Quantitative Assessment of Cytosolic Salmonella in Epithelial Cells

Knodler, Leigh A.; Nair, Vinod; Steele‐Mortimer, Olivia

doi:10.1371/journal.pone.0084681

Cited by 130 publications

(261 citation statements)

References 72 publications

(102 reference statements)

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“…(15,16). As predicted, and consistent with our CHQ R data, almost all WT Shigella (87%), as well as the majority of VP_E.…”

Section: Mt3_e Coli Efficiently Escape From Phagosomes Within Epithesupporting

confidence: 90%

“…Salmonella pathogenicity island 1 (SPI1) and Shigella T3SA belong to the Inv/Mxi-Spa family of T3SS (25), and accordingly, their proteins share a moderate degree of sequence similarity. Interestingly, although most Salmonella enterica serovar Typhimurium (Salmonella) reside within phagosomes, a significant number of Salmonella (∼15-20%) escape from nascent vacuoles to establish an intracytoplasmic replicative niche in epithelial cells in a SPI1 T3SS-dependent manner (15,26). There is evidence to suggest that this vacuole lysis is linked to the SPI1 T3SA itself, rather than seven of its translocated effectors (15).…”

Section: A Single Component Of the Translocon Apparatus Mediates Phagmentioning

confidence: 99%

“…Interestingly, although most Salmonella enterica serovar Typhimurium (Salmonella) reside within phagosomes, a significant number of Salmonella (∼15-20%) escape from nascent vacuoles to establish an intracytoplasmic replicative niche in epithelial cells in a SPI1 T3SS-dependent manner (15,26). There is evidence to suggest that this vacuole lysis is linked to the SPI1 T3SA itself, rather than seven of its translocated effectors (15). Given the observed differences in the ability of the phylogenetically related Shigella and Salmonella T3SA to mediate phagosomal escape, we hypothesized that the degree to which these pathogens enter the cytosol reflects the action of their T3SA, specifically one or more translocator proteins, the only three T3SA proteins that directly contact host cell membranes.…”

Section: A Single Component Of the Translocon Apparatus Mediates Phagmentioning

confidence: 99%

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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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“…(15,16). As predicted, and consistent with our CHQ R data, almost all WT Shigella (87%), as well as the majority of VP_E.…”

Section: Mt3_e Coli Efficiently Escape From Phagosomes Within Epithesupporting

confidence: 90%

Section: A Single Component Of the Translocon Apparatus Mediates Phagmentioning

confidence: 99%

Section: A Single Component Of the Translocon Apparatus Mediates Phagmentioning

confidence: 99%

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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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“…Bacterial phagocytosis and proliferation where not altered by the constitutive expression of mCherry (data not shown) 16 . 1.…”

Section: Preparing Bacteriamentioning

confidence: 87%

Quantification of Cytosolic vs. Vacuolar <em>Salmonella</em> in Primary Macrophages by Differential Permeabilization

Meunier

Brož

2015

JoVE

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Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell. Video LinkThe video component of this article can be found at

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“…This activation has been observed in the context of infections with mutants that aberrantly enter the cytosol (Aachoui et al, 2013) after lysis of the SCV dependent on IFN-induced GTPases (Meunier et al, 2014). However, wild-type S. Typhimurium can also lyse their nascent vacuole following invasion of tissue culture epithelial cells and enter the cytosol where it can eventually hyper-replicate Knodler et al, 2014). Using a semi-quantitative single-cell analysis, Malik-Kale et al,showed that although cytosolic hyper-replication occurs in less than 20% of infected epithelial cells, it accounts for the majority of net intracellular replication (Malik-Kale et al, 2012).…”

Section: Manipulation Of Inflammatory Pathwaysmentioning

confidence: 93%