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Author: Donna Huber

Is reliance on an inaccurate genome sequence sabotaging your experiments?

Donna Huber on September 12, 2019

Advances in genomics have made whole genome studies increasingly feasible across the life sciences. However, new technologies and algorithmic advances do not guarantee flawless genomic sequences or annotation. Bias, errors, and artifacts can enter at any stage of the process from library preparation to annotation. When planning an experiment that utilizes a genome sequence as the basis for the design, there are a few basic checks that, if performed, may better inform the experimental design and ideally help avoid a failed experiment or inconclusive result.

Baptista RP, Kissinger JC (2019) Is reliance on an inaccurate genome sequence sabotaging your experiments? PLoS Pathog 15(9): e1007901. https://doi.org/10.1371/journal.ppat.1007901

Visiting Scholar Fellow: Fernando Sanchez-Valdéz

Donna Huber on August 28, 2019

Fellow: Fernando Sanchez ValdezDr. Fernando Sanchez-Valdéz, from Salta, Argentina, completed a Ph.D. in Molecular Biology at the Faculty of Pharmacy and Biochemistry at the University of Buenos Aires, Argentina in 2014. After his Ph.D., he completed a postdoctoral fellowship in Dr. Rick Tarleton´s laboratory at University of Georgia. In 2018, he obtained a Research Scientist position in the career pathway of the National Research Council in Argentina (CONICET). Earlier this year, he was awarded a fellowship from the CTEGD-Janssen Visiting Scholars Program, which enabled him to return to the Tarleton Research Group.

What is your primary research focus? Why are you interested in this subject?

The main focus of my research has been to uncover the mechanism of drug resistance in the Chagas disease agent, Trypanosoma cruzi. The main question we are trying to answer is why the treatment with highly effective drugs like Benznidazole (the current available treatment for Chagas disease) often fails to cure Chagas disease. By combining ex vivo luminescence assays and tissue-clearing techniques we were able to report, for the first time, the presence of dormant non-replicating amastigotes forms in the chronic phase of the disease. Dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after treatment interruption. T. cruzi‘s capacity to become dormant makes them transiently drug-resistant, suggesting that this phenomenon accounts for the failure of the otherwise highly active compounds such Benznidazole (Sanchez-Valdéz, et al eLife 2018).

Why did you choose UGA?

I returned to Athens in February 2019 to continue working on the findings we made during my postdoctoral training in the Tarleton Laboratory. I initially decided to come UGA based on a colleague’s recommendations and the fact that Tarleton´s lab is one of the reference centers for Chagas disease research. It’s a really motivating environment to do science since the scientific and technical level here is really high as well as diverse including areas as immunology, drug discovery, genetic manipulation, genomics, diagnostics, etc. Also the amount of resources available is impressive not only from the lab but also from the Biomedical Microscopy Core, Cytometry Shared Resource Laboratory and the animal facility at UGA.

What has been your research project while at UGA?

Currently, we are expanding our knowledge about T. cruzi dormancy and trying to interfere T. cruzi dormancy using new compounds or the conventional drugs but in a different treatment schedule. One of the approaches we are testing now involves the evaluation of drug doses and treatment schemes able to kill dormant parasites. For this purpose, we are optimizing a robust platform to detect low levels of parasites in whole clarified mice organs using light-sheet fluorescent microscopy. This technique will allow us the specific detection of low levels of persistent dormant parasites.

How has the CTEGD-Janssen Visiting Scholar Fellowship and your time at UGA impacted your research and professional goals?

I am so glad about the opportunity to continue working on T. cruzi dormancy with such experienced and renowned scientists and particularly using state-of-the-art microscopy techniques currently unavailable in South America. This experience will definitely have a positive impact on my career development and probably in the Chagas disease research field.

Accessing Cryptosporidium Omic and Isolate Data via CryptoDB.org

Donna Huber on August 27, 2019

Cryptosporidium has historically been a difficult organism to work with, and molecular genomic data for this important pathogen have typically lagged behind other prominent protist pathogens. CryptoDB ( http://cryptodb.org/ ) was launched in 2004 following the appearance of draft genome sequences for both C. parvum and C. hominis. CryptoDB merged with the EuPathDB Bioinformatics Resource Center family of databases ( https://eupathdb.org ) and has been maintained and updated regularly since its establishment. These resources are freely available, are web-based, and permit users to analyze their own sequence data in the context of reference genome sequences in our user workspaces. Advances in technology have greatly facilitated Cryptosporidium research in the last several years greatly enhancing and extending the data and types of data available for this genus. Currently, 13 genome sequences are available for 9 species of Cryptosporidium as well as the distantly related Gregarina niphandrodes and two free-living alveolate outgroups of the Apicomplexa, Chromera velia and Vitrella brassicaformis. Recent years have seen several new genome sequences for both existing and new Cryptosporidium species as well as transcriptomics, proteomics, SNP, and isolate population surveys. This chapter introduces the extensive data mining and visualization capabilities of the EuPathDB software platform and introduces the data types and tools that are currently available for Cryptosporidium. Key features are demonstrated with Cryptosporidium-relevant examples and explanations.

Warrenfeltz S, Kissinger JC, EuPathDB Team. Methods Mol Biol. 2020;2052:139-192. doi: 10.1007/978-1-4939-9748-0_10.

Protozoan persister-like cells and drug treatment failure

Donna Huber on August 23, 2019

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmaniaspp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as ‘persister-like cells’. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.

Michael P. Barrett, Dennis E. Kyle, L. David Sibley, Joshua B. Radke & Rick L. Tarleton. Nat Rev Microbiol. 2019 Aug 23. doi: 10.1038/s41579-019-0238-x.

Enteropathogen antibody dynamics and force of infection among children in low-resource settings

Donna Huber on August 19, 2019

Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG responses to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) in cohorts from Haiti, Kenya, and Tanzania. We studied antibody dynamics and force of infection across pathogens and cohorts. Enteropathogens shared common seroepidemiologic features that enabled between-pathogen comparisons of transmission. Overall, exposure was intense: for most pathogens the window of primary infection was <3 years old; for highest transmission pathogens primary infection occurred within the first year. Longitudinal profiles demonstrated significant IgG boosting and waning above seropositivity cutoffs, underscoring the value of longitudinal designs to estimate force of infection. Seroprevalence and force of infection were rank-preserving across pathogens, illustrating the measures provide similar information about transmission heterogeneity. Our findings suggest antibody response can be used to measure population-level transmission of diverse enteropathogens in serologic surveillance.

Benjamin F Arnold, Diana L Martin, Jane Juma, Harran Mkocha, John B Ochieng, Gretchen M Cooley, Richard Omore, E Brook Goodhew, Jamae F Morris, Veronica Costantini, Jan Vinjé, Patrick J Lammie, Jeffrey W Priest. Elife. 2019 Aug 19;8. pii: e45594. doi: 10.7554/eLife.45594.

Five-Year Impact of Different Multi-Year Mass Drug Administration Strategies on Childhood Schistosoma mansoni-Associated Morbidity: A Combined Analysis from the Schistosomiasis Consortium for Operational Research and Evaluation Cohort Studies in the Lake Victoria Regions of Kenya and Tanzania

Donna Huber on August 12, 2019

The WHO recommends mass treatment with praziquantel as the primary approach for Schistosoma mansoni-related morbidity control in endemic populations. The Schistosomiasis Consortium for Operational Research and Evaluation implemented multi-country, cluster-randomized trials to compare effectiveness of community-wide and school-based treatment (SBT) regimens on prevalence and intensity of schistosomiasis. To assess the impact of two different treatment schedules on S. mansoni-associated morbidity in children, cohort studies were nested within the randomized trials conducted in villages in Kenya and Tanzania having baseline prevalence ≥ 25%. Children aged 7-8 years were enrolled at baseline and followed to ages 11-12 years. Infection intensity and odds of infection were reduced both in villages receiving four years of annual community-wide treatment (CWT) and those who received biennial SBT over 4 years. These regimens were also associated with reduced odds of undernutrition and reduced odds of portal vein dilation at follow-up. However, neither hemoglobin levels nor the prevalence of the rare abnormal pattern C liver scores on ultrasound improved. For the combined cohorts, growth stunting worsened in the areas receiving biennial SBT, and maximal oxygen uptake as estimated by fitness testing scores declined under both regimens. After adjusting for imbalance in starting prevalence between study arms, children in villages receiving annual CWT had significantly greater decreases in infection prevalence and intensity than those villages receiving biennial SBT. Although health-related quality-of-life scores improved in both study arms, children in the CWT villages gained significantly more. We conclude that programs using annual CWT are likely to achieve better overall S. mansoni morbidity control than those implementing only biennial SBT.

Ye Shen, Ryan E. Wiegand, Annette Olsen, Charles H. King, Nupur Kittur, Sue Binder, Feng Zhang, Christopher C. Whalen, William Evan Secor, Susan P. Montgomery, Pauline N. M. Mwinzi, Pascal Magnussen, Safari Kinung’hi, Carl H. Campbell Jr. and Daniel G. Colley. Am J Trop Med Hyg. 2019 Aug 12. doi: 10.4269/ajtmh.19-0273.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages

Donna Huber on August 12, 2019

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

Chua ACY, Ong JJY, Malleret B, Suwanarusk R, Kosaisavee V, Zeeman AM, Cooper CA, Tan KSW, Zhang R, Tan BH, Abas SN, Yip A, Elliot A, Joyner CJ, Cho JS, Breyer K, Baran S, Lange A, Maher SP, Nosten F, Bodenreider C, Yeung BKS, Mazier D, Galinski MR, Dereuddre-Bosquet N, Le Grand R, Kocken CHM, Rénia L, Kyle DE, Diagana TT, Snounou G, Russell B, Bifani P. Nat Commun. 2019 Aug 12;10(1):3635. doi: 10.1038/s41467-019-11332-4.

Isolation and characterization of antiplasmodial constituents from the marine sponge Coscinoderma sp.

Donna Huber on August 6, 2019

Six known compounds, namely two halisulfates 1 and 2 and four epidioxy sterols 3–6, were isolated from the marine sponge Coscinoderma sp. The structures of these compounds were confirmed by nuclear magnetic resonance (1H and 13C NMR) spectroscopy, and their antiplasmodial activities were determined against the chloroquine-resistant Dd2 strain of Plasmodium falciparum. The epidioxy steroids 3–6 all showed moderate to weak antiplasmodial activity, with IC50 values of 2.7 μM for (24S)-5α,8α-epidioxy-24-methylcholesta-6-en-3β-ol (3), 11.6 μM for 5α,8α-epidioxycholesta-6,24(28)-dien-3β-o1 (4), 2.33 μM for 5α,8α-epidioxy-24-methylcholesta-6,9(11)-24(28)-trien-3β-ol (5), and between 12 and 24 μM for 5α,8α-epidioxycholesta-6-en-3β-ol (6). In contrast, halisulfate 2 (1) was inactive, and halisulfate 1 (2) had an of IC50 value of about 24 μM.

Jeong H, Latif A, Kong CS, Seo Y, Lee YJ, Dalal SR, Cassera MB, Kingston DGI. Z Naturforsch C. 2019 Aug 6. pii: /j/znc.ahead-of-print/znc-2019-0039/znc-2019-0039.xml. doi: 10.1515/znc-2019-0039.

Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication

Donna Huber on July 26, 2019

By binding to the adaptor protein SKP1 and serving as substrate receptors for the Skp1, Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasmagenome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.

Carlos Gustavo Baptista, Agnieszka Lis, Bowen Deng, Elisabet Gas-Pascual, Ashley Dittmar, Wade Sigurdson, Christopher M. West, Ira J. Blader. PLoS Pathog. 2019 Jul 26;15(7):e1007946. doi: 10.1371/journal.ppat.1007946.