Protein interaction networks in the vasculature prioritize genes and pathways underlying coronary artery disease.

Population-based association studies have identified many genetic risk loci for coronary artery disease (CAD), but it is often unclear how genes within these loci are linked to CAD. Here, we perform interaction proteomics for 11 CAD-risk genes to map their protein-protein interactions (PPIs) in human vascular cells and elucidate their roles in CAD. The resulting PPI networks contain interactions that are outside of known biology in the vasculature and are enriched for genes involved in immunity-related and arterial-wall-specific mechanisms. Several PPI networks derived from smooth muscle cells are significantly enriched for genetic variants associated with CAD and related vascular phenotypes. Furthermore, the networks identify 61 genes that are found in genetic loci associated with risk of CAD, prioritizing them as the causal candidates within these loci. These findings indicate that the PPI networks we have generated are a rich resource for guiding future research into the molecular pathogenesis of CAD.

Using brain cell-type-specific protein interactomes to interpret neurodevelopmental genetic signals in schizophrenia.

Genetics have nominated many schizophrenia risk genes and identified convergent signals between schizophrenia and neurodevelopmental disorders. However, functional interpretation of the nominated genes in the relevant brain cell types is often lacking. We executed interaction proteomics for six schizophrenia risk genes that have also been implicated in neurodevelopment in human induced cortical neurons. The resulting protein network is enriched for common variant risk of schizophrenia in Europeans and East Asians, is down-regulated in layer 5/6 cortical neurons of individuals affected by schizophrenia, and can complement fine-mapping and eQTL data to prioritize additional genes in GWAS loci. A sub-network centered on HCN1 is enriched for common variant risk and contains proteins (HCN4 and AKAP11) enriched for rare protein-truncating mutations in individuals with schizophrenia and bipolar disorder. Our findings showcase brain cell-type-specific interactomes as an organizing framework to facilitate interpretation of genetic and transcriptomic data in schizophrenia and its related disorders.

Protein interaction studies in human induced neurons indicate convergent biology underlying autism spectrum disorders.

Autism spectrum disorders (ASDs) have been linked to genes with enriched expression in the brain, but it is unclear how these genes converge into cell-type-specific networks. We built a protein-protein interaction network for 13 ASD-associated genes in human excitatory neurons derived from induced pluripotent stem cells (iPSCs). The network contains newly reported interactions and is enriched for genetic and transcriptional perturbations observed in individuals with ASDs. We leveraged the network data to show that the ASD-linked brain-specific isoform of ANK2 is important for its interactions with synaptic proteins and to characterize a PTEN-AKAP8L interaction that influences neuronal growth. The IGF2BP1-3 complex emerged as a convergent point in the network that may regulate a transcriptional circuit of ASD-associated genes. Our findings showcase cell-type-specific interactomes as a framework to complement genetic and transcriptomic data and illustrate how both individual and convergent interactions can lead to biological insights into ASDs.

A small bacteriophage protein determines the hierarchy over co-residential jumbo phage in Bacillus thuringiensis serovar israelensis.

Bacillus thuringiensis serovar israelensis is the most widely used biopesticide against insects, including vectors of animal and human diseases. Among several extrachromosomal elements, this endospore-forming entomopathogen harbors two bacteriophages: a linear DNA replicon named GIL01 that does not integrate into the chromosome during lysogeny and a circular-jumbo prophage known as pBtic235. Here, we show that GIL01 hinders the induction of cohabiting prophage pBtic235. The GIL01-encoded small protein, gp7, which interacts with the host LexA repressor, is a global transcription regulator and represses the induction of pBtic235 after DNA damage to presumably allow GIL01 to multiply first. In a complex with host LexA in stressed cells, gp7 down-regulates the expression of more than 250 host and pBtic235 genes, many of which are involved in the cellular functions of genome maintenance, cell-wall transport, and membrane and protein stability. We show that gp7 homologs that are found exclusively in bacteriophages act in a similar fashion to enhance LexA's binding to DNA, while likely also affecting host gene expression. Our results provide evidence that GIL01 influences both its host and its co-resident bacteriophage.

The Small DdrR Protein Directly Interacts with the UmuDAb Regulator of the Mutagenic DNA Damage Response in Acinetobacter baumannii.

Acinetobacter baumannii poses a great threat in health care settings worldwide, with clinical isolates displaying an ever-evolving multidrug resistance. In strains of A. baumannii, expression of multiple error-prone polymerase genes are corepressed by UmuDAb, a member of the LexA superfamily, and a small protein, DdrR. It is currently unknown how DdrR establishes this repression. Here, we used surface plasmon resonance spectrometry to show that DdrR formed a stable complex with the UmuDAb regulator. Our results indicated that the carboxy-terminal dimerization domain of UmuDAb formed the interaction interface with DdrR. Our in vitro data also showed that RecA-mediated inactivation of UmuDAb was inhibited when this transcription factor was bound to its target DNA. In addition, we showed that DdrR interacted with a putative prophage repressor, homologous to LexA superfamily proteins. These data suggested that DdrR modulated DNA damage response and prophage induction in A. baumannii by binding to LexA-like regulators. IMPORTANCE We previously identified a 50-residue bacteriophage protein, gp7, which interacts with and modulates the function of the LexA transcription factor from Bacillus thuringiensis. Here, we present data that indicates that the small DdrR protein from A. baumannii likely coordinates the SOS response and prophage processes by also interacting with LexA superfamily members. We suggest that similar small proteins that interact with LexA-like proteins to coordinate DNA repair and bacteriophage functions may be common to many bacteria that mount the SOS response.

Genoppi is an open-source software for robust and standardized integration of proteomic and genetic data.

Combining genetic and cell-type-specific proteomic datasets can generate biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi (lagelab.org/genoppi) that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We use Genoppi to analyze 16 cell-type-specific protein interaction datasets of four proteins (BCL2, TDP-43, MDM2, PTEN) involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer cell types and one human iPSC-derived neuronal cell type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodegenerative diseases. Importantly, our analyses suggest that human iPSC-derived neurons are a relevant model system for studying the involvement of BCL2 and TDP-43 in amyotrophic lateral sclerosis.

Systematic auditing is essential to debiasing machine learning in biology.

Biases in data used to train machine learning (ML) models can inflate their prediction performance and confound our understanding of how and what they learn. Although biases are common in biological data, systematic auditing of ML models to identify and eliminate these biases is not a common practice when applying ML in the life sciences. Here we devise a systematic, principled, and general approach to audit ML models in the life sciences. We use this auditing framework to examine biases in three ML applications of therapeutic interest and identify unrecognized biases that hinder the ML process and result in substantially reduced model performance on new datasets. Ultimately, we show that ML models tend to learn primarily from data biases when there is insufficient signal in the data to learn from. We provide detailed protocols, guidelines, and examples of code to enable tailoring of the auditing framework to other biomedical applications.

Growth effects of N-acylethanolamines on gut bacteria reflect altered bacterial abundances in inflammatory bowel disease.

Inflammatory bowel diseases (IBD) are associated with alterations in gut microbial abundances and lumenal metabolite concentrations, but the effects of specific metabolites on the gut microbiota in health and disease remain largely unknown. Here, we analysed the influences of metabolites that are differentially abundant in IBD on the growth and physiology of gut bacteria that are also differentially abundant in IBD. We found that N-acylethanolamines (NAEs), a class of endogenously produced signalling lipids elevated in the stool of IBD patients and a T-cell transfer model of colitis, stimulated growth of species over-represented in IBD and inhibited that of species depleted in IBD in vitro. Using metagenomic sequencing, we recapitulated the effects of NAEs in complex microbial communities ex vivo, with Proteobacteria blooming and Bacteroidetes declining in the presence of NAEs. Metatranscriptomic analysis of the same communities identified components of the respiratory chain as important for the metabolism of NAEs, and this was verified using a mutant deficient for respiratory complex I. In this study, we identified NAEs as a class of metabolites that are elevated in IBD and have the potential to shift gut microbiota towards an IBD-like composition.

Structural Insights into Bacteriophage GIL01 gp7 Inhibition of Host LexA Repressor.

Bacteria identify and respond to DNA damage using the SOS response. LexA, a central repressor in the response, has been implicated in the regulation of lysogeny in various temperate bacteriophages. During infection of Bacillus thuringiensis with GIL01 bacteriophage, LexA represses the SOS response and the phage lytic cycle by binding DNA, an interaction further stabilized upon binding of a viral protein, gp7. Here we report the crystallographic structure of phage-borne gp7 at 1.7-Å resolution, and characterize the 4:2 stoichiometry and potential interaction with LexA using surface plasmon resonance, static light scattering, and small-angle X-ray scattering. These data suggest that gp7 stabilizes LexA binding to operator DNA via coordination of the N- and C-terminal domains of LexA. Furthermore, we have found that gp7 can interact with LexA from Staphylococcus aureus, a significant human pathogen. Our results provide structural evidence as to how phage factors can directly associate with LexA to modulate the SOS response.

Bacteroides-Derived Sphingolipids Are Critical for Maintaining Intestinal Homeostasis and Symbiosis.

Sphingolipids are structural membrane components and important eukaryotic signaling molecules. Sphingolipids regulate inflammation and immunity and were recently identified as the most differentially abundant metabolite in stool from inflammatory bowel disease (IBD) patients. Commensal bacteria from the Bacteroidetes phylum also produce sphingolipids, but the impact of these metabolites on host pathways is largely uncharacterized. To determine whether bacterial sphingolipids modulate intestinal health, we colonized germ-free mice with a sphingolipid-deficient Bacteroides thetaiotaomicron strain. A lack of Bacteroides-derived sphingolipids resulted in intestinal inflammation and altered host ceramide pools in mice. Using lipidomic analysis, we described a sphingolipid biosynthesis pathway and revealed a variety of Bacteroides-derived sphingolipids including ceramide phosphoinositol and deoxy-sphingolipids. Annotating Bacteroides sphingolipids in an IBD metabolomic dataset revealed lower abundances in IBD and negative correlations with inflammation and host sphingolipid production. These data highlight the role of bacterial sphingolipids in maintaining homeostasis and symbiosis in the gut.

Author Correction: Gut microbiome structure and metabolic activity in inflammatory bowel disease.

In the Supplementary Tables 2, 4 and 6 originally published with this Article, the authors mistakenly included sample identifiers in the form of UMCGs rather than UMCG IBDs in the validation cohort; this has now been amended.

Gut microbiome structure and metabolic activity in inflammatory bowel disease.

The inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are multifactorial chronic conditions of the gastrointestinal tract. While IBD has been associated with dramatic changes in the gut microbiota, changes in the gut metabolome-the molecular interface between host and microbiota-are less well understood. To address this gap, we performed untargeted metabolomic and shotgun metagenomic profiling of cross-sectional stool samples from discovery (n = 155) and validation (n = 65) cohorts of CD, UC and non-IBD control patients. Metabolomic and metagenomic profiles were broadly correlated with faecal calprotectin levels (a measure of gut inflammation). Across >8,000 measured metabolite features, we identified chemicals and chemical classes that were differentially abundant in IBD, including enrichments for sphingolipids and bile acids, and depletions for triacylglycerols and tetrapyrroles. While > 50% of differentially abundant metabolite features were uncharacterized, many could be assigned putative roles through metabolomic 'guilt by association' (covariation with known metabolites). Differentially abundant species and functions from the metagenomic profiles reflected adaptation to oxidative stress in the IBD gut, and were individually consistent with previous findings. Integrating these data, however, we identified 122 robust associations between differentially abundant species and well-characterized differentially abundant metabolites, indicating possible mechanistic relationships that are perturbed in IBD. Finally, we found that metabolome- and metagenome-based classifiers of IBD status were highly accurate and, like the vast majority of individual trends, generalized well to the independent validation cohort. Our findings thus provide an improved understanding of perturbations of the microbiome-metabolome interface in IBD, including identification of many potential diagnostic and therapeutic targets.

Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue.

The GIL01 bacteriophage is a temperate phage that infects the insect pathogen Bacillus thuringiensis. During the lytic cycle, phage gene transcription is initiated from three promoters: P1 and P2, which control the expression of the early phage genes involved in genome replication and P3, which controls the expression of the late genes responsible for virion maturation and host lysis. Unlike most temperate phages, GIL01 lysogeny is not maintained by a dedicated phage repressor but rather by the host's regulator of the SOS response, LexA. Previously we showed that the lytic cycle was induced by DNA damage and that LexA, in conjunction with phage-encoded protein gp7, repressed P1. Here we examine the lytic/lysogenic switch in more detail and show that P3 is also repressed by a LexA-gp7 complex, binding to tandem LexA boxes within the promoter. We also demonstrate that expression from P3 is considerably delayed after DNA damage, requiring the phage-encoded DNA binding protein, gp6. Surprisingly, gp6 is homologous to LexA itself and, thus, is a rare example of a LexA homologue directly activating transcription. We propose that the interplay between these two LexA family members, with opposing functions, ensures the timely expression of GIL01 phage late genes.

A Common Mechanism Links Activities of Butyrate in the Colon.

Two biological activities of butyrate in the colon (suppression of proliferation of colonic epithelial stem cells and inflammation) correlate with inhibition of the activity of histone deacetylases. Cellular and biochemical studies of molecules similar in structure to butyrate, but different in molecular details (functional groups, chain-length, deuteration, oxidation level, fluorination, or degree of unsaturation), demonstrated that these activities were sensitive to molecular structure, and were compatible with the hypothesis that butyrate acts by binding to the Zn2+ in the catalytic site of histone deacetylases. Structure-activity relationships drawn from a set of 36 compounds offer a starting point for the design of new compounds targeting the inhibition of histone deacetylases. The observation that butyrate was more potent than other short-chain fatty acids is compatible with the hypothesis that crypts evolved (at least in part), to separate stem cells at the base of crypts from butyrate produced by commensal bacteria.

A novel Ruminococcus gnavus clade enriched in inflammatory bowel disease patients.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract that is associated with changes in the gut microbiome. Here, we sought to identify strain-specific functional correlates with IBD outcomes. METHODS: We performed metagenomic sequencing of monthly stool samples from 20 IBD patients and 12 controls (266 total samples). These were taxonomically profiled with MetaPhlAn2 and functionally profiled using HUMAnN2. Differentially abundant species were identified using MaAsLin and strain-specific pangenome haplotypes were analyzed using PanPhlAn. RESULTS: We found a significantly higher abundance in patients of facultative anaerobes that can tolerate the increased oxidative stress of the IBD gut. We also detected dramatic, yet transient, blooms of Ruminococcus gnavus in IBD patients, often co-occurring with increased disease activity. We identified two distinct clades of R. gnavus strains, one of which is enriched in IBD patients. To study functional differences between these two clades, we augmented the R. gnavus pangenome by sequencing nine isolates from IBD patients. We identified 199 IBD-specific, strain-specific genes involved in oxidative stress responses, adhesion, iron-acquisition, and mucus utilization, potentially conferring an adaptive advantage for this R. gnavus clade in the IBD gut. CONCLUSIONS: This study adds further evidence to the hypothesis that increased oxidative stress may be a major factor shaping the dysbiosis of the microbiome observed in IBD and suggests that R. gnavus may be an important member of the altered gut community in IBD.

The Use and Abuse of LexA by Mobile Genetic Elements.

The SOS response is an essential process for responding to DNA damage in bacteria. The expression of SOS genes is under the control of LexA, a global transcription factor that undergoes self-cleavage during stress to allow the expression of DNA repair functions and delay cell division until the damage is rectified. LexA also regulates genes that are not part of this cell rescue program, and the induction of bacteriophages, the movement of pathogenicity islands, and the expression of virulence factors and bacteriocins are all controlled by this important transcription factor. Recently it has emerged that when regulating the expression of genes from mobile genetic elements (MGEs), LexA often does so in concert with a corepressor. This accessory regulator can either be a host-encoded global transcription factor, which responds to various metabolic changes, or a factor that is encoded for by the MGE itself. Thus, the coupling of LexA-mediated regulation to a secondary transcription factor not only detaches LexA from its primary SOS role, but also fine-tunes gene expression from the MGE, enabling it to respond to multiple stresses. Here we discuss the mechanisms of such coordinated regulation and its implications for cells carrying such MGEs.

Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage.

The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription.

Identification of a ligand on the Wip1 bacteriophage highly specific for a receptor on Bacillus anthracis.

Tectiviridae is a family of tailless bacteriophages with Gram-negative and Gram-positive hosts. The family model PRD1 and its close relatives all infect a broad range of enterobacteria by recognizing a plasmid-encoded conjugal transfer complex as a receptor. In contrast, tectiviruses with Gram-positive hosts are highly specific to only a few hosts within the same bacterial species. The cellular determinants that account for the observed specificity remain unknown. Here we present the genome sequence of Wip1, a tectivirus that infects the pathogen Bacillus anthracis. The Wip1 genome is related to other tectiviruses with Gram-positive hosts, notably, AP50, but displays some interesting differences in its genome organization. We identified Wip1 candidate genes for the viral spike complex, the structure located at the capsid vertices and involved in host receptor binding. Phage adsorption and inhibition tests were combined with immunofluorescence microscopy to show that the Wip1 gene product p23 is a receptor binding protein. His-p23 also formed a stable complex with p24, a Wip1 protein of unknown function, suggesting that the latter is involved with p23 in host cell recognition. The narrow host range of phage Wip1 and the identification of p23 as a receptor binding protein offer a new range of suitable tools for the rapid identification of B. anthracis.

Identification of five novel tectiviruses in Bacillus strains: analysis of a highly variable region generating genetic diversity.

Our biosphere is abundant with unique and small genes for which no homologs are known. These genes, often referred to as orphans or ORFans, are commonly found in bacteriophage genomes but their origins remain unclear. We discovered five novel tectivirus-like genetic elements by screening more than five-hundred Bacillus strains. A highly variable region (HVR) of these viruses was shown to harbor ORFans in most of these otherwise well-conserved bacteriophages. Previous studies demonstrated that mutations close to this region dramatically alter bacteriophage gene regulation, suggesting that the acquisition of those ORFans may provide a source of genetic diversity that is then subject to genetic selection during bacteriophage evolution.

Phage-borne factors and host LexA regulate the lytic switch in phage GIL01.

The Bacillus thuringiensis temperate phage GIL01 does not integrate into the host chromosome but exists stably as an independent linear replicon within the cell. Similar to that of the lambdoid prophages, the lytic cycle of GIL01 is induced as part of the cellular SOS response to DNA damage. However, no CI-like maintenance repressor has been detected in the phage genome, suggesting that GIL01 uses a novel mechanism to maintain lysogeny. To gain insights into the GIL01 regulatory circuit, we isolated and characterized a set of 17 clear plaque (cp) mutants that are unable to lysogenize. Two phage-encoded proteins, gp1 and gp7, are required for stable lysogen formation. Analysis of cp mutants also identified a 14-bp palindromic dinBox1 sequence within the P1-P2 promoter region that resembles the known LexA-binding site of Gram-positive bacteria. Mutations at conserved positions in dinBox1 result in a cp phenotype. Genomic analysis identified a total of three dinBox sites within GIL01 promoter regions. To investigate the possibility that the host LexA regulates GIL01, phage induction was measured in a host carrying a noncleavable lexA (Ind(-)) mutation. GIL01 formed stable lysogens in this host, but lytic growth could not be induced by treatment with mitomycin C. Also, mitomycin C induced ß-galactosidase expression from GIL01-lacZ promoter fusions, and induction was similarly blocked in the lexA (Ind(-)) mutant host. These data support a model in which host LexA binds to dinBox sequences in GIL01, repressing phage gene expression during lysogeny and providing the switch necessary to enter lytic development.