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Category: publications

A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites

Donna Huber on May 29, 2024

Hypnozonticidal hit detection and confirmation.
Hypnozonticidal hit detection and confirmation.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent ‘hypnozoite’ forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

S. P. Maher, M. A. Bakowski, A. Vantaux, E. L. Flannery, C. Andolina, M. Gupta, Y. Antonova-Koch, M. Argomaniz, M. Cabrera-Mora, B. Campo, A. T. Chao, A. K. Chatterjee, W. T. Cheng, E. Chuenchob, C. A. Cooper, K. Cottier, M. R. Galinski, A. Harupa-Chung, H. Ji, S. B. Joseph, T. Lenz, S. Lonardi, J. Matheson, S. A. Mikolajczak, T. Moeller, A. Orban, V. Padín-Irizarry, K. Pan, J. Péneau, J. Prudhomme, C. Roesch, A. A. Ruberto, S. S. Sabnis, C. L. Saney, J. Sattabongkot, S. Sereshki, S. Suriyakan, R. Ubalee, Y. Wang, P. Wasisakun, J. Yin, J. Popovici, C. W. McNamara, C. J. Joyner, F. Nosten, B. Witkowski, K. G. Le Roch, D. E. Kyle. 2024. eLife13:RP98221, https://doi.org/10.7554/eLife.98221.1

 

Temporal gene expression during asexual development of the apicomplexan Sarcocystis neurona

Donna Huber on May 29, 2024

Fig 1 Progression of the schizont stages during the intracellular development of Sarcocystis neurona expressing yellow fluorescent protein. Post-invasion, the merozoites convert into schizonts that progressively develop into early-, mid-, and late-schizonts while undergoing a form of asexual reproduction called endopolygeny. In the final step in endopolygeny, the mature schizont forms 64 haploid merozoites fully equipped to egress and invade new host cells. S. neurona, Sn; DAPI-stained host cell nuclei, HCN.
Fig 1 Progression of the schizont stages during the intracellular development of Sarcocystis neurona expressing yellow fluorescent protein. Post-invasion, the merozoites convert into schizonts that progressively develop into early-, mid-, and late-schizonts while undergoing a form of asexual reproduction called endopolygeny. In the final step in endopolygeny, the mature schizont forms 64 haploid merozoites fully equipped to egress and invade new host cells. S. neurona, Sn; DAPI-stained host cell nuclei, HCN.

Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes.

Importance: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.

Sriveny Dangoudoubiyam, Jamie K Norris, Sivaranjani Namasivayam, Rodrigo de Paula Baptista, Naila Cannes do Nascimento, Joseph Camp, Christopher L Schardl, Jessica C Kissinger, Daniel K Howe. mSphere. 2024 May 29:e0011124. doi: 10.1128/msphere.00111-24.

Protein phosphatase PP1 regulation of RNA polymerase II transcription termination and allelic exclusion of VSG genes in trypanosomes

Donna Huber on May 23, 2024

graphical abstract

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

Rudo Kieft, Yang Zhang, Haidong Yan, Robert J Schmitz, Robert Sabatini. Nucleic Acids Res. 2024 May 23:gkae392. doi: 10.1093/nar/gkae392.

The influence of oviposition status on measures of transmission potential in malaria-infected mosquitoes depends on sugar availability

Donna Huber on May 23, 2024

graphical abstract

Background: Like other oviparous organisms, the gonotrophic cycle of mosquitoes is not complete until they have selected a suitable habitat to oviposit. In addition to the evolutionary constraints associated with selective oviposition behavior, the physiological demands relative to an organism’s oviposition status also influence their nutrient requirement from the environment. Yet, studies that measure transmission potential (vectorial capacity or competence) of mosquito-borne parasites rarely consider whether the rates of parasite replication and development could be influenced by these constraints resulting from whether mosquitoes have completed their gonotrophic cycle.

Methods: Anopheles stephensi mosquitoes were infected with Plasmodium berghei, the rodent analog of human malaria, and maintained on 1% or 10% dextrose and either provided oviposition sites (‘oviposited’ herein) to complete their gonotrophic cycle or forced to retain eggs (‘non-oviposited’). Transmission potential in the four groups was measured up to 27 days post-infection as the rates of (i) sporozoite appearance in the salivary glands (‘extrinsic incubation period’ or EIP), (ii) vector survival and (iii) sporozoite densities.

Results: In the two groups of oviposited mosquitoes, rates of sporozoite appearance and densities in the salivary glands were clearly dependent on sugar availability, with shorter EIP and higher sporozoite densities in mosquitoes fed 10% dextrose. In contrast, rates of appearance and densities in the salivary glands were independent of sugar concentrations in non-oviposited mosquitoes, although both measures were slightly lower than in oviposited mosquitoes fed 10% dextrose. Vector survival was higher in non-oviposited mosquitoes.

Conclusions: Costs to parasite fitness and vector survival were buffered against changes in nutritional availability from the environment in non-oviposited but not oviposited mosquitoes. Taken together, these results suggest vectorial capacity for malaria parasites may be dependent on nutrient availability and oviposition/gonotrophic status and, as such, argue for more careful consideration of this interaction when estimating transmission potential. More broadly, the complex patterns resulting from physiological (nutrition) and evolutionary (egg-retention) trade-offs described here, combined with the ubiquity of selective oviposition behavior, implies the fitness of vector-borne pathogens could be shaped by selection for these traits, with implications for disease transmission and management. For instance, while reducing availability of oviposition sites and environmental sources of nutrition are key components of integrated vector management strategies, their abundance and distribution are under strong selection pressure from the patterns associated with climate change.

Justine C Shiau, Nathan Garcia-Diaz, Dennis E Kyle, Ashutosh K Pathak. Parasit Vectors. 2024 May 23;17(1):236. doi: 10.1186/s13071-024-06317-2.

Hepatocytes and the art of killing Plasmodium softly

Donna Huber on May 6, 2024

Figure 1. The gap in our understanding of how hepatocytes eliminate Plasmodium.
Figure 1. The gap in our understanding of how hepatocytes eliminate Plasmodium.

 

The Plasmodium parasites that cause malaria undergo asymptomatic development in the parenchymal cells of the liver, the hepatocytes, prior to infecting erythrocytes and causing clinical disease. Traditionally, hepatocytes have been perceived as passive bystanders that allow hepatotropic pathogens such as Plasmodium to develop relatively unchallenged. However, now there is emerging evidence suggesting that hepatocytes can mount robust cell-autonomous immune responses that target Plasmodium, limiting its progression to the blood and reducing the incidence and severity of clinical malaria. Here we discuss our current understanding of hepatocyte cell-intrinsic immune responses that target Plasmodium and how these pathways impact malaria.

Camila Marques-da-Silva, Clyde Schmidt-Silva, Samarchith P Kurup. Trends Parasitol. 2024 May 6:S1471-4922(24)00086-2. doi: 10.1016/j.pt.2024.04.004.

Identification of a viral gene essential for the genome replication of a domesticated endogenous virus in ichneumonid parasitoid wasps

Donna Huber on April 25, 2024

Fig 6. RNAi knockdown of U16.
RNAi knockdown of U16.

 

Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor “domesticated endogenous viruses” (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs). Large quantities of DNA-containing IV virions are produced in ovary calyx cells during the pupal and adult stages of female wasps. Females parasitize host insects by injecting eggs and virions into the body cavity. After injection, virions rapidly infect host cells which is followed by expression of IV genes that promote the successful development of wasp offspring. IV genomes consist of two components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus that produce virions. In this study, we generated a chromosome-scale genome assembly for Hyposotor didymator that harbors H. didymator ichnovirus (HdIV). We identified a total of 67 HdIV loci that are amplified in calyx cells during the wasp pupal stage. We then focused on an HdIV gene, U16, which is transcribed in calyx cells during the initial stages of replication. Sequence analysis indicated that U16 contains a conserved domain in primases from select other viruses. Knockdown of U16 by RNA interference inhibited virion morphogenesis in calyx cells. Genome-wide analysis indicated U16 knockdown also inhibited amplification of HdIV loci in calyx cells. Altogether, our results identified several previously unknown HdIV loci, demonstrated that all HdIV loci are amplified in calyx cells during the pupal stage, and showed that U16 is required for amplification and virion morphogenesis.

Ange Lorenzi, Fabrice Legeai, Véronique Jouan, Pierre-Alain Girard, Michael R Strand, Marc Ravallec, Magali Eychenne, Anthony Bretaudeau, Stéphanie Robin, Jeanne Rochefort, Mathilde Villegas, Gaelen R Burke, Rita Rebollo, Nicolas Nègre, Anne-Nathalie Volkoff. PLoS Pathog. 2024 Apr 25;20(4):e1011980. doi: 10.1371/journal.ppat.1011980.

 

Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax

Donna Huber on April 8, 2024

Figure 1 Mean IC50 concentrations (nM) of chloroquine, doxycycline, piperaquine, and tafenoquine using 48-, 72- and 96-hour assays.
Mean IC50 concentrations (nM) of chloroquine, doxycycline, piperaquine, and tafenoquine using 48-, 72- and 96-hour assays.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

Peter Christensen, Rosy Cinzah, Rossarin Suwanarusk, Adeline Chiew Yen Chua, Osamu Kaneko, Dennis E Kyle, Htin Lin Aung, Jessica Matheson, Pablo Bifani, Laurent Rénia, Gregory M Cook, Georges Snounou, Bruce Russell. Antimicrob Agents Chemother. 2024 Apr 8:e0028024. doi: 10.1128/aac.00280-24.

The Unfortunate Abundance of Trypanosoma cruzi in Naturally Infected Dogs and Monkeys Provides Unique Opportunities to Advance Solutions for Chagas Disease

Donna Huber on April 4, 2024

Trypanosoma cruzi, the protozoan parasite and cause of Chagas disease, is widely distributed in many vertebrate and triatomine species throughout North, Central, and South America. Variations in housing quality largely determines human infection risk in the Americas. However, the southern U.S. contains widespread, infected triatomine vectors and captive species and domesticated animals with active T. cruzi infection or at high risk of becoming infected and developing Chagas disease. There is a critical need for better detection and intervention strategies, principally focused on human infection throughout the Americas, but mainly in the U.S., for high-value dogs employed in government and other work. In addition to this economic impact, the concentration of largely unavoidable T. cruzi infections in U.S. dogs provides an incomparable opportunity to answer questions related to T. cruzi infection and Chagas disease that are impossible or unethical to address in humans. As the course of T. cruzi infection and Chagas disease, the immune response to infection, and the response to therapeutics are highly similar across the range of mammalian host species, information obtained from studies in other species can directly inform researchers on how to best detect, manage, and treat T. cruzi infection and Chagas disease in humans.

Rick L. Tarleton, Ashley B. Saunders, Bruno Lococo, Maria Gabriela Alvarez Gianni, Susana Laucella, Carolyn L. Hodo, Gregory K. Wilkerson, Sarah A. Hamer. Zoonoses. 2024. Vol. 4(1). DOI: 10.15212/ZOONOSES-2024-0005

What is new in FungiDB: a web-based bioinformatics platform for omics-scale data analysis for fungal and oomycete species

Donna Huber on March 26, 2024

New data in FungiDB since FungiDB Release 37.
New data in FungiDB since FungiDB Release 37.

 

FungiDB (https://fungidb.org) serves as a valuable online resource that seamlessly integrates genomic and related large-scale data for a wide range of fungal and oomycete species. As an integral part of the VEuPathDB Bioinformatics Resource Center (https://veupathdb.org), FungiDB continually integrates both published and unpublished data addressing various aspects of fungal biology. Established in early 2011, the database has evolved to support 674 datasets. The datasets include over 300 genomes spanning various taxa (e.g. Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Mucoromycota, as well as Albuginales, Peronosporales, Pythiales, and Saprolegniales). In addition to genomic assemblies and annotation, over 300 extra datasets encompassing diverse information, such as expression and variation data, are also available. The resource also provides an intuitive web-based interface, facilitating comprehensive approaches to data mining and visualization. Users can test their hypotheses and navigate through omics-scale datasets using a built-in search strategy system. Moreover, FungiDB offers capabilities for private data analysis via the integrated VEuPathDB Galaxy platform. FungiDB also permits genome improvements by capturing expert knowledge through the User Comments system and the Apollo genome annotation editor for structural and functional gene curation. FungiDB facilitates data exploration and analysis and contributes to advancing research efforts by capturing expert knowledge for fungal and oomycete species.

Evelina Y Basenko, Achchuthan Shanmugasundram, Ulrike Böhme, David Starns, Paul A Wilkinson, Helen R Davison, Kathryn Crouch, Gareth Maslen, Omar S Harb, Beatrice Amos, Mary Ann McDowell, Jessica C Kissinger, David S Roos, Andrew Jones. Genetics. 2024 Mar 26:iyae035. doi: 10.1093/genetics/iyae035

Positive clinical outcome using a modified dosing regimen of benznidazole in dogs at high risk for infection or acutely infected with Trypanosoma cruzi

Donna Huber on March 18, 2024

Serum cardiac troponin I results for the 4 dogs.
Serum cardiac troponin I results for the 4 dogs.

 

Trypanosoma cruzi infection in dogs can cause heart failure and sudden death with few treatment options available. A litter of 4 dogs living in a T cruzi endemic area were randomized to prophylaxis and nonprophylaxis groups as part of a study evaluating a modified benznidazole dosing regimen administered twice weekly to prevent T cruzi infection during a vector transmission season. The 2 dogs that received prophylaxis remained healthy without T cruzi infection or cardiac disease for >2 years. One dog that did not receive prophylaxis died unexpectedly with acute T cruzi-induced pancarditis, and the second dog tested positive for T cruzi and developed complex arrhythmias with markedly increased cardiac troponin I and improved with a higher benznidazole treatment dose. Although the small sample size precludes definitive conclusions, we describe the potential clinical benefit of prophylactic and early treatment with modified benznidazole dosing regimens for dogs with T cruzi infection.

Sukjung Lim, Stephanie Collins, Sarah A Hamer, Rick L Tarleton, Ashley B Saunders. J Vet Intern Med. 2024 Mar 18. doi: 10.1111/jvim.17028.