To maximise the likelihood of success, global health programmes need repeated, honest appraisal of their own weaknesses, with research undertaken to address any identified gaps. There is still much to be learned to optimise work against neglected tropical diseases. To facilitate that learning, a comprehensive research and development plan is required. Here, we discuss how such a plan might be developed.
David Mabey, Ellen Agler, John H Amuasi, Leda Hernandez, T Déirdre Hollingsworth, Peter J Hotez, Patrick J Lammie, Mwelecele N Malecela, Sultani H Matendechero, Eric Ottesen, Richard O Phillips, John C Reeder, Célia Landmann Szwarcwald, Joseph P Shott, Anthony W Solomon, Andrew Steer, Soumya Swaminathan. Trans R Soc Trop Med Hyg. 2020 Nov 11;traa114. doi: 10.1093/trstmh/traa114.
Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals O-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing and signaling. Knockout experiments showed that TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC3, 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O-Fuc are important for tachyzoite fitness, O-fucosylation by TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically-expressed proteins normally modified with O-fucose. Intact TgSPY-MYC3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy tachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O-fucosylated proteins, suggest that TgSPY controls the levels of proteins to be held in reserve for response to novel stresses.
Giulia Bandini, Carolina Agop-Nersesian, Hanke van der Wel, Msano Mandalasi , Hyun W Kim, Christopher M West, John Samuelson. J Biol Chem. 2020 Nov 6;jbc.RA120.015883. doi: 10.1074/jbc.RA120.015883.
Accurate and reliable diagnostic tools are an essential requirement for neglected tropical diseases (NTDs) programmes. However, the NTD community has historically underinvested in the development and improvement of diagnostic tools, potentially undermining the successes achieved over the last 2 decades. Recognizing this, the WHO, in its newly released draft roadmap for NTD 2021-2030, has identified diagnostics as one of four priority areas requiring concerted action to reach the 2030 targets. As a result, WHO established a Diagnostics Technical Advisory Group (DTAG) to serve as the collaborative mechanism to drive progress in this area. Here, the purpose and role of the DTAG are described in the context of the challenges facing NTD programmes.
Ashley A Souza, Camilla Ducker, Daniel Argaw, Jonathan D King, Anthony W Solomon, Marco A Biamonte, Rhea N Coler, Israel Cruz, Veerle Lejon, Bruno Levecke, Fabricio K Marchini, Michael Marks, Pascal Millet, Sammy M Njenga, Rahmah Noordin, René Paulussen, Esvawaran Sreekumar, Patrick J Lammie. Trans R Soc Trop Med Hyg. 2020 Nov 9;traa118. doi: 10.1093/trstmh/traa118.
Megna Tiwari is a second-year Ph.D. trainee in the laboratory of Diego Huet. She is originally from Newport Beach, California and completed her undergraduate degree in Cell, Molecular and Developmental Biology at the University of California, Riverside (UCR). While at UCR, she worked as an undergraduate researcher in the fungal genomics lab of Dr. Jason Stajich for 2 years and co-founded Women in STEM Engaging Riverside (WISER). After graduation, she worked as a blood bank lab technician at LifeStream Blood Bank where she screened for and routinely found blood samples positive for understudied pathogenic parasites. Her fascination with pathogenic parasites led her to seek a thesis-based Master of Science in Biology at California State University, Fullerton under the supervision of Dr. Veronica Jimenez. During this period, Megna worked on understanding the functional and structural relationship of mechanosensitive ion channels found in T. cruzi and cemented her passion for molecular parasitology.
Megna has been awarded a CTEGD T32 Training Fellowship. She currently serves as Vice-president of CTEGD’s Graduate Student Association and New Student Liaison for the Department of Cellular Biology’s Graduate Student Association.
Why did you choose UGA?
My master’s research in parasitology reaffirmed my passion for research in unconventional parasitic pathogens. Therefore, I applied for doctoral programs that would allow me to remain in the field of cell and molecular parasitology and the CTEGD at UGA was the perfect place for me to obtain the best possible training as a parasitologist.
What is your research focus/project and why are you interested in the topic?
The over-reaching research goal of the Huet lab is the investigation of the highly divergent metabolic adaptations of apicomplexans. My research interests in the lab have led me to study the role of the ATP synthase in the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis. For my project, I am examining the role of apicomplexan-specific ATP synthase subunits and how they might contribute to the regulation of the ATP synthase function in the parasite.
What are your future professional plans?
Following graduation from UGA, I hope to continue on for a postdoctoral research position in parasitology.
What do you hope to do for your capstone experience?
For my capstone experience, I want to gain an outside perspective and understanding of foreign research culture that I can apply to my own research when I return to the CTEGD.
What is your favorite thing about UGA and/or Athens?
At the CTEGD, I love the collaborative nature. If I am trying to learn a new technique or understand new concepts, I am able to easily walk down the hall to a neighboring lab and get advice. In Athens, for entertainment, I love the endless craft beer scene and I love all the greenery and being able to hike gaps of the Appalachian trail!
Support trainees like Megna by giving today to the Center for Tropical & Emerging Global Diseases.
3D (left) and single slice (right) light sheet microscopy imaging of the heart of a mouse infected with two strains (red and blue) of Trypanosoma cruzi. (Image credit: Fernando Sanchez-Valdez)
Research shows stronger but less frequent drug doses could be key
Researchers in the University of Georgia’s Center for Tropical and Emerging Global Diseases have found that a more intensive, less frequent drug regimen with currently available therapeutics could cure the infection that causes Chagas disease, a potentially life-threatening illness affecting up to 300,000 people in the United States.
Trypanosoma cruzi is a single-celled parasitic organism that causes Chagas disease. At least 6 million people are infected by T. cruzi, mostly in South America. Current drug therapies have been ineffective in completely clearing the infection and are associated with severe adverse side effects.
A single dose of benznidazole has been shown to be highly effective in killing more than 90% of parasites. However, after a CTEGD team found some of the parasites enter into a dormancy stage, the researchers hypothesized that an intermittent treatment schedule could be effective.
Photo credit: Peter Frey/UGA
“Current human trials are only looking at giving lower doses over a shorter time period, which is the exact opposite of what we show works.” — Rick Tarleton
“In this system we can see what a single dose of drug does,” said Rick Tarleton, Regents’ Professor in UGA’s department of cellular biology. “Does it make sense to give a drug twice daily when the remaining dormant parasites are insensitive to it?”
The investigators found that giving as little as two-and-a-half times the typical daily dose of benznidazole, once per week for 30 weeks, completely cleared the infection, whereas giving the standard daily dose once a week for a longer period did not.
“Current human trials are only looking at giving lower doses over a shorter time period, which is the exact opposite of what we show works,” said Tarleton.
Since Tarleton’s team worked with a mouse model, how this change in treatment regimen will translate in humans is yet unknown, as are any potential side effects of the higher doses. Adverse reactions already are a problem with current treatments; the hope is that side effects from a less frequent dosage would be more tolerable.
Significant challenge
Assessing the success of treatments in Chagas disease is a significant challenge. Tissue samples from infected organisms might not be representative of the entire organ or animal, since low numbers of persistent, dormant parasites can be difficult to detect. Therefore, Tarleton’s group used light sheet fluorescence microscopy to view intact whole organs from infected mice.
“With light sheet fluorescence microscopy, you have a broad view of potentially any tissue in the mouse that allows for dependable assessment of parasite load and persistence,” said Tarleton. “It gives you an incredible view of the infection.”
Using this technology, they learned something new about the dormant parasites: Some were still susceptible to drug treatment. This provides hope that new drug therapies could be developed to target these parasites.
“Discovery of new drugs should continue,” Tarleton said. “We still need better drugs.”
Co-led by assistant research scientist Juan Bustamante and research professional Fernando Sanchez-Valdez in Tarleton’s research group, the study’s findings appear in Science Translational Medicine.
A major contributor to treatment failure in Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is that current treatment regimens do not address the drug insensitivity of transiently dormant T. cruzi amastigotes. Here, we demonstrated that use of a currently available drug in a modified treatment regimen of higher individual doses, given less frequently over an extended treatment period, could consistently extinguish T. cruzi infection in three mouse models of Chagas disease. Once per week administration of benznidazole at a dose 2.5 to 5 times the standard daily dose rapidly eliminated actively replicating parasites and ultimately eradicated the residual, transiently dormant parasite population in mice. This outcome was initially confirmed in “difficult to cure” mouse infection models using immunological, parasitological, and molecular biological approaches and ultimately corroborated by whole organ analysis of optically clarified tissues using light sheet fluorescence microscopy (LSFM). This tool was effective for monitoring pathogen load in intact organs, including detection of individual dormant parasites, and for assessing treatment outcomes. LSFM-based analysis also suggested that dormant amastigotes of T. cruzi may not be fully resistant to trypanocidal compounds such as benznidazole. Collectively, these studies provide important information on the phenomenon of dormancy in T. cruzi infection in mice, demonstrate methods to therapeutically override dormancy using a currently available drug, and provide methods to monitor alternative therapeutic approaches for this, and possibly other, low-density infectious agents.
Juan M. Bustamante, Fernando Sanchez-Valdez, Angel M. Padilla, Brooke White, Wei Wang and Rick L. Tarleton. Science Translational Medicine 28 Oct 2020: Vol. 12, Issue 567, eabb7656. DOI: 10.1126/scitranslmed.abb7656
Trypanosoma cruzi is a unicellular parasite and the etiologic agent of Chagas disease. The parasite has a digenetic life cycle alternating between mammalian and insect hosts, where it faces a variety of environmental conditions to which it must adapt in order to survive. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Major environmental changes include temperature, nutrient availability, ionic composition, pH, osmolarity, oxidative stress, contact with host cells and tissues, host immune response, and intracellular life. Some of the signaling pathways and second messengers potentially involved in the response to these changes have been elucidated in recent years and will be the subject of this review.
Glycosylation is a highly diverse set of co- and post-translational modification of proteins. For mammalian glycoproteins, glycosylation is often site-, tissue- and species-specific, and diversified by microheterogeneity. Multitudinous biochemical, cellular, physiological and organismic effects of their glycans have been revealed, either intrinsic to the carrier proteins or mediated by endogenous reader proteins with carbohydrate recognition domains. Furthermore, glycans frequently form the first line of access by or defense from foreign invaders, and new roles for nucleocytoplasmic glycosylation are blossoming. We now know enough to conclude that the same general principles apply in invertebrate animals and unicellular eukaryotes – different branches of which spawned the plants or fungi and animals. The two major driving forces for exploring the glycomes of invertebrates and protists are (i) to understand the biochemical basis of glycan-driven biology in these organisms, especially of pathogens, and (ii) to uncover the evolutionary relationships between glycans, their biosynthetic enzyme genes, and biological functions for new glycobiological insights. With an emphasis on emerging areas of protist glycobiology, here we offer an overview of glycan diversity and evolution, to promote future access to this treasure trove of glycobiological processes.
The microbiota’s influence on host (patho) physiology has gained interest in the context of Gulf War Illness (GWI), a chronic disorder featuring dysregulation of the gut-brain-immune axis. This study examined short- and long-term effects of GWI-related chemicals on gut health and fecal microbiota and the potential benefits of Lacto-N-fucopentaose-III (LNFPIII) treatment in a GWI model. Male C57BL/6J mice were administered pyridostigmine bromide (PB; 0.7 mg/kg) and permethrin (PM; 200 mg/kg) for 10 days with concurrent LNFPIII treatment (35 μg/mouse) in a short-term study (12 days total) and delayed LNFPIII treatment (2×/week) beginning 4 months after 10 days of PB/PM exposure in a long-term study (9 months total). Fecal 16S rRNA sequencing was performed on all samples post-LNFPIII treatment to assess microbiota effects of GWI chemicals and acute/delayed LNFPIII administration. Although PB/PM did not affect species composition on a global scale, it affected specific taxa in both short- and long-term settings. PB/PM elicited more prominent long-term effects, notably, on the abundances of bacteria belonging to Lachnospiraceae and Ruminococcaceae families and the genus Allobaculum. LNFPIII improved a marker of gut health (i.e., decreased lipocalin-2) independent of GWI and, importantly, increased butyrate producers (e.g., Butyricoccus, Ruminococcous) in PB/PM-treated mice, indicating a positive selection pressure for these bacteria. Multiple operational taxonomic units correlated with aberrant behavior and lipocalin-2 in PB/PM samples; LNFPIII was modulatory. Overall, significant and lasting GWI effects occurred on specific microbiota and LNFPIII treatment was beneficial.
Ryan S Mote, Jessica M Carpenter, Rachel L Dockman, Andrew J Steinberger, Garret Suen, Thomas Norberg, Donald A Harn, John J Wagner, Nikolay M Filipov. Int J Environ Res Public Health. 2020 Sep 27;17(19):E7081. doi: 10.3390/ijerph17197081.
