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

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.

The ability of the Chagas disease agent  to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy state in  that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of  to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.

Spontaneous dormancy protects  during extended drug exposure.

Chagas disease agent, Trypanosoma cruzi, is capable to persist after prolonged drug treatment using effective drugs. The reason of treatment failure is not known, but recent development of highly sensible bioluminescence imaging coupled to tissue clarification techniques has made possible the detection of individual amastigotes within chronically infected murine tissues and the study of their replicative status. In this chapter, we provide a step-by-step explanation for these protocols that allowed the visualization of nonproliferating amastigotes in tissues of chronically infected mice for the first time.

In Situ Detection of Dormant Trypanosoma cruzi Amastigotes Using Bioluminescent-Fluorescent Reporters.

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.

Article	Year
Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins. mBio\| PubMed ID: 29114029	2017
Spontaneous dormancy protects during extended drug exposure. eLife\| PubMed ID: 29578409	2018
In Situ Detection of Dormant Trypanosoma cruzi Amastigotes Using Bioluminescent-Fluorescent Reporters. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30868527	2019
A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease. Science translational medicine\| PubMed ID: 33115952	2020

Fernando Sanchez-Valdez

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