Salmonella exploits membrane reservoirs for invasion of host cells
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ABSTRACT: Submitted data contain the results of BioID-LC-MS experiments performed in T-REx HEK293 expressing RAB10 protein (WT and mutants) fused to BirA biotin carboxylase.
Project description:Data deposited contain results from proximity-dependent biotinylation LC-MS (BioID) experiments in which T-REx Flp-In HEK293 cells express NSD2 protein fused to miniTurbo biotin carboxylase with or without treatment with the protac UNC8732.
Project description:Here we investigated the effects of CEBPA transcription factor expression on myeloid NB4 cells. The sequence of rat CEBPA was C-terminally fused to a promiscuous biotin ligase tag (BirA*) and NB4 cell lines were engineered to express the fusion protein under the control of a doxycycline inducible promoter. Three different NB4 cell lines were investigated that expressed (i) BirA* tag alone (ii) full length CEBPA isoform (P42) fused to BirA* (iii) truncated CEBPA isoform (P30) fused to BirA*. Cells were seeded in media supplemented with or without doxycycline.
Project description:Data contains LC-MS data of anti-Flag affinity purification to generate the protein interaction network of SopD protein fused with BirAFlag (C-terminal tag)
and expressed in T-REx HEK293 cells.
Project description:We report the genomic localization of cohesin oligomers in nocodazole arrested yeast cells. Two alleles of SMC3 were expressed in yeast cells, one fused to BirA enzyme and the other tagged with AviTag. Cohesin oligomers were biotinylated and ChIP with streptavidin beads. As control experiments, cohesin localization on chromosome was determined in strains expresses freely diffusable BirA enzyme, where all Smc3 proteins were biotinylated; non-specific ChIP were determined in strains with no BirA.
Project description:Data deposited contains results from proximity-labeling LC-MS experiment performed in T-REx Flp-In HEK293 cells stably expressing RPB9/POLR2I fused with the biotin carboxylase miniTurbo.
Project description:Salmonella attachment to the intestinal epithelium triggers delivery of bacterial effector proteins into the host cytosol through a type III secretion system (T3SS), leading to pronounced membrane ruffling and macropinocytic uptake of attached bacteria. The tip of the T3SS is made up of two proteins, SipB and SipC, which insert into the host plasma membrane, forming a translocation pore. Both the N and C termini of SipC are exposed in the host cytosol and have been shown to directly modulate actin cytoskeleton assembly. We have identified a direct interaction between SipC and Exo70, a component of the exocyst complex, which mediates docking and fusion of exocytic vesicles with the plasma membrane. Here, we show that exocyst components coprecipitate with SipC and accumulate at sites of invasion by Salmonella typhimurium. Exocyst assembly requires activation of the small GTPase RalA, which we show is triggered during Salmonella infection by the translocated effector, SopE. Knockdown of RalA or Sec5 results in reduced membrane ruffling at sites of attachment and impairs bacterial entry into host cells. These findings suggest that S. typhimurium enhances invasion efficiency by promoting localized membrane expansion, directly through SipC-dependent recruitment of the exocyst and indirectly via SopE-dependent activation of RalA.
Project description:Data deposited contain results from proximity-dependent biotinylation LC-MS (BioID) experiments in which 786-O and RCC243 cells expressed PRMT1 protein fused to miniTurbo biotin carboxylase.
Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
Project description:Actin nucleators and their binding partners play crucial roles during Salmonella invasion, but how these factors are dynamically coordinated remains unclear. Here, we show that septins, a conserved family of GTP binding proteins, play a role during the early stages of Salmonella invasion. We demonstrate that septins are rapidly enriched at sites of bacterial entry and contribute to the morphology of invasion ruffles. We found that SEPTIN2, SEPTIN7, and SEPTIN9 are required for efficient bacterial invasion. Septins contributed to the recruitment of ROCK2 kinase during Salmonella invasion, and the downstream activation of the actin nucleating protein FHOD1. In contrast, activation of the ROCK2 substrate myosin II, which is known to be required for Salmonella enterica serovar Typhimurium invasion, did not require septins. Collectively, our studies provide new insight into the mechanisms involved in Salmonella invasion of host cells.
Project description:Pathogenic Salmonella species initiate infection by invading non-phagocytic intestinal epithelial cells (IEC). This invasion is brought about by a number of Salmonella invasion promoting molecules (Sips) encoded by the Salmonella Pathogenicity Island - 1 (SPI-1). Intracellular delivery of some of these molecules also brings about caspase-1 - mediated pyroptotic cell death that contributes to pathogen clearance. These molecules are secreted and delivered inside cells upon contact of Salmonella with one or more host signals whose identity has not been established. We show that lysophosphatidylcholine (LPC) released following activation of caspase-1 in Salmonella - infected cells and abundant in plasma amplifies production of Sips from this pathogen and promotes its cellular invasion. LPC brings about adenylate cyclase and cAMP receptor protein (CRP) - dependent de novo synthesis of SipC that is accompanied by its translocation to bacterial cell surface and release into the outside milieu. Treatment of Salmonella with LPC produces sustained induction of SPI - 1 transcriptional regulator, hilA. Our findings reveal a novel host lipid sensing - driven regulatory mechanism for Salmonella invasion.