Publications by authors named "Ian R Henderson"

160 Publications

Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system.

Elife 2021 Feb 24;10. Epub 2021 Feb 24.

Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia.

Protein acylation is critical for many cellular functions across all domains of life. In bacteria, lipoproteins have important roles in virulence and are targets for the development of antimicrobials and vaccines. Bacterial lipoproteins are secreted from the cytosol via the Sec pathway and acylated on an N-terminal cysteine residue through the action of three enzymes. In Gram-negative bacteria, the Lol pathway transports lipoproteins to the outer membrane. Here we demonstrate that the Aat secretion system is a composite system sharing similarity with elements of a type I secretion systems and the Lol pathway. During secretion, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue. Mutations disrupting glycine acylation interfere with membrane incorporation and trafficking. Our data reveal CexE as the first member of a new class of glycine-acylated lipoprotein, while Aat represents a new secretion system that displays the substrate lipoprotein on the cell surface.
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http://dx.doi.org/10.7554/eLife.63762DOI Listing
February 2021

Meiotic chromosome axis remodelling is critical for meiotic recombination in Brassica rapa.

J Exp Bot 2021 Jan 27. Epub 2021 Jan 27.

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) OT Gatersleben, Corrensstrasse, Seeland, Germany.

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling on crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2 COs are increased towards telomeric regions at the expense of (peri-)centromeric COs compared to WT. Together, in B. rapa axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programs.
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http://dx.doi.org/10.1093/jxb/erab035DOI Listing
January 2021

Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation.

Elife 2020 12 14;9. Epub 2020 Dec 14.

Institute of Microbiology and Infection, University of Birmingham, Edgbaston, United Kingdom.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.
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http://dx.doi.org/10.7554/eLife.62614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806268PMC
December 2020

Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA.

J Bacteriol 2020 Dec 18;203(2). Epub 2020 Dec 18.

Institute of Microbiology and Infection, University of Birmingham, Edgbaston, United Kingdom

The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from , solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid. M48 metalloproteases are widely distributed in all domains of life. possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.
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http://dx.doi.org/10.1128/JB.00434-20DOI Listing
December 2020

MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis.

EMBO J 2020 Nov 16;39(21):e104858. Epub 2020 Sep 16.

Department of Plant Sciences, University of Cambridge, Cambridge, UK.

During meiosis, DNA double-strand breaks undergo interhomolog repair to yield crossovers between homologous chromosomes. To investigate how interhomolog sequence polymorphism affects crossovers, we sequenced multiple recombinant populations of the model plant Arabidopsis thaliana. Crossovers were elevated in the diverse pericentromeric regions, showing a local preference for polymorphic regions. We provide evidence that crossover association with elevated diversity is mediated via the Class I crossover formation pathway, although very high levels of diversity suppress crossovers. Interhomolog polymorphism causes mismatches in recombining molecules, which can be detected by MutS homolog (MSH) mismatch repair protein heterodimers. Therefore, we mapped crossovers in a msh2 mutant, defective in mismatch recognition, using multiple hybrid backgrounds. Although total crossover numbers were unchanged in msh2 mutants, recombination was remodelled from the diverse pericentromeres towards the less-polymorphic sub-telomeric regions. Juxtaposition of megabase heterozygous and homozygous regions causes crossover remodelling towards the heterozygous regions in wild type Arabidopsis, but not in msh2 mutants. Immunostaining showed that MSH2 protein accumulates on meiotic chromosomes during prophase I, consistent with MSH2 regulating meiotic recombination. Our results reveal a pro-crossover role for MSH2 in regions of higher sequence diversity in A. thaliana.
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http://dx.doi.org/10.15252/embj.2020104858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604573PMC
November 2020

Mice Deficient in T-bet Form Inducible NO Synthase-Positive Granulomas That Fail to Constrain .

J Immunol 2020 Aug 26;205(3):708-719. Epub 2020 Jun 26.

Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom;

Clearance of intracellular infections caused by Typhimurium (STm) requires IFN-γ and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-γ mice succumb rapidly to STm infections, T-bet mice do not. In this study, we assess the anatomy of immune responses and the relationship with bacterial localization in the spleens and livers of STm-infected IFN-γ and T-bet mice. In IFN-γ mice, there is deficient granuloma formation and inducible NO synthase (iNOS) induction, increased dissemination of bacteria throughout the organs, and rapid death. The provision of a source of IFN-γ reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-γ. T-bet mice induce significant levels of IFN-γ after challenge. Moreover, T-bet mice have augmented IL-17 and neutrophil numbers, and neutralizing IL-17 reduces the neutrophilia but does not affect numbers of bacteria detected. Surprisingly, T-bet mice exhibit surprisingly wild-type-like immune cell organization postinfection, including extensive iNOS granuloma formation. In wild-type mice, most bacteria are within iNOS granulomas, but in T-bet mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-γ-dependent iNOS granulomas and prevent dissemination.
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http://dx.doi.org/10.4049/jimmunol.2000089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372318PMC
August 2020

and Maintain the Obligate Crossover in Wheat Despite Stepwise Gene Loss following Polyploidization.

Plant Physiol 2020 08 11;183(4):1545-1558. Epub 2020 Jun 11.

Department of Genetics and Genome Biology, University of Leicester, Leicester LE1 7RH, United Kingdom

Crossovers (COs) ensure accurate chromosome segregation during meiosis while creating novel allelic combinations. Here, we show that allotetraploid (AABB) durum wheat ( ssp. ) utilizes two pathways of meiotic recombination. The class I pathway requires MSH4 and MSH5 (MutSγ) to maintain the obligate CO/chiasma and accounts for ∼85% of meiotic COs, whereas the residual ∼15% are consistent with the class II CO pathway. Class I and class II chiasmata are skewed toward the chromosome ends, but class II chiasmata are significantly more distal than class I chiasmata. Chiasma distribution does not reflect the abundance of double-strand breaks, detected by proxy as RAD51 foci at leptotene, but only ∼2.3% of these sites mature into chiasmata. MutSγ maintains the obligate chiasma despite a 5.4-kb deletion in rendering it nonfunctional, which occurred early in the evolution of tetraploid wheat and was then domesticated into hexaploid (AABBDD) common wheat (), as well as an 8-kb deletion in in hexaploid wheat, predicted to create a nonfunctional pseudogene. Stepwise loss of and following hybridization and whole-genome duplication may have occurred due to gene redundancy (as functional copies of , , and are still present in the tetraploid and , , , and are present in the hexaploid) or as an adaptation to modulate recombination in allopolyploid wheat.
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http://dx.doi.org/10.1104/pp.20.00534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401138PMC
August 2020

ASY1 acts as a dosage-dependent antagonist of telomere-led recombination and mediates crossover interference in .

Proc Natl Acad Sci U S A 2020 06 4;117(24):13647-13658. Epub 2020 Jun 4.

Department of Plant Sciences, University of Cambridge, CB2 3EA Cambridge, United Kingdom

During meiosis, interhomolog recombination produces crossovers and noncrossovers to create genetic diversity. Meiotic recombination frequency varies at multiple scales, with high subtelomeric recombination and suppressed centromeric recombination typical in many eukaryotes. During recombination, sister chromatids are tethered as loops to a polymerized chromosome axis, which, in plants, includes the ASY1 HORMA domain protein and REC8-cohesin complexes. Using chromatin immunoprecipitation, we show an ascending telomere-to-centromere gradient of ASY1 enrichment, which correlates strongly with REC8-cohesin ChIP-seq data. We mapped crossovers genome-wide in the absence of ASY1 and observe that telomere-led recombination becomes dominant. Surprisingly, heterozygotes also remodel crossovers toward subtelomeric regions at the expense of the pericentromeres. Telomeric recombination increases in occur in distal regions where ASY1 and REC8 ChIP enrichment are lowest in wild type. In wild type, the majority of crossovers show interference, meaning that they are more widely spaced along the chromosomes than expected by chance. To measure interference, we analyzed double crossover distances, MLH1 foci, and fluorescent pollen tetrads. Interestingly, while crossover interference is normal in , it is undetectable in mutants, indicating that ASY1 is required to mediate crossover interference. Together, this is consistent with ASY1 antagonizing telomere-led recombination and promoting spaced crossover formation along the chromosomes via interference. These findings provide insight into the role of the meiotic axis in patterning recombination frequency within plant genomes.
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http://dx.doi.org/10.1073/pnas.1921055117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306779PMC
June 2020

Iron is a ligand of SecA-like metal-binding domains .

J Biol Chem 2020 05 2;295(21):7516-7528. Epub 2020 Apr 2.

School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In , the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron .
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http://dx.doi.org/10.1074/jbc.RA120.012611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247292PMC
May 2020

Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface.

Nat Commun 2020 02 12;11(1):851. Epub 2020 Feb 12.

Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.
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http://dx.doi.org/10.1038/s41467-020-14655-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015928PMC
February 2020

Interacting Genomic Landscapes of REC8-Cohesin, Chromatin, and Meiotic Recombination in Arabidopsis.

Plant Cell 2020 04 5;32(4):1218-1239. Epub 2020 Feb 5.

Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom

Meiosis recombines genetic variation and influences eukaryote genome evolution. During meiosis, DNA double-strand breaks (DSBs) enter interhomolog repair to yield crossovers and noncrossovers. DSB repair occurs as replicated sister chromatids are connected to a polymerized axis. Cohesin rings containing the REC8 kleisin subunit bind sister chromatids and anchor chromosomes to the axis. Here, we report the genomic landscape of REC8 using chromatin immunoprecipitation sequencing (ChIP-seq) in Arabidopsis (). REC8 associates with regions of high nucleosome occupancy in multiple chromatin states, including histone methylation at H3K4 (expressed genes), H3K27 (silent genes), and H3K9 (silent transposons). REC8 enrichment is associated with suppression of meiotic DSBs and crossovers at the chromosome and fine scales. As REC8 enrichment is greatest in transposon-dense heterochromatin, we repeated ChIP-seq in H3K9me2 mutants. Surprisingly, REC8 enrichment is maintained in heterochromatin and no defects in centromeric cohesion were observed. REC8 occupancy within genes anti-correlates with transcription and is reduced in transposons that reactivate expression in Abnormal axis structures form in that recruit DSB-associated protein foci and undergo synapsis, which is followed by chromosome fragmentation. Therefore, REC8 occupancy correlates with multiple chromatin states and is required to organize meiotic chromosome architecture and interhomolog recombination.
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http://dx.doi.org/10.1105/tpc.19.00866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145502PMC
April 2020

Understanding Infection-Induced Thrombosis: Lessons Learned From Animal Models.

Front Immunol 2019 5;10:2569. Epub 2019 Nov 5.

Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Thrombosis is a common consequence of infection that is associated with poor patient outcome. Nevertheless, the mechanisms by which infection-associated thrombosis is induced, maintained and resolved are poorly understood, as is the contribution thrombosis makes to host control of infection and pathogen spread. The key difference between infection-associated thrombosis and thrombosis in other circumstances is a stronger inflammation-mediated component caused by the presence of the pathogen and its products. This inflammation triggers the activation of platelets, which may accompany damage to the endothelium, resulting in fibrin deposition and thrombus formation. This process is often referred to as thrombo-inflammation. Strikingly, despite its clinical importance and despite thrombi being induced to many different pathogens, it is still unclear whether the mechanisms underlying this process are conserved and how we can best understand this process. This review summarizes thrombosis in a variety of models, including single antigen models such as LPS, and infection models using viruses and bacteria. We provide a specific focus on Typhimurium infection as a useful model to address all stages of thrombosis during infection. We highlight how this model has helped us identify how thrombosis can appear in different organs at different times and thrombi be detected for weeks after infection in one site, yet largely be resolved within 24 h in another. Furthermore, we discuss the observation that thrombi induced to Typhimurium are largely devoid of bacteria. Finally, we discuss the value of different therapeutic approaches to target thrombosis, the potential importance of timing in their administration and the necessity to maintain normal hemostasis after treatment. Improvements in our understanding of these processes can be used to better target infection-mediated mechanisms of thrombosis.
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http://dx.doi.org/10.3389/fimmu.2019.02569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848062PMC
November 2020

Chromatin Immunoprecipitation of Meiotically Expressed Proteins from Arabidopsis thaliana Flowers.

Methods Mol Biol 2020 ;2061:219-236

Department of Plant Sciences, University of Cambridge, Cambridge, UK.

During meiosis recombination occurs between homologous chromosomes which can result in reciprocal exchanges of genetic information, called crossovers. Crossover rate is heterogeneous within the genome, with local regions having a significantly higher recombination rate relative to the genome average. These regions are termed hotspots and typically occur with widths of kilobases. Therefore, there is a need to profile recombination factors at a similar resolution during meiosis via techniques such as chromatin immunoprecipitation (ChIP). Here we describe a ChIP protocol, combined with high throughput sequencing (ChIP-seq) optimised for analysis of meiotically expressed proteins in Arabidopsis thaliana flowers. We provide methods to (1) isolate nuclei and prepare the chromatin for shearing, (2) immunoprecipitate DNA molecules cross-linked to a protein of interest, (3) to size-select and purify immunoprecipitated DNA molecules, and (4) to prepare DNA sequencing libraries suitable for high-throughput sequencing. Together, these methods allow the detection of binding sites for meiotic proteins in the Arabidopsis genome at high resolution, which will provide insights into relationships between meiotic chromosome organization, chromatin and recombination.
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http://dx.doi.org/10.1007/978-1-4939-9818-0_16DOI Listing
December 2020

DeepTetrad: high-throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana.

Plant J 2020 01 22;101(2):473-483. Epub 2019 Oct 22.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, Republic of Korea.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.
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http://dx.doi.org/10.1111/tpj.14543DOI Listing
January 2020

An Ultra High-Density Crossover Map That Refines the Influences of Structural Variation and Epigenetic Features.

Genetics 2019 11 16;213(3):771-787. Epub 2019 Sep 16.

Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany.

Many environmental, genetic, and epigenetic factors are known to affect the frequency and positioning of meiotic crossovers (COs). Suppression of COs by large, cytologically visible inversions and translocations has long been recognized, but relatively little is known about how smaller structural variants (SVs) affect COs. To examine fine-scale determinants of the CO landscape, including SVs, we used a rapid, cost-effective method for high-throughput sequencing to generate a precise map of >17,000 COs between the Col-0 and L accessions of COs were generally suppressed in regions with SVs, but this effect did not depend on the size of the variant region, and was only marginally affected by the variant type. CO suppression did not extend far beyond the SV borders and CO rates were slightly elevated in the flanking regions. Disease resistance gene clusters, which often exist as SVs, exhibited high CO rates at some loci, but there was a tendency toward depressed CO rates at loci where large structural differences exist between the two parents. Our high-density map also revealed in fine detail how CO positioning relates to genetic (DNA motifs) and epigenetic (chromatin structure) features of the genome. We conclude that suppression of COs occurs over a narrow region spanning large- and small-scale SVs, representing an influence on the CO landscape in addition to sequence and epigenetic variation along chromosomes.
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http://dx.doi.org/10.1534/genetics.119.302406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827372PMC
November 2019

Natural Variation in TBP-ASSOCIATED FACTOR 4b Controls Meiotic Crossover and Germline Transcription in Arabidopsis.

Curr Biol 2019 08 1;29(16):2676-2686.e3. Epub 2019 Aug 1.

Department of Plant Sciences, Downing Street, University of Cambridge, Cambridge CB2 3EA, UK. Electronic address:

Meiotic crossover frequency varies within genomes, which influences genetic diversity and adaptation. In turn, genetic variation within populations can act to modify crossover frequency in cis and trans. To identify genetic variation that controls meiotic crossover frequency, we screened Arabidopsis accessions using fluorescent recombination reporters. We mapped a genetic modifier of crossover frequency in Col × Bur populations of Arabidopsis to a premature stop codon within TBP-ASSOCIATED FACTOR 4b (TAF4b), which encodes a subunit of the RNA polymerase II general transcription factor TFIID. The Arabidopsis taf4b mutation is a rare variant found in the British Isles, originating in South-West Ireland. Using genetics, genomics, and immunocytology, we demonstrate a genome-wide decrease in taf4b crossovers, with strongest reduction in the sub-telomeric regions. Using RNA sequencing (RNA-seq) from purified meiocytes, we show that TAF4b expression is meiocyte enriched, whereas its paralog TAF4 is broadly expressed. Consistent with the role of TFIID in promoting gene expression, RNA-seq of wild-type and taf4b meiocytes identified widespread transcriptional changes, including in genes that regulate the meiotic cell cycle and recombination. Therefore, TAF4b duplication is associated with acquisition of meiocyte-specific expression and promotion of germline transcription, which act directly or indirectly to elevate crossovers. This identifies a novel mode of meiotic recombination control via a general transcription factor.
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http://dx.doi.org/10.1016/j.cub.2019.06.084DOI Listing
August 2019

Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation.

Sci Rep 2019 07 31;9(1):11100. Epub 2019 Jul 31.

Université Clermont Auvergne, INRA, UMR454 MEDiS, 63000, Clermont-Ferrand, France.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.
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http://dx.doi.org/10.1038/s41598-019-47608-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668479PMC
July 2019

The Type III Secretion System (T3SS)-Translocon of Atypical Enteropathogenic (aEPEC) Can Mediate Adherence.

Front Microbiol 2019 9;10:1527. Epub 2019 Jul 9.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.

The intimin protein is the major adhesin involved in the intimate adherence of atypical enteropathogenic (aEPEC) strains to epithelial cells, but little is known about the structures involved in their early colonization process. A previous study demonstrated that the type III secretion system (T3SS) plays an additional role in the adherence of an strain. Therefore, we assumed that the T3SS could be related to the adherence efficiency of aEPEC during the first stages of contact with epithelial cells. To test this hypothesis, we examined the adherence of seven aEPEC strains and their (intimin) isogenic mutants in the standard HeLa adherence assay and observed that all wild-type strains were adherent while five isogenic mutants were not. The two mutant strains that remained adherent were then used to generate the / double mutants (encoding intimin and the T3SS ATPase, respectively) and after the adherence assay, we observed that one strain lost its adherence capacity. This suggested a role for the T3SS in the initial adherence steps of this strain. In addition, we demonstrated that this strain expressed the T3SS at significantly higher levels when compared to the other wild-type strains and that it produced longer translocon-filaments. Our findings reveal that the T3SS-translocon can play an additional role as an adhesin at the beginning of the colonization process of aEPEC.
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http://dx.doi.org/10.3389/fmicb.2019.01527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629874PMC
July 2019

Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system.

Nat Microbiol 2019 10 24;4(10):1692-1705. Epub 2019 Jun 24.

School of Biosciences, University of Birmingham, Birmingham, UK.

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.
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http://dx.doi.org/10.1038/s41564-019-0481-yDOI Listing
October 2019

Bacterial flagellin promotes viral entry via an NF-kB and Toll Like Receptor 5 dependent pathway.

Sci Rep 2019 05 27;9(1):7903. Epub 2019 May 27.

Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK.

Viruses and bacteria colonize hosts by invading epithelial barriers. Recent studies have shown that interactions between the microbiota, pathogens and the host can potentiate infection through poorly understood mechanisms. Here, we investigated whether diverse bacterial species could modulate virus internalization into host cells, often a rate-limiting step in establishing infections. Lentiviral pseudoviruses expressing influenza, measles, Ebola, Lassa or vesicular stomatitis virus envelope glycoproteins enabled us to study entry of viruses that exploit diverse internalization pathways. Salmonella Typhimurium, Escherichia coli and Pseudomonas aeruginosa significantly increased viral uptake, even at low bacterial frequencies. This did not require bacterial contact with or invasion of host cells. Studies determined that the bacterial antigen responsible for this pro-viral activity was the Toll-Like Receptor 5 (TLR5) agonist flagellin. Exposure to flagellin increased virus attachment to epithelial cells in a temperature-dependent manner via TLR5-dependent activation of NF-ΚB. Importantly, this phenotype was both long lasting and detectable at low multiplicities of infection. Flagellin is shed from bacteria and our studies uncover a new bystander role for this protein in regulating virus entry. This highlights a new aspect of viral-bacterial interplay with significant implications for our understanding of polymicrobial-associated pathogenesis.
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http://dx.doi.org/10.1038/s41598-019-44263-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536546PMC
May 2019

Complete genome sequence of Helicobacter pylori B128 7.13 and a single-step method for the generation of unmarked mutations.

Helicobacter 2019 Aug 7;24(4):e12587. Epub 2019 May 7.

Institute of Microbiology and Infection College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Background: Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction-modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium.

Materials And Methods: Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter-selection marker, galactokinase from Escherichia coli.

Results: We show that this mutagenesis strategy can be used to generate in-frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori-specific DUF874 family protein of unknown function.

Conclusions: This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium.
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http://dx.doi.org/10.1111/hel.12587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618122PMC
August 2019

Meiotic recombination within plant centromeres.

Curr Opin Plant Biol 2019 04 5;48:26-35. Epub 2019 Apr 5.

Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom. Electronic address:

Meiosis is a conserved eukaryotic cell division that increases genetic diversity in sexual populations. During meiosis homologous chromosomes pair and undergo recombination that can result in reciprocal genetic exchange, termed crossover. The frequency of crossover is highly variable along chromosomes, with hot spots and cold spots. For example, the centromeres that contain the kinetochore, which attach chromosomes to the microtubular spindle, are crossover cold spots. Plant centromeres typically consist of large tandemly repeated arrays of satellite sequences and retrotransposons, a subset of which assemble CENH3-variant nucleosomes, which bind to kinetochore proteins. Although crossovers are suppressed in centromeres, there is abundant evidence for gene conversion and homologous recombination between repeats, which plays a role in satellite array change. We review the evidence for recombination within plant centromeres and the implications for satellite sequence evolution. We speculate on the genetic and epigenetic features of centromeres that may influence meiotic recombination in these regions. We also highlight unresolved questions relating to centromere function and sequence change and how the advent of new technologies promises to provide insights.
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http://dx.doi.org/10.1016/j.pbi.2019.02.008DOI Listing
April 2019

Interhomolog polymorphism shapes meiotic crossover within the Arabidopsis RAC1 and RPP13 disease resistance genes.

PLoS Genet 2018 12 13;14(12):e1007843. Epub 2018 Dec 13.

Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom.

During meiosis, chromosomes undergo DNA double-strand breaks (DSBs), which can be repaired using a homologous chromosome to produce crossovers. Meiotic recombination frequency is variable along chromosomes and tends to concentrate in narrow hotspots. We mapped crossover hotspots located in the Arabidopsis thaliana RAC1 and RPP13 disease resistance genes, using varying haplotypic combinations. We observed a negative non-linear relationship between interhomolog divergence and crossover frequency within the hotspots, consistent with polymorphism locally suppressing crossover repair of DSBs. The fancm, recq4a recq4b, figl1 and msh2 mutants, or lines with increased HEI10 dosage, are known to show increased crossovers throughout the genome. Surprisingly, RAC1 crossovers were either unchanged or decreased in these genetic backgrounds, showing that chromosome location and local chromatin environment are important for regulation of crossover activity. We employed deep sequencing of crossovers to examine recombination topology within RAC1, in wild type, fancm, recq4a recq4b and fancm recq4a recq4b backgrounds. The RAC1 recombination landscape was broadly conserved in the anti-crossover mutants and showed a negative relationship with interhomolog divergence. However, crossovers at the RAC1 5'-end were relatively suppressed in recq4a recq4b backgrounds, further indicating that local context may influence recombination outcomes. Our results demonstrate the importance of interhomolog divergence in shaping recombination within plant disease resistance genes and crossover hotspots.
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http://dx.doi.org/10.1371/journal.pgen.1007843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307820PMC
December 2018

Draft Whole-Genome Sequences of 10 Atypical Enteropathogenic Escherichia coli Strains Isolated in Brazil.

Microbiol Resour Announc 2018 Dec 6;7(22). Epub 2018 Dec 6.

Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

The number of diarrhea cases caused by atypical enteropathogenic Escherichia coli (aEPEC) has been increasing worldwide. Here, we report the draft whole-genome sequences of 10 aEPEC strains isolated in Brazil. These sequences will provide an important source for future studies concerning aEPEC pathogenicity and genetic markers of potentially virulent strains.
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http://dx.doi.org/10.1128/MRA.01432-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284090PMC
December 2018

Serovar Typhimurium Travels to Mesenteric Lymph Nodes Both with Host Cells and Autonomously.

J Immunol 2019 01 28;202(1):260-267. Epub 2018 Nov 28.

Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom.

infection is a globally important cause of gastroenteritis and systemic disease and is a useful tool to study immune responses in the intestine. Although mechanisms leading to immune responses against have been extensively studied, questions remain about how bacteria travel from the intestinal mucosa to the mesenteric lymph nodes (MLN), a key site for Ag presentation. In this study, we used a mouse model of infection with serovar Typhimurium (STM) to identify changes in intestinal immune cells induced during early infection. We then used fluorescently labeled STM to identify interactions with immune cells from the site of infection through migration in lymph to the MLN. We show that viable STM can be carried in the lymph by any subset of migrating dendritic cells but not by macrophages. Moreover, approximately half of the STM in lymph are not associated with cells at all and travel autonomously. Within the MLN, STM associates with dendritic cells and B cells but predominantly with MLN-resident macrophages. In conclusion, we describe the routes used by STM to spread systemically in the period immediately postinfection. This deeper understanding of the infection process could open new avenues for controlling it.
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http://dx.doi.org/10.4049/jimmunol.1701254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305795PMC
January 2019

Organization and architecture of AggR-dependent promoters from enteroaggregative Escherichia coli.

Mol Microbiol 2019 02 18;111(2):534-551. Epub 2018 Dec 18.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.

Enteroaggregative Escherichia coli (EAEC), is a diarrhoeagenic human pathogen commonly isolated from patients in both developing and industrialized countries. Pathogenic EAEC strains possess many virulence determinants, which are thought to be involved in causing disease, though, the exact mechanism by which EAEC causes diarrhoea is unclear. Typical EAEC strains possess the transcriptional regulator, AggR, which controls the expression of many virulence determinants, including the attachment adherence fimbriae (AAF) that are necessary for adherence to human gut epithelial cells. Here, using RNA-sequencing, we have investigated the AggR regulon from EAEC strain 042 and show that AggR regulates the transcription of genes on both the bacterial chromosome and the large virulence plasmid, pAA2. Due to the importance of fimbriae, we focused on the two AAF/II fimbrial gene clusters in EAEC 042 (afaB-aafCB and aafDA) and identified the promoter elements and AggR-binding sites required for fimbrial expression. In addition, we examined the organization of the fimbrial operon promoters from other important EAEC strains to understand the rules of AggR-dependent activation. Finally, we generated a series of semi-synthetic promoters to define the minimal sequence required for AggR-mediated activation and show that the correct positioning of a single AggR-binding site is sufficient to confer AggR-dependence.
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http://dx.doi.org/10.1111/mmi.14172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392122PMC
February 2019

Hotspots for Initiation of Meiotic Recombination.

Front Genet 2018 5;9:521. Epub 2018 Nov 5.

Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom.

Homologous chromosomes must pair and recombine to ensure faithful chromosome segregation during meiosis, a specialized type of cell division that occurs in sexually reproducing eukaryotes. Meiotic recombination initiates by programmed induction of DNA double-strand breaks (DSBs) by the conserved type II topoisomerase-like enzyme SPO11. A subset of meiotic DSBs are resolved as crossovers, whereby reciprocal exchange of DNA occurs between homologous chromosomes. Importantly, DSBs are non-randomly distributed along eukaryotic chromosomes, forming preferentially in permissive regions known as hotspots. In many species, including plants, DSB hotspots are located within nucleosome-depleted regions. DSB localization is governed by interconnected factors, including -regulatory elements, transcription factor binding, and chromatin accessibility, as well as by higher-order chromosome architecture. The spatiotemporal control of DSB formation occurs within a specialized chromosomal structure characterized by sister chromatids organized into linear arrays of chromatin loops that are anchored to a proteinaceous axis. Although SPO11 and its partner proteins required for DSB formation are bound to the axis, DSBs occur preferentially within the chromatin loops, which supports the "tethered-loop/axis model" for meiotic recombination. In this mini review, we discuss insights gained from recent efforts to define and profile DSB hotspots at high resolution in eukaryotic genomes. These advances are deepening our understanding of how meiotic recombination shapes genetic diversity and genome evolution in diverse species.
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http://dx.doi.org/10.3389/fgene.2018.00521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237102PMC
November 2018

-induced thrombi in mice develop asynchronously in the spleen and liver and are not effective bacterial traps.

Blood 2019 02 6;133(6):600-604. Epub 2018 Nov 6.

Institute of Immunology and Immunotherapy and.

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.
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http://dx.doi.org/10.1182/blood-2018-08-867267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474721PMC
February 2019

Intestinal CD103CD11b cDC2 Conventional Dendritic Cells Are Required for Primary CD4 T and B Cell Responses to Soluble Flagellin.

Front Immunol 2018 17;9:2409. Epub 2018 Oct 17.

Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Systemic immunization with soluble flagellin (sFliC) from Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103CD11b cDC2 contribute to this process, the relationship between these is unclear in the early activation of CD4 T cells and the development of antigen-specific B cell responses. In this work, we use TLR5-deficient mice and mice (which have reduced numbers of cDC2, particularly intestinal CD103CD11b cDCs), to address these points by studying the responses concurrently in the spleen and the mesenteric lymph nodes (MLN). We show that CD103CD11b cDC2 respond rapidly and accumulate in the MLN after immunization with sFliC in a TLR5-dependent manner. Furthermore, we identify that whilst CD103CD11b cDC2 are essential for the induction of primary T and B cell responses in the mucosa, they do not play such a central role for the induction of these responses in the spleen. Additionally, we show the involvement of CD103CD11b cDC2 in the induction of Th2-associated responses. mice showed a reduced primary FliC-specific Th2-associated IgG1 responses, but enhanced Th1-associated IgG2c responses. These data expand our current understanding of the mucosal immune responses promoted by sFliC and highlights the potential of this adjuvant for vaccine usage by taking advantage of the functionality of mucosal CD103CD11b cDC2.
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http://dx.doi.org/10.3389/fimmu.2018.02409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199373PMC
October 2019

YraP Contributes to Cell Envelope Integrity and Virulence of Salmonella enterica Serovar Typhimurium.

Infect Immun 2018 11 25;86(11). Epub 2018 Oct 25.

Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom

Mutations in σ-regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and during infection. YraP is conserved across a number of Gram-negative pathogens, including , where the homolog is a component of the Bexsero meningococcal group B vaccine. Investigations using laboratory-adapted K-12 have shown that mutants have elevated sensitivity to a range of compounds, including detergents and normally ineffective antibiotics. In this study, we investigate the role of the outer membrane lipoprotein YraP in the pathogenesis of serovar Typhimurium. We show that mutations in Typhimurium result in a defective outer membrane barrier with elevated sensitivity to a range of compounds. This defect is associated with attenuated virulence in an oral infection model and during the early stages of systemic infection. We show that this attenuation is not a result of defects in lipopolysaccharide and O-antigen synthesis, changes in outer membrane protein levels, or the ability to adhere to and invade eukaryotic cell lines .
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http://dx.doi.org/10.1128/IAI.00829-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204713PMC
November 2018