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Category: publications

Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes

Most mosquito species are anautogenous, which means they must blood feed on a vertebrate host to produce eggs, while a few are autogenous and can produce eggs without blood feeding. Egg formation is best understood in the anautogenous mosquito Aedes aegypti where insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) interact to regulate gonadotrophic cycles. Circulating hemocytes also approximately double in abundance in conjunction with a gonadotrophic cycle but the factors responsible for stimulating this increase remain unclear. Focusing on Ae. aegypti, we determined that hemocyte abundance similarly increased in intact blood-fed females and decapitated blood-fed females that were injected with ILP3, whereas OEH, 20E, or heat-killed bacteria had no stimulatory activity. ILP3 upregulated insulin-insulin growth factor signaling in hemocytes but few genes, including almost no transcripts for immune factors, were differentially expressed. ILP3 also stimulated circulating hemocytes to increase in two other anautogenous (Anopheles gambiae and Culex quinquefasciatus) and two facultatively autogenous mosquitoes (Aedes atropalpus and Culex pipiens molestus), but had no stimulatory activity in the obligately autogenous mosquito Toxorhynchites amboinensis. Altogether, our results identify ILPs as the primary regulators of hemocyte proliferation in association with egg formation, but also suggest this response has been lost in the evolution of obligate autogeny.

Ellen O Martinson, Kangkang Chen, Luca Valzania, Mark R Brown, Michael R Strand. J Exp Biol. 2022 Feb 7;jeb.243460. doi: 10.1242/jeb.243460.

CRISPR/Cas9-induced disruption of Bodo saltans paraflagellar rod-2 gene reveals its importance for cell survival

Developing transfection protocols for marine protists is an emerging field that will allow the functional characterization of protist genes and their roles in organism responses to the environment. We developed a CRISPR/Cas9 editing protocol for Bodo saltans, a free-living kinetoplastid with tolerance to both marine and freshwater conditions, and a close non-parasitic relative of trypanosomatids. Our results show that SaCas9/sgRNA ribonucleoprotein (RNP) complex-mediated disruption of the paraflagellar rod 2 gene (BsPFR2) was achieved using electroporation-mediated transfection. The use of CRISPR/Cas9 genome editing can increase the efficiency of targeted homologous recombination when a repair DNA template is provided. Based on sequence analysis, two mechanisms for repairing double-strand breaks (DSB) in B. saltans are active; homologous directed repair (HDR) utilizing an exogenous DNA template that carries an antibiotic resistance gene, and non-homologous end joining (NHEJ). However, HDR was only achieved when a single (vs. multiple) SaCas9 RNP complex was provided. Further, the biallelic knockout of BsPFR2 was detrimental for the cell, highlighting its essential role for cell survival because it facilitates the movement of food particles into the cytostome. Our Cas9/sgRNA RNP complex protocol provides a new tool for assessing gene functions in B. saltans, and perhaps similar protists with polycistronic transcription. This article is protected by copyright. All rights reserved.

Fatma Gomaa, Zhu-Hong Li, David J Beaudoin, Heba Alzan, Peter R Girguis, Roberto Docampo, Virginia P Edgcomb. Environ Microbiol. 2022 Jan 31. doi: 10.1111/1462-2920.15918.

Structure-activity and structure-property relationship studies of spirocyclic chromanes with antimalarial activity

Malaria is a prevalent and lethal disease. The fast emergence and spread of resistance to current therapies is a major concern and the development of a novel line of therapy that could overcome, the problem of drug resistance, is imperative. Screening of a set of compounds with drug/natural product-based sub-structural motifs led to the identification of spirocyclic chroman-4-one 1 with promising antimalarial activity against the chloroquine-resistant Dd2 and chloroquine-sensitive 3D7 strains of the parasite. Extensive structure-activity and structure-property relationship studies were conducted to identify the essential features necessary for its activity and properties.

Iredia D Iyamu, Yingzhao Zhao, Prakash T Parvatkar, Bracken F Roberts, Debora R Casandra, Lukasz Wojtas, Dennis E Kyle, Debopam Chakrabarti, Roman Manetsch. Bioorg Med Chem. 2022 Jan 14;57:116629. doi: 10.1016/j.bmc.2022.116629.

The complete genome of Chelonus insularis reveals dynamic arrangement of genome components in parasitoid wasps that produce bracoviruses

Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (family Braconidae). Microgastroid wasps have coopted nudivirus genes to produce replication-defective virions that females use to transfer virulence genes to parasitized hosts. The microgastroid complex further consists of six subfamilies and ∼50,000 species but current understanding of BV gene inventories and organization primarily derives from analysis of two wasp species in the subfamily Microgastrinae (Microplitis demolitor and Cotesia congregata) that produce M. demolitor BV (MdBV) and C. congregata BV (CcBV). Notably, several genomic features of MdBV and CcBV remain conserved since divergence of M. demolitor and C. congregata ∼53 million years ago (MYA). However, it is unknown whether these conserved traits more broadly reflect BV evolution, because no complete genomes exist for any microgastroid wasps outside of the Microgastrinae. In this regard, the subfamily Cheloninae is of greatest interest because it diverged earliest from the Microgastrinae (∼85 MYA) after endogenization of the nudivirus ancestor. Here, we present the complete genome of Chelonus insularis, which is an egg-larval parasitoid in the Cheloninae that produces C. insularis BV (CinsBV). We report that the inventory of nudivirus genes in C. insularis is conserved but are dissimilarly organized when compared to M. demolitor and C. congregata. Reciprocally, CinsBV proviral segments share organizational features with MdBV and CcBV but virulence gene inventories exhibit almost no overlap. Altogether, our results point to the functional importance of a conserved inventory of nudivirus genes and a dynamic set of virulence genes for the successful parasitism of hosts. Our results also suggest organizational features previously identified in MdBV and CcBV are likely not essential for BV virion formation.

Significance Bracoviruses are a remarkable example of virus endogenization, because large sets of genes from a nudivirus ancestor continue to produce virions that thousands of wasp species rely upon to parasitize hosts. Understanding how these genes interact and have been coopted by wasps for novel functions is of broad interest in the study of virus evolution. This manuscript characterizes bracovirus genome components in the parasitoid wasp Chelonus insularis, which together with existing wasp genomes captures a large portion of the diversity among wasp species that produce bracoviruses. Results provide new information about how bracovirus genome components are organized in different wasps while also providing additional insights on key features required for function.

Meng Mao, Michael R Strand, Gaelen R Burke. J Virol. 2022 Jan 5;JVI0157321. doi: 10.1128/JVI.01573-21.

Antimalarial Natural Products

Natural products have made a crucial and unique contribution to human health, and this is especially true in the case of malaria, where the natural products quinine and artemisinin and their derivatives and analogues, have saved millions of lives. The need for new drugs to treat malaria is still urgent, since the most dangerous malaria parasite, Plasmodium falciparum, has become resistant to quinine and most of its derivatives and is becoming resistant to artemisinin and its derivatives. This volume begins with a short history of malaria and follows this with a summary of its biology. It then traces the fascinating history of the discovery of quinine for malaria treatment and then describes quinine’s biosynthesis, its mechanism of action, and its clinical use, concluding with a discussion of synthetic antimalarial agents based on quinine’s structure. The volume then covers the discovery of artemisinin and its development as the source of the most effective current antimalarial drug, including summaries of its synthesis and biosynthesis, its mechanism of action, and its clinical use and resistance. A short discussion of other clinically used antimalarial natural products leads to a detailed treatment of other natural products with significant antiplasmodial activity, classified by compound type. Although the search for new antimalarial natural products from Nature’s combinatorial library is challenging, it is very likely to yield new antimalarial drugs. The chapter thus ends by identifying over ten natural products with development potential as clinical antimalarial agents.

Kingston D.G.I., Cassera M.B. (2022) Antimalarial Natural Products. In: Kinghorn A.D., Falk H., Gibbons S., Asakawa Y., Liu JK., Dirsch V.M. (eds) Antimalarial Natural Products. Progress in the Chemistry of Organic Natural Products, vol 117. Springer, Cham. https://doi.org/10.1007/978-3-030-89873-1_1

Clinical recovery of Macaca fascicularis infected with Plasmodium knowlesi

Background: Kra monkeys (Macaca fascicularis), a natural host of Plasmodium knowlesi, control parasitaemia caused by this parasite species and escape death without treatment. Knowledge of the disease progression and resilience in kra monkeys will aid the effective use of this species to study mechanisms of resilience to malaria. This longitudinal study aimed to define clinical, physiological and pathological changes in kra monkeys infected with P. knowlesi, which could explain their resilient phenotype.

Methods: Kra monkeys (n = 15, male, young adults) were infected intravenously with cryopreserved P. knowlesi sporozoites and the resulting parasitaemias were monitored daily. Complete blood counts, reticulocyte counts, blood chemistry and physiological telemetry data (n = 7) were acquired as described prior to infection to establish baseline values and then daily after inoculation for up to 50 days. Bone marrow aspirates, plasma samples, and 22 tissue samples were collected at specific time points to evaluate longitudinal clinical, physiological and pathological effects of P. knowlesi infections during acute and chronic infections.

Results: As expected, the kra monkeys controlled acute infections and remained with low-level, persistent parasitaemias without anti-malarial intervention. Unexpectedly, early in the infection, fevers developed, which ultimately returned to baseline, as well as mild to moderate thrombocytopenia, and moderate to severe anaemia. Mathematical modelling and the reticulocyte production index indicated that the anaemia was largely due to the removal of uninfected erythrocytes and not impaired production of erythrocytes. Mild tissue damage was observed, and tissue parasite load was associated with tissue damage even though parasite accumulation in the tissues was generally low.

Conclusions: Kra monkeys experimentally infected with P. knowlesi sporozoites presented with multiple clinical signs of malaria that varied in severity among individuals. Overall, the animals shared common mechanisms of resilience characterized by controlling parasitaemia 3-5 days after patency, and controlling fever, coupled with physiological and bone marrow responses to compensate for anaemia. Together, these responses likely minimized tissue damage while supporting the establishment of chronic infections, which may be important for transmission in natural endemic settings. These results provide new foundational insights into malaria pathogenesis and resilience in kra monkeys, which may improve understanding of human infections.

Mariko S Peterson, Chester J Joyner, Jessica A Brady, Jennifer S Wood, Monica Cabrera-Mora, Celia L Saney, Luis L Fonseca, Wayne T Cheng, Jianlin Jiang, Stacey A Lapp, Stephanie R Soderberg, Mustafa V Nural, Jay C Humphrey, Allison Hankus, Deepa Machiah, Ebru Karpuzoglu, Jeremy D DeBarry, MaHPIC-Consortium; Rabindra Tirouvanziam, Jessica C Kissinger, Alberto Moreno, Sanjeev Gumber, Eberhard O Voit, Juan B Gutiérrez, Regina Joice Cordy, Mary R Galinski. Clinical recovery of Macaca fascicularis infected with Plasmodium knowlesiMalar J 20, 486 (2021). https://doi.org/10.1186/s12936-021-03925-6

Generation of human long-lived plasma cells by developmentally regulated epigenetic imprinting

Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1CASP3, and CASP8 Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

Chester J Joyner, Ariel M Ley, Doan C Nguyen, Mohammad Ali, Alessia Corrado, Christopher Tipton, Christopher D Scharer, Tian Mi, Matthew C Woodruff, Jennifer Hom, Jeremy M Boss, Meixue Duan, Greg Gibson, Danielle Roberts, Joel Andrews, Sagar Lonial, Inaki Sanz, F Eun-Hyung Lee. Life Sci Alliance. 2021 Dec 24;5(3):e202101285. doi: 10.26508/lsa.202101285.

Bracoviruses, ichnoviruses, and virus-like particles from parasitoid wasps retain many features of their virus ancestors

Animal genomes commonly contain genes or sequences that have been acquired from different types of viruses. The vast majority of these endogenous virus elements (EVEs) are inactive or consist of only a small number of components that show no evidence of cooption for new functions or interaction. Unlike most EVEs, bracoviruses (BVs), ichnoviruses (IVs) and virus-like particles (VLPs) in parasitoid wasps have evolved through retention and interaction of many genes from virus ancestors. Here, we discuss current understanding of BV, IV and VLP evolution along with associated implications for what constitutes a virus. We suggest that BVs and IVs are domesticated endogenous viruses (DEVs) that differ in several important ways from other known EVEs.

Jean-Michel Drezen, Annie Bézier, Gaelen R Burke, Michael R Strand. Curr Opin Insect Sci. 2021 Dec 22;S2214-5745(21)00132-2. doi: 10.1016/j.cois.2021.12.003.

A Phenotypic Screen for the Liver Stages of Plasmodium vivax

Control of malaria caused by Plasmodium vivax can be improved by the discovery and development of novel drugs against the parasite’s liver stage, which includes relapse-causing hypnozoites. Several recent reports describe breakthroughs in the culture of the P. vivax liver stage in 384-well microtiter plates, with the goal of enabling a hypnozoite-focused drug screen. Herein we describe assay details, protocol developments, and different assay formats to interrogate the chemical sensitivity of the P. vivax liver stage in one such medium-throughput platform. The general assay protocol includes seeding of primary human hepatocytes which are infected with P. vivax sporozoites generated from the feeding of Anopheles dirus mosquitoes on patient isolate bloodmeals. This protocol is unique in that, after source drug plates are supplied, all culture-work steps have been optimized to preclude the need for automated liquid handling, thereby allowing the assay to be performed within resource-limited laboratories in malaria-endemic countries. Throughput is enhanced as complex culture methods, such as extracellular matrix overlays, multiple cell types in co-culture, or hepatic spheroids, are excluded as the workflow consists entirely of routine culture methods for adherent cells. Furthermore, installation of a high-content imager at the study site enables assay data to be read and transmitted with minimal logistical delays. Herein we detail distinct assay improvements which increase data quality, provide a means to limit the confounding effect of hepatic metabolism on assay data, and detect activity of compounds with a slow-clearance phenotype.

Steven P. Maher, Amélie Vantaux, Caitlin A. Cooper, Nathan M. Chasen, Wayne T. Cheng, Chester J. Joyner, Roman Manetsch, Benoît Witkowski, Dennis Kyle. 2021. Bio-Protocol. 11(23): DOI: 10.21769/BioProtoc.4253

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

Yong Fu, Kevin M Brown, Nathaniel G Jones, Silvia Nj Moreno, L David Sibley. Elife. 2021 Dec 3;10:e73011. doi: 10.7554/eLife.73011.