Publications
27 results found
May DA, Taha F, Child MA, et al., 2023, How colonization bottlenecks, tissue niches, and transmission strategies shape protozoan infections, Trends in Parasitology, Vol: 39, Pages: 1074-1086, ISSN: 0169-4758
Protozoan pathogens such as Plasmodium spp., Leishmania spp., Toxoplasma gondii, and Trypanosoma spp. are often associated with high-mortality, acute and chronic diseases of global health concern. For transmission and immune evasion, protozoans have evolved diverse strategies to interact with a range of host tissue environments. These interactions are linked to disease pathology, yet our understanding of the association between parasite colonization and host homeostatic disruption is limited. Recently developed techniques for cellular barcoding have the potential to uncover the biology regulating parasite transmission, dissemination, and the stability of infection. Understanding bottlenecks to infection and the in vivo tissue niches that facilitate chronic infection and spread has the potential to reveal new aspects of parasite biology.
Benns HJ, Storch M, Falco JA, et al., 2022, CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery., Nat Microbiol
Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.
Kolli SK, Molina-Cruz A, Araki T, et al., 2022, Malaria parasite evades mosquito immunity by glutaminyl cyclase-mediated posttranslational protein modification., Proc Natl Acad Sci U S A, Vol: 119
Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.
Wincott CJ, Sritharan G, Benns HJ, et al., 2022, Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization, Cell Reports Methods, Vol: 2, Pages: 1-16, ISSN: 2667-2375
Cellular barcoding techniques are powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been broadly applied to protozoan parasites, which have unique genomic structures and virulence strategies compared with viral and bacterial pathogens. Here, we present a CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. Using libraries of barcoded T. gondii, we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes representing a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. These data establish a cellular barcoding approach for protozoa and evidence that the blood-brain barrier is not a major bottleneck to colonization by T. gondii.
Jeffers V, Child MA, 2022, No acetyl-CoA keeps <i>Plasmodium</i> at bay, CELL CHEMICAL BIOLOGY, Vol: 29, Pages: 174-176, ISSN: 2451-9456
Alves E, Benns H, Magnus L, et al., 2021, An extracellular redox signal triggers calcium release and impacts the asexual development of Toxoplasma gondii, Frontiers in Cellular and Infection Microbiology, Vol: 11, Pages: 1-14, ISSN: 2235-2988
The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in apicomplexan parasites such as Toxoplasma gondii. The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 58 genes containing domains related to canonical antioxidant function, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H2O2 to human fibroblasts infected with T.gondii triggers a Ca2+ flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H2O2 is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression a glutaredoxin-roGFP2 redox sensor fusion protein in the parasitophorous vacuole severely impacts parasite replication. These data highlight the rich redox network that exists in T. gondii, evidencing a link betweenextracellular redox and intracellular Ca2+ signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasitegrowth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.
Alves E, Benns HJ, Magnus L, et al., 2021, An extracellular redox signal triggers calcium release and impacts the asexual development of <i>Toxoplasma gondii</i>
<jats:title>Abstract</jats:title><jats:p>The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in apicomplexan parasites such as <jats:italic>Toxoplasma gondii</jats:italic>. The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 58 genes containing domains related to canonical antioxidant function, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> to human fibroblasts infected with <jats:italic>T. gondii</jats:italic> triggers a Ca<jats:sup>2+</jats:sup> flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression a glutaredoxin-roGFP2 redox sensor fusion protein in the parasitophorous vacuole severely impacts parasite replication. These data highlight the rich redox network that exists in <jats:italic>T. gondii</jats:italic>, evidencing a link between extracellular redox and intracellular Ca<jats:sup>2+</jats:sup> signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasite growth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.</jats:p>
Benns HJ, Storch M, Falco J, et al., 2021, Prioritization of antimicrobial targets by CRISPR-based oligo recombineering
<jats:title>Summary</jats:title><jats:p>Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen<jats:italic>Toxoplasma gondii</jats:italic>and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.</jats:p>
Wincott CJ, Sritharan G, Benns HJ, et al., 2020, Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of <i>Toxoplasma gondii</i> host colonization
<jats:title>Abstract</jats:title><jats:p>Molecular barcoding techniques have emerged as powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been extended to protozoan parasites, which have unique genomic structures and virulence strategies compared to viral and bacterial pathogens. Here, we present a versatile CRISPR-based method to barcode protozoa, which we successfully apply to <jats:italic>Toxoplasma gondii</jats:italic> and <jats:italic>Trypanosoma brucei</jats:italic>. The murine brain is an important transmission niche for <jats:italic>T. gondii</jats:italic>, and brain persistence is a clinically untreatable feature of infection. The blood-brain barrier is expected to physically restrict parasite colonization of this niche, resulting in a selection bottleneck. Using libraries of barcoded <jats:italic>T. gondii</jats:italic> we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one-month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes that represented a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. Together, these data establish the first, robust molecular barcoding approach for protozoa and evidence that the blood-brain barrier does not represent a major bottleneck to colonization by <jats:italic>T. gondii</jats:italic>.</jats:p>
Benns HJ, Wincott CJ, Tate EW, et al., 2020, Activity- and reactivity-based proteomics: Recent technological advances and applications in drug discovery., Current Opinion in Chemical Biology, Vol: 60, Pages: 20-29, ISSN: 1367-5931
Activity-based protein profiling (ABPP) is recognized as a powerful and versatile chemoproteomic technology in drug discovery. Central to ABPP is the use of activity-based probes to report the activity of specific enzymes or reactivity of amino acid types in complex biological systems. Over the last two decades, ABPP has facilitated the identification of new drug targets and discovery of lead compounds in human and infectious disease. Furthermore, as part of a sustained global effort to illuminate the druggable proteome, the repertoire of target classes addressable with activity-based probes has vastly expanded in recent years. Here, we provide an overview of ABPP and summarise the major technological advances with an emphasis on probe development.
Anderson DP, Benns HJ, Tate EW, et al., 2020, CRISPR-TAPE: protein-centricCRISPRguide design for targeted proteome engineering, MOLECULAR SYSTEMS BIOLOGY, Vol: 16, ISSN: 1744-4292
Anderson DP, Benns HJ, Tate EW, et al., 2020, CRISPR-TAPE: protein-centric CRISPR guide design for targeted proteome engineering., Mol Syst Biol, Vol: 16
Rational molecular engineering of proteins with CRISPR-based approaches is challenged by the gene-centric nature of gRNA design tools. To address this, we have developed CRISPR-TAPE, a protein-centric gRNA design algorithm that allows users to target specific residues, or amino acid types within proteins. gRNA outputs can be customized to support maximal efficacy of homology-directed repair for engineering purposes, removing time-consuming post hoc curation, simplifying gRNA outputs and reducing CPU times.
Benns HJ, Tate EW, Child MA, 2018, Activity-Based Protein Profiling for the Study of Parasite Biology., Curr Top Microbiol Immunol, Vol: 420, Pages: 155-174, ISSN: 0070-217X
Parasites exist within most ecological niches, often transitioning through biologically and chemically complex host environments over the course of their parasitic life cycles. While the development of technologies for genetic engineering has revolutionised the field of functional genomics, parasites have historically been less amenable to such modification. In light of this, parasitologists have often been at the forefront of adopting new small-molecule technologies, repurposing drugs into biological tools and probes. Over the last decade, activity-based protein profiling (ABPP) has evolved into a powerful and versatile chemical proteomic platform for characterising the function of enzymes. Central to ABPP is the use of activity-based probes (ABPs), which covalently modify the active sites of enzyme classes ranging from serine hydrolases to glycosidases. The application of ABPP to cellular systems has contributed vastly to our knowledge on the fundamental biology of a diverse range of organisms and has facilitated the identification of potential drug targets in many pathogens. In this chapter, we provide a comprehensive review on the different forms of ABPP that have been successfully applied to parasite systems, and highlight key biological insights that have been enabled through their application.
Garland M, Schulze CJ, Foe IT, et al., 2018, Development of an activity-based probe for acyl-protein thioesterases, PLOS ONE, Vol: 13, ISSN: 1932-6203
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- Citations: 16
Child MA, Garland M, Foe I, et al., 2017, Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1, mBio, Vol: 8, ISSN: 2150-7511
Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii.IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of the
Foe IT, Child MA, Majmudar JD, et al., 2015, Global analysis of palmitoylated proteins in Toxoplasma gondii, Cell Host and Microbe, Vol: 18, Pages: 501-511, ISSN: 1931-3128
Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.
Bender KO, Garland M, Ferreyra JA, et al., 2015, A small-molecule antivirulence agent for treating Clostridium difficile infection, Science Translational Medicine, Vol: 7, Pages: 1-11, ISSN: 1946-6234
Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.
van der Linden WA, Segal E, Child MA, et al., 2015, Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase, CHEMISTRY & BIOLOGY, Vol: 22, Pages: 995-1001, ISSN: 1074-5521
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- Citations: 8
Treeck M, Sanders JL, Gaji RY, et al., 2014, The Calcium-Dependent Protein Kinase 3 of <i>Toxoplasma</i> Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis, PLOS PATHOGENS, Vol: 10, ISSN: 1553-7366
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- Citations: 49
Child MA, 2013, Chemical biology approaches for the study of apicomplexan parasites, MOLECULAR AND BIOCHEMICAL PARASITOLOGY, Vol: 192, Pages: 1-9, ISSN: 0166-6851
Child MA, Hall CI, Beck JR, et al., 2013, Small-molecule inhibition of a depalmitoylase enhances <i>Toxoplasma</i> host-cell invasion, NATURE CHEMICAL BIOLOGY, Vol: 9, Pages: 651-656, ISSN: 1552-4450
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- Citations: 46
Child MA, Harris PK, Collins CR, et al., 2013, Molecular Determinants for Subcellular Trafficking of the Malarial Sheddase PfSUB2, TRAFFIC, Vol: 14, Pages: 1053-1064, ISSN: 1398-9219
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- Citations: 9
Child MA, Hall CI, Beck JR, et al., 2013, Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion., Nat Chem Biol, Vol: 9, Pages: 651-656
Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.
Li H, Child MA, Bogyo M, 2012, Proteases as regulators of pathogenesis: Examples from the Apicomplexa, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, Vol: 1824, Pages: 177-185, ISSN: 1570-9639
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- Citations: 47
Hall CI, Reese ML, Weerapana E, et al., 2011, Chemical genetic screen identifies <i>Toxoplasma</i> DJ-1 as a regulator of parasite secretion, attachment, and invasion, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 10568-10573, ISSN: 0027-8424
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- Citations: 48
Child MA, Epp C, Bujard H, et al., 2010, Regulated maturation of malaria merozoite surface protein-1 is essential for parasite growth, MOLECULAR MICROBIOLOGY, Vol: 78, Pages: 187-202, ISSN: 0950-382X
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- Citations: 32
Koussis K, Withers-Martinez C, Yeoh S, et al., 2009, A multifunctional serine protease primes the malaria parasite for red blood cell invasion, EMBO JOURNAL, Vol: 28, Pages: 725-735, ISSN: 0261-4189
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- Citations: 111
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