Pathogen infection triggers host innate defenses which may result in the activation of regulated cell death (RCD) pathways such as apoptosis. Given a vital role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptosis. Yet, it is unclear whether components of this emerging RCD pathway display signatures associated with pathogen conflict that are rare in combination but common to key host defense factors, namely, rapid evolution, viral homolog (virolog), and cytokine induction. We leveraged evolutionary sequence analysis that examines rates of amino acid replacement, which revealed: 1) strong and recurrent signatures of positive selection for primate and bat RIPK3 and MLKL, and 2) elevated rates of amino acid substitution on multiple RIPK3/MLKL surfaces suggestive of past antagonism with multiple, distinct pathogen-encoded inhibitors. Furthermore, our phylogenomics analysis across poxvirus genomes illuminated volatile patterns of evolution for a recently described MLKL viral homolog. Specifically, poxviral MLKLs have undergone numerous gene replacements mediated by duplication and deletion events. In addition, MLKL protein expression is stimulated by interferons in human and mouse cells. Thus, MLKL displays all three hallmarks of pivotal immune factors of which only a handful of factors like OAS1 exhibit. These data support the hypothesis that over evolutionary time MLKL functions—which may include execution of necroptosis—have served as a major determinant of infection outcomes despite gene loss in some host genomes.

The 16S ribosomal RNA (rRNA) gene is one of the scaffolding molecules of the prokaryotic ribosome. Because this gene is slow to evolve and has very well conserved regions, this gene is used to reconstruct phylogenies in prokaryotes. Universal primers can be used to amplify the gene in prokaryotes including bacteria and archaea. To determine the microbial composition in microbial communities using high‐throughput short‐read sequencing techniques, primers are designed to span two or three of the nine variable regions of the gene. Mothur, developed in 2009, is a suite of tools to study the composition and structure of bacterial communities. This package is freely available from the developers (https://www.mothur.org). This protocol will show how to (1) perform preprocessing of sequences to remove errors, (2) perform operational taxonomic unit (OTU) analysis to determine alpha and beta diversity, and (3) determine the taxonomic profile of OTUs and the environmental sample. © 2019 The Authors.

Pathogen infection triggers host innate defenses which can lead to the activation of regulated cell death (RCD) pathways such as apoptosis. Given a key role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptotic signaling. However, whether this emerging inflammatory RCD pathway functions primarily as a back-up or fundamental response remains inconclusive. We hypothesized that if necroptosis is an instrumental defense, then its effectors should display specific signatures associated with pathogen conflict that are rare in combination: rapid evolution, viral homolog hereafter virolog, and induction by cytokines (e.g. interferons). Our rapid evolution analysis across the necroptosis pathway revealed: 1) strong signatures of positive selection for RIPK3 and MLKL in primate genomes and to a lesser extent DAI/ZBP1, 2) elevated rates of amino acid substitution on multiple surfaces including the RIPK3/MLKL binding interface and 3) evidence supporting a means of activating RIPK3 independent of homotypic RHIM domain interactions. Interestingly, a poxvirus MLKL homolog has recently been identified that acts as a RIPK3 pseudosubstrate. Our findings indicate that poxvirus MLKLs are also subject to similar but distinct volatile patterns of evolution comparable to host necroptotic factors. Specifically, viral MLKLs have undergone numerous gains and losses in poxvirus evolution with some species harboring three distinct copies. Furthermore, we confirm that MLKL can be induced by cytokines like interferon gamma. In summary, MLKL displays all three hallmarks of pivotal immune factors of which only OAS1, but not other factors like cGAS, APOBEC3G, or PKR, exhibits. These data support the hypothesis that over evolutionary time, necroptosis has served as a key battleground during infection and is therefore, not an auxiliary response.

The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication. We show that early viral gene expression triggers nuclear accumulation of specialized, SUMOylated Spt16 subunits of FACT required for expression of ETS-1, a downstream transcription factor that activates a virus restriction program. However, poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting SSRP1 for binding to SUMOylated Spt16 in the cytosol and by tethering SUMOylated Spt16 to microtubules. Moreover, we show that A51R antagonism of FACT enhances both poxvirus replication in human cells and viral virulence in mice. Finally, we demonstrate that FACT also restricts unrelated RNA viruses, suggesting a broad role for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 Antiviral Response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

The double-cut-and-join (DCJ) operation, introduced by Yancopoulos et al., allows minimum edit distance to be computed by modeling all possible classical rearrangement operations, such as inversions, fusions, fissions, translocations and transpositions, in linear-time between two genomes. However, there is lack of visualization tool that can effectively present DCJ operations that will help biologists to use DCJ operation. In this paper, a new visualization program is introduced, DCJVis, to create a diagram of each DCJ operation necessary to transform between the genomes of two distinct organisms by describing a possible sequence of genome graphs based on the selected gene adjacency on the source genome for the DCJ operation. Our program is the first visualization tool for DCJ operations using circular layout. Specifically, the genomes of Saccharomyces cerevisiae and Candida albicans are used to demonstrate the functionality of this program and provide an example of the type of problem this program can solve for biologists.