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

Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

Donna Huber on March 24, 2020

Plasmodium vivax infects hepatocytes to form schizonts that cause blood infection, or dormant hypnozoites that can persist for months in the liver before leading to relapsing blood infections. The molecular processes that drive P. vivax schizont and hypnozoite survival remain largely unknown, but they likely involve a rich network of host-pathogen interactions, including those occurring at the host-parasite interface, the parasitophorous vacuole membrane (PVM). Using a recently developed P. vivax liver-stage model system we demonstrate that host aquaporin-3 (AQP3) localizes to the PVM of schizonts and hypnozoites within 5 days after invasion. This recruitment is also observed in P. vivax-infected reticulocytes. Chemical treatment with the AQP3 inhibitor auphen reduces P. vivax liver hypnozoite and schizont burden, and inhibits P. vivax asexual blood-stage growth. These findings reveal a role for AQP3 in P. vivax liver and blood stages and suggest that the protein may be targeted for therapeutic treatment.

Dora Posfai, Steven P. Maher, Camille Roesch, Amélie Vantaux, Kayla Sylvester, Julie Péneau, Jean Popovici, Dennis E. Kyle, Benoît Witkowski, Emily R. Derbyshire. Cell Chem Biol. 2020 Mar 24. pii: S2451-9456(20)30083-0. doi: 10.1016/j.chembiol.2020.03.009.

Acute Plasmodium Infection Promotes Interferon-Gamma-Dependent Resistance to Ebola Virus Infection

Donna Huber on March 24, 2020

During the 2013-2016 Ebola virus (EBOV) epidemic, a significant number of patients admitted to Ebola treatment units were co-infected with Plasmodium falciparum, a predominant agent of malaria. However, there is no consensus on how malaria impacts EBOV infection. The effect of acute Plasmodium infection on EBOV challenge was investigated using mouse-adapted EBOV and a biosafety level 2 (BSL-2) model virus. We demonstrate that acute Plasmodium infection protects from lethal viral challenge, dependent upon interferon gamma (IFN-γ) elicited as a result of parasite infection. Plasmodium-infected mice lacking the IFN-γ receptor are not protected. Ex vivo incubation of naive human or mouse macrophages with sera from acutely parasitemic rodents or macaques programs a proinflammatory phenotype dependent on IFN-γ and renders cells resistant to EBOV infection. We conclude that acute Plasmodium infection can safeguard against EBOV by the production of protective IFN-γ. These findings have implications for anti-malaria therapies administered during episodic EBOV outbreaks in Africa.

Kai J Rogers, Olena Shtanko, Rahul Vijay, Laura N Mallinger, Chester J Joyner, Mary R Galinski, Noah S Butler, Wendy Maury. Cell Rep. 2020 Mar 24;30(12):4041-4051.e4. doi: 10.1016/j.celrep.2020.02.104.

Trainee Spotlight: Edwin Pierre Louis

Donna Huber on March 23, 2020

Edwin Pierre Louis is a pre-doctoral trainee in the laboratory of Dr. Drew Etheridge. Originally from Haiti, he immigrated to the US to attend the University of Florida (UF), where he graduated with a Bachelor of Science in Biochemistry Molecular Biology. After earning his degree at UF, Edwin accepted a position as a biological scientist in the UF Center of Excellence for Regenerative Health Biotechnology, with a focus on gene and cell based therapeutic development, where he worked for three years. There, he first discovered his love of host-pathogen interactions as a biological scientist working under the supervision of Dr. Richard Snyder for the component Florida Biologix at this center and later merged to create Brammer Bio which was subsequently acquired by Thermo Fisher Scientific. During this time in industry, he realized that to improve his scientific capacities he would need to continue his studies by pursuing a graduate degree. As part of his preparations to apply to a graduate program, he joined the UGA post-baccalaureate PREP program whose mission is to prepare students interested in a graduate degree for the application process. During this time, he was granted the opportunity to join Dr. Michael Terns’ laboratory for a year where he investigated the molecular mechanism of CRISPR-Cas based viral defense in Streptococcus thermophilus as well as prime adaptation events in the type II-A CRISPR-Cas system.

Since attending UGA, Edwin has been awarded both the Gateway to Graduate School Bridge Program and the Graduate Scholars Leadership, Engagement and Development Program (GS LEAD) scholarships sponsored by the National Science Foundation (NSF).

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

Broadly, my key research interests center around how organisms like viruses and parasites manipulate their host cell in order to grow and propagate. My current project is focused on elucidating how the protozoan pathogen Toxoplasma gondii is able to use secreted protein effectors to manipulate its host cells functions.

Why did you choose UGA?

I chose to study at the University of Georgia, in part, because of my excellent post-baccalaureate experience in the PREP program. It was evident from my interactions that UGA excels at fostering a productive relationship between students and faculty. Regardless of any faculty member’s relationship to the students, there was a sustained willingness for faculty to give of their time in order to see the students succeed. I also decided to pursue my PhD at UGA because of the cutting-edge research and in particular the collection of outstanding parasitologists that is uniquely found in the Center for Tropical and Emerging Global Diseases (CTEGD).

What are your future professional plans?

As I continue my graduate studies on host pathogen interaction, I plan to do some post-doctoral trainings to augment my apprenticeship and ultimately become an independent scientist to lead my own research group. I also hope to be able to give back to the local community that has contributed so much to my own personal success by donating my time and knowledge to mentor young budding scientists especially those from underprivileged homes and/or underdeveloped countries.

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

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The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans

Donna Huber on March 17, 2020

Mitochondrial Ca²⁺ transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that Trypanosoma brucei MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the TbMCUC subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and P_i transport with ATP synthesis, a process that is stimulated by Ca²⁺ in trypanosomes and human cells.

IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei, the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells.

Guozhong Huang, Roberto Docampo. mBio. 2020 Mar 17;11(2). pii: e00268-20. doi: 10.1128/mBio.00268-20.

Update on Cryptosporidium spp.: highlights from the Seventh International Giardia and Cryptosporidium Conference

Donna Huber on March 13, 2020

While cryptosporidiosis is recognized as being among the most common causes of human parasitic diarrhea in the world, there is currently limited knowledge on Cryptosporidium infection mechanisms, incomplete codification of diagnostic methods, and a need for additional therapeutic options. In response, the Seventh International Giardia and Cryptosporidium Conference (IGCC 2019) was hosted from 23 to 26 June 2019, at the Rouen Normandy University, France. This trusted event brought together an international delegation of researchers to synthesize recent advances and identify key research questions and knowledge gaps. The program of the interdisciplinary conference included all aspects of host-parasite relationships from basic research to applications to human and veterinary medicine, and environmental issues associated with waterborne parasites and their epidemiological consequences. In relation to Cryptosporidium and cryptosporidiosis, the primary research areas for which novel findings and the most impressive communications were presented and discussed included: Cryptosporidium in environmental waters, seafood, and fresh produce; Animal epidemiology; Human cryptosporidiosis and epidemiology; Genomes and genomic evolution encompassing: Comparative genomics of Cryptosporidium spp., Genomic insights into biology, Acquiring and utilizing genome sequences, Genetic manipulation; Host-parasite interaction (immunology, microbiome); and Diagnosis and treatment. High quality presentations discussed at the conference reflected decisive progress and identified new opportunities that will engage investigators and funding agencies to spur future research in a “one health” approach to improve basic knowledge and the clinical and public health management of zoonotic cryptosporidiosis.

Giovanni Widmer, David Carmena, Martin Kváč, Rachel M. Chalmers, Jessica C. Kissinger, Lihua Xiao, Adam Sateriale, Boris Striepen, Fabrice Laurent, Sonia Lacroix-Lamandé, Gilles Gargala and Loïc Favennec. Parasite. 2020;27:14. doi: 10.1051/parasite/2020011.

Battling Malaria

Donna Huber on February 28, 2020

UGA is developing new drugs to fight a lethal parasite. Dennis Kyle discusses what his lab is doing to fight malaria in this brief (2:30) video.

HELP BATTLE DEADLY DISEASES

Donate to the Center for Tropical and Emerging Global Diseases

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Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes

Donna Huber on February 28, 2020

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3′ extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3′ adenylation, uridine insertion/deletion editing, and 3′ A/U-tailing. The rRNAs and gRNAs are 3′ uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Inna Aphasizheva, Juan Alfonzo, Jason Carnes, Igor Cestari, Jorge Cruz-Reyes, H. Ulrich Göringer, Stephen Hajduk, Julius Lukeš, Susan Madison-Antenucci, Dmitri A. Maslov, Suzanne M. McDermott, Torsten Ochsenreiter, Laurie K. Read, Reza Salavati, Achim Schnaufer, André Schneider, Larry Simpson, Kenneth Stuart, Vyacheslav Yurchenko, Z. Hong Zhou, Alena Zíková, Liye Zhang, Sara Zimmer, Ruslan Aphasizhev. Trends Parasitol. 2020 Apr;36(4):337-355. doi: 10.1016/j.pt.2020.01.006. Epub 2020 Feb 28.

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi

Donna Huber on February 27, 2020

Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.

Noelia Lander, Teresa Cruz-Bustos, and Roberto Docampo. Front Cell Infect Microbiol. 2020 Feb 27;10:68. doi: 10.3389/fcimb.2020.00068. eCollection 2020.

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

Donna Huber on February 25, 2020

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum endoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.

IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.

Manuel A. Fierro, Beejan Asady, Carrie F. Brooks, David W. Cobb, Alejandra Villegas, Silvia N. J. Moreno, Vasant Muralidharan. mBio. 2020 Feb 25;11(1). pii: e03078-19. doi: 10.1128/mBio.03078-19.

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

Donna Huber on February 21, 2020

Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3’-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.

Rudo Kieft, Yang Zhang, Alexandre P. Marand, Jose Dagoberto Moran, Robert Bridger, Lance Wells, Robert J. Schmitz, Robert Sabatini. PLoS Genet. 2020 Feb 21;16(2):e1008390. doi: 10.1371/journal.pgen.1008390.

Author: Donna Huber