Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1β processing within the Salmonella-infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella-infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro-IL-1β substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1β processing during Salmonella infection.innate immunity | bacteria | caspase-1 | caspase-8 | ASC
Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
Viral glycoprotein-mediated membrane fusion is an essential step for productive infection of host cells by enveloped viruses; however, due to its rarity and challenges in detection, little is known about the details of fusion events at the single particle level. Here, we have developed dual-color foamy viruses (FVs) composed of eGFP-tagged prototype FV (PFV) Gag and mCherry-tagged Env of either PFV or macaque simian FV (SFVmac) origin that have been optimized for detection of the fusion process. Using our recently developed tracking imaging correlation (TrIC) analysis, we were able to detect the fusion process for both PFV and SFVmac Env containing virions. PFV Env-mediated fusion was observed both at the plasma membrane as well as from endosomes, whereas SFVmac Env-mediated fusion was only observed from endosomes. PFV Env-mediated fusion was observed to happen more often and more rapidly than as for SFVmac Env. Strikingly, using the TrIC method, we detected a novel intermediate state where the envelope and capsids are still tethered but separated by up to 400 nm before final separation of Env and Gag occurred.
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