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Author: Donna Huber

Trainee Spotlight: Ciro Cordeiro

Donna Huber on August 3, 2018

 

Dr. Ciro D. Cordeiro recently completed his Ph.D. training in Roberto Docampo‘s laboratory.

I was born in Brazil and grew up in a small city in the heart of the country, a place of great natural beauty dominated by savannas and agriculture. I have always been fascinated by nature and curious about all forms of life. My interest in science motivated me to enroll in biology as an undergraduate at the University of Brasilia. As I learned the current challenges of modern biology, I became aware of how parasitic diseases are still a prevalent burden in my home country and abroad. After graduating, I enrolled in a master’s program where I studied the parasite that causes Chagas disease, Trypanosoma cruzi, and its vectors, the Triatominae bugs. Then I decided to study the pathogens’ cells at the molecular level, so I enrolled at The University of Georgia.

Why did you choose UGA?

I wanted to learn about parasitic diseases that affect tropical countries and UGA has one of the most complete and competent group of researchers working on tropical and neglected diseases. Here, I knew I would have many options of interesting labs, many of them with world-renowned researchers. Additionally, I wanted to be exposed to this rich research environment to learn about the work being developed in different model organisms.

What is your research focus/project and why are you interested in the topic?

I am studying phosphate and polyphosphate regulation in Trypanosoma brucei. Recently, I worked on the cell signaling pathway named inositol phosphates and looked at how they regulate phosphate homeostasis. Phosphate is essential for all living cells, but there is little information on how parasites’ and other unicellular organisms regulate it. One important molecule for phosphate storage is the ubiquitous polymer named polyphosphate. I believe that understanding phosphate and polyphosphate regulation in eukaryotic parasites may lead to a better understanding of the parasites’ biology. The study of phosphate regulation may also help us understand cellular biology processes of other organisms.

What are your future professional plans? 

I intend to continue my training in a top-caliber research institution where I can keep studying the biology of parasites and learn about other cellular biology model organisms. I look forward to participating in interdisciplinary collaborations to address the core challenges related to parasitic diseases, while also serving as a mentor for students.

Have you worked with any collaborators outside of UGA during your training?

I was supported by the EMBO and CTEGD’s Training Innovations in Parasitologic Studies Fellowship to visit the lab of Dr. Adolfo Saiardi at the University College London to perform experiments during my Ph.D. training. We had a productive collaboration that resulted in a successful publication. This great experience enabled me to learn many new techniques that are now routinely used in our lab. Since then, Dr. Saiardi’s lab has published new interesting findings on polyphosphate, which are relevant to many of the current projects of our lab. If possible, I would like to visit Dr. Saiardi’s lab again to continue our collaborative projects.

What is your favorite thing about UGA?

My favorite thing about the experience in UGA was the diverse environment we encounter here. I met people from all continents and learned about their home countries, cultures, and their work. This was an exceptional opportunity to be exposed to new ideas and discover research topics unrelated to my own.

Any advice for a student interested in this field?

I think it is important to carefully choose where you want to study during graduate school. It is imperative to know the program you are enrolling in, when it comes to the research they are performing, the facilities available and whether that aligns with your expectations and ambitions.

 

Your finiancial donations to CTEGD support the research efforts of students like Ciro Cordeiro. Give today!

Characterization of Two EF-hand Domain-containing Proteins from Toxoplasma gondii

Donna Huber on July 31, 2018

The universal role of calcium (Ca2+ ) as a second messenger in cells depends on a large number of Ca2+ -binding proteins (CBP), which are able to bind Ca2+ through specific domains. Many CBPs share a type of Ca2+ -binding domain known as the EF-hand. The EF-hand motif has been well studied and consists of a helix-loop-helix structural domain with specific amino acids in the loop region that interact with Ca2+ . In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF-hand motif. The majority of these genes have not been characterized. We report the characterization of two EF-hand motif-containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca2+ measurements showed that Δtg216620 cells did not respond to extracellular Ca2+ as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca2+ influx while TgGT1_280480 may be playing a different role in the rhoptries. This article is protected by copyright. All rights reserved.

Trainee Adds New Tool to the Trypanosome Toolbox

Donna Huber on July 19, 2018

Trypanosoma brucei stained with mCLING
mCLING-staining of membrane (green) and DAPI staining of DNA (magenta) in trypanosomes in various life cycle stages. Flagella appear as tube-like structures along the length of the cell body. Nanotubes can be seen projecting outward from the periphery of the cells.

When Ph.D. trainee Justin Wiedeman started investigating the role of protein kinase TbCK1.2, an enzyme found near the flagellum of Trypanosoma brucei, he quickly ran into a problem common to parasitologists. He needed a better tool for visualizing the membranes of this parasite. Since none of the membrane probes on the market quite did the job, he looked at how he could modify one for his purpose. He found a successful candidate in Synaptic Systems’ mCLING.

What is Trypanosoma brucei?

Trypanosoma brucei is a single cell parasite that causes Human African Trypanosomiasis (HAT), which is also known as African sleeping sickness. HAT occurs in 36 sub-Saharan countries where tsetse flies transmit the parasite to people and livestock. In cattle, the disease is known as nagana. Tsetse fly control efforts have drastically reduced the number of cases. According to the World Health Organization, in 2015, there were around 2,800 cases. However, a person can be infected for months or even years without symptoms. By the time symptoms become evident, the person is in the advanced stages of the disease and their central nervous system is impaired.

New tools are needed to study trypanosomes

There is still much to be learned about the parasite that could lead to better detection and more effective treatment. A major obstacle to the study of this tiny organism is the lack of tools and technology. Kojo Mensa-Wilmot’s research group in the Center for Tropical and Emerging Global Diseases at The University of Georgia has been instrumental in developing techniques and tools to increase the research community’s understanding of T. brucei. Now, Wiedeman has added a new tool to the trypanosome biology toolbox – a general method of outlining trypanosomes in fluorescence microscopy experiments.

“We are the first group to solve this general problem in super-resolution microscopy of T. brucei,” said Wiedeman. “mCLING is a highly versatile tool for studying trypanosome biology – it can be used with live or fixed trypanosomes.”

Fluorescent microscopy has been a leading method of studying T. brucei; however, there are limitations to this technology. Super-resolution microscopy offers great advantages over standard fluorescence microscopy. By employing several techniques to increase resolution, it allows for the observation of objects smaller than what can be seen with visible light.  Yet, it is not without its own limitations, most notably the inability to determine the periphery of cells. Without knowing the outer edges of the parasite, orientation of organelles and other structures within the cell is difficult.

“For Trypanosoma brucei, most of the membrane probes available do not work well in fixed trypanosomes,” said Wiedeman. “Researchers have been forced to use crude methods to outline trypanosomes in fluorescence microscopy.”

These “crude methods” include superimposing a transmitted light image or hand-drawing the outline. However, this workaround only allows for a two-dimensional study of the cell. Therefore, Wiedeman turned to a dye called mCLING that has been developed to track the membranes of neurons using super-resolution microscopy to see if he could adapt the technology to T. brucei membranes.

mCLING allows for the visualization of T. brucei membranes

“mCLING labels the flagellum and plasma membrane vividly, sometimes providing details of cell structure that rivals images obtained with scanning electron microscopy,” said Wiedeman.

Using a combination of standard-resolution and super-resolution fluorescence microscopy, he was able to confirm mCLING labels the plasma and flagellar membranes of T. brucei. Furthermore, using the Zeiss ELYRA S1 super-resolution microscopy in the Biomedical Microscopy Core, mCLING allowed for a 3D reconstruction of the parasite. This is the first time such an image has been reported. Finally, using the new ImageStream X Mark II in the CTEGD Cytometry Shared Resource Laboratory, he discovered mCLING could be used to track endocytosis (the process of importing molecules into the cell) in real time.

Recognizing mCLING’s potential to inform other studies of trypanosome biology, Weideman optimized protocols for using it with immunofluorescence assays and thus making possible what had been impossible with the overlay technique – visualizing the location of organelles in the vertical dimension relative to the cell body.

“It is especially well-suited for studying flagellar membrane biogenesis as well as kinetically tracking uptake of the plasma membrane into vesicles inside trypanosomes,” said Wiedeman. Other laboratories have already implemented these protocols in their own research. Steve Hajduk’s group, also in the Center for Tropical and Emerging Global Diseases, is using mCLING to study nanotubes in T. brucei.

This tool will allow for the study of trypanosomes in finer detail than ever before and the Mensa-Wilmot Research Group anticipates unlocking previously unseen secrets in T. brucei.

 

The full published study is available online: Wiedeman J, Mensa-Wilmot K (2018). A fixable probe for visualizing flagella and plasma membranes of the African trypanosome. PLoS One 13(5):e0197541. https://doi.org/10.1371/journal.pone.0197541