Donna Huber, Author at Center for Tropical and Emerging Global Diseases

Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers

Donna Huber on September 15, 2020

Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewis^x antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewis^x antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM^®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.

Jessica Ramadhin, Vanessa Silva-Moraes, Thomas Norberg, Donald Harn. Antibodies 2020, 9, 48. https://doi.org/10.3390/antib9030048

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii

Donna Huber on September 15, 2020

In animals, the response to chronic hypoxia is mediated by prolyl-hydroxylases (PHDs) that regulate the levels of hypoxia inducible transcription factor a (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non-HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-Phase Kinase Associated Protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, TgPhyA informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

Tongri Liu, Martine I Abboud, Rasheduzzaman Chowdhury, Anthony Tumber, Adam P Hardy, Kerstin Lippl, Christopher T Lohans, Elisabete Pires, James Wickens, Michael A McDonough, Christopher M West, Christopher J Schofield. J Biol Chem. 2020 Sep 15;jbc.RA120.013998. doi: 10.1074/jbc.RA120.013998.

mSphere of Influence: Tweaking Organellar Purification Approaches

Donna Huber on September 9, 2020

Diego Huet studies the organelles involved in the metabolic adaptations of the apicomplexan parasite Toxoplasma gondii. In this mSphere of Influence article, he reflects on how the paper “Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism” by Chen et al. (W. W. Chen, E. Freinkman, T. Wang, K. Birsoy, and D. M. Sabatini, Cell 166:1324–1337.e11, 2016, https://doi.org/10.1016/j.cell.2016.07.040) shaped his research by providing an approach for rapidly and specifically isolating mitochondria to probe the metabolism of these organelles.

Diego Huet . mSphere Sep 2020, 5 (5) e00690-20; DOI: 10.1128/mSphere.00690-20

Probing the B- & C-rings of the antimalarial tetrahydro-β-carboline MMV008138 for steric and conformational constraints

Donna Huber on September 5, 2020

The antimalarial candidate MMV008138 (1a) is of particular interest because its target enzyme (IspD) is absent in human. To achieve higher potency, and to probe for steric demand, a series of analogs of 1a were prepared that featured methyl-substitution of the B- and C-rings, as well as ring-chain transformations. X-ray crystallography, NMR spectroscopy and calculation were used to study the effects of these modifications on the conformation of the C-ring and orientation of the D-ring. Unfortunately, all the B- and C-ring analogs explored lost in vitro antimalarial activity. The possible role of steric effects and conformational changes on target engagement are discussed.

Sha Ding, Maryam Ghavami, Joshua H.Butler, Emilio F. Merino, Carla Slebodnick, Maria B. Cassera, Paul R. Carlier. Bioorg Med Chem Lett. 2020 Sep 5;127520. doi: 10.1016/j.bmcl.2020.127520

p53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo

Donna Huber on September 4, 2020

CRISPR/Cas9 technology has revolutionized rapid and reliable gene editing in cells. Although many cell types have been subjected to CRISPR/Cas9-mediated gene editing, there is no evidence of success in genetic alteration of Ag-experienced memory CD8 T cells. In this study, we show that CRISPR/Cas9-mediated gene editing in memory CD8 T cells precludes their proliferation after Ag re-encounter in vivo. This defect is mediated by the proapoptotic transcription factor p53, a sensor of DNA damage. Temporarily inhibiting p53 function offers a window of opportunity for the memory CD8 T cells to repair the DNA damage, facilitating robust recall responses on Ag re-encounter. We demonstrate this by functionally altering memory CD8 T cells using CRISPR/Cas9-mediated targeted gene disruption under the aegis of p53siRNA in the mouse model. Our approach thus adapts the CRISPR/Cas9 technology for memory CD8 T cells to undertake gene editing in vivo, for the first time, to our knowledge.

Samarchith P. Kurup, Steven J. Moioffer, Lecia L. Pewe and John T. Harty. J Immunol. 2020 Sep 4;ji2000654. doi: 10.4049/jimmunol.2000654.

IP3 receptor-mediated Ca2+ release from acidocalcisomes regulates mitochondrial bioenergetics and prevents autophagy in Trypanosoma cruzi

Donna Huber on September 2, 2020

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIP₃R) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIP₃R is a Ca²⁺ release channel gated by IP₃ when expressed in DT40 cells knockout for all vertebrate IP₃ receptors, and is required for Ca²⁺ uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O₂ consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIP₃R by CRISPR/Cas9 genome editing caused: a) a reduction in O₂ consumption rate and citrate synthase activity; b) decreased mitochondrial Ca²⁺ transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIP₃R overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIP₃R-mediated acidocalcisome Ca²⁺ release on cell bioenergetics in T. cruzi.

Miguel A. Chiurillo, Noelia Lander, Anibal E. Vercesi, Roberto Docampo, 2020. Cell Calcium; 92:102284, https://doi.org/10.1016/j.ceca.2020.102284.

Fine-scale heterogeneity in Schistosoma mansoni force of infection measured through antibody response

Donna Huber on August 31, 2020

Schistosomiasis is among the most common parasitic diseases in the world, with over 142 million people infected in low- and middle-income countries. Measuring population-level transmission is centrally important in guiding schistosomiasis control programs. Traditionally, human Schistosoma mansoni infections have been detected using stool microscopy, which is logistically difficult at program scale and has low sensitivity when people have low infection burdens. We compared serological measures of transmission based on antibody response to S. mansoni soluble egg antigen (SEA) with stool-based measures of infection among 3,663 preschool-age children in an area endemic for S. mansoni in western Kenya. We estimated force of infection among children using the seroconversion rate and examined how it varied geographically and by age. At the community level, serological measures of transmission aligned with stool-based measures of infection (ρ = 0.94), and serological measures provided more resolution for between-community differences at lower levels of infection. Force of infection showed a clear gradient of transmission with distance from Lake Victoria, with 94% of infections and 93% of seropositive children in communities <1.5 km from the lake. Force of infection increased through age 3 y, by which time 65% (95% CI: 53%, 75%) of children were SEA positive in high-transmission communities—2 y before they would be reached by school-based deworming programs. Our results show that serologic surveillance platforms represent an important opportunity to guide and monitor schistosomiasis control programs, and that in high-transmission settings preschool-age children represent a key population missed by school-based deworming programs.

Benjamin F. Arnold, Henry Kanyi, Sammy M. Njenga, Fredrick O. Rawago, Jeffrey W. Priest, W. Evan Secor, Patrick J. Lammie, Kimberly Y. Won, and Maurice R. Odiere. Proc Natl Acad Sci U S A. 2020 Aug 31;202008951. doi: 10.1073/pnas.2008951117.

Noncoding RNAs in Apicomplexan Parasites: An Update

Donna Huber on August 20, 2020

Illustration of Long Noncoding RNA (lncRNA) Functions in Apicomplexan Parasites.

Recent breakthroughs in high-throughput technologies, transcriptomics, and advances in our understanding of gene regulatory networks have enhanced our perspective on the complex interplay between parasite and host. Noncoding RNA molecules have been implicated in critical roles covering a broad range of biological processes in the Apicomplexa. Processes that are affected range from parasite development to host–parasite interactions and include interactions with epigenetic machinery and other regulatory factors. Here we review recent progress involving noncoding RNAs and their functions in the Apicomplexa, with a focus on three parasites: Plasmodium, Toxoplasma, and Cryptosporidium. We discuss the limitations and challenges of current methods applied to apicomplexan noncoding RNA study and discuss future directions in this exciting field.

Yiran Li, Rodrigo P. Baptista, Jessica C. Kissinger. Trends Parasitol. 2020 Aug 19;S1471-4922(20)30189-6. https://doi.org/10.1016/j.pt.2020.07.006

Trainee Spotlight: Alona Botnar

Donna Huber on August 18, 2020

T32 trainee Alona Botnar is entering her fifth year as a Ph.D. candidate in Dr. Dennis Kyle’s laboratory. She is from Doylestown, Pennsylvania and completed her B.S. in Chemistry with a minor in Biochemistry at the University of Georgia in 2015. During her undergraduate career, she also worked at Janssen, a pharmaceutical company of Johnson & Johnson as a Biologics R&D co-op.

As a graduate student, she was able to spend three semesters teaching Anatomy and Physiology labs here at UGA, and in 2019, she was awarded the Outstanding Teaching Assistant Award sponsored by the Office of the Vice President for Instruction. She also received a graduate school travel award to attend the 2018 annual meeting of the American Society of Tropical Medicine and Hygiene in New Orleans, Louisiana, and an Office for Vice President and Research travel award to attend the 2020 Molecular Approaches to Malaria meeting held in Lorne, Victoria, Australia.

Why did you choose UGA?

Having been at UGA for my undergraduate degree, I was already in love with UGA and Athens and all that they have to offer. I was attracted to the Integrated Life Sciences program because it gives incoming graduate students the freedom to explore a wide range of research topics among 14 different departments before choosing the lab they would ultimately like to join. Furthermore, I found the interdisciplinary approach of the program appealing. I love the Center for Tropical and Emerging Global Diseases because of all the resources available to us as scientists. Not only do we have state of the art microscopy and flow cytometry cores, but we also have very collaborative labs that are happy to share equipment, supplies, and expertise.

What is your research focus?

My research is focused on malaria and addressing significant problems at the stages of development at which the malaria parasite enters a drug-induced dormant period and evades the antimalarial. The mechanism by which the parasite enters drug-induced dormancy and later recrudesces to continue development is currently unknown.

Half the world’s population is at risk of malaria with about half a million people dying each year from it. A majority of these deaths are in children under the age of 5, located mainly in sub-Saharan Africa. While there are 5 species that can infect humans, Plasmodium falciparum is the most lethal and is responsible for a majority of the deaths. Our current arsenal of malaria drugs is failing at an alarming rate as drug-resistant strains of the parasite continue to emerge.

Thus I chose this research project because it is vital that we respond to the challenge of antimalarial drug resistance by not only developing novel drugs but also by understanding the mechanisms the parasite is using to evade the drugs.

What are your future career goals?

I plan on continuing my work in the field of infectious diseases. I am leaning towards industry research but I’m keeping an open mind. Lately, I have been interested in alternative careers available to life science Ph.D.’s such as consulting and being a medical science liaison.

What is your favorite thing about UGA?

UGA football. There’s nothing quite like a fall Saturday in Athens between the hedges. GO DAWGS!

Do you have any advice for a student interested in this field?

It’s never too early or too late to get into the field. Don’t be afraid to send those emails and get involved in research. And always ask questions.

Support trainees like Alona by giving today to the Center for Tropical & Emerging Global Diseases.

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Age influences thermal tolerance in Asian malaria mosquito

Donna Huber on August 11, 2020

Environmental portrait of Courtney Murdock in her laboratory at UGA’s School of Veterinary Medicine (Dorothy Kozlowski)

Malaria disease transmission models are important tools for controlling and eliminating disease spread. However, a model is only as good as the assumptions about the various variables. Dr. Courtney Murdock, a member of the UGA’s Center for Tropical and Emerging Global Diseases and professor at Cornell University, has been studying how various biological and environmental factors influence mosquito survival. In a study recently published in the Proceedings of the Royal Society B led by graduate student Kerri Miazgowicz, Murdock and her colleagues examined several life traits, such as biting, feeding, and egg production, over the course of the life span of the mosquito Anopheles stephensi in hopes of providing better data for the models.

“Due to a lack of high-quality entomological data in general, researchers are often forced to input data from multiple disease systems to inform models in a given system or use approximations of key model components,” said Murdock.

An. stephensi is the primary mosquito species that transmits malaria in India. While most of the focus on malaria is most often associated with sub-Saharan Africa, it is widespread in the Indian subcontinent and throughout southeast Asia. Several Plasmodium species cause malaria, but P. falciparum is the deadliest of them. It has also shown drug resistance to current treatments. Control of the mosquito population is an important component in malaria control and elimination programs. Researchers need to be able to more accurately predict where mosquito populations will occur as climate changes and current territories become unsuitable living and breeding grounds. Program managers need to be prepared to incorporate more northern regions in their control efforts.

trainee field work — Kerri Miazgowicz, a graduate student in the Murdock Laboratory at the University of Georgia, led the study on the effects of age on thermal tolerances.

Current models rely on data that are only snapshots in time and often from multiple mosquito species, particularly the African mosquitoes, which are vectors for different malaria species. Miazgowicz, Murdock, and colleagues wanted to determine if the data from a single species of mosquito and parasite, over the course of its entire lifespan, significantly influenced current models in determining disease transmission in hopes of creating more accurate models.

The single most important factor driving current models is temperature. Mosquitoes are cold-blooded animals and therefore rely on their environment to regulate their body temperature. However, temperature is not the only factor influencing life traits. Currently, data are only available as snapshots in time. These incomplete data do not take into account for changes in mosquito behavior and life traits that occur over the course of the mosquito’s life. Murdock and her colleagues have recorded changes in biological function as the mosquito ages. Just as people slow down biologically as they age – metabolism slows, reproduction ability declines, etc. – the same is true for mosquitos. They also found that various traits peak at different times depending on temperature. Importantly they found that temperature and age significantly affected the number of females taking a blood meal (this is the means in which malaria parasites are transmitted to humans) on a given day, average daily egg production, and ultimately survival.

The findings in this study indicated that the addition of An. stephensi data yielded qualitatively different temperature-transmission suitability relationships compared to models that included multiple malaria vectors. With An. stephensi data, the model predicted a broader geographical range of temperature suitability.

“Accounting for these age and species effects in models of transmission potential alters how much of South Asia is predicted to be suitable for malaria relative to models that do not account for these factors,” said Murdock.

These findings can lead to improved malaria transmission models. However, more study outside the laboratory is needed to truly understand the impact mosquito age has on life traits and thermal tolerance.

“This study highlights a critical need for more research in natural settings characterizing the effects of age on mosquito biology to improve predictions of current and future risk,” concluded Murdock.

Author: Donna Huber