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Author: Donna Huber

Pseudokinase NRP1 facilitates endocytosis of transferrin in the African trypanosome

Donna Huber on November 3, 2022

Trypanosoma brucei causes human African trypanosomiasis (HAT) and nagana in cattle. During infection of a vertebrate, endocytosis of host transferrin (Tf) is important for viability of the parasite. The majority of proteins involved in trypanosome endocytosis of Tf are unknown. Here we identify pseudokinase NRP1 (Tb427tmp.160.4770) as a regulator of Tf endocytosis. Genetic knockdown of NRP1 inhibited endocytosis of Tf without blocking uptake of bovine serum albumin. Binding of Tf to the flagellar pocket was not affected by knockdown of NRP1. However the quantity of Tf per endosome dropped significantly, consistent with NRP1 promoting robust capture and/or retention of Tf in vesicles. NRP1 is involved in motility of Tf-laden vesicles since distances between endosomes and the kinetoplast were reduced after knockdown of the gene. In search of possible mediators of NRP1 modulation of Tf endocytosis, the gene was knocked down and the phosphoproteome analyzed. Phosphorylation of protein kinases forkhead, NEK6, and MAPK10 was altered, in addition to EpsinR, synaptobrevin and other vesicle-associated proteins predicted to be involved in endocytosis. These candidate proteins may link NRP1 functionally either to protein kinases or to vesicle-associated proteins.

Gaurav Kumar, Bryanna Thomas, Kojo Mensa-Wilmot. Sci Rep. 2022 Nov 3;12(1):18572. doi: 10.1038/s41598-022-22054-x.

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure

Donna Huber on October 31, 2022

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.

Juan M Bustamante, Angel M Padilla, Brooke White, Lisa D Auckland, Rachel E Busselman, Stephanie Collins, Elizabeth L Malcolm, Briana F Wilson, Ashley B Saunders, Sarah A Hamer, Rick L Tarleton. PLoS Negl Trop Dis. 2022 Oct 31;16(10):e0010688. doi: 10.1371/journal.pntd.0010688.

Assessing seroprevalence and associated risk factors for multiple infectious diseases in Sabah, Malaysia using serological multiplex bead assays

Donna Huber on October 25, 2022

Background: Infectious diseases continue to burden populations in Malaysia, especially among rural communities where resources are limited and access to health care is difficult. Current epidemiological trends of several neglected tropical diseases in these populations are at present absent due to the lack of habitual and efficient surveillance. To date, various studies have explored the utility of serological multiplex beads to monitor numerous diseases simultaneously. We therefore applied this platform to assess population level exposure to six infectious diseases in Sabah, Malaysia. Furthermore, we concurrently investigated demographic and spatial risk factors that may be associated with exposure for each disease.

Methods: This study was conducted in four districts of Northern Sabah in Malaysian Borneo, using an environmentally stratified, population-based cross-sectional serological survey targeted to determine risk factors for malaria. Samples were collected between September to December 2015, from 919 villages totaling 10,100 persons. IgG responses to twelve antigens of six diseases (lymphatic filariasis- Bm33, Bm14, BmR1, Wb123; strongyloides- NIE; toxoplasmosis-SAG2A; yaws- Rp17 and TmpA; trachoma- Pgp3, Ct694; and giardiasis- VSP3, VSP5) were measured using serological multiplex bead assays. Eight demographic risk factors and twelve environmental covariates were included in this study to better understand transmission in this community.

Results: Seroprevalence of LF antigens included Bm33 (10.9%), Bm14+ BmR1 (3.5%), and Wb123 (1.7%). Seroprevalence of Strongyloides antigen NIE was 16.8%, for Toxoplasma antigen SAG2A was 29.9%, and Giardia antigens GVSP3 + GVSP5 was 23.2%. Seroprevalence estimates for yaws Rp17 was 4.91%, for TmpA was 4.81%, and for combined seropositivity to both antigens was 1.2%. Seroprevalence estimates for trachoma Pgp3 + Ct694 were 4.5%. Age was a significant risk factors consistent among all antigens assessed, while other risk factors varied among the different antigens. Spatial heterogeneity of seroprevalence was observed more prominently in lymphatic filariasis and toxoplasmosis.

Conclusions: Multiplex bead assays can be used to assess serological responses to numerous pathogens simultaneously to support infectious disease surveillance in rural communities, especially where prevalences estimates are lacking for neglected tropical diseases. Demographic and spatial data collected alongside serosurveys can prove useful in identifying risk factors associated with exposure and geographic distribution of transmission.

YuYen L Chan, Catriona L Patterson, Jeffrey W Priest, Gillian Stresman, Timothy William, Tock H Chua, Kevin Tetteh, Patrick Lammie, Chris Drakeley, Kimberly M Fornace. Front Public Health. 2022 Oct 25;10:924316. doi: 10.3389/fpubh.2022.924316. eCollection 2022.

The mystery of massive mitochondrial complexes: the apicomplexan respiratory chain

Donna Huber on October 24, 2022

The mitochondrial respiratory chain is an essential pathway in most studied eukaryotes due to its roles in respiration and other pathways that depend on mitochondrial membrane potential. Apicomplexans are unicellular eukaryotes whose members have an impact on global health. The respiratory chain is a drug target for some members of this group, notably the malaria-causing Plasmodium spp. This has motivated studies of the respiratory chain in apicomplexan parasites, primarily Toxoplasma gondii and Plasmodium spp. for which experimental tools are most advanced. Studies of the respiratory complexes in these organisms revealed numerous novel features, including expansion of complex size. The divergence of apicomplexan mitochondria from commonly studied models highlights the diversity of mitochondrial form and function across eukaryotic life.

Andrew E Maclean, Jenni A Hayward, Diego Huet, Giel G van Dooren, Lilach Sheiner. Trends Parasitol. 2022 Oct 24;S1471-4922(22)00219-7. doi: 10.1016/j.pt.2022.09.008. Online ahead of print.

Spindly is a nucleocytosolic O-fucosyltransferase in Dictyostelium and related proteins are widespread in protists and bacteria

Donna Huber on October 17, 2022

O-GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants, and is mediated by a single O-GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii, and both enzymes are now recognized as O-fucosyltransferases (OFTs). Here we investigate the evolution of spy-like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium – a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma, the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation and shared monoglycosylation targets suggest overlapping roles in physiological regulation.

Hanke Wel, Ana Maria Garcia, Elisabet Gas-Pascual, Macy M Willis, Hyun W Kim, Giulia Bandini, Maissa Mareme Gaye, Catherine E Costello, John Samuelson, Christopher M West. Glycobiology. 2022 Oct 17;cwac071. doi: 10.1093/glycob/cwac071.

The Toxoplasma Plant-Like Vacuolar Compartment (PLVAC)

Donna Huber on October 11, 2022

Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. T. gondii shares several characteristics with plants including a non-photosynthetic plastid termed apicoplast and a multi-vesicular organelle that was named the plant-like vacuole (PLV) or vacuolar compartment (VAC). The name plant-like vacuole was selected based on its resemblance in composition and function to plant vacuoles. The name VAC represents its general vacuolar characteristics. We will refer to the organelle as PLVAC in this review. New findings in recent years have revealed that the PLVAC represents the lysosomal compartment of T. gondii which has adapted peculiarities to fulfill specific Toxoplasma needs. In this review, we discuss the composition and functions of the PLVAC highlighting its roles in ion storage and homeostasis, endocytosis, exocytosis, and autophagy.

Andrew J Stasic, Silvia N J Moreno, Vern B Carruthers, Zhicheng Dou. J Eukaryot Microbiol. 2022 Oct 11;e12951. doi: 10.1111/jeu.12951.

Hypothesis-generating proteome perturbation to identify NEU-4438 and acoziborole modes of action in the African Trypanosome

Donna Huber on October 7, 2022

NEU-4438 is a lead for the development of drugs against Trypanosoma brucei, which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, we present a cell perturbome workflow that compares NEU-4438’s molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments.

Amrita Sharma, Michael Cipriano, Lori Ferrins, Stephen L Hajduk, Kojo Mensa-Wilmot. iScience. 2022 Oct 7;25(11):105302. doi: 10.1016/j.isci.2022.105302. eCollection 2022 Nov 18.

Liver-stage fate determination in Plasmodium vivax parasites: Characterization of schizont growth and hypnozoite fating from patient isolates

Donna Huber on September 23, 2022

Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite’s lifecycle.

Amélie Vantaux, Julie Péneau, Caitlin A Cooper, Dennis E Kyle, Benoit Witkowski, Steven P Maher. Front Microbiol. 2022 Sep 23;13:976606. doi: 10.3389/fmicb.2022.976606.

UGA researcher uncovers humans’ natural weapon against malaria

Donna Huber on September 22, 2022

UGA’s Samarchith “Sam” Kurup, assistant professor of cellular biology, has been awarded a five-year National Institutes of Health grant to study the natural immune response to the Plasmodium parasite—which causes malaria—in liver cells. (photo credit: Lauren Corcino)

Samarchith “Sam” Kurup grew up in India, and he’s always been aware of the impact of malaria.

In 2020 there were an estimated 241 million cases of malaria worldwide and an estimated 627,000 deaths, according to a recently released World Health Organization Fact Sheet. Eighty percent of the malaria-related deaths in Africa are children under the age of 5. The relapsing nature of the disease leads to educational and employment loss that has long-term economic impacts for both the individual as well as society.

“Malaria is huge global problem,” said Kurup, a member of UGA’s Center for Tropical and Emerging Global Diseases. “Almost half of the world’s population is currently at risk of contracting malaria.”

Kurup began his training in veterinary medicine in India, where he became hooked on parasitology, then continued his studies at UGA. While pursuing his Ph.D. he worked in Rick Tarleton’s lab, studying a parasitic disease that affects both animals and humans—his first introduction to human immunology. He continued his training in immunology as a postdoctoral researcher in John Harty’s lab at the University of Iowa.

Combining parasitology with immunology prepared him to tackle malaria.

Malaria is one of the most studied parasitic diseases, yet the Plasmodium parasite that causes it keeps evading attempts to treat the infection in humans. This is largely due to its complex life cycle and the ability of the parasite to evolve drug resistance. In addition to life stages that occur in the mosquito, which transmits the Plasmodium parasite to humans, there are two life stages in humans—a short phase of initial development in the liver, followed by an infection of the blood cells that causes clinical disease.

“A lot of research has been focused on the blood stage in humans, as this is when a person is symptomatic,” said Kurup, assistant professor of cellular biology in the Franklin College of Arts and Sciences. “But we now recognize that if we want to stop malaria, we need to stop it in its tracks in the liver before accessing the blood, and for that we need to understand the liver stage.”

Kurup, a member of UGA’s Center for Tropical and Emerging Global Diseases, trained in parasitology and immunology. He hopes that uncovering how the human immune system naturally fights malaria in the liver stage will lead to an effective malaria vaccine. (photo credit: Lauren Corcino)

Kurup has been awarded a five-year National Institutes of Health grant to study the natural immune response to the Plasmodium parasite in liver cells.

“The liver stage is short and can be difficult to study in the laboratory,” he said. “There are also practical and ethical limitations to studying the liver stage of malaria in humans. We are hoping to tease apart the basic principles of immune responses during this stage using the mouse model.”

Kurup’s preliminary studies have shown that a group of signaling proteins called type 1 interferons play a role in the destruction of Plasmodium parasites in the liver. His newly funded project will fill a gap in the malaria knowledge base by using a combination of in vitro study and in vivo experiments to determine the molecular processes that eliminate Plasmodium parasites in liver cells. His group recently developed a transgenic parasite line that can be used to genetically alter its host cell.

“This strain is a game changer for our line of research because we can now determine how our liver cells would naturally eliminate the parasite, and maybe why it sometimes fails,” he said.

In a study recently published in Cell Reports, Kurup and colleagues used the genetically altered parasite to inhibit signaling by type 1 interferons and showed that this protein has a direct role in the control of malaria. Their study also revealed that other natural immune mechanisms may be active in controlling malaria in liver cells. The project funded by the new grant will delve further into these mechanisms.

“In addition to taking us a step closer to the control and possible eradication of malaria, this project will expand our knowledge so that we can better reduce the burdens of this illness in our society,” he said.

Kurup is hopeful that uncovering how the human immune system naturally fights malaria in the liver stage will lead to an effective malaria vaccine.

“I really believe that bringing together our knowledge in parasitology and approaches in immunology is key to uncovering new information on this elusive life stage in malaria,” he said. “There is no better place to do this, considering the intellectual and material resources we have at our disposal at UGA and the CTEGD.”

 

This story was first published at https://research.uga.edu/news/uga-researcher-uncovers-humans-natural-weapon-against-malaria/