The long and winding road of reverse genetics in Trypanosoma cruzi

Chiurillo, Miguel Ángel; Lander, Noelia

doi:10.15698/mic2021.09.758

Cited by 10 publications

(10 citation statements)

References 41 publications

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“…This ability to make endocytic‐null mutants, it turns out, may be extremely fortuitous, as the lack of a conditional knockdown system in T . cruzi continues to make the study of essential gene function exceedingly difficult (Chiurillo & Lander, 2021). This, therefore, opens up the possibility of using CRISPR/Cas9 gene‐editing tools to begin ablating SPC components to directly assess their role in the endocytic process without compromising parasite fitness (Lander et al, 2017, 2019; Peng et al, 2014).…”

Section: How Is Cargo Propelled Down the Spc? Speculations On The Ide...mentioning

confidence: 99%

Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome‐cytopharynx complex of Trypanosoma cruzi

Etheridge

2022

J Eukaryotic Microbiology

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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.

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Section: How Is Cargo Propelled Down the Spc? Speculations On The Ide...mentioning

confidence: 99%

Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome‐cytopharynx complex of Trypanosoma cruzi

Etheridge

2022

J Eukaryotic Microbiology

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“…In this work we have provided evidence demonstrating the specific disruption of the TcP21 locus in KO cell lines following a CRISPR/Cas9 strategy that has been successfully used in several studies (reviewed in Chiurillo and Lander, 2021). To further investigate the physiological function of TcP21 and its role in host cell infection, complementation studies should be performed by restoring the WT P21 gene in KO parasites to confirm and validate our results.…”

Section: Discussionmentioning

confidence: 62%

Ablation of the P21 Gene of Trypanosoma cruzi Provides Evidence of P21 as a Mediator in the Control of Epimastigote and Intracellular Amastigote Replication

Teixeira

Chiurillo

Lander

et al. 2022

Front. Cell. Infect. Microbiol.

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P21 is an immunomodulatory protein expressed throughout the life cycle of Trypanosoma cruzi, the etiologic agent of Chagas disease. In vitro and in vivo studies have shown that P21 plays an important role in the invasion of mammalian host cells and establishment of infection in a murine model. P21 functions as a signal transducer, triggering intracellular cascades in host cells and resulting in the remodeling of the actin cytoskeleton and parasite internalization. Furthermore, in vivo studies have shown that P21 inhibits angiogenesis, induces inflammation and fibrosis, and regulates intracellular amastigote replication. In this study, we used the CRISPR/Cas9 system for P21 gene knockout and investigated whether the ablation of P21 results in changes in the phenotypes associated with this protein. Ablation of P21 gene resulted in a lower growth rate of epimastigotes and delayed cell cycle progression, accompanied by accumulation of parasites in G1 phase. However, P21 knockout epimastigotes were viable and able to differentiate into metacyclic trypomastigotes, which are infective to mammalian cells. In comparison with wild-type parasites, P21 knockout cells showed a reduced cell invasion rate, demonstrating the role of this protein in host cell invasion. However, there was a higher number of intracellular amastigotes per cell, suggesting that P21 is a negative regulator of amastigote proliferation in mammalian cells. Here, for the first time, we demonstrated the direct correlation between P21 and the replication of intracellular amastigotes, which underlies the chronicity of T. cruzi infection.

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“…By taking advantage of the fact that T. cruzi is both genetically tractable and does not require SPC function for in vitro growth we can examine the molecular components of this organelle without negatively impacting parasite fitness in culture. This, in the end, is a fortuitous finding since T. cruzi continues to lack the necessary genetic tools to study the function of essential genes (47). An array of important questions regarding the formation and regulation of the SPC organelle clearly still remain and future studies will no doubt continue to provide important insight into the molecular mechanisms undergirding this fundamental process of protozoan phagotrophy.…”

Section: Discussionmentioning

confidence: 99%

Endocytosis inTrypanosoma cruziDepends on Proper Recruitment and Regulation of Functionally Redundant Myosin Motors

Chasen

Etheridge

Campbell

et al. 2022

Preprint

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The cytostome-cytopharynx complex (SPC) is an endocytic organelle absent from all human trypanosomatid pathogens save Trypanosoma cruzi. Building upon our previous work identifying the myosin motor MyoF as the first enzymatic component of the T. cruzi SPC, we sought to expand our understanding of this organelle by identifying additional protein machinery which contribute to endocytosis. While deletion of MyoF alone did not fully ablate endocytosis, we found that deletion of both MyoF and the similarly localized MyoC produced an endocytic-null phenotype that was rescued upon complementation. To identify potential regulatory components of this motor complex, we pulled down MyoF and identified an SPC-targeted protein that was conserved across a wide range of protozoan lineages. Deletion of this myosin associated protein (MyAP) alone was sufficient to produce an endocytic-null phenotype, which we were able to fully rescue via complementation. The deletion of MyAP also caused the mis-localization of both cytopharynx myosins to the cytosol. While MyAP lacking the EF-hand domain was unable to complement endocytosis, it was sufficient to restore proper myosin localization. This suggested that MyAP plays two distinct roles, one in targeting myosins to the SPC and a second in regulating myosin motor activity. Lipidomic analysis subsequently revealed a dramatic reduction in the scavenged cholesterol content in the endocytic-null mutants. Overall, this work showcases the first viable endocytic-null mutants generated in T. cruzi through specific gene deletion and highlights the feasibility of leveraging this strategy towards a full dissection of the endocytic machinery and biogenesis of the SPC.

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The long and winding road of reverse genetics in Trypanosoma cruzi

Cited by 10 publications

References 41 publications

Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome‐cytopharynx complex of Trypanosoma cruzi

Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome‐cytopharynx complex of Trypanosoma cruzi

Ablation of the P21 Gene of Trypanosoma cruzi Provides Evidence of P21 as a Mediator in the Control of Epimastigote and Intracellular Amastigote Replication

Endocytosis inTrypanosoma cruziDepends on Proper Recruitment and Regulation of Functionally Redundant Myosin Motors

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