Biology subjects

Whitney, J.

Publications and source records attributed to Whitney, J..

8 recordsLinked to original sources

bioRxiv2025

GWAS SVatalog: a visualization tool to aid fine-mapping of GWAS loci with structural variations

BackgroundGenome-wide association studies (GWAS) have been successful in identifying single nucleotide polymorphisms (SNPs) associated with phenotypic traits. However, SNPs form an incomplete set of variation across the genome and since a large percentage of GWAS-significant SNPs lie in non-coding regions, their impact on a given trait is difficult to decipher. Recognizing whether these SNPs are tagging other polymorphisms, like structural variations (SV), is an important step towards understanding the putative causal variation at GWAS loci. ResultsHere, we develop GWAS SVatalog (https://svatalog.research.sickkids.ca/), a novel open-source web tool that computes and visualizes linkage disequilibrium (LD) between SVs and GWAS-associated SNPs throughout the human genome. The tool combines GWAS Catalogs SNP-trait association data across 14,479 phenotypes with LD statistics calculated between 35,732 SVs and 116,870 SNPs identified in 101 whole-genome long-read sequences. We use GWAS SVatalog to identify SVs that may explain GWAS loci for iron levels, refractive error, and Alzheimers disease, where previously SNPs were unable to provide a causal explanation. ConclusionsGWAS SVatalog advances the fine-mapping of GWAS loci with structural variations, enabling researchers to associate 35,732 common SVs with 14,479 phenotypes, accelerating the understanding of disease etiology.

bioinformatics↗

bioRxiv2025

Rapidly evolving orphan immunity genes protect human gut bacteria from intoxication by the type VI secretion system

Bacteria encode diverse mechanisms for mediating interbacterial antagonism through the exchange of toxic effector proteins. Although the structure, function, and regulation of these pathways has been well established for many organisms, an understanding of their ecological and evolutionary dynamics lags behind. Type VI secretion systems (T6SS) deliver effectors between competing Gram-negative bacteria, including among mammalian gut Bacteroidales, resulting in the evolution of elaborate defense mechanisms that protect against T6SS attack. One such mechanism is the recombinase-associated acquired interbacterial defence (rAID) system, which harbors arrays of orphan immunity genes that diverge in sequence from T6SS-associated cognate immunity genes. It is not known if such sequence divergence impacts rAID orphan immunity function, or how rAID distribution across microbiomes relates to the T6SS. Here, we show that divergent rAID orphan immunity factors that possess SUKH domains allow bacteria to survive intoxication by cognate effectors. Such protection is due to high affinity protein-protein interactions between orphan immunity and effector that are comparable to that of cognate effector-immunity. Unlike other examples of T6SS effector-immunity interactions, we find that the binding interface is comprised of electrostatic interactions with a high degree of redundancy underlying its protective capacity. Finally, we quantify orphan immunity and effector gene abundance and dynamics across human gut metagenomes, revealing patterns of co-occurrence indicative of positive selection. Population genetic analyses of longitudinal data suggests that orphan immunity genes accumulate non-synonymous mutations that lie at the predicted effector-immunity interface. Together, our findings establish rAID orphan immunity genes as important bacterial fitness determinants in the human gut.

microbiology↗

bioRxiv2024

The structure of the Tad pilus alignment complex reveals a periplasmic conduit for pilus extension

The Tad (Tight adherence) pilus is a bacterial appendage implicated in bacterial virulence, cell-cell aggregation, and biofilm formation. Despite its homology to the well-characterised Type IV pilus, the structure and assembly mechanism of the Tad pilus are poorly understood. Here, we investigate the role of the protein RcpC from Pseudomonas aeruginosa. Our analyses reveal that RcpC forms a dodecameric periplasmic complex, anchored to the inner membrane by a transmembrane helix, and interacting with the outer membrane secretin RcpA. We use single-particle Cryo-EM to elucidate the structure of this RcpC dodecamer, and cell-based assays to demonstrate that the RcpC-RcpA complex is essential for Tad-mediated cell-cell aggregation. Collectively, these data demonstrate that RcpC forms the Tad pilus alignment complex, which provides a conduit across the periplasm for the Tad pilus filament to access the extracellular milieu. Our experimental data and structure-based models allow us to propose a mechanism for Tad plus assembly.

microbiology↗

bioRxiv2022

Dual targeting factors are required for LXG toxin export by the bacterial type VIIb secretion system

Bacterial type VIIb secretion systems (T7SSb) are multi-subunit integral membrane protein complexes found in Firmicutes that play a role in both bacterial competition and virulence by secreting toxic effector proteins. The majority of characterized T7SSb effectors adopt a polymorphic domain architecture consisting of a conserved N-terminal Leu-X-Gly (LXG) domain and a variable C-terminal toxin domain. Recent work has started to reveal the diversity of toxic activities exhibited by LXG effectors; however, little is known about how these proteins are recruited to the T7SSb apparatus. In this work, we sought to characterize genes encoding domains of unknown function (DUFs) 3130 and 3958, which frequently co-occur with LXG effector-encoding genes. Using coimmunoprecipitation-mass spectrometry analyses, in vitro copurification experiments and T7SSb secretion assays, we find that representative members of these protein families form heteromeric complexes with their cognate LXG domain and in doing so, function as targeting factors that promote effector export. Additionally, an X-ray crystal structure of a representative DUF3958 protein, combined with predictive modelling of DUF3130 using AlphaFold2, reveals structural similarity between these protein families and the ubiquitous WXG100 family of T7SS effectors. Interestingly, we identify a conserved FxxxD motif within DUF3130 that is reminiscent of the YxxxD/E "export arm" found in Mycobacterial T7SSa substrates and mutation of this motif abrogates LXG effector secretion. Overall, our data experimentally link previously uncharacterized bacterial DUFs to type VIIb secretion and reveal a molecular signature required for LXG effector export. Significance statementType VIIb secretion systems (T7SSb) are protein secretion machines used by an array of Gram-positive bacterial genera including Staphylococcus, Streptococcus, Bacillus, and Enterococcus. These bacteria use the T7SSb to facilitate interbacterial killing and pathogenesis through the secretion of toxins. Although the modes of toxicity for a number of these toxins have been investigated, the mechanisms by which they are recognized and secreted by T7SSb remains poorly understood. The significance of this work is the discovery of two new protein families, termed Lap1 and Lap2, that directly interact with these toxins and are required for their secretion. Overall, Lap1 and Lap2 represent two widespread families of proteins that function as targeting factors that participate in T7SSb-dependent toxin release from Gram-positive bacteria.

microbiology↗

bioRxiv2022

High Quality Phasing Using Linked-Read Whole Genome Sequencing of Patient Cohorts Informs Genetic Understanding of Complex Traits

Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. For population studies, phase is often inferred from external data but read-based phasing approaches that span long genomic distances would be more accurate because they enable both genotype and phase to be obtained from a single dataset. To demonstrate how read-based phasing can provide functional insights, we sequenced 477 individuals with Cystic Fibrosis (CF) using linked-read sequencing. We benchmark read-based phasing with different short- and long-read sequencing technologies, prioritize linked-read technology as the most informative and produce a benchmark phase call set from reference sample HG002 for the community. The 477 samples display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, which facilitates understanding of complex CF alleles. Fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus demonstrates a 20 kb deletion and a PRSS2 missense variant p.Thr8Ile (rs62473563) independently contribute to meconium ileus risk (p=0.0028, p=0.011, respectively) and are PRSS2 pancreas eQTLs (p=9.5e-7 and p=1.4e-4, respectively), explaining the mechanism by which these polymorphisms contribute to CF. Phase enables access to haplotypes that can be used for genome graph or reference panel construction, identification of cis-effects, and for understanding disease associated loci. The phase information from linked-reads provides a causal explanation for variation at a CF-relevant locus which also has implications for the genetic basis of non-CF pancreatitis to which this locus has been reported to contribute.

genetics↗

bioRxiv2021

Structure of a bacterial Rhs effector exported by the type VI secretion system

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 [A] resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise {beta}-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs cocoon where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal plug domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

biochemistry↗

bioRxiv2020

Polymorphic SINEC_Cf Retrotransposons in the Genome of the Dog (Canis familiaris)

The dog is an exciting genetic system in which many simple and complex traits have now been mapped. For many traits the causal mutation is a polymorphic SINE. To investigate the genome-wide pattern of young SINEC_Cf insertions, we sampled 62 dogs representing 59 breeds and sequenced libraries enriched for SINE flanks. In each dog we detect an average of 10,423 polymorphic loci and all together the libraries identify 81,747 putative polymorphic SINEs. We validated 184 SINEs inserted in protein-coding exons, untranslated regions, introns and intergenic sequence. In dogs both SINEC_Cf and LINEs exhibit a strand bias in introns where antisense copies are more frequent. Antisense polymorphic SINEs also have a higher density in introns. Both SINEs and LINEs drop to very low density near exons. Both sense and antisense polymorphic SINEs also drop to low density upstream of coding exons but not downstream. Antisense polymorphic SINEC_Cfs upstream of coding exons are known to cause narcolepsy, merle, and progressive retinal atrophy in dogs. In other mammals SINE pairs in inverted orientation disrupt gene expression. We find inverted pairs of SINEC_Cf are rare in both introns and intergenic sequence when the two SINEs are separated by less than 100 bp. The lack of inverted pairs is even more pronounced when the SINEs have high sequence identity. Intronic and intergenic LINE pairs show similar patterns. Polymorphic SINEs rarely pair with either SINEC_Cf or SINEC_Cf2. Overall, the high insertion rate of SINEC_Cf provides a natural mutagenesis screen in the dog genome.

genomics↗

bioRxiv2020

Structure of the extracellular region of the bacterial type VIIb secretion system subunit EsaA

Gram-positive bacteria use type VII secretion systems (T7SSs) to export effector proteins that manipulate the physiology of nearby prokaryotic and eukaryotic cells. Several mycobacterial T7SSs have established roles in virulence. By contrast, recent work has demonstrated that the genetically distinct T7SSb pathway found in Firmicutes bacteria more often functions to mediate interbacterial competition. A lack of structural information on the T7SSb has limited the understanding of effector export by this protein secretion apparatus. In this work, we present the 2.4[A] crystal structure of the extracellular region of the elusive T7SSb subunit EsaA from Streptococcus gallolyticus. Our structure reveals that homodimeric EsaA is an elongated, arrow-shaped protein with a surface-accessible tip, which serves as a receptor for lytic bacteriophages in some species of bacteria. Because it is the only T7SSb subunit large enough to traverse the thick peptidoglycan layer of Firmicutes bacteria, we propose that EsaA plays a critical role in transporting effectors across the entirety of the Gram-positive cell envelope.

microbiology↗