocation could be entirely accounted for by the decrease in host cell attachment.

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Cytoskeletal mechanisms regulating attaching/effacing bacteria interactions with host cells: It takes a village to build the pedestal

Naydenov,

Marino‐Melendez,

Campellone

et al. 2024

BioEssays

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The actin cytoskeleton is a key cellular structure subverted by pathogens to infect and survive in or on host cells. Several pathogenic strains of Escherichia coli, such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), developed a unique mechanism to remodel the actin cytoskeleton that involves the assembly of actin filament‐rich pedestals beneath the bacterial attachment sites. Actin pedestal assembly is driven by bacterial effectors injected into the host cells, and this structure is important for EPEC and EHEC colonization. While the interplay between bacterial effectors and the actin polymerization machinery of host cells is well‐understood, how other mechanisms of actin filament remodelling regulate pedestal assembly and bacterial attachment are poorly investigated. This review discusses the gaps in our understanding of the complexity of the actin cytoskeletal remodelling during EPEC and EHEC infection. We describe possible roles of actin depolymerizing, crosslinking and motor proteins in pedestal dynamics, and bacterial interactions with the host cells. We also discuss the biological significance of pedestal assembly for bacterial infection.

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The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment

Cited by 25 publications

References 96 publications

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation

Actin Pedestal Formation by Enterohemorrhagic Escherichia coli Enhances Bacterial Host Cell Attachment and Concomitant Type III Translocation

Cytoskeletal mechanisms regulating attaching/effacing bacteria interactions with host cells: It takes a village to build the pedestal

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