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Background The quantity of genomic data is expanding at an increasing rate. Tools for phylogenetic analysis which scale to the quantity of available data are required. To address this need, we present cognac, a user-friendly software package to rapidly generate concatenated gene alignments for phylogenetic analysis. Results We illustrate that cognac is able to rapidly identify phylogenetic marker genes using a data driven approach and efficiently generate concatenated gene alignments for very large genomic datasets. To benchmark our tool, we generated core gene alignments for eight unique genera of bacteria, including a dataset of over 11,000 genomes from the genus Escherichia producing an alignment with 1353 genes, which was constructed in less than 17 h. Conclusions We demonstrate that cognac presents an efficient method for generating concatenated gene alignments for phylogenetic analysis. We have released cognac as an R package ( https://github.com/rdcrawford/cognac ) with customizable parameters for adaptation to diverse applications.

To identify genetic variation underlying atrial fibrillation, the most common cardiac arrhythmia, we performed a genome-wide association study of >1,000,000 people, including 60,620 atrial fibrillation cases and 970,216 controls. We identified 142 independent risk variants at 111 loci and prioritized 151 functional candidate genes likely to be involved in atrial fibrillation. Many of the identified risk variants fall near genes where more deleterious mutations have been reported to cause serious heart defects in humans ( GATA4, MYH6 , NKX2-5 , PITX2 , TBX5 ) 1 , or near genes important for striated muscle function and integrity (for example, CFL2 , MYH7 , PKP2, RBM20, SGC G, SSPN ). Pathway and functional enrichment analyses also suggested that many of the putative atrial fibrillation genes act via cardiac structural remodeling, potentially in the form of an ‘atrial cardiomyopathy’ 2 , either during fetal heart development or as a response to stress in the adult heart. Large-scale association analyses identify 142 independent risk variants for atrial fibrillation. Pathway and functional enrichment analyses suggest that many of the putative risk genes act via cardiac structural remodeling.

A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPSs), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPSs have an impact on Bacteroides –phage interactions, we isolated 71 Bacteroides thetaiotaomicron -infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPSs, we show that CPSs dictate host tropism for these phages and that expression of non-permissive CPS variants is selected under phage predation, enabling survival. In the absence of CPSs, B. thetaiotaomicron escapes bacteriophage predation by altering expression of eight distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPSs and other cell surface structures that allow these bacteria to persist under bacteriophage predation, and hold important implications for using bacteriophages therapeutically to target gut symbionts. Isolation of phages associated with the gut commensal Bacteroides thetaiotaomicron reveals a link between cell surface structures, including phase-variable capsular polysaccharides, lipoproteins and S-layer proteins, and susceptibility to phage infection.

Horizontal Gene Transfer is a powerful force shaping microbial evolution. This constant process by which genes are acquired into and excised from bacterial genomes enables an enormous capacity for rapid phenotypic evolution. HGT enables the dissemination of clinically important genes, including antibiotic resistance genes, genes mediating virulence, environmental persistence genes, and metabolic genes. Acquisition of these genes potentiates phenotypic evolution in several important contexts: increasing the capacity for transmission, enhancing the ability for infection, limiting the efficacy of antibiotic therapies, and facilitating the metabolism of new substrates. Methods to characterize the pathways by which these genes spread through bacterial populations are critical for understanding the evolution of these phenotypes and their implications for public health. In this dissertation, I develop a novel computational approach to generate core gene alignments for large numbers of bacterial genomes and implement two methods to characterize HGT events from bacterial whole-genome sequences. I then apply these methods to understand the dissemination of antimicrobial resistance genes and the evolution of carbohydrate utilization phenotypes in the microbiome. First, we developed cognac (Core Gene Alignment Concatenation), an open-source R package for generating concatenated, core gene alignments for microbial genomes. cognac rapidly identifies shared phylogenetic marker genes, creates gene alignments, and concatenates them into a single alignment for downstream phylogenetic analysis. We demonstrate that this method can efficiently handle extremely large whole-genome sequencing datasets of diverse bacterial lineages. Second, we sought to trace the spread of the KPC gene, a carbapenemase conferring broad-spectrum resistance to commonly used antibiotics for treating infections caused by Enterobacterales. Using comprehensive collections of clinical isolates from regional healthcare networks in three US states, we quantify the role of importation, clonal dissemination, and HGT on the total burden of KPC in these regions. To identify HGT events, we implemented a novel marker gene-based approach that enabled us to track KPC plasmid transfer using short-read data and identify HGT events occurring between circulating strains in the same region. Using this approach, we show that while the horizontal transfer of KPC frequently occurs in all three states, the strains and species involved and the overall contribution to the regional burden of KPC-carrying organisms differ substantially across the three states. Third, we investigated the role of HGT in common members of the human gut microbiome. We developed a novel method to identify ancestral HGT loci by identifying core genes with significantly greater than expected divergence from the assigned species and greater similarity to the putative donor species. We then characterized HGT loci with conserved synteny and collinearity between donor and recipient species that have enabled pan-genome expansion and evolution of new phenotypes. This approach illustrates that HGT is common between two closely related species of Bacteroides, with many loci exhibiting evidence of HGT. These data, in conjunction with molecular data, provide insight into the breadth and complexity of metabolism in the microbiome and the underlying genomic events that enable the evolution of complex phenotypes. In summary, this body of work establishes computational tools with broad application in computational genomics and genomic epidemiology: enabling phylogenetic analysis of large genomic datasets, identifying recent plasmid-mediated transfer occurring within and across regional healthcare networks, and identification of ancestral HGT loci carried on the chromosome mediating the development of complex phenotypes in the microbiome.

Abstract The quantity of genomic data is expanding at an increasing rate. Tools for phylogenetic analysis which scale to the quantity of available data are required. We present cognac, a user-friendly software package to rapidly generate concatenated gene alignments for phylogenetic analysis. We applied this tool to generate core gene alignments for very large genomic datasets, including a dataset of over 11,000 genomes from the genus Escherichia containing 1,353 genes, which was constructed in less than 17 hours. We have released cognac as an R package (https://github.com/rdcrawford/cognac) with customizable parameters for adaptation to diverse applications. Competing Interest Statement The authors have declared no competing interest.

Abstract A variety of cell surface structures, including capsular polysaccharides (CPS), dictate interactions between bacteria and their environment including their viruses (bacteriophages). Members of the prominent human gut Bacteroidetes characteristically produce several phase-variable CPS, but their contributions to bacteriophage interactions are unknown. We used engineered strains of the human symbiont Bacteroides thetaiotaomicron, which differ only in the CPS they express, to isolate bacteriophages from two locations in the United States. Testing each of 71 bacteriophages against a panel of strains that express wild-type phase-variable CPS, one of eight different single CPS, or no CPS at all, revealed that each phage infects only a subset of otherwise isogenic strains. Deletion of infection-permissive CPS from B. thetaiotaomicron was sufficient to abolish infection for several individual bacteriophages, while infection of wild-type B. thetaiotaomicron with either of two different bacteriophages rapidly selected for expression of non-permissive CPS. Surprisingly, acapsular B. thetaiotaomicron also escapes complete killing by these bacteriophages, but surviving bacteria exhibit increased expression of 8 distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Finally, both wild-type and acapsular B. thetaiotaomicron were able to separately co-exist with one bacteriophage for over two months in the mouse gut, suggesting that phase-variation promotes resistance but also generates sufficient numbers of susceptible revertants to allow bacteriophage persistence. Our results reveal important roles for Bacteroides CPS and other cell surface structures that allow these bacteria to persist despite bacteriophage predation and hold important implications for using bacteriophages therapeutically to target gut symbionts.

To understand the genetic variation underlying atrial fibrillation (AF), the most common cardiac arrhythmia, we performed a genome-wide association study (GWAS) of > 1 million people, including 60,620 AF cases and 970,216 controls. We identified 163 independent risk variants at 111 loci and prioritized 165 candidate genes likely to be involved in AF. Many of the identified risk variants fall near genes where more deleterious mutations have been reported to cause serious heart defects in humans or mice (MYH6, NKX2-5, PITX2, TBC1D32, TBX5), or near genes important for striated muscle function and integrity (e.g. MYH7, PKP2, SSPN, SGCA). Experiments in rabbits with heart failure and left atrial dilation identified a heterogeneous distributed molecular switch from MYH6 to MYH7 in the left atrium, which resulted in contractile and functional heterogeneity and may predispose to initiation and maintenance of atrial arrhythmia.

Aim: The distribution of marine predators is driven by the distribution and abundance of their prey; areas preferred by multiple marine predator species should therefore indicate areas of ecological significance. The Southern Ocean supports large populations of seabirds and marine mammals and is undergoing rapid environmental change. The management and conservation of these predators and their environment relies on understanding their distribution and its link with the biophysical environment, as the latter determines the distribution and abundance of prey. We addressed this issue using tracking data from 14 species of marine predators to identify important habitat. Location: Indian Ocean sector of the Southern Ocean. Methods: We used tracking data from 538 tag deployments made over a decade at the Subantarctic Prince Edward Islands. For each real track, we simulated a set of pseudo-tracks that allowed a presence-availability habitat modelling approach that estimates an animal's habitat preference. Using model ensembles of boosted regression trees and random forests, we modelled these tracks as a response to a set of 17 environmental variables. We combined the resulting species-specific models to evaluate areas of mean importance. Results: Real tracking locations covered 39.75 million km², up to 7,813 km from the Prince Edward Islands. Areas of high mean importance were located broadly from the Subtropical Zone to the Polar Frontal Zone in summer and from the Subantarctic to Antarctic Zones in winter. Areas of high mean importance were best predicted by factors including wind speed, sea surface temperature, depth and current speed. Main conclusions: The models and predictions developed here identify important habitat of marine predators around the Prince Edward Islands and can support the largescale conservation and management of Subantarctic ecosystems and the marine predators they sustain. The results also form the basis of future efforts to predict the consequences of environmental change.

Ketone bodies are elevated in response to fasting, a low-carbohydrate ketogenic diet or high-intensity exercise. Vishwa Deep Dixit and colleagues report that one metabolite, β-hydroxybutyrate, inhibits the NLRP3 inflammasome. In vivo , β-hydroxybutyrate is anti-inflammatory and suppresses NLRP3-mediated inflammatory disease. The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP 1 . BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet 2 . Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown 2 , 3 , 4 , 5 , 6 . We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K + efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein–coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome–mediated interleukin (IL)-1β and IL-18 production in human monocytes. In vivo , BHB or a ketogenic diet attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases such as Muckle–Wells syndrome, familial cold autoinflammatory syndrome and urate crystal–induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.

Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and hypothesized to drive the loss of immune tolerance to self-antigens in autoimmunity. Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines. Here, we investigate the hypothesis that citrullination exposes cryptic peptides by modifying protein structure and proteolytic cleavage. We show that citrullination alters processing and presentation of autoantigens, resulting in the generation of a unique citrullination-dependent repertoire composed primarily of native sequences. This repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than controls. The generation of this unique repertoire is achieved through altered protease cleavage and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA. Antibodies directed against citrullinated proteins are commonly found in patients with rheumatoid arthritis. Here, the authors show that citrullination alters the peptide repertoire presented to T cells by altering protease cleavage and inducing protein destabilization, thereby exposing cryptic epitopes.