Project description:U2OS cells were co-transfected with PiggyBac (PB) transposon plasmid pPB-SB-CMV-puro-SD3 and the transposase p-hyPBASE. The modified PiggyBac (PB) transposon, which has a constitutively active CMV promoter which can stimulate or disrupt expression of neighboring genes, depending on insertional orientation. For each library, between 10e7-10e8 cells were transfected, cultured with the addition of 3 µg/ml puromycin for one week to select cells that had incorporated the transposon, and then used for screens of resistance to alpha-toxin-induced cell death with minimal further expansion. Mutagenized cells were treated twice with alpha-toxin (500 ng/ml) for 48h to ensure the selection of only highly resistant cells. Genomic DNA (gDNA) was isolated from 5-10 X 10e6 cells and transposon insertion sites were mapped using high throughput sequencing.
Project description:Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we unveil that bacterial membrane pore-forming toxin (PFT) induces autophagy through an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.
Project description:The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) was proposed as a mechanism to eliminate PFTs pores and restore PM integrity. We show here that indeed PFTs are at least partially eliminated through EV release and hypothesized that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different PFTs, Listeriolysin O and Pneumolysin, and determined their proteomic repertoire by LC-MS/MS analysis. Intoxicated cells release similar EVs irrespectively of the PFT used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 76 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further studies. We reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.
Project description:To understand the transcriptional response of mammalian cells to plasma membrane stress we induced partial loss of plasmid membrane integrity by chemogenetic induction of pore forming proteins (Gasderimin and MLKL) or treatment with the detergent digitonin. We monitored transcription with mRNAseq in the immediate hours after plasma membrane damage and identified conserved transcriptional programs.
Project description:This study searched for cell-type specific effectors of endocytic escape in dendritic cells and identified a pore-forming protein, perforin-2 (encoded by Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Whole cell proteomics identified differences in the abundance of tryptic and semi-tryptic (cleaved) perforin-2 peptides between control MutuDCs and cells treated with BafA, CpG or with knockout of asparagine endopeptidase (AEP). This revealed how perforin-2 undergoes proteolytic cleavage releasing its pore forming domain into the organellar lumen.
Project description:In this study, we characterized the proteomic composition of extracellular vesicles released from human THP-1 monocytes challenged with the pneumococcal pore-forming toxin, pneumolysin. We found that the vesicles shed from pneumolysin challenged cells are selectively enriched in key inflammatory host proteins relative to vesicles from untreated cells.
Project description:The success of many pathogens relies on their ability to circumvent the innate and adaptive immune defenses. How bacterial pathogens subvert host responses is not clear. Cholesterol-dependent cytolysins (CDCs) represent an expansive family of homologous pore-forming toxins produced by more than 20 Gram-positive bacterial species. Here we show that listeriolysin O (LLO), a prototype CDC produced by Listeria monocytogenes, inhibits antigen receptor-induced T cell proliferation. In vivo proliferation of OT II T cells was highly diminished in the presence of wild type but not the LLO-deficient bacteria. T cells pre-exposed to LLO ex vivo were also impaired in proliferation upon TCR activation in vivo and in vitro. Our results suggest that LLO-induced T cell unresponsiveness is due to the sub-threshold activation of T cells via the induction of a calcium-NFAT dependent transcriptional program that drives the expression of negative regulators of TCR signaling. Keywords: Listerilysin O, Ionomycin, 3 hours stimulation Pooled primary CD4+ T cells isolated from spleen and mesenteric lymph nodes were incubated in either control (Ctrl) medium or medium containing 0.25 ug/ml listeriolysin O (LLO) or 1 uM Ionomycin (Iono) for 3 hours. Thereafter, cells were washed and RNA was prepared.
Project description:The success of many pathogens relies on their ability to circumvent the innate and adaptive immune defenses. How bacterial pathogens subvert host responses is not clear. Cholesterol-dependent cytolysins (CDCs) represent an expansive family of homologous pore-forming toxins produced by more than 20 Gram-positive bacterial species. Here we show that listeriolysin O (LLO), a prototype CDC produced by Listeria monocytogenes, inhibits antigen receptor-induced T cell proliferation. In vivo proliferation of OT II T cells was highly diminished in the presence of wild type but not the LLO-deficient bacteria. T cells pre-exposed to LLO ex vivo were also impaired in proliferation upon TCR activation in vivo and in vitro. Our results suggest that LLO-induced T cell unresponsiveness is due to the sub-threshold activation of T cells via the induction of a calcium-NFAT dependent transcriptional program that drives the expression of negative regulators of TCR signaling. Keywords: Listerilysin O, Ionomycin, 3 hours stimulation
Project description:Conventional dendritic cells (cDCs) generate protective cytotoxic T lymphocyte (CTL) responses against pathogens and tumours. This is achieved through a process known as cross-presentation (XP) and, despite its obvious biological importance, the mechanism(s) driving XP remain unclear. Here, we show that a cDC-specific pore-forming protein called apolipoprotein L 7C (APOL7C) is upregulated in response to innate immune stimuli and is recruited to phagosomes. Strikingly, its association with phagosomes leads to phagosomal rupture. This allows for the escape of engulfed antigens to the cytosol where they can be processed via the endogenous MHC class I antigen processing pathway. Accordingly, mice deficient in APOL7C do not efficiently prime CD8+ T cells in response to immunization with bead-bound and cell-associated antigens. Altogether, our data indicate the presence of dedicated apolipoproteins that mediate the delivery of phagocytosed proteins to the cytosol of activated cDCs to facilitate XP.
Project description:Perforation of cellular membranes by pore-forming proteins can affect cell physiology, tissue integrity, or immune response. Since many pore-forming proteins are toxins or highly potent virulence factors, they represent an attractive target for the development of molecules that neutralize their actions with high efficacy. There has been an assortment of inhibitors developed to specifically obstruct the activity of pore-forming proteins, in addition to vaccination and antibiotics that serve as a plausible treatment for the majority of diseases caused by bacterial infections. Here we review a wide range of potential inhibitors that can specifically and effectively block the activity of pore-forming proteins, from small molecules to more specific macromolecular systems, such as synthetic nanoparticles, antibodies, antibody mimetics, polyvalent inhibitors, and dominant negative mutants. We discuss their mechanism of inhibition, as well as advantages and disadvantages.