Background: A drawback in the treatment of chronic Chagas disease (American trypanosomiasis) is the long time required to achieve complete loss of serological reactivity, the standard for determining treatment efficacy.
Methods: Antibody-secreting and memory B cells specific for Trypanosoma cruzi and their degree of differentiation were evaluated in adult and pediatric subjects with chronic Chagas disease prior to and after etiological treatment.
Results: Trypanosoma cruzi-specific antibody-secreting cells disappeared from the circulation in benznidazole or nifurtimox-treated subjects with declining parasite-specific antibody levels posttreatment, whereas B cells in most subjects with unaltered antibody levels were low prior to treatment and did not change after treatment. The timing of the decay in parasite-specific antibody-secreting B cells was similar to that in parasite-specific antibodies as measured by a Luminex-based assay, but preceded the decay in antibody levels detected by conventional serology. The phenotype of total B cells returned to a non-infection profile after successful treatment.
Conclusions: T. cruzi-specific antibodies in the circulation of chronically T. cruzi-infected subjects likely derive from both antigen-driven plasmablasts, that disappear following successful treatment, and long-lived plasma cells that persist and account for the low frequency and long course to complete seronegative conversion in successfully treated subjects.
G Cesar, M A Natale, M C Albareda, M G Alvarez, B Lococo, Ana María De Rissio, Marisa Fernandez, M Castro Eiro, G Bertocchi, B E White, F Zabaleta, R Viotti, R L Tarleton, S A Laucella.J Infect Dis. 2022 Dec 26;jiac495. doi: 10.1093/infdis/jiac495. Online ahead of print.
Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.
Juan M Bustamante, Angel M Padilla, Brooke White, Lisa D Auckland, Rachel E Busselman, Stephanie Collins, Elizabeth L Malcolm, Briana F Wilson, Ashley B Saunders, Sarah A Hamer, Rick L Tarleton. PLoS Negl Trop Dis. 2022 Oct 31;16(10):e0010688. doi: 10.1371/journal.pntd.0010688.
The mitochondrial Ca2+ uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca2+ signaling, is mediated via the calcium uniporter complex (MCUC). In animal cells the MCUC is regulated by the mitochondrial calcium uptake 1 and 2 dimer (MICU1/MICU2), which has been proposed to act as gatekeeper preventing mitochondrial Ca2+ overload at low cytosolic Ca2+ levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca2+ uptake at low extramitochondrial Ca2+ concentrations ([Ca2+]ext) and it was though that in the absence of one MICU the other would replace its role. However, previous attempts to knockout both genes were unsuccessful. Here, we designed a strategy to generate TcMICU1/TcMICU2 double knockout cell lines using CRISPR/Cas9 genome editing. Ablation of both genes was confirmed by PCR and Southern blot analyses. The absence of both proteins did not allow Ca2+ uptake at low [Ca2+]ext, significantly decreased the mitochondrial Ca2+ uptake at different [Ca2+]ext, without dissipation of the mitochondrial membrane potential, and increased the [Ca2+]ext set point needed for Ca2+ uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg2+ was found to be a negative regulator of MCUC-mediated mitochondrial Ca2+ uptake at different [Ca2+]ext. Occlusion of the MCUC pore by Mg2+ could partially explain the lack of mitochondrial Ca2+ uptake at low [Ca2+]ext in TcMICU1/TcMICU2-KO cells. In addition, TcMICU1/TcMICU2-KO epimastigotes had a lower growth rate, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes.
The skeletal muscle of a mouse infected with Trypanosoma cruzi is shown under a microscope. (Submitted by Fernando Sanchez)
The condition affects tens of millions across the Americas but lacks effective treatments
Researchers from the University of Georgia have discovered a potential treatment for Chagas disease, marking the first medication with promise to successfully and safely target the parasitic infection in more than 50 years.
Human clinical trials of the drug, an antiparasitic compound known as AN15368, will hopefully begin in the next few years.
Distinguished Research Professor Dr. Rick Tarleton of the Center for Tropical and Emerging Global Diseases at the Paul D. Coverdell Center for Biomedical and Health Sciences on Thursday, May 8, 2008. Dr. Tarleton is researching the effects of drug treatments on Chagas’ disease.
“I’m very optimistic,” said Rick Tarleton, corresponding author of the study and a UGA Athletic Association Distinguished Professor in the Franklin College of Arts and Sciences and member of the Center for Tropical and Emerging Global Diseases. “I think it has a really strong chance of being a real solution, not just a stand-in for something that works better than the drugs we currently have.”
The new drug works by targeting the parasite that causes the disease, Trypanosoma cruzi, also known as T. cruzi.
Nearly all people infected with the parasite experience flu-like symptoms such as fever, headaches and vomiting. However, after their immune response kicks in, their symptoms may subside.
But for 30% to 40% of patients, the infection can result in severe heart damage that can be both debilitating and life-threatening.
New drug is 100% effective in eliminating T. cruzi
Published in Nature Microbiology, the study found the new medication was 100% effective in curing mice, as well as non-human primates that were naturally infected by the parasite at a research facility in Texas. The animals also experienced no significant side effects from exposure to the drug.
Over the past several decades, previous treatment candidates went straight from experimental infections in mice to human clinical trials, where they failed to cure the infection. The new drug’s efficacy in non-human primates bodes well for how it will perform in humans.
“We’ve got something that is as close to effective as it can be in what is as close to a human as it could be, and there aren’t any side effects. That really de-risks it by a lot going into humans,” Tarleton said. “It doesn’t make it fail-safe, but it moves it much further along.”
Current medications to treat T. cruzi infection not ideal
T. cruzi is carried by blood-sucking insects known as kissing bugs. The insects can be found throughout North, Central and South America.
In addition to a nasty bite, the creatures carry the T. cruzi parasite, which is transmitted through their fecal matter. Victims can become infected when they unknowingly rub the insect’s feces into their eyes, nose or an open wound.
The infection may also be transmitted through organ transplants, from a pregnant person to their fetus or through contaminated food. However, infections from these pathways are less common.
The go-to medications used to treat Chagas aren’t terrible, Tarleton said, but they’re not ideal. They can pack some serious side effects and they’re not reliably effective, but they’re currently the only treatment option.
Patients also have to take the drugs for two months. And even the common but mild side effects like headache or nausea get old after a few weeks. As a result, about one in five people being treated for the disease stop taking their medications before they have a chance to cure the infection.
“Plus they have variable efficacy, and it’s not predictable,” Tarleton said. “I think most physicians in Latin America have to say, ‘We have a drug. It’s going to make you feel bad, and two months later after we finish it, we’re not really going to be able to tell you if it worked or not.’
“It’s really not a good inducement to take the medication.”
Chagas disease common in Latin American countries
Tens of millions of people across the Americas are infected with the parasite that causes Chagas disease. But it doesn’t get much media attention.
It’s most common in Latin American countries, particularly in low-income areas where housing isn’t ideal. Some of the countries with the highest rates of the disease include Bolivia, Venezuela, Argentina, Chile, Mexico and Brazil.
In homes with thatched roofs, mud walls or inadequate protection from the elements, kissing bugs thrive, making infection more likely.
Chagas disease poses significant risk to pets
The Centers for Disease Control and Prevention estimates around 300,000 people infected with the parasite currently live in the U.S. But because the condition isn’t a huge threat in places with good housing options, Chagas disease treatment and prevention doesn’t get much research funding.
There is growing concern about the T. cruzi infection rate among outdoor pets in the U.S., however. Working dogs and other pets that spend extended periods of time outside are contracting the parasite at an alarming rate.
“There are areas where the infection rates are 20% to 30% new infections per year,” Tarleton said. “Those tend to be severe infections where the dogs either die or develop a disease that makes them unable to work.”
Tarleton hopes to partner with veterinary pharmaceutical companies in the future to create a drug to treat the infection in pets as a means of funding diagnostics and medication purchases in Latin America.
For the present study, Tarleton partnered with colleagues at Anacor Pharmaceuticals, Texas A&M University, the University of Texas, the University of Kansas and Pfizer. Angel Padilla, Wei Wang, Dylan Orr, Brooke White, Arlene George and Huifeng Shen from UGA’s Center for Tropical and Emerging Global Diseases and the Department of Cellular Biology are co-authors on the paper.
Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.
Angel M. Padilla, Wei Wang, Tsutomu Akama, David S. Carter, Eric Easom, Yvonne Freund, Jason S. Halladay, Yang Liu, Sarah A. Hamer, Carolyn L. Hodo, Gregory K. Wilkerson, Dylan Orr, Brooke White, Arlene George, Huifeng Shen, Yiru Jin, Michael Zhuo Wang, Susanna Tse, Robert T. Jacobs & Rick L. Tarleton. Nat Microbiol (2022). https://doi.org/10.1038/s41564-022-01211-y
Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi(T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease’s complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.
Fernando Sanchez-Valdez, Ángel M Padilla, Juan M Bustamante, Caleb W D Hawkins, Rick L Tarleton. J Vis Exp. 2022 Jun 23;(184). doi: 10.3791/63919.
Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of TcBDF2 in T. cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results point to TcBDF2 as a druggable target against T. cruzi.
Alejandro Pezza, Luis E Tavernelli, Victoria L Alonso, Virginia Perdomo, Raquel Gabarro, Rab Prinjha, Elvio Rodríguez Araya, Inmaculada Rioja, Roberto Docampo, Felix Calderón, Julio Martin, Esteban Serra. ACS Infect Dis. 2022 Apr 28. doi: 10.1021/acsinfecdis.2c00057.
Eating is fundamental and from this basic principle, living organisms have evolved innumerable strategies to capture energy and nutrients from their environment. As part of the world’s aquatic ecosystems, the expansive family of heterotrophic protozoans uses self-generated currents to funnel prokaryotic prey into an ancient, yet highly enigmatic, oral apparatus known as the cytostome-cytopharynx complex prior to digestion. Despite its near ubiquitous presence in protozoans, little is known mechanistically about how this feeding organelle functions. Intriguingly, one class of these flagellated phagotrophic predators known as the kinetoplastids gave rise to a lineage of obligate parasitic protozoa, the trypanosomatids, that can infect a wide variety of organisms ranging from plants to humans. One parasitic species of humans, Trypanosoma cruzi, has retained this ancestral organelle much like its free-living relatives and continues to use it as its primary mode of endocytosis. In this review, we will highlight foundational observations made regarding the cytostome-cytopharynx complex and examine some of the most pressing questions regarding the mechanistic basis for its function. We propose that T. cruzi has the potential to serve as an excellent model system to dissect the enigmatic process of protozoal phagotrophy and thus enhance our overall understanding of fundamental eukaryotic biology.
Trypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca2+ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca2+ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH3) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism.
IMPORTANCETrypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Release of these molecules, which are necessary for growth and cell signaling, is induced by alkalinization, but a physiological mechanism for acidocalcisome alkalinization was unknown. In this work, we demonstrate that a histidine ammonia lyase localizes to acidocalcisomes and is responsible for their alkalinization.
Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 (TcAEK1) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line (TcAEK1-SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome. IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.
