Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells

Klein, Jessica A.; Powers, TuShun R.; Knodler, Leigh A.

doi:10.1007/978-1-4939-6581-6_19

Cited by 24 publications

(38 citation statements)

References 35 publications

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“…Assays were performed similarly to those described before ( Klein et al., 2017 , Thurston et al., 2016 ). THP1 GSDMDmiR cells (7.5 × 10 4 /well) were seeded in triplicate in 96-well plates and differentiated for 48 h, after which medium without PMA or antibiotics was used.…”

Section: Methodsmentioning

confidence: 99%

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages

et al. 2019

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Summary Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.

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Section: Methodsmentioning

confidence: 99%

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages

et al. 2019

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“…Intra-vacuolar and cytosolic Salmonella populations are distinguished by a variety of assays. We refer the reader to excellent recent reports that cover in depth these techniques (Knodler, 2015 ; Klein et al, 2017 ). Here, we briefly comment the bases and main features of the most commonly used assays.…”

Section: Methods To Identify Cytosolic and Intra-vacuolar Smentioning

confidence: 99%

“…The chloroquine resistance assay was used for the first time to quantify cytosolic Salmonella in a study conducted by Knodler and colleagues in multiple epithelial cell lines (Knodler et al, 2014b ). This assay exploits the lysosomotrophic nature of CHQ, which accumulates in vacuolar acidic compartments (Knodler et al, 2014b ; Klein et al, 2017 ). Due to this property, CHQ targets intra-vacuolar but not cytosolic bacteria and allows the selective quantification of cytosolic bacteria through enumeration of viable counts following exposure of infected cells to the drug.…”

Section: Methods To Identify Cytosolic and Intra-vacuolar Smentioning

confidence: 99%

Salmonella Populations inside Host Cells

Castanheira

Portillo

2017

Front. Cell. Infect. Microbiol.

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Bacteria of the Salmonella genus cause diseases ranging from gastroenteritis to life-threatening typhoid fever and are among the most successful intracellular pathogens known. After the invasion of the eukaryotic cell, Salmonella exhibits contrasting lifestyles with different replication rates and subcellular locations. Although Salmonella hyper-replicates in the cytosol of certain host cell types, most invading bacteria remain within vacuoles in which the pathogen proliferates at moderate rates or persists in a dormant-like state. Remarkably, these cytosolic and intra-vacuolar intracellular lifestyles are not mutually exclusive and can co-exist in the same infected host cell. The mechanisms that direct the invading bacterium to follow the cytosolic or intra-vacuolar “pathway” remain poorly understood. In vitro studies show predominance of either the cytosolic or the intra-vacuolar population depending on the host cell type invaded by the pathogen. The host and pathogen factors controlling phagosomal membrane integrity and, as consequence, the egress into the cytosol, are intensively investigated. Other aspects of major interest are the host defenses that may affect differentially the cytosolic and intra-vacuolar populations and the strategies used by the pathogen to circumvent these attacks. Here, we summarize current knowledge about these Salmonella intracellular subpopulations and discuss how they emerge during the interaction of this pathogen with the eukaryotic cell.

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“…We used gentamicin at the same dose as a control. This is the reference antimicrobial agent used in protection assays (Klein et al, 2017). Compared with the untreated control, tilmicosin had a bacteriostatic activity against S. aureus from 2 up to the 24 h evaluated (P < 0.01).…”

Section: Antimicrobial Activity Of Tilmicosin Against S Aureusmentioning

confidence: 97%

Tilmicosin modulates the innate immune response and preserves casein production in bovine mammary alveolar cells duringStaphylococcus aureusinfection1

Martínez-Cortés

Acevedo-Domínguez

Olguín‐Alor

et al. 2018

Journal of Animal Science

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Tilmicosin is an antimicrobial agent used to treat intramammary infections against Staphylococcus aureus and has clinical anti-inflammatory effects. However, the mechanism by which it modulates the inflammatory process in the mammary gland is unknown. We evaluated the effect of tilmicosin treatment on the modulation of the mammary innate immune response after S. aureus infection and its effect on casein production in mammary epithelial cells. To achieve this goal, we used immortalized mammary epithelial cells (MAC-T), pretreated for 12 h or treated with tilmicosin after infection with S. aureus (ATCC 27543). Our data showed that tilmicosin decreases intracellular infection (P < 0.01) and had a protective effect on MAC-T reducing apoptosis after infection by 80% (P < 0.01). Furthermore, tilmicosin reduced reactive oxygen species (ROS) (P < 0.01), IL-1β (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.05) production. In an attempt to investigate the signaling pathways involved in the immunomodulatory effect of tilmicosin, mitogen-activated protein kinase (MAPK) phosphorylation was measured by fluorescent-activated cell sorting. Pretreatment with tilmicosin increased ERK1/2 (P < 0.05) but decreased P38 phosphorylation (P < 0.01). In addition, the anti-inflammatory effect of tilmicosin helped to preserve casein synthesis in mammary epithelial cells (P < 0.01). This result indicates that tilmicosin could be an effective modulator inflammation in the mammary gland. Through regulation of MAPK phosphorylation, ROS production and pro-inflammatory cytokine secretion tilmicosin can provide protection from cellular damage due to S. aureus infection and help to maintain normal physiological functions of the bovine mammary epithelial cell.

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Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells

Cited by 24 publications

References 35 publications

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages

Salmonella Populations inside Host Cells

Tilmicosin modulates the innate immune response and preserves casein production in bovine mammary alveolar cells duringStaphylococcus aureusinfection1

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