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Tag: Vasant Muralidharan

Plasmodium falciparum cGMP-dependent protein kinase interacts with a subunit of the parasite proteasome

Donna Huber on October 15, 2018

ABSTRACT

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

K. Govindasamy, R. Khan, M. Snyder, H. J. Lou, P. Du, H. M. Kudyba, V. Muralidharan, B. E. Turk, P. Bhanot. 2018. Infection and Immunity. https://doi.org/10.1128/IAI.00523-18

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

 

Trainee Spotlight: Anat Florentin

Donna Huber on May 30, 2018

trainee Anat Florentin

Anat Florentin, a post-doctoral associate in Vasant Muralidharan‘s laboratory, is originally from Israel. She received her BSc degree from Tel-Aviv University and MSc from the Weizmann Institute of Science. She obtained her Ph.D. also from the Weizmann Institute where she studied programmed cell death mechanisms using the fruit fly as a model organism. Dr. Florentin moved to the United States 4 years ago when she joined the Muralidharan Research Group. During her time at UGA, she has received a number of awards in recognition of her research:

  • American Heart Association Postdoctoral Fellowship (2018-2020)
  • Postdoctoral Research Award, UGA Office of Research (2018)
  • Foreign travel award, UGA Office of Research (2018)
  • Best Poster Presentation award at the UGA GSPS Research Day (2016)
  • Best Postdoctoral Poster award at the 2015 UGA Conference on Drug Discovery (2015)
Why did you choose UGA?

Since my background is in basic cell biology and genetics, I knew very little of the biology of parasites but was determined to study malaria. While I was looking into different places in Europe and the US, I met with another Israeli, Lilach Sheiner who, at the time, was doing her postdoctoral training with Dr. Boris Striepen at UGA. She told me very good things of CTEGD and of a great newly recruited faculty who studies malaria. I came for a visit, and was impressed by the engaging scientific community, the super friendly atmosphere and the variety of different parasites and approaches to study tropical neglected diseases. I am so glad I made this decision!

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

The goal of my research is to understand the unique cell biology of malaria parasites and to identify potential drug targets. In order to do that I develop and apply genetic and molecular tools that are used to manipulate the genome of the parasite. During my years in the lab I was involved in several projects; One of them studies mechanisms by which the parasite transports proteins into the host red blood cell. Another interesting project focuses on a conserved complex from bacterial origin that resides within a unique parasite organelle called the apicoplast. Lastly, I am looking for genes that might be involved in programmed cell death processes in the parasites.

What are your future professional plans?

I want to establish my own research lab, conduct independent research and train the next generation of future scientists.

Have you done any field work or is there a collaborator/field site that you would like to visit in order to enhance your training?

Although we use field samples in our studies, I have never been to any field site, and would absolutely love to visit one. I am positive it will enhance my training and will add another layer to the work that I am doing. I am sure that visiting any field site in a malaria endemic area, such as Africa or Southeast Asia would be an enriching experience that would underline the significance of our work.

What is your favorite thing about UGA and Athens, GA?

Many things… At CTEGD I cherish the collaborative atmosphere, the variety of parasitism-related topics, the strong basic science that goes together with field studies and translational research. I am highly appreciative of the fact that I have access to a huge amount of knowledge by working side by side with top experts in these fields.

Athens is also great. Moving here in 2014 with a family of 2 young kids couldn’t go smoother! We found here a great community of friends, great public schools, and amazing nature. I love the mountains, the trees and the wildlife around us!

Any advice for students interested in this field?

There is still so much to do and learn in the field of parasitology and every discovery that you make may impact the life of the millions that suffer from these diseases. Don’t hesitate if you don’t know much about parasites. No matter what your background is, you can use the tools and knowledge that you acquired and apply them to this challenging but rewarding research!

 

Your financial gift to the CTEGD Fund helps provide field research opportunities to trainees like Anat Florentin through The CTEGD Training Innovations in Parasitological Studies Fellowship.

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Anat Florentin receives 2018 Postdoctoral Research Award

Donna Huber on April 23, 2018

Anat Florentin, a postdoctoral researcher in Vasant Muralidharan‘s laboratory at the Center for Tropical and Emerging Global Diseases, studies molecular mechanisms that drive life stages of Plasmodium falciparum, the deadliest of parasite species that infect humans with malaria. During her exceptionally productive years at UGA, she has advanced two related areas of research to learn more about the functions of P. falciparum gene and metabolic pathways. First, she established a highly efficient, markerless system to create mutants more rapidly using the powerful CRISPR-Cas9-based genetic editing tool. Her data from this project was published in the high-impact journal mSphere. Second, she used the CRISPR-Cas9 tool to understand P. falciparum’s unique plastid known as the apicoplast, which harbors essential metabolic pathways for the parasite’s growth and whose biological processes could be ideal parasite-specific drug targets. This work has been recognized by multiple invitations to present her work and a first author publication in Cell Reports.

Created in 2011, the Postdoctoral Research Award recognizes the remarkable contributions of postdoctoral research scholars to the UGA research enterprise. The UGA Research Foundation funds up to two awards a year to current scholars.

UGA Researcher Seeks to Unlock Secrets of Malaria Parasite

Donna Huber on January 31, 2018

malaria parasites
Super-resolution microscopy showing malaria parasites infecting human red blood cells. Image credit: Muthugapatti Kandasamy, Biomedical Microscopy Core

Vasant Muralidharan and his research team at the University of Georgia’s Center for Tropical and Emerging Global Diseases are making great strides in understanding how the malaria parasite hijacks red blood cells to cause disease but many of the parasite’s strategies remain elusive.  A new $1.875 million grant from the National Institutes of Health will allow them to continue this research.

Malaria is a parasitic disease that infects nearly 220 million people and kills nearly half a million people every year. Almost all the deaths occur in young children and primarily in sub-Saharan Africa. The parasite Plasmodium falciparum invades human red blood cells which directly leads to malaria symptoms that include headaches, muscle pain, periodic fevers with shivering, severe anemia, trouble breathing, and kidney failure. The parasite can also cause the most severe forms of malaria, such as cerebral malaria which can lead to brain damage, coma and death, and placental malaria, which occurs in pregnancy and can be life-threatening to both the mother and fetus.

Complete control of the infected red blood cell is required for parasites to grow and spread. The malaria parasite remodels the host cell by exporting hundreds of parasite proteins across numerous membranes that transform all aspects of infected red blood cells to suit its needs. The export of these proteins by P. falciparum to the host red blood cells is a unique parasite-driven process that is associated with many of the clinical manifestations of malaria, including death. The mechanisms which these proteins are exported are unknown.

“Exported proteins, many of them absolutely essential for the growth of the parasite, are recognized and sorted throughout the trafficking process by dedicated machinery that we have only now begun to understand,” said Muralidharan, assistant professor in the department of cellular biology.

His lab hopes to reveal unique protein trafficking mechanisms of P. falciparum that may be targets for antimalarial drug development.

 “We expect that this project will significantly advance our understanding of the protein export pathway in P. falciparum and how key decisions are made within the parasite that usher exported proteins to their site of action in the infected red blood cells,” concluded Muralidharan.

National Institutes of Health Award R01 AI130139 “Elucidating the trafficking mechanisms of effector proteins to the Plasmodium infected red blood cell.”

An ancient bacterial protein complex in human malaria parasites is essential for parasite growth

Donna Huber on November 14, 2017

Vasant Muralidharan and Anat Florentin

Several species of Plasmodium parasites cause malaria in humans and results in nearly 450,000 deaths annually. The deadliest of these species is Plasmodium falciparum. Unfortunately, it is also drug resistance to many of the currently available treatments. Vasant Muralidharan, assistant professor in the department of cellular biology, and his research group at The Center for Tropical and Emerging Global Diseases at The University of Georgia reported on an essential protein in hopes of identifying new drug targets.

Plasmodium parasites contain an organelle known as the apicoplast that evolved via the endosymbiosis of a red alga. The apicoplast produces several essential metabolites required for parasite growth and survival. Therefore, drugs that target the apicoplast are clinically effective. However, there is still not a lot known about this organelle. Understanding the function, structure, and biogenesis of the apicoplast provides a gold mine of antimalarial drug targets.

The role of Clp proteins in Plasmodium apicoplast

Clp (Caseinolytic Proteases) are conserved prokaryotic proteins that serve a wide variety of biological functions in bacteria, the evolutionary ancestors of the apicoplast. Several Clp proteins have been reported to localize in the apicoplast of the parasite but their biological functions were unknown.

The research team used different genetic tools to conditionally inhibit the function of various apicoplast-Clp proteins. “It is similar to understanding the role of a single card in holding up a house of cards by removing it from the structure,” said Muralidharan.

Their data show that the Clp chaperone PfClpC is essential for parasite viability and that its inhibition resulted in morphological defects, and loss of the apicoplast. They also revealed that the chaperone activity is required to stabilize a Clp Protease, PfClpP, suggesting that, similar to bacteria and plants chloroplasts, these two proteins form a proteolytic complex. These data may be relevant to the function of bacterial and plant Clp complexes. “Our findings shed light on the biological roles of the apicoplast Clp Proteins and their involvement in apicoplast replication,” said Dr. Anat Florentin, lead author on the study.

Significance of the findings

The role that bacterial Clp proteins play in cell division, stress response and ability to cause disease have placed them at the center of several drug discovery programs. The new understanding of Clp proteins in Plasmodium provides an avenue for drug development in malaria in which highly active antibacterial compounds can be repurposed as effective anti-malarial agents.

 

An online version of this study is available: A. Florentin,  D.W. Cobb, J.D. Fishburn, M. J. Cipriano, P.S. Kim, M.A. Fierro, B. Striepen, V. Muralidharan. 2017. PfClpC is an essential Clp chaperone required for plastid integrity and Clp protease stability in Plasmodium falciparum. Cell Reports 21, 1 – 11. http://dx.doi.org/10.1016/j.celrep.2017.10.081