Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9

ABSTRACT Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excel-lent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special ge-netic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 con-tributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. IMPORTANCE Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. How-ever, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to suc

Functional analysis of rhomboid proteases during Toxoplasma invasion

Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii

Canvass: a crowd-sourced, natural-product screening library for exploring biological space

NCATS thanks Dingyin Tao for assistance with compound characterization. This research was supported by the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH). R.B.A. acknowledges support from NSF (CHE-1665145) and NIH (GM126221). M.K.B. acknowledges support from NIH (5R01GM110131). N.Z.B. thanks support from NIGMS, NIH (R01GM114061). J.K.C. acknowledges support from NSF (CHE-1665331). J.C. acknowledges support from the Fogarty International Center, NIH (TW009872). P.A.C. acknowledges support from the National Cancer Institute (NCI), NIH (R01 CA158275), and the NIH/National Institute of Aging (P01 AG012411). N.K.G. acknowledges support from NSF (CHE-1464898). B.C.G. thanks the support of NSF (RUI: 213569), the Camille and Henry Dreyfus Foundation, and the Arnold and Mabel Beckman Foundation. C.C.H. thanks the start-up funds from the Scripps Institution of Oceanography for support. J.N.J. acknowledges support from NIH (GM 063557, GM 084333). A.D.K. thanks the support from NCI, NIH (P01CA125066). D.G.I.K. acknowledges support from the National Center for Complementary and Integrative Health (1 R01 AT008088) and the Fogarty International Center, NIH (U01 TW00313), and gratefully acknowledges courtesies extended by the Government of Madagascar (Ministere des Eaux et Forets). O.K. thanks NIH (R01GM071779) for financial support. T.J.M. acknowledges support from NIH (GM116952). S.M. acknowledges support from NIH (DA045884-01, DA046487-01, AA026949-01), the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program (W81XWH-17-1-0256), and NCI, NIH, through a Cancer Center Support Grant (P30 CA008748). K.N.M. thanks the California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board for support. B.T.M. thanks Michael Mullowney for his contribution in the isolation, elucidation, and submission of the compounds in this work. P.N. acknowledges support from NIH (R01 GM111476). L.E.O. acknowledges support from NIH (R01-HL25854, R01-GM30859, R0-1-NS-12389). L.E.B., J.K.S., and J.A.P. thank the NIH (R35 GM-118173, R24 GM-111625) for research support. F.R. thanks the American Lebanese Syrian Associated Charities (ALSAC) for financial support. I.S. thanks the University of Oklahoma Startup funds for support. J.T.S. acknowledges support from ACS PRF (53767-ND1) and NSF (CHE-1414298), and thanks Drs. Kellan N. Lamb and Michael J. Di Maso for their synthetic contribution. B.S. acknowledges support from NIH (CA78747, CA106150, GM114353, GM115575). W.S. acknowledges support from NIGMS, NIH (R15GM116032, P30 GM103450), and thanks the University of Arkansas for startup funds and the Arkansas Biosciences Institute (ABI) for seed money. C.R.J.S. acknowledges support from NIH (R01GM121656). D.S.T. thanks the support of NIH (T32 CA062948-Gudas) and PhRMA Foundation to A.L.V., NIH (P41 GM076267) to D.S.T., and CCSG NIH (P30 CA008748) to C.B. Thompson. R.E.T. acknowledges support from NIGMS, NIH (GM129465). R.J.T. thanks the American Cancer Society (RSG-12-253-01-CDD) and NSF (CHE1361173) for support. D.A.V. thanks the Camille and Henry Dreyfus Foundation, the National Science Foundation (CHE-0353662, CHE-1005253, and CHE-1725142), the Beckman Foundation, the Sherman Fairchild Foundation, the John Stauffer Charitable Trust, and the Christian Scholars Foundation for support. J.W. acknowledges support from the American Cancer Society through the Research Scholar Grant (RSG-13-011-01-CDD). W.M.W.acknowledges support from NIGMS, NIH (GM119426), and NSF (CHE1755698). A.Z. acknowledges support from NSF (CHE-1463819). (Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH); CHE-1665145 - NSF; CHE-1665331 - NSF; CHE-1464898 - NSF; RUI: 213569 - NSF; CHE-1414298 - NSF; CHE1361173 - NSF; CHE1755698 - NSF; CHE-1463819 - NSF; GM126221 - NIH; 5R01GM110131 - NIH; GM 063557 - NIH; GM 084333 - NIH; R01GM071779 - NIH; GM116952 - NIH; DA045884-01 - NIH; DA046487-01 - NIH; AA026949-01 - NIH; R01 GM111476 - NIH; R01-HL25854 - NIH; R01-GM30859 - NIH; R0-1-NS-12389 - NIH; R35 GM-118173 - NIH; R24 GM-111625 - NIH; CA78747 - NIH; CA106150 - NIH; GM114353 - NIH; GM115575 - NIH; R01GM121656 - NIH; T32 CA062948-Gudas - NIH; P41 GM076267 - NIH; R01GM114061 - NIGMS, NIH; R15GM116032 - NIGMS, NIH; P30 GM103450 - NIGMS, NIH; GM129465 - NIGMS, NIH; GM119426 - NIGMS, NIH; TW009872 - Fogarty International Center, NIH; U01 TW00313 - Fogarty International Center, NIH; R01 CA158275 - National Cancer Institute (NCI), NIH; P01 AG012411 - NIH/National Institute of Aging; Camille and Henry Dreyfus Foundation; Arnold and Mabel Beckman Foundation; Scripps Institution of Oceanography; P01CA125066 - NCI, NIH; 1 R01 AT008088 - National Center for Complementary and Integrative Health; W81XWH-17-1-0256 - Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program; P30 CA008748 - NCI, NIH, through a Cancer Center Support Grant; California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board; American Lebanese Syrian Associated Charities (ALSAC); University of Oklahoma Startup funds; 53767-ND1 - ACS PRF; PhRMA Foundation; P30 CA008748 - CCSG NIH; RSG-12-253-01-CDD - American Cancer Society; RSG-13-011-01-CDD - American Cancer Society; CHE-0353662 - National Science Foundation; CHE-1005253 - National Science Foundation; CHE-1725142 - National Science Foundation; Beckman Foundation; Sherman Fairchild Foundation; John Stauffer Charitable Trust; Christian Scholars Foundation)Published versionSupporting documentatio

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (−)-2(S)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016). Keywords: PHGDH; inhibitor; serineNational Institutes of Health (U.S.) (Grant U54MH084681)National Institutes of Health (U.S.) (R37 AI047389)National Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (R01 CA129105)National Institutes of Health (U.S.) (R37 AI047389)National Institutes of Health (U.S.) (K22 CA212059)Mary Kay Foundation (017-32)V foundation (V2017-004

Assessing inhibitors of mutant isocitrate dehydrogenase using a suite of pre-clinical discovery assays

Abstract Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). We present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors. This involved biochemical, cell-based, and tier-one ADME techniques

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH)

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as <b>63</b> exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug–target residence time was determined via SPR. Analysis of these data suggests that drug–target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target