Shigella Subverts the Host Recycling Compartment to Rupture Its Vacuole

Mellouk, Nora; Weiner, Allon; Aulner, Nathalie; Schmitt, Christine; Elbaum, Michael; Shorte, Spencer; Danckært, Anne; Enninga, Jost

doi:10.1016/j.chom.2014.09.005

Cited by 100 publications

(164 citation statements)

References 46 publications

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“…The 31-kb region of Shigella DNA present in mT3_E. coli encodes proteins needed to form a functional T3SA and four effectors, three of which-IpgB1, IpgD, and IpaA-play roles in mediating host cell invasion and one-IpgD-that promotes phagosomal escape (4). Furthermore, studies with purified IpaB and IpaC have implicated these proteins in mediating vacuole lysis (5-7); however, given the absolute requirement of the IpaBIpaC translocon for the delivery of effectors into host cells, a direct physiologic role for either protein in phagosomal escape has not yet been possible to assess.…”

Section: Mt3_e Coli Efficiently Escape From Phagosomes Within Epithementioning

confidence: 99%

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

Reeves

Klein

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon—Shigella IpaC, Salmonella SipC, or Chromobacterium CipC—determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.

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Section: Mt3_e Coli Efficiently Escape From Phagosomes Within Epithementioning

confidence: 99%

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

Reeves

Klein

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Our method may be used in conjunction with time-lapse studies (19), fluorescence-activated cell sorter analysis (35), and highthroughput screening (26), allowing deciphering of which factors are associated with L. monocytogenes entry into host cells and identification of factors driving vacuolar rupture. We have generated a reporter system that can be adapted to studies in other Grampositive bacteria for which internalization in host cells has been reported (36).…”

Section: Discussionmentioning

confidence: 99%

“…Images from replicate wells were automatically acquired and analyzed using a customized image analysis algorithm (26). We observed a strong reduction in the 450 nm/535 nm fluorescence ratio of the c-Met siRNA-treated cells compared to the scrambled siRNA-treated cells that was dependent on the MOI used (Fig.…”

Section: Construction Of Reportermentioning

confidence: 99%

“…Columbus analysis building block routines were used for feature extraction and automated scoring of the fluorescence signal for each individual cell. Statistical analysis of the results was performed as previously described (26). The strictly standardized mean difference (SSMD) statistical tests were used for quality control (QC) analysis (26).…”

Section: Construction Of An Lpxtg-␤-lactamase Fusion Proteinmentioning

confidence: 99%

“…Statistical analysis of the results was performed as previously described (26). The strictly standardized mean difference (SSMD) statistical tests were used for quality control (QC) analysis (26).…”

Section: Construction Of An Lpxtg-␤-lactamase Fusion Proteinmentioning

confidence: 99%

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A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape

Quereda

Pizarro‐Cerdá

Balestrino

et al. 2016

Appl Environ Microbiol

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e Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a ␤-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface ␤-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes. Listeria monocytogenes is a Gram-positive pathogen responsible for listeriosis, a human disease characterized by gastroenteritis in immunocompromised individuals, miscarriage in pregnant women, and meningitis in newborns (1). L. monocytogenes is able to promote its own internalization in mammalian cells due to interaction of the bacterial surface invasion proteins InlA and InlB with their host cell plasma membrane receptors, the epithelial adhesion molecule E-cadherin and the hepatocyte growth factor receptor c-Met, respectively (2). This interaction leads to the activation of signaling cascades triggering clathrin recruitment (3, 4) and cytoskeletal rearrangements for bacterial engulfment (5-8).Once inside host cells, secretion of the cholesterol-dependent pore-forming toxin listeriolysin O (LLO) and of two bacterial phospholipases, PlcA and PlcB, leads to disruption of the L. monocytogenes internalization vacuole and pathogen escape into the cytoplasm (9). An actin-based motility system dependent on activation of the Arp2/3 complex by the bacterial surface protein ActA allows L. monocytogenes to spread to neighboring cells (2, 10).While the cell invasion process by L. monocytogenes has been extensively investigated (9), cellular components and signaling pathways leading to vacuolar escape are less well characterized. In macrophages, the small GTPase Rab5 and its interactor RABEP1 have been reported to negatively modulate L. monocytogenes survival in the vacuole (11,12), and the heat shock protein 70 was reported to inhibit vacuolar escape (13). Furthermore, the gamma-interferon-inducible lysosomal thiol reductase (GILT) activates LLO and favors vacuolar rupture (14), while the cystic ...

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Type III Secreted Virulence Factors Manipulating Signaling to Actin Dynamics

Stradal

Costa

2016

Current Topics in Microbiology and Immunology

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A key aspect of bacterial pathogenesis is the colonization and persistence within the host and, later on, its dissemination to new niches. During evolution, bacteria developed a myriad of virulence mechanisms to usurp the host's sophisticated defense mechanisms in order to establish their colonization niche. Elucidation of the highly dynamic and complex interactions between host and pathogens remains an important field of study. Here, we highlight the conserved manipulation of the actin cytoskeleton by some Gram-negative gastrointestinal pathogens, addressing the role of type III secreted bacterial GEFs at the different steps of pathogenesis. As a final topic, we review cytoskeleton dynamics induced by EPEC/EHEC strains for pedestal formation.

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Shigella Subverts the Host Recycling Compartment to Rupture Its Vacuole

Cited by 100 publications

References 46 publications

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape

Type III Secreted Virulence Factors Manipulating Signaling to Actin Dynamics

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