Publications
37 results found
Ramlaul K, Feng Z, Canavan C, et al., 2023, A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate, Journal of Structural Biology, Vol: 215, Pages: 1-12, ISSN: 1047-8477
While recent advances in cryo-EM, coupled with single particle analysis, have thepotential to allow structure determination in a near-native state from vanishingly few individualparticles, this vision has yet to be realised in practise. Requirements for particle numbers thatcurrently far exceed the theoretical lower limits, challenges with the practicalities of achievinghigh concentrations for difficult-to-produce samples, and inadequate sample-dependent imagingconditions, all result in significant bottlenecks preventing routine structure determination usingcryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks bydeveloping affinity purification of samples on-grid; at once obviating the need to produce largeamounts of protein, as well as more directly controlling the variable, and sample-dependent,process of grid preparation.In this proof-of-concept study, we demonstrate a further practical step towards thisparadigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification fromraw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-celldevice can be interfaced directly with routinely-used laboratory equipment such as liquidchromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16”) connectors,and can be used to allow binding of samples to affinity grids in a controlled environment priorto the extensive washing required to remove impurities. Furthermore, by designing a devicewhich can be 3D printed and coupled to routinely used laboratory equipment, we hope toincrease the accessibility of the techniques presented herein to researchers working towardssingle-particle macromolecular structures.
Robinson C, Short N, Riglar D, 2022, Achieving spatially precise diagnosis and therapy in the mammalian gut using synthetic microbial gene circuits, Frontiers in Bioengineering and Biotechnology, Vol: 10, Pages: 1-10, ISSN: 2296-4185
The mammalian gut and its microbiome form a temporally dynamic and spatially heterogeneous environment. The inaccessibility of the gut and the spatially restricted nature of many gut diseases translate into difficulties in diagnosis and therapy for which novel tools are needed. Engineered bacterial whole-cell biosensors and therapeutics have shown early promise at addressing these challenges. Natural and engineered sensing systems can be repurposed in synthetic genetic circuits to detect spatially specific biomarkers during health and disease. Heat, light, and magnetic signals can also activate gene circuit function with externally directed spatial precision. The resulting engineered bacteria can report on conditions in situ within the complex gut environment or produce biotherapeutics that specifically target host or microbiome activity. Here, we review the current approaches to engineering spatial precision for in vivo bacterial diagnostics and therapeutics using synthetic circuits, and the challenges and opportunities this technology presents.
Bullen HE, Sanders PR, Dans MG, et al., 2022, The <i>Plasmodium falciparum</i> parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture, MOLECULAR MICROBIOLOGY, Vol: 117, Pages: 1245-1262, ISSN: 0950-382X
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- Citations: 4
Riglar DT, Richmond DL, Potvin-Trottier L, et al., 2021, Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator (vol 10, 4665, 2019), NATURE COMMUNICATIONS, Vol: 12, ISSN: 2041-1723
Morgan A, Vander Broek C, 2021, Microbiome Strategic Roadmap, Publisher: KTN
Lee ED, Aurand ER, Friedman DC, 2020, Engineering Microbiomes-Looking Ahead, ACS SYNTHETIC BIOLOGY, Vol: 9, Pages: 3181-3183, ISSN: 2161-5063
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- Citations: 5
Riglar DT, Richmond DL, Potvin-Trottier L, et al., 2019, Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator, NATURE COMMUNICATIONS, Vol: 10
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- Citations: 31
Ziesack M, Gibson T, Oliver JKW, et al., 2019, Engineered Interspecies Amino Acid Cross-Feeding Increases Population Evenness in a Synthetic Bacterial Consortium, MSYSTEMS, Vol: 4, ISSN: 2379-5077
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- Citations: 29
Naydich AD, Nangle SN, Bues JJ, et al., 2019, Synthetic Gene Circuits Enable Systems-Level Biosensor Trigger Discovery at the Host-Microbe Interface., mSystems, Vol: 4, ISSN: 2379-5077
Engineering synthetic circuits into intestinal bacteria to sense, record, and respond to in vivo signals is a promising new approach for the diagnosis, treatment, and prevention of disease. However, because the design of disease-responsive circuits is limited by a relatively small pool of known biosensors, there is a need for expanding the capacity of engineered bacteria to sense and respond to the host environment. Here, we apply a robust genetic memory circuit in Escherichia coli to identify new bacterial biosensor triggers responding in the healthy and diseased mammalian gut, which may be used to construct diagnostic or therapeutic circuits. We developed a pipeline for rapid systems-level library construction and screening, using next-generation sequencing and computational analysis, which demonstrates remarkably reliable identification of responsive biosensor triggers from pooled libraries. By testing libraries of potential triggers-each consisting of a promoter and ribosome binding site (RBS)-and using RBS variation to augment the range of trigger sensitivity, we identify and validate triggers that selectively activate our synthetic memory circuit during transit through the gut. We further identify biosensor triggers with increased response in the inflamed gut through comparative screening of one of our libraries in healthy mice and those with intestinal inflammation. Our results demonstrate the power of systems-level screening for the identification of novel biosensor triggers in the gut and provide a platform for disease-specific screening that is capable of contributing to both the understanding and clinical management of intestinal illness.IMPORTANCE The gut is a largely obscure and inaccessible environment. The use of live, engineered probiotics to detect and respond to disease signals in vivo represents a new frontier in the management of gut diseases. Engineered probiotics have also shown promise as a novel mechanism for drug delivery. However, the desig
Riglar DT, Silver PA, 2018, Engineering bacteria for diagnostic and therapeutic applications, Nature Reviews Microbiology, Vol: 16, Pages: 214-225, ISSN: 1740-1526
Our ability to generate bacterial strains with unique and increasingly complex functions has rapidly expanded in recent times. The capacity for DNA synthesis is increasing and costing less; new tools are being developed for fast, large-scale genetic manipulation; and more tested genetic parts are available for use, as is the knowledge of how to use them effectively. These advances promise to unlock an exciting array of 'smart' bacteria for clinical use but will also challenge scientists to better optimize preclinical testing regimes for early identification and validation of promising strains and strategies. Here, we review recent advances in the development and testing of engineered bacterial diagnostics and therapeutics. We highlight new technologies that will assist the development of more complex, robust and reliable engineered bacteria for future clinical applications, and we discuss approaches to more efficiently evaluate engineered strains throughout their preclinical development.
Riglar DT, Giessen TW, Baym M, et al., 2017, Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation, Nature Biotechnology, Vol: 35, Pages: 653-658, ISSN: 1087-0156
Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.
Baum J, Richard D, Riglar DT, 2017, Malaria parasite invasion: achieving superb resolution., Cell Host and Microbe, Vol: 21, Pages: 294-296, ISSN: 1931-3128
It is only in the last decade that sub-cellular resolution of red cell invasion by the malaria parasite Plasmodium falciparum has been possible. Here we look back on the development of methodologies that led to this possibility and the subsequent advancements made in understanding this key event in malaria disease.
Riglar DT, Baym M, Kerns SJ, et al., 2016, Long-term monitoring of inflammation in the mammalian gut using programmable commensal bacteria
<jats:title>Abstract</jats:title><jats:p>Inflammation in the gut, caused by infection and autoimmunity, remains challenging to effectively detect, monitor, and treat. Here, we engineer a commensal mouse <jats:italic>E. coli</jats:italic> strain to record exposure to tetrathionate, a downstream product of reactive oxygen species generated during inflammation. Using these programmed bacteria to sense <jats:italic>in situ</jats:italic> levels we show that tetrathionate accompanies inflammation during Salmonella-induced colitis in mice and is elevated in an inflammatory bowel disease mouse model. We demonstrate long-term genetic stability and associated robust function of synthetic genetic circuits in bacteria colonizing the mammalian gut. These results demonstrate the potential for engineered bacteria to stably and reliably probe pathophysiological processes for which traditional diagnostics may not be feasible or cost-effective.</jats:p><jats:sec><jats:title>One sentence summary</jats:title><jats:p>Engineered bacteria record an inflammatory response in an IBD mouse model and are genetically stable during long-term growth in the mouse gut.</jats:p></jats:sec>
Riglar DT, Whitehead L, Cowman AF, et al., 2016, Localization-based imaging of malarial antigens during red cell entry reaffirms role for AMA1 but not MTRAP in invasion, Journal of Cell Science, Vol: 129, Pages: 228-242, ISSN: 1477-9137
Microscopy-based localisation of proteins during malaria parasite invasion of the erythrocyte is widely used for tentative assignment of protein function. To date, however, imaging has been limited by the rarity of invasion events and poor resolution available, given micron size of the parasite, which leads to a lack of quantitative measures for definitive localisation. Here, using computational image analysis we have attempted to assign relative protein localisation during invasion using wide-field deconvolution microscopy. By incorporating three-dimensional information we present a detailed assessment of known parasite effectors predicted to function during entry but as yet untested or for which data is equivocal. Our method, longitudinal intensity profiling, resolves confusion surrounding localisation of apical membrane antigen (AMA1) at the merozoite-erythrocyte junction and predicts that the merozoite thrombospondin related anonymous protein (MTRAP) is unlikely to play a direct role in the mechanics of entry, an observation supported with additional biochemical evidence. This approach sets a benchmark for imaging of complex micron-scale events and cautions against simplistic interpretations of small numbers of representative images for assignment of protein function or prioritisation of candidates as therapeutic targets.
Olshina MA, Angrisano F, Marapana DS, et al., 2015, Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites, Malaria Journal, Vol: 14, ISSN: 1475-2875
BackgroundGliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin.MethodsHere the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion.ResultsA combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely medi
Olshina M, Bane K, Angrisano F, et al., 2014, Coronin organizes actin filament polarity required for directional gliding motility in the malaria parasite., ASCB/IFCB Meeting, Publisher: AMER SOC CELL BIOLOGY, ISSN: 1059-1524
Dasgupta S, Auth T, Gov NS, et al., 2014, Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte, BIOPHYSICAL JOURNAL, Vol: 107, Pages: 43-54, ISSN: 0006-3495
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- Citations: 60
Healer J, Thompson JK, Riglar DT, et al., 2013, Vaccination with Conserved Regions of Erythrocyte-Binding Antigens Induces Neutralizing Antibodies against Multiple Strains of <i>Plasmodium falciparum</i>, PLOS ONE, Vol: 8, ISSN: 1932-6203
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- Citations: 42
Hanssen E, Dekiwadia C, Riglar DT, et al., 2013, Electron tomography of <i>Plasmodium falciparum</i> merozoites reveals core cellular events that underpin erythrocyte invasion, CELLULAR MICROBIOLOGY, Vol: 15, Pages: 1457-1472, ISSN: 1462-5814
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- Citations: 57
Lemgruber L, Kudryashev M, Dekiwadia C, et al., 2013, Cryo-electron tomography reveals four-membrane architecture of the <i>Plasmodium</i> apicoplast, MALARIA JOURNAL, Vol: 12, ISSN: 1475-2875
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- Citations: 37
Riglar DT, Rogers KL, Hanssen E, et al., 2013, Spatial association with PTEX complexes defines regions for effector export into <i>Plasmodium falciparum</i>-infected erythrocytes, NATURE COMMUNICATIONS, Vol: 4, ISSN: 2041-1723
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- Citations: 66
Riglar DT, Baum J, 2013, Static and dynamic imaging of erythrocyte invasion and early intra-erythrocytic development in Plasmodium falciparum., Methods Mol Biol, Vol: 923, Pages: 269-280
Cellular imaging has reemerged in recent years as a powerful approach to provide researchers with a direct measure of essential molecular events in a cell's life, ranging in scale from broad morphological observations of whole cells to intricate single molecule imaging. When combined with quantitative image analysis, the available imaging techniques can act as a critical means to confirm hypotheses, drive the formation of new theories or provide accurate determination of protein localization at subcellular and nanometer scales. Here, we describe two methodological approaches for imaging the transient step of malaria parasite invasion of the human erythrocyte. When applied to image the most virulent human malaria parasite, Plasmodium falciparum, the first approach, using live time-lapse wide-field microscopy, allows the capture of transient events during invasion and postinvasion intra-erythrocytic development, while the second, using immunofluorescence assay (IFA) of fixed samples, allows high-definition exploration of parasite architecture on multiple platforms.
Zuccala ES, Gout AM, Dekiwadia C, et al., 2012, Subcompartmentalisation of Proteins in the Rhoptries Correlates with Ordered Events of Erythrocyte Invasion by the Blood Stage Malaria Parasite, PLOS ONE, Vol: 7, ISSN: 1932-6203
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- Citations: 39
Bullen HE, Charnaud SC, Kalanon M, et al., 2012, Biosynthesis, Localization, and Macromolecular Arrangement of the <i>Plasmodium falciparum</i> Translocon of Exported Proteins (PTEX), JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 7871-7884, ISSN: 0021-9258
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- Citations: 97
Angrisano F, Riglar DT, Sturm A, et al., 2012, Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite, PLOS One23/1/12, Vol: 7, ISSN: 1932-6203
Chen L, Lopaticki S, Riglar DT, et al., 2011, An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by <i>Plasmodium falciparum</i>, PLOS PATHOGENS, Vol: 7, ISSN: 1553-7366
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- Citations: 108
Wong W, Skau CT, Marapana DS, et al., 2011, Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 9869-9874, ISSN: 0027-8424
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- Citations: 36
Triglia T, Chen L, Lopaticki S, et al., 2011, <i>Plasmodium falciparum</i> Merozoite Invasion Is Inhibited by Antibodies that Target the PfRh2a and b Binding Domains, PLOS PATHOGENS, Vol: 7, ISSN: 1553-7366
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- Citations: 37
Riglar DT, Richard D, Wilson DW, et al., 2011, Super-Resolution Dissection of Coordinated Events during Malaria Parasite Invasion of the Human Erythrocyte, CELL HOST & MICROBE, Vol: 9, Pages: 9-20, ISSN: 1931-3128
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- Citations: 253
Riglar D, Richard D, Boyle M, et al., 2010, Stepwise dissection of Plasmodium falciparum merozoite invasion of the human erythrocyte, Malaria Journal, Vol: 9
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