University of Georgia
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors

Author: Donna Huber

Inorganic Polyphosphate Interacts with Nucleolar and Glycosomal Proteins in Trypanosomatids

Donna Huber on September 19, 2018

Summary

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high‐energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over‐representation of nucleolar and glycosomal proteins. Using the polyphosphate‐binding domain (PPBD) of Escherichia coliexopolyphosphatase (PPX), we localized long‐chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre‐incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration‐dependent and chain length‐dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Raquel S. Negreiros, Noelia Lander, Guozhong Huang, Ciro D. Cordeiro, Stephanie A. Smith, James H. Morrissey, Roberto Docampo. 2018. Molecular Microbiology; 110(6):973-994. https://doi.org/10.1111/mmi.14131

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi

Donna Huber on September 19, 2018

Abstract

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation–dephosphorylation cycle for the E1α subunit of PDH from non-vertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and Trypanosoma brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP’s role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP:ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting culture host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity and energy metabolism in T. cruzi.  Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.

Noelia Lander, Miguel A. Chiurillo, Mayara S. Bertolini, Melissa Storey, Anibal E. Vercesi and Roberto Docampo. 2018. Journal of Biological Chemistry; 293(45):17402-17417. doi: 10.1074/jbc.RA118.004498

Trainee Spotlight: Josh Butler

Donna Huber on September 18, 2018

Josh Butler

New T32 trainee Josh Butler is a third year Ph.D. student in Belen Cassera‘s laboratory. He is from Front Royal, Virginia and completed his B.S. in chemistry at James Madison University in Harrisonburg, Virginia.

Butler decided to pursue his graduate degree at the University of Georgia because of the Integrate Life Sciences program which offers the opportunity to explore a range of research topics. The same interdisciplinary aspect is what he found appealing about the Center for Tropical and Emerging Global Diseases and ultimately why he joined a lab within this department.

“There is no shortage of resources here, ranging from state of the art instrumentation and core facilities to people that are willing to mentor and train successful scientists,” said Butler. “Coming from a smaller institution, I had never really seen anything to this scale and I knew it was something I wanted to experience and become a part of.”

Research Focus

Broadly, Butler’s research is focused on antimalarial drug discovery. More specifically, he is using antimalarial natural products as tools to discover novel drug targets in the malaria parasite Plasmodium falciparum.

Nearly 220 million people have malaria, and it kills nearly half a million people each year. Plasmodium falciparum causes the most severe forms of malaria, such as cerebral malaria, which can lead to brain damage, coma, and death, and placental malaria, which can be life-threatening to both mother and fetus.

“I chose this research because not only does it contribute positively to the global campaign of malaria eradication, but from a training standpoint it would also provide a solid foundation for a career further researching and developing antimicrobial therapies in general.”

Capstone Experience

Each T32 trainee is provided with the opportunity to pursue a capstone experience. Butler hopes to do an internship with a pharmaceutical industry research group that is actively performing anti-parasitic research to experience how the type research he does as a graduate student can translate outside the realm of academia.

“Private-public collaboration in malaria research has really driven drug discovery research in a positive direction and  I would like the opportunity to experience that first hand and develop acumen to engage in that type of research in the next stage of my career.”

Future Career Goals

“I would like to continue working in a field of scientific research which can positively impact people’s lives, whether it be through a biomedical or biotechnical avenue.”

Advice for Aspiring Scientists

“Don’t be afraid to fail or be wrong. Learn from it and use it to keep pushing forward. Try to find positives in the negatives.”

 

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

[button size=’large’ style=” text=’Give Online’ icon=” icon_color=’#b80d32′ link=’https://ctegd.uga.edu/give/’ target=’_self’ color=” hover_color=” border_color=” hover_border_color=” background_color=” hover_background_color=” font_style=” font_weight=” text_align=’center’ margin=”]

The Mitochondrial Ca2+ Uniporter Complex (MCUC) of Trypanosoma brucei Is a Hetero-oligomer That Contains Novel Subunits Essential for Ca2+ Uptake

Donna Huber on September 18, 2018

ABSTRACT

The mitochondrial calcium uniporter complex (MCUC) is a highly selective channel that conducts calcium ions across the organelle inner membrane. We previously characterized Trypanosoma brucei’s MCU (TbMCU) as an essential component of the MCUC required for parasite viability and infectivity. In this study, we characterize its paralog T. brucei MCUb (TbMCUb) and report the identification of two novel components of the complex that we named TbMCUc and TbMCUd. These new MCUC proteins are unique and conserved only in trypanosomatids. In situ tagging and immunofluorescence microscopy revealed that they colocalize with TbMCU and TbMCUb to the mitochondria of T. brucei. Blue Native PAGE and immunodetection analyses indicated that the MCUC proteins exist in a large protein complex with a molecular weight of approximately 380 kDa. RNA interference (RNAi) or overexpression of the TbMCUc and TbMCUd genes significantly reduced or enhanced mitochondrial Ca2+uptake in T. brucei, respectively, without affecting the mitochondrial membrane potential, indicating that they are essential components of the MCUC of this parasite. The specific interactions of TbMCU with TbMCUb, TbMCUc, or TbMCUd were confirmed by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Furthermore, combining mutagenesis analysis with MYTH assays revealed that transmembrane helices (TMHs) were determinant of the interactions between TbMCUC subunits. In summary, our study has identified two novel essential components of the MCUC of T. brucei and defined their direct physical interactions with the other subunits that result in a hetero-oligomeric MCUC.

Guozhong Huang, Roberto Docampo. 2018. Molecular Biology and Physiology. DOI: 10.1128/mBio.01700-18

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Donna Huber on September 18, 2018

ABSTRACT

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

 

Kudyba, H. M., Cobb, D. W., Florentin, A., Krakowiak, M., Muralidharan, V. 2018. J. Vis. Exp. (139), e57747, doi:10.3791/57747

 

Steve Hajduk to give keynote address at Molecular Parasitology Meeting

Donna Huber on September 11, 2018

Hajduk Stephen

Dr. Stephen Hajduk is an Emeritus Professor at the University of Georgia, USA. He has had a long and distinguished career making significant contributions to our understanding of the underlying mechanisms and function of gene expression, RNA editing, human innate immunity to trypanosome infection and the role of membrane nanotubes and extracellular vesicles (EVs) in communication between trypanosomes and with host cells.

He will give the keynote lecture entitled “The Hidden Life of African Trypanosomes” during the 29th Annual Molecular Parasitology Meeting which is currently being held at the Marine Biological Laboratory at Woods Hole, MA. The address will be on Wednesday, September 12 at 5:30 pm in the Lillie Auditorium.

CTEGD opens lab doors to undergraduates at area schools

Donna Huber on September 10, 2018

Sharon Keller and Makayla Yang

by Donna Huber

When UGA alumna Dr. Sharon Keller accepted the position of Assistant Professor at Georgia Gwinnett College (GGC) she wasn’t ready to say goodbye to the research project she started in graduate school. Since GGC is not a research-intensive institution, Keller approached her Ph.D. mentor Dr. Silvia Moreno, distinguished research professor in the department of cellular biology and member of the Center for Tropical and Emerging Global Diseases (CTEGD) at the University of Georgia, about continuing the project in her free time. It was during this conversation, that they came up with a way of not only keeping Keller in the lab but helping to solve a problem that many undergraduates in the biological sciences face – gaining independent research experience in an NIH-funded laboratory.

“The opportunity to be involved in a collaborative research project was very exciting and an experience that I knew would be attractive to GGC students who might be interested in this type of research,” said Keller.

In 2014, with matching support from Franklin College’s Office of the Dean, the Center for Tropical and Emerging Global Diseases sought to increase diversity in graduate education by offering independent research opportunities to faculty from local non-research intensive institutions. Originally, it was meant to be a 1-month summer sabbatical for these faculty. However, during her conversation with Keller, Moreno saw an opportunity to expand the program.

“Students get very excited when they experience independent research,” said Moreno. “But at some of the smaller schools in the area, they don’t have the opportunity to experience this level of research.”

Many undergraduate teaching labs consist of well-prepared protocols and information on the expected result. The experiments they conduct are supposed to work. That’s not how it happens in real life. In a research lab, a student develops a different skill set from those they learn in a teaching lab. They use critical thinking, problem-solving, and result analysis skills to tackle real-world problems.

“As an instructor, I see the disconnect students can have between a learned concept and the application of that concept,” said Keller.

Keller agreed it was a great idea to include her undergraduates and began the process of selecting students shortly after their initial conversation. Makayla Yang, a student lab assistant in the biology department at GGC, was one of two students selected to accompany Keller 2 – 4 times a week to the laboratory of Dr. Roberto Docampo, the Barbara and Sanford Orkin –GRA Eminent Scholar in Tropical and Emerging Global Diseases.

“Working with trypanosomes at UGA was one of the greatest learning opportunities I have experienced,” said Yang. “It made me see the bigger picture of my work that it was not just an undergraduate research opportunity, but it was a contribution to science and what could be a future health solution.”

It is the hope of the program that by giving faculty and undergraduates from area schools real-life research experience more students will apply to graduate school and have the same competitive edge as students from research-intensive institutions.

For Yang, the experience has her thinking more about pursuing research and a Ph.D.  “I was able to discover my true passion for lab bench work,” concluded Yang.

 

If you are a faculty member at a Georgia non-research intensive institution and are interested in participating in this program, please contact Silvia Moreno (moreno@uga.edu).

 

Your financial gift will help us expand this program, giving even more area undergraduates hands-on research experience.

[button size=’large’ style=” text=’Give Today’ icon=” icon_color=’#b80d32′ link=’https://ctegd.uga.edu/give/’ target=’_self’ color=” hover_color=” border_color=” hover_border_color=” background_color=” hover_background_color=” font_style=” font_weight=” text_align=’center’ margin=”]

Young Adults in Endemic Areas: An Untreated Group in Need of School-Based Preventive Chemotherapy for Schistosomiasis Control and Elimination

Donna Huber on September 5, 2018

Abstract

Parasitologic surveys of young adults in college and university settings are not commonly done, even in areas known to be endemic for schistosomiasis and soil-transmitted helminths. We have done a survey of 291 students and staff at the Kisumu National Polytechnic in Kisumu, Kenya, using the stool microscopy Kato-Katz (KK) method and the urine point-of-care circulating cathodic antigen (POC-CCA) test. Based on three stools/two KK slides each, in the 208 participants for whom three consecutive stools were obtained, Schistosoma mansoni prevalence was 17.8%. When all 291 individuals were analyzed based on the first stool, as done by the national neglected tropical disease (NTD) program, and one urine POC-CCA assay (n = 276), the prevalence was 13.7% by KK and 23.2% by POC-CCA. Based on three stools, 2.5% of 208 participants had heavy S. mansoni infections (≥400 eggs/gram feces), with heavy S. mansoni infections making up 13.5% of the S. mansoni cases. The prevalence of the soil-transmitted helminths (STH: Ascaris lumbricoides, Trichuris trichiura and hookworm) by three stools was 1.4%, 3.1%, and 4.1%, respectively, and by the first stool was 1.4%, 2.4% and 1.4%, respectively. This prevalence and intensity of infection with S. mansoni in a college setting warrants mass drug administration with praziquantel. This population of young adults is ‘in school’ and is both approachable and worthy of inclusion in national schistosomiasis control and elimination programs.

Harrison K. Korir, Diana K. Riner, Emmy Kavere, Amos Omondi, Jasmine Landry, Nupur Kittur, Eric M. Ndombi, Bartholomew N. Ondigo, W. Evan Secor, Diana M. S. Karanja and Daniel G. Colley. 2018. Trop. Med. Infect. Dis.; 3(3):100. https://doi.org/10.3390/tropicalmed3030100

Antiplasmodial flavanones and a stilbene from Carpha glomerata

Donna Huber on September 4, 2018

graphical abstract

Abstract

Bioassay-guided fractionation of an extract of Carpha glomerata (Cyperaceae) led to the isolation of seven compounds. Compounds 1 (carphorin A), 3 (carphorin C), 4(carphorin D), and 5 (carphabene) are new compounds, and compound 2 (8-(3″-hydroxyisoamyl)-naringenin) was isolated for the first time as a natural product. All structures were elucidated based on analyses of their HR-ESIMS and 1D and 2D NMR data. Compounds 1, 2, and 6, which have prenyl or hydroxyprenyl side chains, exhibited antiplasmodial activities with IC50 values of 5.2 ± 0.6, 3.4 ± 0.4, and 6.7 ± 0.8 µM against the drug-resistant Dd2 strain of Plasmodium falciparum. In addition the prenylated stilbene 5 also showed good activity, with IC50 5.8 ± 0.7 µM.

Namki Cho, Ana Lisa Valenciano, Yongle Du, Jason Clement, Maria B. Cassera, Michael Goetz, David G. I. Kingston. 2018. Bioorganic & Medicinal Chemistry Letters; 28(20):3368-3371. https://doi.org/10.1016/j.bmcl.2018.09.003