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Category: publications

Polymorphic Molecular Signatures in Variable Regions of the Plasmodium falciparum var2csa DBL3x Domain Are Associated with Virulence in Placental Malaria

The Plasmodium falciparum protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood. The deep sequencing of the var2csa DBL3X domain in placental blood from forty-nine primigravid and multigravid women living in malaria-endemic western Kenya revealed numerous unique sequences within individuals in association with chronic PM but not gravidity. Additional analysis unveiled four distinct sequence types that were variably present in mixed proportions amongst the study population. An analysis of the abundance of each of these sequence types revealed that one was inversely related to infant gestational age, another was inversely related to placental parasitemia, and a third was associated with chronic PM. The categorization of women according to the type to which their dominant sequence belonged resulted in the segregation of types as a function of gravidity: two types predominated in multigravidae whereas the other two predominated in primigravidae. The univariate logistic regression analysis of sequence type dominance further revealed that gravidity, maternal age, placental parasitemia, and hemozoin burden (within maternal leukocytes), reported a lack of antimalarial drug use, and infant gestational age and birth weight influenced the odds of membership in one or more of these sequence predominance groups. Cumulatively, these results show that unique var2csa sequences differentially appear in women with different PM exposure histories and segregate to types independently associated with maternal factors, infection parameters, and birth outcomes. The association of some var2csa sequence types with indicators of pathogenesis should motivate vaccine efforts to further identify and target VAR2CSA epitopes associated with maternal morbidity and poor birth outcomes.

Eldin Talundzic, Stephen Scott, Simon O Owino, David S Campo, Naomi W Lucchi, Venkatachalam Udhayakumar, Julie M Moore, David S Peterson. Pathogens. 2022 Apr 28;11(5):520. doi: 10.3390/pathogens11050520

Essential Bromodomain Tc BDF2 as a Drug Target against Chagas Disease

Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of TcBDF2 in T. cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results point to TcBDF2 as a druggable target against T. cruzi.

Alejandro Pezza, Luis E Tavernelli, Victoria L Alonso, Virginia Perdomo, Raquel Gabarro, Rab Prinjha, Elvio Rodríguez Araya, Inmaculada Rioja, Roberto Docampo, Felix Calderón, Julio Martin, Esteban Serra. ACS Infect Dis. 2022 Apr 28. doi: 10.1021/acsinfecdis.2c00057.

Improving in vitro continuous cultivation of Plasmodium cynomolgi, a model for P. vivax

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Peter Christensen, Annie Racklyeft, Kurt E Ward, Jessica Matheson, Rossarin Suwanarusk, Adeline C Y Chua, Osamu Kaneko, Htin Lin Aung, Laurent Rénia, Nadia Amanzougaghene, Victor Magneron, Julien Lemaitre, Roger Le Grand, Dennis Kyle, Pablo Bifani, Gregory M Cook, Georges Snounou, Bruce Russell. Parasitol Int. 2022 Apr 22;89:102589. doi: 10.1016/j.parint.2022.102589. Online ahead of print.

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp

Graphical Abstract

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite’s ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesized that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalyzed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

Alona Botnar, Grant Lawrence, Steven P Maher, Amélie Vantaux, Benoît Witkowski, Justine C Shiau, Emilio F Merino, David De Vore, Christian Yang, Cameron Murray, Maria B Cassera, James W Leahy, Dennis E Kyle. Int J Parasitol. 2022 Apr 18;S0020-7519(22)00066-2. doi: 10.1016/j.ijpara.2022.03.003.

Ad libitum consumption of protein- or peptide-sucrose solutions stimulates egg formation by prolonging the vitellogenic phase of oogenesis in anautogenous mosquitoes

Background: Anautogenous mosquitoes commonly consume nectars and other solutions containing sugar but are thought to only produce eggs in discrete gonadotrophic cycles after blood-feeding on a vertebrate host. However, some anautogenous species are known to produce eggs if amino acids in the form of protein are added to a sugar solution. Unclear is how different sources of amino acids in sugar solutions affect the processes that regulate egg formation and whether responses vary among species. In this study, we addressed these questions by focusing on Aedes aegypti and conducting some comparative assays with Aedes albopictus, Anopheles gambiae, Anopheles stephensi and Culex quinquefasciatus.

Methods: Adult female mosquitoes were fed sugar solutions containing amino acids, peptides or protein. Markers for activation of a gonadotrophic cycle including yolk deposition into oocytes, oviposition, ovary ecdysteroidogenesis, expression of juvenile hormone and 20-hydroxyecdysone-responsive genes, and adult blood-feeding behavior were then measured.

Results: The five anautogenous species we studied produced eggs when fed two proteins (bovine serum albumin, hemoglobin) or a mixture of peptides (tryptone) in 10% sucrose but deposited only small amounts of yolk into oocytes when fed amino acids in 10% sucrose. Focusing on Ae. aegypti, cultures were maintained for multiple generations by feeding adult females protein- or tryptone-sugar meals. Ad libitum access to protein- or tryptone-sugar solutions protracted production of ecdysteroids by the ovaries, vitellogenin by the fat body and protease activity by the midgut albeit at levels that were lower than in blood-fed females. Females also exhibited semi-continual oogenesis and repressed host-seeking behavior.

Conclusions: Several anautogenous mosquitoes produce eggs when provided ad libitum access to protein- or peptide-sugar meals, but several aspects of oogenesis also differ from females that blood-feed.

Ruby E Harrison, Kangkang Chen, Lilith South, Ange Lorenzi, Mark R Brown, Michael R Strand. Parasit Vectors. 2022 Apr 12;15(1):127. doi: 10.1186/s13071-022-05252-4.

Polychlorinated cyclopentenes from a marine derived Periconia sp. (strain G1144)

Studies on an organic extract of a marine fungus, Periconia sp. (strain G1144), led to the isolation of three halogenated cyclopentenes along with the known and recently reported rhytidhyester D; a series of spectrometric and spectroscopic techniques were used to elucidate these structures. Interestingly, two of these compounds represent tri-halogenated cyclopentene derivatives, which have been observed only rarely from Nature. The relative and absolute configurations of the compounds were established via mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, Mosher’s esters method, optical rotation and GIAO NMR calculations, including correlation coefficient calculations and the use of both DP4+ and dJ DP4 analyses. Several of the isolated compounds were tested for activity in anti-parasitic, antimicrobial, quorum sensing inhibition, and cytotoxicity assays and were shown to be inactive.

Kristóf B Cank, Robert A Shepherd, Sonja L Knowles, Manuel Rangel-Grimaldo, Huzefa A Raja, Zoie L Bunch, Nadja B Cech, Christopher A Rice, Dennis E Kyle, Joseph O Falkinham 3rd, Joanna E Burdette, Nicholas H Oberlies. Phytochemistry . 2022 Apr 11;113200. doi: 10.1016/j.phytochem.2022.113200

Anopheles gambiae strain (Ag55) cultured cells originated from Anopheles coluzzii and are phagocytic with hemocyte-like gene expression

Anopheles gambiae and Anopheles coluzzii are closely related species that are predominant vectors of malaria in Africa. Recently, A. gambiae form M was renamed A. coluzzii and we now conclude on the basis of a diagnostic PCR-restriction fragment length polymorphism assay that Ag55 cells were derived from A. coluzzii. We established an Ag55 cell transcriptome, and KEGG pathway analysis showed that Ag55 cells are enriched in phagosome pathway transcripts. The Ag55 transcriptome has an abundance of specific transcripts characteristic of mosquito hemocytes. Functional E. coli bioparticle uptake experiments visualized by fluorescence microscopy and confocal microscopy and quantified by flow cytometry establish the phagocytic competence of Ag55 cells. Results from this investigation of Ag55 cell properties will guide researchers in the use and engineering of the Ag55 cell line to better enable investigations of Plasmodium, other microbes, and insecticidal toxins. Graphical abstract: Anopheles gambiae cultured Ag55 cells originated from Anopheles coluzzi, have a hemocyte-like transcriptome and are phagocytic. This article is protected by copyright. All rights reserved.

Ruchir Mishra, Gang Hua, Ujwal R Bagal, Donald E Champagne, Michael J Adang. Insect Sci. 2022 Mar 31. doi: 10.1111/1744-7917.13036.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12)

The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.

Yuxiang Dong, Yogesh Sonawane, Steven P Maher, Anne-Marie Zeeman, Victor Chaumeau, Amélie Vantaux, Caitlin A Cooper, Francis C K Chiu, Eileen Ryan, Jenna McLaren, Gong Chen, Sergio Wittlin, Benoît Witkowski, François Nosten, Kamaraj Sriraghavan, Dennis E Kyle, Clemens H M Kocken, Susan A Charman, Jonathan L Vennerstrom. ACS Omega. 2022 Mar 30;7(14):12401-12411. doi: 10.1021/acsomega.2c01099.

Parasite Powerhouse: a Review of the Toxoplasma gondii Mitochondrion

Toxoplasma gondii is a member of the apicomplexan phylum, a group of single-celled eukaryotic parasites that cause significant human morbidity and mortality around the world. T. gondii harbors two organelles of endosymbiotic origin: a non-photosynthetic plastid, known as the apicoplast, and a single mitochondrion derived from the ancient engulfment of an α-proteobacterium. Due to excitement surrounding the novelty of the apicoplast, the T. gondii mitochondrion was, to a certain extent, overlooked for about two decades. However, recent work has illustrated that the mitochondrion is an essential hub of apicomplexan-specific biology. Development of novel techniques, such as cryo-electron microscopy, complexome profiling, and next-generation sequencing have led to a renaissance in mitochondrial studies. This review will cover what is currently known about key features of the T. gondii mitochondrion, ranging from its genome to protein import machinery and biochemical pathways. Particular focus will be given to mitochondrial features that diverge significantly from the mammalian host, along with discussion of this important organelle as a drug target.

Madelaine M. Usey, Diego Huet. J Eukaryot Microbiol. 2022 Mar 21;e12906. doi: 10.1111/jeu.12906.

Development and Introduction of the Filariasis Test Strip: A New Diagnostic Test for the Global Program to Eliminate Lymphatic Filariasis

A key component to achieving the global goal of elimination of lymphatic filariasis (LF) is the availability of appropriate tools for disease mapping, monitoring, and surveillance. However, the development of these tools for a neglected disease such as LF can be a challenge. The lack of a commercial market and low familiarity with these diseases leave little incentive for diagnostic manufacturers to invest in this space. The Filarial Test Strip (FTS) development story provides a case study on how a multi-stakeholder, public-private partnership model facilitated the development, evaluation, and introduction of a new monitoring and surveillance tool for LF. This paper will reflect on the experience with the FTS and document the process from development of the target product profile to adoption and scale-up in country programs. Lessons learned from both the successes and challenges experienced during this process may help inform future efforts to develop and introduce new diagnostic or surveillance tools for neglected diseases.

Anastasia Pantelias, Jonathan D King, Patrick Lammie, Gary J Weil. Am J Trop Med Hyg. 2022 Mar 15;tpmd210990. doi: 10.4269/ajtmh.21-0990.