single-cell-measurements-vacuolar-rupture-caused-intracellular

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Sterol regulatory element-binding proteins (SREBPs) are transcription factors involved in the synthesis of cholesterol and fatty acids. In adults, the isoform SREBP-1c is the predominant transcript in the liver of fed animals, and it activates triglyceride production from glucose when diet is enriched in carbohydrates. Studies have shown that SREBP-1c expression is dependent on insulin but also on the availability of oxysterols, ligands of the nuclear liver X receptor (LXR). The aim of this study was to investigate the regulation of the hepatic SREBP-1c expression in vivo in situations where drastic nutritional and hormonal changes occur, from the gestation to the weaning period. In this paper, we report the discovery of LXR-independent SREBP-1c transcriptional activity during late gestation. In utero insulin injection prior to the natural rise in insulin in late gestation triggers SREBP-1c mRNA elevation, nuclear SREBP-1c binding activity, and expression of its target genes independently of LXR transactivation. On the other hand, during suckling, we observed strong SREBP-1c mRNA expression despite very low plasma insulin, an expression that may be due to LXR transactivation. In contrast to insulin, LXR is not sufficient to trigger nuclear SREBP-1c binding activity and target gene induction. This could be due to the concomitant induction of INSIG-2a by LXR and subsequent retention of SREBP-1c in the endoplasmic reticulum.

Differential regulation of sterol regulatory element-binding protein 1c transcriptional activity by insulin and liver X receptor during liver development.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors central to the regulation of lipid metabolism. The SREBPs are synthesized as precursor proteins that require proteolytic processing to become transcriptionally active. Whereas the regulation of SREBP-1a and -2 cleavage by cellular sterol content is well defined, much less is known about the regulation of SREBP-1c, the predominant SREBP isoform in the liver. Both insulin and liver X receptor alpha (LXRalpha) induce SREBP-1c transcription; however, the respective roles of these factors and the mechanism responsible for proteolytic cleavage of this SREBP isoform are not known. In this study, we compare the effects of insulin and LXR agonist TO-901317 on SREBP-1c expression and transcriptional activity in isolated rat hepatocytes. We report that full induction of the mature and transcriptionally active form of SREBP-1c protein requires insulin. Although activation of LXR leads to the induction of SREBP-1c gene expression and precursor protein, it has a very poor effect in inducing the mature nuclear form of the transcription factor. This may be due to the induction of insulin-induced gene-2a mRNA and protein by LXR activation. The LXR-induced SREBP-1c precursor, however, is rapidly cleaved on acute exposure to insulin via a phosphatidylinositol 3-kinase-dependent mechanism. Finally, we show through experiments in suckling mice that this acute action of insulin to stimulate the proteolytic processing of SREBP-1c is functional in vivo.

Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c.

[A new role for insulin in the control of energetic metabolism in liver].

This study examines the role of insulin and angiotensin II in high-density lipoprotein (HDL) metabolism by focusing on the regulation and function of scavenger receptor type-BI (SR-BI) in adipose tissue.

In vivo evidence for a role of adipose tissue SR-BI in the nutritional and hormonal regulation of adiposity and cholesterol homeostasis.

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high-content and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.

Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time.

A common strategy of microbial pathogens is to invade host cells during infection. The invading microbes explore different intracellular compartments to find their preferred niche.

Spotting the right location- imaging approaches to resolve the intracellular localization of invasive pathogens.

Invasive bacterial pathogens such as Shigella flexneri force their uptake into non-phagocytic host cells. Upon internalization, they rupture the endocytic vacuole and escape into the host cell cytoplasm. Recent studies applying fluorescence resonance energy transfer (FRET) based methods to track host-pathogen interactions have provided insights into the process of bacterial infection at the single cell level. We have previously reported that the vacuolar escape of invasive bacteria into the host cellular cytosol can be tracked by fluorescence microscopy using a FRET CCF4/Î²-Lactamase reporter assay. Here, we show that our vacuolar rupture assay can also be analyzed by flow cytometry constituting an important alternative to data acquisition by microscopy. Whereas analysis of our assay by fluorescence microscopy offers precise spatiotemporal resolution, flow cytometry analysis represents a high-throughput method that allows efficient and fast quantification of a large number of events and can further improve future research on vacuolar escape.

Monitoring Shigella flexneri vacuolar escape by flow cytometry.

Antigen (Ag) crosspresentation by dendritic cells (DCs) involves the presentation of internalized Ags on MHC class I molecules to initiate CD8+ T cell-mediated immunity in response to certain pathogens and tumor cells. Here, we identify the SNARE Sec22b as a specific regulator of Ag crosspresentation. Sec22b localizes to the ER-Golgi intermediate compartment (ERGIC) and pairs to the plasma membrane SNARE syntaxin 4, which is present in phagosomes (Phgs). Depletion of Sec22b inhibits the recruitment of ER-resident proteins to Phgs and to the vacuole containing the Toxoplasma gondii parasite. In Sec22b-deficient DCs, crosspresentation is compromised after Ag phagocytosis or endocytosis and after invasion by T. gondii. Sec22b silencing inhibited Ag export to the cytosol and increased phagosomal degradation by accelerating lysosomal recruitment. Our findings provide insight into an intracellular traffic pathway required for crosspresentation and show that Sec22b-dependent recruitment of ER proteins to Phgs critically influences phagosomal functions in DCs.

Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells.

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisÎ”RD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.

Article	Year
Differential regulation of sterol regulatory element-binding protein 1c transcriptional activity by insulin and liver X receptor during liver development. The Journal of biological chemistry\| PubMed ID: 15509573	2005
Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15637161	2005
[A new role for insulin in the control of energetic metabolism in liver]. MÃ©decine sciences : M/S\| PubMed ID: 15985191	2005
In vivo evidence for a role of adipose tissue SR-BI in the nutritional and hormonal regulation of adiposity and cholesterol homeostasis. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 17363694	2007
Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time. Cellular microbiology\| PubMed ID: 20070313	2010
Spotting the right location- imaging approaches to resolve the intracellular localization of invasive pathogens. Biochimica et biophysica acta\| PubMed ID: 21029766	2011
Monitoring Shigella flexneri vacuolar escape by flow cytometry. Virulence\| PubMed ID: 21317555	2011
Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells. Cell\| PubMed ID: 22153078	2011
Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS pathogens\| PubMed ID: 22319448	2012

Alexandre Bobard

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