Real-time imaging of type III secretion:
            <i>Salmonella</i>
            SipA injection into host cells

Schlumberger, Markus C.; Müller, Andreas J.; Ehrbar, Kristin; Winnen, Brit; Duss, Iwan; Stecher, Bärbel; Hardt, Wolf‐Dietrich

doi:10.1073/pnas.0503407102

Cited by 179 publications

(209 citation statements)

References 31 publications

(21 reference statements)

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“…This observation was further strengthened by the results of Salmonella binding assays, in which a significant increase in the number of bacteria per infected cell was observed in cells pre‐exposed to arsenite or anisomycin, compared to control cells (Fig 5D and E, and Appendix Fig S3A). To confirm that this phenotype was related to bacterial binding, we used an invasion‐deficient but binding proficient Salmonella mutant strain ( Salmonella Δ4), which lacks four effector proteins (SopE, SopE2, SopA, and SipB) essential for invasion (Schlumberger et al , 2005; Lara‐Tejero & Galan, 2009; Misselwitz et al , 2011b). Similar to WT Salmonella , in conditions of cellular stress the Δ4 mutant strain infected a lower number of cells than in the absence of stress, but also accumulated in specific sites, resulting in a higher number of bacteria per infected cell (Fig 5D and F, and Appendix Fig S3A).…”

Section: Resultsmentioning

confidence: 99%

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

et al. 2018

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While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress‐dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re‐infection by this and other non‐motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress‐responsive cell‐autonomous defense mechanism that protects epithelial cells from infection by non‐motile bacterial pathogens.

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Section: Resultsmentioning

confidence: 99%

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

et al. 2018

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“…Notwithstanding, considering Salmonella and Shigella, no surface-localized effector proteins have yet been found, and the few studies that have assayed effector proteins in these pathogens have indicated that these proteins are present in the cytoplasm of the bacteria before contact with target cells (15,38,39). In an investigation of S. typhimurium, Schlumberger et al (15) performed real-time imaging of SipA translocation and showed that 26% of the bacterial cells contained detectable amounts of SipA in their cytosol before they came in contact with host cells.…”

Section: Discussionmentioning

confidence: 99%

“…The Yop effectors, in turn, are believed to be transferred into the host-cell cytoplasm through this pore during the translocation step (13). Investigators using different methods have previously demonstrated the occurrence of polarized, T3SS-dependent translocation of proteins into target cells (2,(14)(15)(16), but the detailed mechanisms underlying that process are still largely unknown.…”

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confidence: 99%

Translocation of surface-localized effectors in type III secretion

Akopyan

Edgren

Wang-Edgren

et al. 2011

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

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Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model.

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“…Bacterial invasion and co-localization with LAMP-1 was quantified by evaluating 100 randomly selected macrophages. Intracellular IpaB was stained after permeabilization of the bacteria with lysozyme as described by Schlumberger et al (2005).…”

Section: Methodsmentioning

confidence: 99%

“…During cell-to-cell spread the contact with the protrusion membrane likely triggers type III secretion. By contrast, the factor triggering type III secretion in the macrophage cytoplasm is currently unknown.Type III secretion by S. flexneri (Enninga et al, 2005) and Salmonella enterica serovar Typhimurium (Schlumberger et al, 2005) was recently analysed on a single-cell level in real time using time-lapse fluorescence microscopy. In these studies, the depletion of tetracysteine-tagged S. flexneri effectors (IpaB, IpaC) labelled with a fluoresceinconjugated biarsenical dye was analysed, or the depletion of Sal.…”

mentioning

confidence: 99%

Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death

2007

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The Gram-negative bacterium Shigella flexneri triggers pro-inflammatory apoptotic cell death in macrophages, which is crucial for the onset of an acute inflammatory diarrhoea termed bacillary dysentery. The Mxi-Spa type III secretion system promotes bacterial uptake and escape into the cytoplasm, where, dependent on the translocator/effector protein IpaB, caspase-1 [interleukin (IL)-1b-converting enzyme] and its substrate IL-1b are activated. Here, we show that in the course of a macrophage infection, IpaB is secreted intracellularly for more than 1 h post-infection and progressively accumulates in aggregates on the bacterial surface. Concomitantly, the bacterial pool of IpaB is gradually depleted. The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dose-dependently inhibited the Mxi-Spa-dependent secretion of IpaB triggered by the dye Congo red in vitro and abolished translocation of IpaB into the host-cell cytoplasm of S. flexneri-infected macrophages. CCCP specifically inhibited S. flexneri-triggered macrophage death in a dose-dependent manner, even if added up to 60 min post-infection. Addition of CCCP 15 min after infection blocked macrophage cell death, the activation of caspase-1 and the maturation of IL-1b, without affecting uptake or escape of S. flexneri from the phagosome. By contrast, CCCP used at the same concentration had no effect on ATP-induced caspase-1 activation or staurosporine-induced apoptosis. Our results indicate that under the conditions used, CCCP rapidly and specifically blocks bacterial type III secretion, and thus, intracellular type III secretion promotes cytotoxicity of S. flexneri. INTRODUCTIONMany symbiotic or pathogenic Gram-negative bacteria employ a type III secretion system (T3SS) to establish a replicative niche (Cornelis, 2006;Galan & Wolf-Watz, 2006). The complex T3SSs are evolutionarily related to flagellar secretion systems and composed of a basal structure spanning the two bacterial membranes, a hollow needle (or pilus/filament) and a hydrophobic translocator complex inserted into eukaryotic target membranes. Some T3SSs deliver more than 20 different 'effector' proteins into target cells, where they subvert cellular functions in favour of the bacteria. In the eukaryotic host cells, the bacterial effectors target phosphoinositide metabolism, GTP cycles, phosphoprotein signalling, cytoskeletal and vesicle dynamics, or apoptotic pathways (Cossart & Sansonetti, 2004;Hilbi, 2006;Rosenberger & Finlay, 2003;Schlumberger & Hardt, 2006). Shigella flexneri, the causative agent of a severe diarrhoea termed bacillary dysentery (shigellosis), employs a T3SS to invade non-phagocytic epithelial cells and to kill macrophages (Cossart & Sansonetti, 2004). The S. flexneri T3SS is encoded by the mxi and spa genes on the 214 kb virulence plasmid (Buchrieser et al., 2000). Investigations by electron microscopy revealed that the Mxi-Spa needle complex is a canonical T3SS composed of a basal body and a needle, the major subunit of which (MxiH) is required for the secretion of eff...

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Real-time imaging of type III secretion: Salmonella SipA injection into host cells

Cited by 179 publications

References 31 publications

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

Translocation of surface-localized effectors in type III secretion

Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death

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