quantitative-3d-imaging-trypanosoma-cruzi-infected-cells-dormant

Quantitative 3D Imaging of <em>Trypanosoma cruzi</em>-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...

University of Georgia

Mice infected with Trypanosoma cruzi Tulahuen strain or SGO-Z12 isolate were treated at 180 days post infection (p.i.) (i.e. chronic phase) with benznidazole (for 30 days) or thioridazine (for 12 days). Both drugs produced a decrease in electrocardiographic alterations, fewer modifications in the affinity and density of cardiac beta-receptors, and few isolated areas of fibrosis in the heart, whereas untreated mice presented areas of necrosis and fibre fragmentation 350 days p.i. (P<0.01). Survival in treated mice was 100% for benznidazole and 88% for thioridazine, independent of the parasite strain; survival for untreated mice was 30% and 40% for Tulahuen strain and SGO-Z12 isolate, respectively (P<0.01). No cardiotoxic effects of thioridazine were detected at the dose and treatment schedule used. These results show the benefit of treatment in the chronic phase of Chagas disease and that thioridazine should be considered as a promising agent for the treatment of Chagas disease.

Treatment with benznidazole or thioridazine in the chronic phase of experimental Chagas disease improves cardiopathy.

In this study, we document the development of stable, antigen-independent CD8+ T cell memory after drug-induced cure of a chronic infection. By establishing a system for drug cure of chronic Trypanosoma cruzi infection, we present the first extensively documented case of total parasite clearance after drug treatment of this infection. Cure resulted in the emergence of a stable, parasite-specific CD8+ T cell population with the characteristics of central memory cells, based upon expression of CD62L, CCR7, CD127, CD122, Bcl-2 and a reduced immediate in vivo CTL function. CD8+ T cells from treated and cured mice also expanded more rapidly and provided greater protection following challenge than those from chronically infected mice. These results show that complete pathogen clearance results in stable, antigen-independent and protective T cell memory, despite the potentially exhausting effects of prior long-term exposure to antigen in this chronic infection.

Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease.

CD8(+) T cells have emerged as crucial players in the control of a number of protozoan pathogens, including Trypanosoma cruzi, the agent of human Chagas disease. The recent identification of the dominant targets of T. cruzi-specific T cells has allowed investigators to follow the generation of and document the functionality of T cell responses in both mice and humans. Although slow to develop in the early stages of the infection, T. cruzi-specific CD8(+) T cells reach prodigious levels and remain highly functional throughout chronic infections in mice. Following drug-induced cure during either the acute or chronic stage, these immunodominant T cells persist as stable, antigen-independent memory populations. T. cruzi-specific CD8(+) T cells in humans are less-well-studied but appear to lose functionality and decline in numbers in these decades-long infections. Changes in the frequency of parasite-specific T cell upon therapeutic treatment in humans may provide a new metric for determining treatment efficacy.

CD8+ T cells in Trypanosoma cruzi infection.

The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Trypanosoma cruzi infects millions of people in Latin America and often leads to the development of Chagas disease. T. cruzi infection can be acquired at or near the bite site of the triatomine vector, but per os infection is also a well-documented mode of transmission, as evidenced by recent microepidemics of acute Chagas disease attributed to the consumption of parasite-contaminated foods and liquids. It would also be convenient to deliver vaccines for T. cruzi by the oral route, particularly live parasite vaccines intended for the immunization of reservoir hosts. For these reasons, we were interested in better understanding immunity to T. cruzi following oral infection or oral vaccination, knowing that the route of infection and site of antigen encounter can have substantial effects on the ensuing immune response. Here, we show that the route of infection does not alter the ability of T. cruzi to establish infection in muscle tissue nor does it impair the generation of a robust CD8(+) T cell response. Importantly, oral vaccination with attenuated parasites provides protection against wild-type (WT) T. cruzi challenge. These results strongly support the development of whole-organism-based vaccines targeting reservoir species as a means to alleviate the burden of Chagas disease in affected regions.

Oral exposure to Trypanosoma cruzi elicits a systemic CD8⁺ T cell response and protection against heterotopic challenge.

INTRODUCTION: Chagas disease is the highest impact human infectious disease in Latin America, and the leading worldwide cause of myocarditis. Despite the availability of several compounds that have demonstrated efficacy in limiting the effects of T. cruzi, these compounds are rarely used due to their variable efficacy, substantial side effects and the lack of methodologies for confirming their effectiveness. Furthermore, the development of more efficacious compounds is challenged by limitations of systems for assessing drug efficacy in vitro and in vivo. AREAS COVERED: Herein, the authors review the development of Chagas disease drug discovery methodology, focusing on recent developments in high throughput screening, in vivo testing methods and assessments of efficacy in humans. Particularly, this review documents the significant progress that has taken place over the last 5 years that have paved the way for both target-focused and high-throughput screens of compound libraries. EXPERT OPINION: The tools for in vitro and in vivo screening of anti-T. cruzi compounds have improved dramatically in the last few years and there are now a number of excellent in vivo testing models available; this somewhat alleviates the bottleneck issue of quickly and definitively demonstrating in vivo efficacy in a relevant host animal system. These advances emphasize the potential for additional progress resulting in new treatments for Chagas disease in the coming years. That being said, national and international agencies must improve the coordination of research and development efforts in addition to cultivating the funding sources for the development of these new treatments.

Methodological advances in drug discovery for Chagas disease.

Chagas disease is an infectious disease with the highest impact in Latin America and a growing worldwide problem. Chagas disease is the result of long-term, persistent infection with the protozoan parasite Trypanosoma cruzi. The current therapies for treating T. cruzi infection and thus preventing Chagas disease often have adverse effects, unpredictable efficacy and require long courses of treatment. Development of new therapies has been very limited, in part due to lack of interest but also as a result of poor support and inappropriate models for discovering and evaluating candidate drugs. The Chagas Drug Discovery Consortium (CDDC) was created with funding from the US National Institutes of Health to help address some of these issues. The goals of the CDDC are to discover and evaluate new candidate drugs and develop rigorous assays of drug efficacy. This report summarizes the second meeting of the CDDC in November 2010.

Report of the 2nd Chagas Drug Discovery Consortium meeting, held on 3 November 2010; Atlanta GA, USA.

The development of treatment protocols with reduced toxicity and equivalent or improved efficacy for Trypanosoma cruzi infection is a priority. We tested the effectiveness of benznidazole (BZ), nifurtimox (NFX), other prospective drugs in intermittent and combined treatment protocols to cure T. cruzi infection initiated with susceptible and drug-resistant parasite strains. A 40-day course of BZ, NFX, or the oxaborale AN4169 cured 100% of mice, whereas posaconazole (POS), and NTLA-1 (a nitro-triazole) cured approximately 90% and 20% of mice, respectively. Reducing the overall dosage of BZ or NFX by using an intermittent (once every 5 days) schedule or combining 5 daily doses of POS with 7 intermittent doses of BZ also provided approximately 100% cure. T. cruzi strains resistant to BZ were also found to be resistant to other drugs (POS), and extending the time of treatment or combining drugs did not increase cure rates with these isolates. Thus, dosing schedules for anti-T. cruzi compounds should be determined empirically, and compounds targeting different pathways may be combined to yield effective therapies with reduced toxicity. This work also suggests that standard treatment protocols using BZ and NFX may be significantly overdosing patients, perhaps contributing to the adverse events. 

New, combined, and reduced dosing treatment protocols cure Trypanosoma cruzi infection in mice.

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PPi ) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF-hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PPi ) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP-OE) resulted in a significant decrease in cytosolic PPi , and short and long-chain polyP levels. Additionally, the TcVSP-OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PPi and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection. 

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence.

Trypanosoma cruzi infection drives the expansion of remarkably focused CD8(+) T cell responses targeting epitopes encoded by variant trans-sialidase (TS) genes. Infection of C57BL/6 mice with T. cruzi results in up to 40% of all CD8(+) T cells committed to recognition of the dominant TSKB20 and subdominant TSKB18 TS epitopes. However, despite this enormous response, these mice fail to clear T. cruzi infection and subsequently develop chronic disease. One possible reason for the failure to cure T. cruzi infection is that immunodomination by these TS-specific T cells may interfere with alternative CD8(+) T cell responses more capable of complete parasite elimination. To address this possibility, we created transgenic mice that are centrally tolerant to these immunodominant epitopes. Mice expressing TSKB20, TSKB18, or both epitopes controlled T. cruzi infection and developed effector CD8(+) T cells that maintained an activated phenotype. Memory CD8(+) T cells from drug-cured TSKB-transgenic mice rapidly responded to secondary T. cruzi infection. In the absence of the response to TSKB20 and TSKB18, immunodominance did not shift to other known subdominant epitopes despite the capacity of these mice to expand epitope-specific T cells specific for the model antigen ovalbumin expressed by engineered parasites. Thus, CD8(+) T cell responses tightly and robustly focused on a few epitopes within variant TS antigens appear to neither contribute to, nor detract from, the ability to control T. cruzi infection. These data also indicate that the relative position of an epitope within a CD8(+) immunodominance hierarchy does not predict its importance in pathogen control.

Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominant trans-Sialidase-Specific CD8+ T Cells.

Trypanosomatids (order Kinetoplastida), including the human pathogens  (agent of Chagas disease), , (African sleeping sickness), and  (leishmaniasis), affect millions of people and animals globally.  is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from  (SaCas9), but not from the more routinely used  Cas9 (SpCas9), and -transcribed single guide RNAs (sgRNAs) results in rapid gene edits in  and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of , as well as in  and  RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens. Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in  and  spp., that have eluded investigators for decades.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a debilitating condition that affects over 10 million humans in the American continents. In addition to its traditional mode of human entry via the "kissing bug" in endemic areas, the infection can also be spread in non-endemic countries through blood transfusion, organ transplantation, eating food contaminated with the parasites, and from mother to fetus. Previous NMR-based studies established that the parasite expresses a variety of strain-specific and developmentally-regulated O-glycans that may contribute to virulence. In this report, we describe five synthetic O-glycan analytical standards and show their potential to enable a more facile analysis of native O-glycan isomers based on mass spectrometry.

Trypanosoma cruzi 13C-labeled O-Glycan standards for mass spectrometry.

Malaria, leishmaniasis and trypanosomiasis are arthropod-borne, parasitic diseases that constitute a major global health problem. They are generally found in developing countries, where lack of access to preventive tools and treatment hinders their management. Because these parasites share an increased demand on glucose consumption with most cancer cells, six compounds used in anti-tumoral research were selected to be tested as antiparasitic agents in in vitro models of Leishmania infantum, Trypanosoma brucei, T. cruzi, and Plasmodium falciparum: dichloroacetic acid (DCA), 3-bromopyruvic acid (3BP), 2-deoxy-D-glucose (2DG), lonidamine (LND), metformin (MET), and sirolimus (SIR). No parasite-killing activity was found in L. infantum promastigotes, whereas DCA and 3BP reduced the burden of intra-macrophagic amastigotes. For T. brucei all selected compounds, but 2DG, decreased parasite survival. DCA, 2DG, LND and MET showed parasite-killing activity in T. cruzi. Finally, anti-plasmodial activity was found for DCA, 2DG, LND, MET and SIR. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with ATP production, proliferation, and survival cell strategies might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing.

Repurposing bioenergetic modulators against protozoan parasites responsible for tropical diseases.

A major contributor to treatment failure in Chagas disease, caused by infection with the protozoan parasite , is that current treatment regimens do not address the drug insensitivity of transiently dormant  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  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  may not be fully resistant to trypanocidal compounds such as benznidazole. Collectively, these studies provide important information on the phenomenon of dormancy in  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.

A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease.

Chagas disease, caused by the parasite , affects more than six million people worldwide, with its greatest burden in Latin America. Available treatments present frequent toxicity and variable efficacy at the chronic phase of the infection, when the disease is usually diagnosed. Hence, development of new therapeutic strategies is urgent. Repositioning of licensed drugs stands as an attractive fast-track low-cost approach for the identification of safer and more effective chemotherapies. With this purpose we screened 32 licensed drugs for different indications against . We used a primary in vitro assay of Vero cells infection by . Five drugs showed potent activity rates against it (IC < 4 µmol L), which were also specific (selectivity index >15) with respect to host cells.  inhibitory activity of four of them was confirmed by a secondary anti-parasitic assay based on NIH-3T3 cells. Then, we assessed toxicity to human HepG2 cells and anti-amastigote specific activity of those drugs progressed. Ultimately, atovaquone-proguanil, miltefosine, and verapamil were tested in a mouse model of acute  infection. Miltefosine performance in vitro and in vivo encourages further investigating its use against .

Article	Year
Treatment with benznidazole or thioridazine in the chronic phase of experimental Chagas disease improves cardiopathy. International journal of antimicrobial agents\| PubMed ID: 17395432	2007
Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease. Nature medicine\| PubMed ID: 18425131	2008
CD8+ T cells in Trypanosoma cruzi infection. Current opinion in immunology\| PubMed ID: 19646853	2009
In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds. PLoS neglected tropical diseases\| PubMed ID: 20644616	2010
Oral exposure to Trypanosoma cruzi elicits a systemic CD8⁺ T cell response and protection against heterotopic challenge. Infection and immunity\| PubMed ID: 21628516	2011
Methodological advances in drug discovery for Chagas disease. Expert opinion on drug discovery\| PubMed ID: 21712965	2011
Report of the 2nd Chagas Drug Discovery Consortium meeting, held on 3 November 2010; Atlanta GA, USA. Expert opinion on drug discovery\| PubMed ID: 22646217	2011
New, combined, and reduced dosing treatment protocols cure Trypanosoma cruzi infection in mice. The Journal of infectious diseases\| PubMed ID: 23945371	2014
Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence. Molecular microbiology\| PubMed ID: 24033456	2013
Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominant trans-Sialidase-Specific CD8+ T Cells. Infection and immunity\| PubMed ID: 27354447	2016
Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins. mBio\| PubMed ID: 29114029	2017
Trypanosoma cruzi 13C-labeled O-Glycan standards for mass spectrometry. Glycobiology\| PubMed ID: 30649355	2019
Repurposing bioenergetic modulators against protozoan parasites responsible for tropical diseases. International journal for parasitology. Drugs and drug resistance\| PubMed ID: 32829099	2020
A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease. Science translational medicine\| PubMed ID: 33115952	2020
Identification of Growth Inhibitors with Activity In Vivo within a Collection of Licensed Drugs. Microorganisms\| PubMed ID: 33669310	2021

Juan M. Bustamante

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