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Category: publications

Cryogenically preserved RBCs support gametocytogenesis of Plasmodium falciparum in vitro and gametogenesis in mosquitoes

Donna Huber on December 6, 2018

Abstract

Background: The malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is standard membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of “infectious” gametocytes requires 10–12 days with considerable physico-chemical demands imposed on host RBCs and thus, “fresh” RBCs that are ≤ 1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs (“cryo-preserved RBCs”) is accepted by European and US FDAs as an alternative to refrigeration (4 °C) for preserving RBC “quality” and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for > 4 days.

Results: Using the standard laboratory strain, P. falciparum NF54, 11 SMFAs were performed with RBCs from four separate donors to demonstrate that RBCs cryo-preserved in the gaseous phase of liquid nitrogen (− 196 °C) supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensimosquitoes. Overall levels of sporogony in the mosquito, as measured by oocyst and sporozoite prevalence, as well as oocyst burden, from each of the four donors thawed after varying intervals of cryopreservation (1, 4, 8, and 12 weeks) were comparable to using ≤ 1-week old refrigerated RBCs. Lastly, the potential for cryo-preserved RBCs to serve as a suitable alternative substrate is demonstrated for a Cambodian isolate of P. falciparum across two independent SMFAs.

Conclusions: Basic guidelines are presented for integrating cryo-preserved RBCs into an existing laboratory/insectary framework for P. falciparum SMFAs with significant potential for reducing running costs while achieving greater reliability. Lastly, scenarios are discussed where cryo-preserved RBCs may be especially useful in enhancing the understanding and/or providing novel insights into the patterns and processes underlying human-to-mosquito transmission.

Ashutosh K. Pathak, Justine C. Shiau, Matthew B. Thomas and Courtney C. Murdock. 2018. Malaria Journal; 17:457. https://doi.org/10.1186/s12936-018-2612-y

Proximal Remote Sensing to Non-destructively Detect and Diagnose Physiological Responses by Host Insect Larvae to Parasitism

Donna Huber on December 4, 2018

As part of identifying and characterizing physiological responses and adaptations by insects, it is paramount to develop non-destructive techniques to monitor individual insects over time. Such techniques can be used to optimize the timing of when in-depth (i.e., destructive sampling of insect tissue) physiological or molecular analyses should be deployed. In this article, we present evidence that hyperspectral proximal remote sensing can be used effectively in studies of host responses to parasitism. We present time series body reflectance data acquired from individual soybean loopers (Chrysodeixis includens) without parasitism (control) or parasitized by one of two species of parasitic wasps with markedly different life histories: Microplitis demolitor, a solitary larval koinobiont endoparasitoid and Copidosoma floridanum, a polyembryonic (gregarious) egg-larval koinobiont endoparasitoid. Despite considerable temporal variation in reflectance data 1–9 days post-parasitism, the two parasitoids caused uniquely different host body reflectance responses. Based on reflectance data acquired 3–5 days post-parasitism, all three treatments (control larvae, and those parasitized by either M. demolitor or C. floridanum) could be classified with >85 accuracy. We suggest that hyperspectral proximal imaging technologies represent an important frontier in insect physiology, as they are non-invasive and can be used to account for important time scale factors, such as: minutes of exposure or acclimation to abiotic factors, circadian rhythms, and seasonal effects. Although this study is based on data from a host-parasitoid system, results may be of broad relevance to insect physiologists. Described approaches provide a non-invasive and rapid method that can provide insights into when to destructively sample tissue for more detailed mechanistic studies of physiological responses to stressors and environmental conditions.

Christian Nansen and Michael R. Strand. 2018. Frontiers in Physiology. https://doi.org/10.3389/fphys.2018.01716

Anilinoquinoline based inhibitors of trypanosomatid proliferation

Donna Huber on November 26, 2018

Abstract

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.

Lori Ferrins, Amrita Sharma, Sarah M. Thomas, Naimee Mehta, Jessey Erath, Scott Tanghe, Susan E. Leed, Ana Rodriguez, Kojo Mensa-Wilmot, Richard J. Sciotti, Kirsten Gillingwater, Michael P. Pollastri. 2018. PLOS Neglected Tropical Diseases. https://doi.org/10.1371/journal.pntd.0006834

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Donna Huber on November 21, 2018

Abstract

Infection with the protozoan parasite Toxoplasma gondii is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, Toxoplasma propagates in intracellular parasitophorous vacuoles, but as for nearly all other eukaryotes, Toxoplasma glycosylates many cellular proteins and lipids and assembles polysaccharides. Toxoplasma glycans resemble those of other eukaryotes but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The Toxoplasma genome encodes a suite of likely glycogenes expected to assemble N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in Toxoplasma, here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc0-2-Man6-GlcNAc2–type N-glycans, a novel HexNAc-GalNAc–mucin-type O-glycan, and Tn-antigen, identified the glycosyltransferases for assembling a novel nuclear O-Fuc–type and cell surface Glc-Fuc–type O-glycans, and showed that they are important for in vitro growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes.

Elisabet Gas-Pascual, Hiroshi Travis Ichikawa, Mohammed Osman Sheikh, Mariam Isabella Serji, Bowen Deng, Msano Mandalasi, Giulia Bandini, John Samuelson, Lance Wells and Christopher M. West. 2018. Journal of Biological Chemistry. 294: 1104-1125. doi: 10.1074/jbc.RA118.006072

Highly competent, non-exhausted CD8+ T cells continue to tightly control pathogen load throughout chronic Trypanosoma cruzi infection

Donna Huber on November 12, 2018

Abstract

Trypanosoma cruzi infection is characterized by chronic parasitism of non-lymphoid tissues and is rarely eliminated despite potent adaptive immune responses. This failure to cure has frequently been attributed to a loss or impairment of anti-T. cruzi T cell responses over time, analogous to the T cell dysfunction described for other persistent infections. In this study, we have evaluated the role of CD8+ T cells during chronic T. cruzi infection (>100 dpi), with a focus on sites of pathogen persistence. Consistent with repetitive antigen exposure during chronic infection, parasite-specific CD8+ T cells from multiple organs expressed high levels of KLRG1, but exhibit a preferential accumulation of CD69+ cells in skeletal muscle, indicating recent antigen encounter in a niche for T. cruzi persistence. A significant proportion of CD8+ T cells in the muscle also produced IFNγ, TNFα and granzyme B in situ, an indication of their detection of and functional response to T. cruzi in vivo. CD8+ T cell function was crucial for the control of parasite burden during chronic infection as exacerbation of parasite load was observed upon depletion of this population. Attempts to improve T cell function by blocking PD-1 or IL-10, potential negative regulators of T cells, failed to increase IFNγ and TNFα production or to enhance T. cruzi clearance. These results highlight the capacity of the CD8+ T cell population to retain essential in vivo function despite chronic antigen stimulation and support a model in which CD8+ T cell dysfunction plays a negligible role in the ability of Trypanosoma cruzi to persist in mice.

Angela D. Pack, Matthew H. Collins, Charles S. Rosenberg, Rick L. Tarleton. 2018. PLOS Pathogens. https://doi.org/10.1371/journal.ppat.1007410

Genetic conservation of Cytauxzoon felis antigens and mRNA expression in the schizont life-stage

Donna Huber on November 6, 2018

Cytauxzoon felis schizont formation in a feline splenic vessel

Fig. 1. Cytauxzoon felis schizont formation in a feline splenic vessel. A. Hematoxylin and eosin stained splenic tissue demonstrating schizonts forming a parasitic thrombus and completely occluding a splenic vessel, 20× objective, B. Hematoxylin and eosin stained schizont with developing merozoites, 40× objective.

Abstract

Cytauxzoonosis is a highly fatal disease of domestic cats caused by the apicomplexan protozoan Cytauxzoon felis, which is most closely related to Theileria spp. The growing prevalence, high morbidity and mortality, and treatment cost of cytauxzoonosis emphasize the need for vaccine development. Traditional approaches for vaccine development, however, have been hindered by the inability to culture C. felis in vitro. Recent availability of the annotated C. felis genome combined with genome-based vaccine design and protein microarray immunoscreening allowed for high-throughput identification of C. felis antigens that could serve as vaccine candidates. This study assessed the suitability of three of these vaccine candidates (cf30, cf63, cf58) in addition to a previously reported vaccine candidate (cf76) based on two criteria: genetic conservation among diverse C. felis geographic isolates and expression in tissues containing the C. felis schizont life stage, which has been previously associated with the development of a protective immune response. A comparison of seventeen C. felis isolates across seven states demonstrated high sequence identity (99–100%) for cf30, cf63, and cf58, similar to the degree of conservation previously reported for cf76. RNAscope® in situ hybridization using acutely infected feline splenic tissue revealed robust levels of all transcripts in the schizont life stage of the parasite. These data support the suitability of these three antigens for further investigation as vaccine candidates against cytauxzoonosis.

Daven B. Khana, David S. Peterson, James B. Stanton, Megan E. Schreeg, Adam J. Birkenheuer, Jaime L.Tarigo. 2018. Veterinary Parasitology; 263:49-53. https://doi.org/10.1016/j.vetpar.2018.10.007

Anthelmintics – From Discovery to Resistance III (Indian Rocks Beach, FL, 2018)

Donna Huber on November 5, 2018

Abstract

The third scientific meeting in the series “Anthelmintics: From Discovery to Resistance” was held in Indian Rocks Beach, Florida, at the end of January 2018. The meeting focused on a variety of topics related to the title, including the identification of novel targets and new leads, the mechanism of action of existing drugs and the genetic basis of resistance against them. Throughout there was an emphasis on the exploitation of new technologies and methods to further these aims. The presentations, oral and poster, covered basic, veterinary and medical science with strong participation by both academic and commercial researchers. This special issue contains selected papers from the meeting.

Adrian J. Wolstenholme, Richard J. Martin. 2018. International Journal of Parasitology: Drugs and Drug Resistance; 8(3):494-495. https://doi.org/10.1016/j.ijpddr.2018.11.002

First evidence of polychaete intermediate hosts for Neospirorchis spp. marine turtle blood flukes (Trematoda: Spirorchiidae)

Donna Huber on October 25, 2018

Abstract

Graphical abstract

Life cycles of spirorchiids that infect the vascular system of turtles are poorly understood. Few life cycles of these blood flukes have been elucidated and all intermediate hosts reported are gastropods (Mollusca), regardless of whether the definitive host is a freshwater or a marine turtle. During a recent survey of blood fluke larvae in polychaetes on the coast of South Carolina, USA, spirorchiid-like cercariae were found to infect the polychaetes Amphitrite ornata (Terebellidae) and Enoplobranchus sanguineus (Polycirridae). Cercariae were large, furcate, with a ventral acetabulum, but no eyespots were observed. Partial sequences of D1–D2 domains of the large ribosomal subunit, the internal transcribed spacer 2, and the mitochondrial cytochrome oxidase 1 genes allowed the identification of sporocysts and cercariae as belonging to two unidentified Neospirorchis species reported from the green turtle, Chelonia mydas, in Florida: Neospirorchis sp. (Neogen 13) in A. ornata and Neospirorchis sp. (Neogen 14) in E. sanguineus. Phylogenetic analysis suggests that infection of annelids by blood flukes evolved separately in aporocotylids and spirorchiids. Our results support the contention that the Spirorchiidae is not a valid family and suggest that Neospirorchis is a monophyletic clade within the paraphyletic Spirorchiidae. Since specificity of spirorchiids for their intermediate hosts is broader than it was thus far assumed, surveys of annelids in turtle habitats are necessary to further our understanding of the life history of these pathogenic parasites.

Isaure de Buron, Beatrice L. Colon, Sasha V. Siegel, Jenna Oberstaller, Andrea Rivero, Dennis E. Kyle. 2018. International Journal for Parasitology; 48(14):1097-1106. https://doi.org/10.1016/j.ijpara.2018.08.002

Phenotypic screens reveal posaconazole as rapidly cidal combination partner for treatment of Primary Amoebic Meningoencephalitis

Donna Huber on October 24, 2018

Abstract

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration–approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 μM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri–infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.

Beatrice L Colon, Christopher A Rice, R Kiplin Guy, Dennis E Kyle. 2018. The Journal of Infectious Diseases. https://doi.org/10.1093/infdis/jiy622

Phloroglucinols from the Roots of Garcinia dauphinensis and Their Antiproliferative and Antiplasmodial Activities

Donna Huber on October 24, 2018

Graphica abstract

Abstract

Garcinia dauphinensis is a previously uninvestigated endemic plant species of Madagascar. The new phloroglucinols dauphinols A–F and 3′-methylhyperjovoinol B (1–7) and six known phloroglucinols (8–13) together with tocotrienol 14 and the three triterpenoids 15–17 were isolated from an ethanolic extract of G. dauphinensis roots using various chromatographic techniques. The structures of the isolated compounds were elucidated by NMR, MS, optical rotation, and ECD data. Theoretical ECD spectra and specific rotations for 2 were calculated and compared to experimental data in order to assign its absolute configuration. Among the compounds tested, 1showed the most promising growth inhibitory activity against A2870 ovarian cancer cells, with IC50= 4.5 ± 0.9 μM, while 2 had good antiplasmodial activity against the Dd2 drug-resistant strain of Plasmodium falciparum, with IC50 = 0.8 ± 0.1 μM.

Rolly G. Fuentes, Kirk C. Pearce, Yongle Du, Andriamalala Rakotondrafara, Ana L. Valenciano, Maria B. Cassera, Vincent E. Rasamison, T. Daniel Crawford, and David G. I. Kingston. 2018. Journal of Natural Products.
DOI: 10.1021/acs.jnatprod.8b00379