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Author: Donna Huber

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

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)

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)

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

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

Graphica abstract

Abstract

Garcinia dauphinensis is a previously uninvestigated endemic plant species of Madagascar. The new phloroglucinols dauphinols A–F and 3′-methylhyperjovoinol B (17) and six known phloroglucinols (813) together with tocotrienol 14 and the three triterpenoids 1517 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

Daniel Colley: The Schisto Kid

By John H. Tibbetts

Daniel Colley
Daniel Colley visits a car wash in Kisumu, Kenya, one of his study sites for more than 20 years. Workers at the car wash drive vehicles into Lake Victoria, infecting and reinfecting themselves with schistosomiasis. (Photo courtesy of SCORE)

One day Daniel Colley raised his hand to volunteer, setting in motion five decades of scientific adventures. It was 1969, and Colley’s postdoctoral adviser, Byron Waksman, a renowned immunologist at Yale University School of Medicine, had stepped into the laboratory and asked if anyone wanted to go to Brazil.

“I have no idea why my hand shot up,” says Colley. “I didn’t know anything about Brazil. My wife and I didn’t even have passports. I asked Byron about the nature of the research, and he said, ‘Schistosomiasis.’ My response was, ‘What’s that?’”

Colley, today a UGA immunologist and Fellow of the American Association for the Advancement of Science, became fascinated by schistosomiasis, a parasitic worm infection plaguing poverty-stricken communities in sub-Saharan Africa and around the world. Globally more than 250 million people are infected via contact with water that carries the parasites.

The waterborne worms penetrate human skin and take up residence in blood vessels. About 5 to 10 percent of infections progress to life-threatening disease over decades. But most people experience more subtle symptoms such as fatigue, anemia, wasting, malnutrition and impaired cognitive development.

“Children playing in the water are picking up these chronic parasitic infections,” he says, “so they are sick and don’t do as well in school. If kids don’t receive what they need to develop early in life, it can become a lifelong disability.”

Kids in water in Niger
This irrigation channel in a village in Niger is a schistosomiasis transmission site. The children exposed there were tested and treated as part of the Schistosomiasis Consortium for Operational Research and Evaluation program, led by Colley. (Photo by Amadou Garba/SCORE)

After his Brazil sojourn, Colley arrived at Vanderbilt University in 1971, setting up a lab and beginning his career-long effort to understand the immunological paradox of schistosomiasis (or “schisto,” in the vernacular).

“The more I learned about schisto, the more interesting it became,” says Colley, who tweets as @SchistoKid. “It has a bizarre life cycle. Here’s a worm that can live inside your blood vessels for up to 40 years, though more typically it lasts for five to 10 years. Why doesn’t your immune system get rid of this creature sooner? That was a very intriguing question.”

In infected human blood vessels, the female worms produce eggs that the male fertilizes. Many of the eggs escape the human body in urine or feces. When people urinate or defecate in or near fresh water, the eggs can infect freshwater snails, where the parasite develops and rapidly multiplies. When worms re-enter fresh water, they can find human victims.

Meanwhile, the body’s remaining worm eggs are swept by the bloodstream into the gut wall and the liver or bladder, where they become lodged. The immune system fights these egg intruders with a delicate, two-pronged effort: First, masses of cells called granulomas wall off the eggs, isolating them from surrounding tissue and reducing disease. But the immune system must also regulate granuloma growth. For most people, this regulatory response keeps granulomas relatively small, but some grow over decades, eventually causing fibrosis and blocking blood flow through the liver, causing internal bleeding.

“Schisto is a very complex puzzle for an immunologist,” Colley says. “If you fail to have the initial immune response against the egg, you die. But if you fail to regulate this immune response against the egg over time, you die. How our immune system has co-evolved with schisto is fascinating to me, and I still haven’t figured out how it’s done.”

In 1992, he joined the Centers for Disease Control and Prevention and a year later was promoted to director of its Division of Parasitic Diseases. “I learned about a lot of other parasitic diseases. It was an incredibly broadening experience that became useful in my later work at UGA. From my colleagues, I gained knowledge in epidemiology—the incidence and prevalence of diseases and detecting the sources and causes of epidemics.”

He arrived at UGA in 2001 as professor of microbiology and director of the Center for Tropical and Emerging Global Diseases, created only three years before. “UGA started the center and took risks by investing in it,” he says. “Now it’s globally famous for its work in parasitic diseases and has 23 principal investigators.”

During the past decade, Colley has been director of UGA’s Schistosomiasis Consortium for Operational Research and Evaluation (SCORE), a program supported by the Bill & Melinda Gates Foundation. SCORE scientists study strategies used in eight sub-Saharan African countries to control and eventually eliminate schistosomiasis. Today, most sub-Saharan African governments collaborate with the World Health Organization and a pharmaceutical company to provide a free drug, praziquantel, that treats existing infections and can significantly reduce new cases.

“SCORE has shown that mass interventions with praziquantel do work, and they are best done every year,” he says. SCORE researchers also helped develop a more rapid and precise diagnostic test for schistosomiasis, discovering many more cases in children than previously thought.

“The main message I’ve learned in my career is that diseases such as schisto are diseases of poverty,” he says. “Poverty contributes to these diseases, and poverty is also the result of them. If you are a stunted kid, and you have anemia, and your cognitive development is not great because of a parasite, it’s harder to succeed.

“People are the same everywhere—they all want a better life, and in some places, that’s not happening. Fighting these infections is an important part of making lives better.”

 

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Originally published at UGA Research.

A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages

Abstract

Background:Plasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available.

Results: Here, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite.

Conclusions: The α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Carola Schafer, Nicholas Dambrauskas, Ryan W. Steel, Sara Carbonetti, Vorada Chuenchob, Erika L. Flannery, Vladimir Vigdorovich, Brian G. Oliver, Wanlapa Roobsoong, Steven P. Maher, Dennis Kyle, Jetsumon Sattabongkot, Stefan H. I. Kappe, Sebastian A. Mikolajczak and D. Noah Sather. 2018. Malaria Journal; 17:370. https://doi.org/10.1186/s12936-018-2519-7

Plasmodium falciparum cGMP-dependent protein kinase interacts with a subunit of the parasite proteasome

ABSTRACT

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

K. Govindasamy, R. Khan, M. Snyder, H. J. Lou, P. Du, H. M. Kudyba, V. Muralidharan, B. E. Turk, P. Bhanot. 2018. Infection and Immunity. https://doi.org/10.1128/IAI.00523-18

5-Diphosphoinositol Pentakisphosphate (5-IP7) Regulates Phosphate Release from Acidocalcisomes and Yeast Vacuoles

Abstract

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate sodium symporter (TbPho91, and Pho91p, in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation, and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi -loaded giant vacuoles prepared from wild type or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ strains, or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Evgeniy Potapenko, Ciro D Cordeiro, Guozhong Huang, Melissa Storey, Christopher Wittwer, Amit K Dutta, Henning J. Jessen, Vincent J. Starai and Roberto Docampo. 2018. Journal of Biological Chemistry; 293:19101-19112.
doi: 10.1074/jbc.RA118.005884