Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.
Ramakrishnan S, Unger LM, Baptista RP, Cruz-Bustos T, Docampo R (2021) Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication. PLoS Pathog 17(3): e1009399. https://doi.org/10.1371/journal.ppat.1009399
Background: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease.
Methods: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months.
Results: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption.
Conclusions: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.
Melisa D Castro Eiro, María A Natale, María G Alvarez, Huifeng Shen, Rodolfo Viotti, Bruno Lococo, Jacqueline Bua, Myriam Nuñez, Graciela L Bertocchi, María C Albareda, Gonzalo Cesar, Rick L Tarleton, Susana A Laucella. J Antimicrob Chemother. 2021 Mar 7;dkab054. doi: 10.1093/jac/dkab054
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.
“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.
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.
Introduction. In a pilot study, we showed that intermittent administration of benznidazole in chronic Chagas disease patients resulted in a low rate of treatment suspension and therapeutic failure, as assessed by qPCR at the end of treatment. Herein, a three-year post-treatment follow-up study of the same cohort of patients is presented.
Methods. The treatment scheme consisted of 12 doses of benznidazole at 5 mg/kg/day in two daily doses every 5 days. Parasite load, T. cruzi-specific antibodies and serum chemokine levels were measured prior to treatment and after a median follow-up of 36 months post-treatment by kDNA and SatDNA qPCR methods, conventional serological techniques and a Luminex-based assay with recombinant T. cruzi protein, and a cytometric bead array, respectively.
Results. At the end of follow-up, 14 of 17 (82%) patients had negative qPCR findings, whereas three of 17 (18%) had detectable nonquantifiable findings by at least one of the qPCR techniques. A decline in parasite-specific antibodies at 12 months post-treatment was confirmed by conventional serological tests and the Luminex assays. Monocyte chemoattractant protein-1 (MCP-1) levels increased after treatment, whereas monokine induced by gamma interferon (MIG) levels decreased. New post-treatment electrocardiographic abnormalities were observed in only one patient who had cardiomyopathy prior to treatment.
Conclusions. Altogether, these data strengthen our previous findings by showing that the intermittent administration of benznidazole results in a low rate of treatment suspension, with comparable treatment efficacy to that of a daily dose of 5mg/kg for 60 days.
María Gabriela Álvarez, Juan Carlos Ramírez, Graciela Bertocchi, Marisa Fernández, Yolanda Hernández, Bruno Lococo, Constanza Lopez-Albizu, Alejandro Schijman, Carolina Cura, Marcelo Abril, Susana Laucella, Rick L Tarleton, María Ailen Natale, Melisa Castro Eiro, Sergio Sosa-Estani, Rodolfo Viotti. Antimicrob Agents Chemother. 2020 Jun 22;AAC.00439-20. doi: 10.1128/AAC.00439-20.
Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.
Dr. Fernando Sanchez-Valdéz, from Salta, Argentina, completed a Ph.D. in Molecular Biology at the Faculty of Pharmacy and Biochemistry at the University of Buenos Aires, Argentina in 2014. After his Ph.D., he completed a postdoctoral fellowship in Dr. Rick Tarleton´s laboratory at University of Georgia. In 2018, he obtained a Research Scientist position in the career pathway of the National Research Council in Argentina (CONICET). Earlier this year, he was awarded a fellowship from the CTEGD-Janssen Visiting Scholars Program, which enabled him to return to the Tarleton Research Group.
What is your primary research focus? Why are you interested in this subject?
The main focus of my research has been to uncover the mechanism of drug resistance in the Chagas disease agent, Trypanosoma cruzi. The main question we are trying to answer is why the treatment with highly effective drugs like Benznidazole (the current available treatment for Chagas disease) often fails to cure Chagas disease. By combining ex vivo luminescence assays and tissue-clearing techniques we were able to report, for the first time, the presence of dormant non-replicating amastigotes forms in the chronic phase of the disease. Dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after treatment interruption. T. cruzi‘s capacity to become dormant makes them transiently drug-resistant, suggesting that this phenomenon accounts for the failure of the otherwise highly active compounds such Benznidazole (Sanchez-Valdéz, et al eLife 2018).
Why did you choose UGA?
I returned to Athens in February 2019 to continue working on the findings we made during my postdoctoral training in the Tarleton Laboratory. I initially decided to come UGA based on a colleague’s recommendations and the fact that Tarleton´s lab is one of the reference centers for Chagas disease research. It’s a really motivating environment to do science since the scientific and technical level here is really high as well as diverse including areas as immunology, drug discovery, genetic manipulation, genomics, diagnostics, etc. Also the amount of resources available is impressive not only from the lab but also from the Biomedical Microscopy Core, Cytometry Shared Resource Laboratory and the animal facility at UGA.
What has been your research project while at UGA?
Currently, we are expanding our knowledge about T. cruzi dormancy and trying to interfere T. cruzi dormancy using new compounds or the conventional drugs but in a different treatment schedule. One of the approaches we are testing now involves the evaluation of drug doses and treatment schemes able to kill dormant parasites. For this purpose, we are optimizing a robust platform to detect low levels of parasites in whole clarified mice organs using light-sheet fluorescent microscopy. This technique will allow us the specific detection of low levels of persistent dormant parasites.
How has the CTEGD-Janssen Visiting Scholar Fellowship and your time at UGA impacted your research and professional goals?
I am so glad about the opportunity to continue working on T. cruzi dormancy with such experienced and renowned scientists and particularly using state-of-the-art microscopy techniques currently unavailable in South America. This experience will definitely have a positive impact on my career development and probably in the Chagas disease research field.
Researchers at the University of Georgia have discovered that dormancy of the parasite Trypanosoma cruzi prevents effective drug treatment for Chagas disease, which kills more than 50,000 people each year in Central and South America and is a growing threat in the United States and Europe.
The disease infects an estimated 6 million to 7 million people, according to the World Health Organization, although some scientists estimate the number could be as high as 20 million. Chagas disease causing irreparable damage to the heart and digestive system, and effective prevention and treatment methods are virtually nonexistent.
In a new study published in eLife, Rick Tarleton and his research team at the Center for Tropical and Emerging Global Diseases sought to determine why drug treatments such as benzimidazole frequently fail.
“Benzimidazole has been shown to be particularly effective in reducing parasite infection,” said Tarleton, Regents’ Professor in the department of cellular biology. “A single dose can eliminate nearly 90 percent of parasites within 48 hours, but we didn’t know why it didn’t kill 100 percent of the parasites.”
For the first time, they show that a small proportion of T. cruzi parasites halt replication within 24 hours of invading the host cell. These dormant parasites are resistant to extended drug treatment and can resume replication after treatment ends, thus re-establishing a growing infection.
The researchers don’t know why some of the parasites exhibit this behavior, but they are hopeful that future studies into this mechanism will shed more light on the way T. cruzi evades the host’s immune response.
“This isn’t drug resistance in the classical way we think of resistance,” said Tarleton. “The parasites aren’t dormant because of the presence of the drug.”
In fact, while treatment continued they saw some of the dormant parasites “wake up” and then become susceptible to the treatment. The team believes the key to effective treatment will be to catch the parasite as they resume replication, continuing medication until no parasites remain in the host.
“This discovery really offers a solution for current drugs to be used in a more effective way,” said Tarleton. “A longer, less concentrated dosing schedule could lead to a cure.”