Presence of SopE and mode of infection result in increased<i>Salmonella</i>‐containing vacuole damage and cytosolic release during host cell infection by<scp><i>Salmonella enterica</i></scp>

Röder, Jennifer; Hensel, Michael

doi:10.1111/cmi.13155

Cited by 35 publications

(55 citation statements)

References 66 publications

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“…Some bacteria were still associated with luminal marker dextran‐Alexa647 and positive for sfGFP expression. These observations are in line with our previous findings on SCV integrity (Röder & Hensel, ) and indicate that bacteria are in contact with the cytosol with partially damaged SCV compartments. The fluid tracer dextran‐Alexa647 may be too large to diffuse through smaller pores in the SCV membrane, or other mechanisms repair transient damages prior to loss of luminal content.…”

Section: Resultssupporting

confidence: 93%

“…In order to detect all intracellular Salmonella , the reporter also expresses a constitutive red fluorophore, i.e. DsRed (Röder & Hensel, ).…”

Section: Resultsmentioning

confidence: 99%

“…HeLa cells either CRIPSR/Cas9‐treated or nontreated were seeded in surface‐treated 12‐well plates (TPP). Cells were infected with Salmonella WT strain harbouring the cytosolic reporter plasmid p4889 (Table , Röder & Hensel, ) as described above at MOI of 5 for 25 min after centrifugation for 5 min and 500 × g . Cells were detached and fixed with 3% paraformaldehyde (PFA) after 4, 8, and 16 hr.…”

Section: Methodsmentioning

confidence: 99%

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Role of the ESCRT‐III complex in controlling integrity of the Salmonella ‐containing vacuole

Göser

Kehl

Röder

et al. 2020

Cellular Microbiology

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Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2-encoded type III secretion system (SPI2-T3SS). Fusion events of endosomal compartments with the Salmonella-containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonellamodified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT-III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT-III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2-T3SS-deficient mutant strain was partially abrogated. ESCRT-dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT-III recruitment in Salmonellainfected host cells.

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Section: Resultssupporting

confidence: 93%

“…In order to detect all intracellular Salmonella , the reporter also expresses a constitutive red fluorophore, i.e. DsRed (Röder & Hensel, ).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Role of the ESCRT‐III complex in controlling integrity of the Salmonella ‐containing vacuole

Göser

Kehl

Röder

et al. 2020

Cellular Microbiology

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“…The use of a dual reporter with P uhpT ∷DsRed and P pstS ∷sfGFP provided information on phosphate concentration over time in distinct habitats in HeLa cells ( Figure 5 A, Figure S 5A). P uhpT induction indicates cytosolic presence of the bacteria (Röder & Hensel, 2020). After invasion, most bacteria are still in a vacuole and therefore P uhpT -negative.…”

Section: Resultsmentioning

confidence: 99%

Single cell analyses reveal phosphate availability as critical factor for nutrition ofSalmonella entericawithin mammalian host cells

Röder

Hensel

2020

Preprint

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Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that resides in a specialized membrane-bound compartment termed Salmonella-containing vacuole (SCV). Essential for survival and proliferation in the SCV is Salmonella pathogenicity island II (SPI2) that encodes a type III secretion system (T3SS). The SPI2-T3SS and the effector translocation maintain SCV integrity and formation of specific tubular membrane compartments, called Salmonella-induced filaments (SIFs). The SCV/SIF continuum allows STM to bypass nutritional restriction in the intracellular environment by acquiring nutrients from the host cell. Phosphate is one of the most abundant elements in living organisms and in STM, inorganic phosphate (Pi) homeostasis is mediated by the two-component regulatory system PhoBR, resulting in expression of the high affinity phosphate transporter pstSCAB-phoU. Using fluorescent protein reporters, we investigate Pi availability for STM at single cell level over time within the intracellular habitats of different host cells. We observed that the pstSCAB-phoU encoded phosphate uptake system is essential for intracellular replication of STM because there is a Pi ion concentration less 10 micromolar within the SCV. Additionally, the demand and consumption of Pi correlates with intracellular proliferation of STM and we identify a dependency of SPI2 activity and Pi starvation.

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“…We aimed to analyze, on the level of single bacterial cells, the sensing and response of stress by intracellular STM. For this, the basic design of dual color fluorescence reporters was applied (15) as shown in Because various FP have divergent maturation times, we analyzed the correlation of growth and fluorescence dilution for a set of commonly used FP. For this, STM WT harbored plasmids for constitutive expression of DsRed, tagRFP-T, mCherry, mCherry2, or sfGFP under control of P EM7 .…”

Section: Design Of Fluorescent Protein-based Reporters For Stress Resmentioning

confidence: 99%

Fluorescent protein-based reporters reveal stress response of intracellularSalmonella entericaon single cell level

Schulte

Olschewski

Hensel

2020

Preprint

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Intracellular bacteria such as Salmonella enterica are confronted with a broad array of defense mechanisms of their mammalian host cells. The ability to sense host cell-imposed damages, and to mount efficient stress responses are crucial for survival and proliferation of intracellular pathogens. The various combinations of host defense mechanisms acting on intracellular bacteria and their individual response also explain the occurrence of distinct subpopulations of intracellular S. enterica such as dormant or persisting, slowly or rapidly replicating cells. Here we describe a set of fluorescence protein (FP)-based reporter strains that were used to monitor the expression of cytoplasmic or periplasmic stress response systems on a single cell level. This is mediated by a fast maturing FP as reporter for induction of stress response genes. We evaluated slower maturing FPs for a second function, i.e. the analyses of the status of intracellular proliferation of pathogens. The combination of two FPs allows, on a single cell level, the interrogation of stress response and intracellular proliferation. Application of these reporters to S. enterica allowed us to detect and quantify distinct intracellular subpopulations with different levels of stress response and proliferation.ImportanceSensing of, and responding to host-mediated damages are important defensive virulence traits of bacterial pathogens. Intracellular pathogens such as Salmonella enterica are exposed to various types of antimicrobial host cell defenses that impose, among other, periplasmic and cytosolic stresses. Intracellular S. enterica form distinct subpopulations that differ in proliferation rate, metabolic activity and persister formation. Here we deploy fluorescence protein-based reporter strains to monitor, on a single cell level, the response of intracellular S. enterica to periplasmic or cytoplasmic stress. A second fluorescent protein reports the biosynthetic capacity of individual intracellular S. enterica. The dual fluorescence reporters can be deployed to characterize by flow cytometry phenotypically diverse subpopulations and stress responses in intracellular bacteria.

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Presence of SopE and mode of infection result in increasedSalmonella‐containing vacuole damage and cytosolic release during host cell infection bySalmonella enterica

Cited by 35 publications

References 66 publications

Role of the ESCRT‐III complex in controlling integrity of the Salmonella ‐containing vacuole

Role of the ESCRT‐III complex in controlling integrity of the Salmonella ‐containing vacuole

Single cell analyses reveal phosphate availability as critical factor for nutrition ofSalmonella entericawithin mammalian host cells

Fluorescent protein-based reporters reveal stress response of intracellularSalmonella entericaon single cell level

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