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Journal articleChee MSJ, Serrano E, Chiang YN, et al., 2023,
Dual pathogenicity island transfer by piggybacking lateral transduction
, Cell, Vol: 186, Pages: 3414-3426.e16, ISSN: 0092-8674Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry.
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Journal articleZeden MS, Schuster CF, Gründling A, 2023,
Staphylococcus aureus Colony Polymerase Chain Reaction.
, Cold Spring Harb Protoc, Vol: 2023Here, we describe a protocol for a colony polymerase chain reaction (PCR) method for Staphylococcus aureus The methodology involves the preparation of small S. aureus lysates by using the enzyme lysostaphin to degrade the peptidoglycan layer. These lysates are prepared using a small patch of bacteria grown on LB agar plates, and the lysates can subsequently be used for PCR analyses.
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Journal articleZeden MS, Schuster CF, Gründling A, 2023,
Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli.
, Cold Spring Harb Protoc, Vol: 2023We present a protocol for the generation of a gene-deletion allelic-exchange plasmid and its recovery in Escherichia coli for the purpose of constructing an in-frame gene deletion in Staphylococcus aureus Here, we present detailed methodologies for (i) the primer design (using the S. aureus tagO gene as our specific example); (ii) PCR amplification of the required gene fragments; (iii) preparation of the cloning vector (using the S. aureus allelic-exchange vector pIMAY* as an example); (iv) the Gibson assembly cloning method; (v) introduction of the plasmid into E. coli; (vi) confirmation of the plasmid insert in E. coli by colony PCR; and, finally, (vii) confirmation of the insert by sequencing. We also consider the long-term storage of the E. coli strains containing the desired plasmid.
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Journal articleZeden MS, Schuster CF, Gründling A, 2023,
Allelic-Exchange Procedure in Staphylococcus aureus.
, Cold Spring Harb Protoc, Vol: 2023This protocol continues a series of methods for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for S. aureus We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, then introduced into electrocompetent S. aureus cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (pheS* gene) and confers chloramphenicol resistance to S. aureus As a specific example, we present the construction of strain RN4220*ΔtagO from strain RN4220 carrying the pIMAY*-ΔtagO plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.
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Journal articleZeden MS, Schuster CF, Gründling A, 2023,
Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus.
, Cold Spring Harb Protoc, Vol: 2023Here we describe an allelic-exchange procedure for the construction of an unmarked gene deletion in the bacterium Staphylococcus aureus As a practical example, we outline the construction of a tagO gene deletion in S. aureus using the allelic-exchange plasmid pIMAY*. We first present the general principles of the allelic-exchange method, along with information on counterselectable markers. Furthermore, we summarize relevant cloning procedures, such as the splicing by overhang extension (SOE) polymerase chain reaction (PCR) and Gibson assembly methods, and we conclude by giving some general consideration to performing genetic modifications in S. aureus.
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Journal articleFantuzzi A, Haniewicz P, Farci D, et al., 2023,
Bicarbonate activation of the monomeric photosystem II-PsbS/Psb27 complex
, Plant Physiology, Vol: 192, Pages: 2656-2671, ISSN: 0032-0889In thylakoid membranes, Photosystem II (PSII) monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while PSII monomers from granal regions (PSIIm) lack these subunits. Here, we have isolated and characterised these two types of Photosystem II complexes in tobacco (Nicotiana tabacum). PSIIm-S/27 showed enhanced fluorescence, the near-absence of oxygen evolution, as well as limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•– couple, which controls the charge recombination route, and this limits chlorophyll triplet mediated 1O2 formation. These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit using a bicarbonate-mediated switch and protective mechanism.
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Journal articleZeden MS, Schuster CF, Gründling A, 2023,
Preparation of Electrocompetent Staphylococcus aureus Cells and Plasmid Transformation.
, Cold Spring Harb Protoc, Vol: 2023This protocol is part of a series of methodologies for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. Having previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, we now present details of the next steps in this method-the preparation of electrocompetent S. aureus cells and introduction of the tagO mutant plasmid DNA into the S. aureus cells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.
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Conference paperYe F, Gao F, Liu X, et al., 2023,
The macromolecular machine that opens up double-stranded DNA for gene transcription: from structures to functions
, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: C188-C188, ISSN: 2053-2733 -
Journal articleChaukimath P, Frankel G, Visweswariah SS, 2023,
The metabolic impact of bacterial infection in the gut
, FEBS JOURNAL, Vol: 290, Pages: 3928-3945, ISSN: 1742-464X- Cite
- Citations: 1
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Journal articleHoose A, Vellacott R, Storch M, et al., 2023,
DNA synthesis technologies to close the gene writing gap (vol 7, pg 144, 2023)
, NATURE REVIEWS CHEMISTRY, Vol: 7, Pages: 590-590 -
Journal articleLiang P, Lister K, Yates L, et al., 2023,
Phosphoregulation of DNA repair via the Rad51 auxiliary factor Swi5-Sfr1
, Journal of Biological Chemistry, Vol: 299, Pages: 1-15, ISSN: 0021-9258Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks, the most severe form of DNA damage. The Rad51 protein is central to HR, but multiple auxiliary factors regulate its activity. The heterodimeric Swi5-Sfr1 complex is one such factor. It was previously shown that two sites within the intrinsically disordered domain of Sfr1 are critical for the interaction with Rad51. Here, we show that phosphorylation of five residues within this domain regulates the interaction of Swi5-Sfr1 with Rad51. Biochemical reconstitutions demonstrated that a phosphomimetic mutant version of Swi5-Sfr1 is defective in both the physical and functional interaction with Rad51. This translated to a defect in DNA repair, with the phosphomimetic mutant yeast strain phenocopying the previously established interaction mutant. Interestingly, a strain in which Sfr1 phosphorylation was blocked also displayed sensitivity to DNA damage. Taken together, we propose that controlled phosphorylation of Sfr1 is important for the role of Swi5-Sfr1 in promoting Rad51-dependent DNA repair.
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Journal articleSaenz Cavazos P, Hunter-Sellars E, Iacomi P, et al., 2023,
Evaluating solid sorbents for CO2 capture: linking material properties and process efficiency via adsorption performance
, Frontiers in Energy Research, Vol: 11, Pages: 1-22, ISSN: 2296-598XExpanding populations and growing economies result in higher energy needs. Meeting this increasing demand, while lowering carbon emissions, calls for a broad energy mix and commercial deployment of solutions like carbon capture and carbon removal technologies. The scale-up of these solutions is partially hindered by the lack of materials-related information, particularly in the case of solid adsorption-based carbon capture technologies. Furthermore, experimental measurement parameters used and how data is presented lack uniformity, which makes material comparisons extremely difficult. This review examines the current state of solid sorbent characterization for carbon capture, exploring physical and chemical properties, performance parameters, and process indicators. Adsorbent performance parameters demonstrate to be the crucial link between intrinsic material properties and the overall adsorption process effectiveness and therefore are the focus of this work. This paper outlines the relevant techniques used to measure Key Performance Indicators (KPIs) related to adsorption performance such as CO2 adsorption capacity, selectivity, kinetics, ease of regeneration, stability, adsorbent cost, and environmental impact. Additionally, this study highlights the relevant experimental conditions for diluted versus concentrated CO2 streams. Lastly, efforts in harmonizing experimental data sets are considered, and an outlook on solid sorbent characterization for carbon capture processes is presented. Overall, the aim of this work is to provide the reader a critical understanding of KPIs from atomic to process scale, highlighting the importance of experimental data throughout.
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Journal articleKaur A, Darvill D, Xiang S, et al., 2023,
Development of nanopackaging for storage and transport of loaded lipid nanoparticles
, Nano Letters, Vol: 23, Pages: 6760-6767, ISSN: 1530-6984Easily deploying new vaccines globally to combat disease outbreaks has been highlighted as a major necessity by the World Health Organization. RNA-based vaccines using lipid nanoparticles (LNPs) as a drug delivery system were employed to great effect during the recent COVID-19 pandemic. However, LNPs are still unstable at room temperature and agglomerate over time during storage, rendering them ineffective for intracellular delivery. We demonstrate the suitability of nanohole arrays (nanopackaging) as patterned surfaces to separate and store functionalized LNPs (fLNPs) in individual recesses, which can be expanded to other therapeutics. Encapsulating calcein as a model drug, we show through confocal microscopy the effective loading of fLNPs into our nanopackaging for both wet and dry systems. We prove quantifiably pH-mediated capture and subsequent unloading of over 30% of the fLNPs using QCM-D on alumina surfaces altering the pH from 5.5 to 7, displaying controllable storage at the nanoscale.
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Journal articleFadini A, Hutchison CDM, Morozov D, et al., 2023,
Serial femtosecond crystallography reveals that photoactivation in a fluorescent protein proceeds via the hula twist mechanism
, Journal of the American Chemical Society, Vol: 145, Pages: 15796-15808, ISSN: 0002-7863Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.
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Journal articleMurooka Y, Bryan W, Clarke J, et al., 2023,
The Design of Relativistic Ultrafast Electron Diffraction and Imaging (RUEDI) Facility for Materials in Extremes.
, Microsc Microanal, Vol: 29, Pages: 1487-1488 -
Journal articleMansueto S, Fusco G, De Simone A, 2023,
α-Synuclein and biological membranes: the danger of loving too much
, CHEMICAL COMMUNICATIONS, Vol: 59, Pages: 8769-8778, ISSN: 1359-7345- Cite
- Citations: 8
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Journal articleMalpartida-Cardenas K, Baum J, Cunnington A, et al., 2023,
A dual paper-based nucleic acid extraction method from blood in under ten minutes for point-of-care diagnostics
, The Analyst, Vol: 148, Pages: 3036-3044, ISSN: 0003-2654Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution (eluted disk) as well as direct placement of the disk in the LAMP reaction (in situ disk). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually
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Journal articleJia L, Heng JYY, Cui P, et al., 2023,
Isostructurality or not: a deep insight into the selective formation of spironolactone solvates
, Crystal Growth and Design, Vol: 23, Pages: 4721-4727, ISSN: 1528-7483Isostructurality is a potential tool to design crystals with tailored arrangements, especially for multicomponent systems, but research into its formation mechanism is still limited to date. Herein, a family of spironolactone (SPI) isostructural and non-isostructural solvates was investigated to explore the selective formation of isostructural crystals. The SPI framework, dominated by van der Waals forces, is robust and flexible, with different solvent molecules in its channels playing the same structural role. The formation of the SPI isostructural solvate is a delicate energy balance between the energy loss caused by the expansion of the SPI framework and the addition of SPI···solvent interactions. Besides the size and functional groups of solvent molecules, the directionality of the SPI···solvent interactions should also be carefully considered in the isostructurality-based crystal design. In addition, weak interactions seem to be regarded as potential design elements for organic host architectures.
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Journal articleKondor A, Burnett DJ, Bismarck A, et al., 2023,
Correct specific retention volume determination in inverse gas chromatography
, JOURNAL OF CHROMATOGRAPHY A, Vol: 1700, ISSN: 0021-9673- Cite
- Citations: 4
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Journal articleVoisin TB, Couves EC, Tate EW, et al., 2023,
Dynamics and molecular interactions of GPI-anchored CD59
, Toxins, Vol: 15, ISSN: 2072-6651CD59 is a GPI-anchored cell surface receptor that serves as a gatekeeper to controlling pore formation. It is the only membrane-bound inhibitor of the complement membrane attack complex (MAC), an immune pore that can damage human cells. While CD59 blocks MAC pores, the receptor is co-opted by bacterial pore-forming proteins to target human cells. Recent structures of CD59 in complexes with binding partners showed dramatic differences in the orientation of its ectodomain relative to the membrane. Here, we show how GPI-anchored CD59 can satisfy this diversity in binding modes. We present a PyLipID analysis of coarse-grain molecular dynamics simulations of a CD59-inhibited MAC to reveal residues of complement proteins (C6:Y285, C6:R407 C6:K412, C7:F224, C8β:F202, C8β:K326) that likely interact with lipids. Using modules of the MDAnalysis package to investigate atomistic simulations of GPI-anchored CD59, we discover properties of CD59 that encode the flexibility necessary to bind both complement proteins and bacterial virulence factors.
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Conference paperChadha D, Heng JYY, Sun E, 2023,
Equality, Diversity and Inclusion (EDI) in the Chemical Engineering curriculum: working in partnership with students to create sustainable practices
, 2023 ASEE Annual Conference & Exposition, Publisher: American Society for Engineering EducationHaving suitable provisions in place to support equality, diversity and inclusion (EDI) has become increasingly important throughout higher education. In our chemical engineering department, we are currently exploring strategies that would actively enhance and promote EDI whilst aligning the provisions to the curriculum. In this paper, we discuss some of the work we have been doing in partnership with our student body on developing sustainable practices - that builds on earlier work which focused on defining and identifying EDI practices. We have identified 5 key areas of particular interest to us: improvements to our student-to-student peer review system, a departmental charter for staff and students, constructive changes to the programme handbook, a resource developed by students for students, and changes to the content and delivery of certain modules that could accommodate EDI, or the identification of modules/courses that do. As part of the research/verification process, a limited amount of survey data was collected from students to help us establish underlying issues and how suggested sustainable changes might be perceived. In conducting this work, certain challenges have arisen as well as opportunities.
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Journal articleAlmusaimi O, Williams D, 2023,
Methods of Chemical Synthesis of Peptides - GB2309538.3
, Patent -
Journal articleVassallo N, Ongeri S, De Simone A, 2023,
Editorial: Oligomers in amyloid-associated diseases-structural properties and toxicity
, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 10- Author Web Link
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- Citations: 1
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Journal articleArredondo-Alonso S, Blundell-Hunter G, Fu Z, et al., 2023,
Evolutionary and functional history of the Escherichia coli K1 capsule
, Nature Communications, Vol: 14, Pages: 1-17, ISSN: 2041-1723Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.
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Journal articleMasuda T, Beckova M, Turóczy Z, et al., 2023,
Accumulation of cyanobacterial photosystem II containing the “rogue” D1 subunit is controlled by FtsH protease and synthesis of the standard D1 protein
, Plant and Cell Physiology, ISSN: 0032-0781 -
Journal articleMohamed NAN, Han Y, Harcourt-Vernon S, et al., 2023,
Effects of surfactant head group modification on vertically oriented mesoporous silica produced by the electrochemically assisted surfactant assembly method
, NANOSCALE ADVANCES, Vol: 5, Pages: 3316-3325, ISSN: 2516-0230 -
Journal articleMoussaoui D, Robblee JP, Robert-Paganin J, et al., 2023,
Mechanism of small molecule inhibition of <i>Plasmodium falciparum</i> myosin A informs antimalarial drug design
, NATURE COMMUNICATIONS, Vol: 14- Cite
- Citations: 7
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Journal articleErrington E, Guo M, Heng JYY, 2023,
Synthetic amorphous silica: environmental impacts of current industry and the benefit of biomass-derived silica
, Green Chemistry, Vol: 25, Pages: 4244-4259, ISSN: 1463-9262The production of Synthetic Amorphous Silica (SAS) is a billion-dollar industry. However, very little is shared publicly on the environmental impact of SAS production. This work provides the first complete treatment for the environmental impacts of SAS produced via the existing ‘dry’ and ‘wet’ industrial methods using Life Cycle Assessment (LCA). To provide a more robust method, this includes an evaluation of 8 environmental impact indicators and consideration for uncertainty during process comparison. Predictions are then used to compare the impact of the existing dry and wet methods as well as theoretical methods in which rice husk (RH) is used as a biomass-derived feedstock alternative. Results highlight cases in which using RH as an alternative feedstock is likely to be beneficial. However, it is demonstrated that these benefits are highly dependent on specifics of the process, region, and feedstock characteristics rather than the inherent “green-ness” of RH alone. Findings are therefore of significance to those interested in the existing SAS industry and the sustainable development of SAS. Moreover, findings also have potential implications for wider policy.
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Journal articleMurdoch J, Baum J, 2023,
A sting in the tail-are antibodies against the C-terminus of <i>Plasmodium falciparum</i> circumsporozoite protein protective?
, EMBO MOLECULAR MEDICINE, Vol: 15, ISSN: 1757-4676- Author Web Link
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- Citations: 3
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Journal articleRamakrishnan C, Fort C, Marques SR, et al., 2023,
Radial spoke protein 9 is necessary for axoneme assembly in Plasmodium but not in trypanosomatid parasites.
, J Cell Sci, Vol: 136Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.
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