Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection

Pacheco-Lugo, Lisandro; Díaz-Olmos, Yirys; Saenz-Garcia, Jose L.; Probst, Christian M.; DaRocha, Wanderson D.

doi:10.1016/j.parint.2017.01.019

Cited by 23 publications

(18 citation statements)

References 19 publications

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“…CL Brener epimastigotes (3–5 × 10 7 parasites ml −1 ) were first transfected with pLEW13 (Wirtz et al, ) or the derivative p Tc rRNA‐T7Tet (Taylor & Kelly, ) using the Nucleofector 2b (Amaxa) X‐014 setting: 10 8 cells were washed with phosphate‐buffered saline (PBS) and resuspended in 100 μl of transfection buffer (Pacheco‐Lugo, Díaz‐Olmos, Sáenz‐García, Probst, & DaRocha, ), mixed with ~30 μg of plasmid DNA and submitted to electroporation, after which cells were transferred into 15 ml of LIT medium with 15% of FBS and diluted in a 24‐well plate. After 24 hr, geneticin (G418) was added at a concentration of 100 μg/ml to obtain semi‐clonal cultures.…”

Section: Methodsmentioning

confidence: 99%

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

Ramirez-Barrios

Susa

Smoniewski

et al. 2020

Molecular Microbiology

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The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalianinfectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism. K E Y W O R D S gene expression, life cycle stages, organelles, ribonuclease, RNA editing, trypanosomiasis S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Ramirez-Barrios R, Susa EK, Smoniewski CM, Faacks SP, Liggett CK, Zimmer SL. A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi. Mol Microbiol.

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Section: Methodsmentioning

confidence: 99%

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

Ramirez-Barrios

Susa

Smoniewski

et al. 2020

Molecular Microbiology

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“…Highest transfection efficiency, 7.4 ±0.8%, was achieved by X-14 program, whereas maximum survival rate was observed with X-01 and X-06 programs (S2 Table). We selected X-14 program as our standard protocol for EA transfection, as it is also suited for epimastigote transfection[38].…”

Section: Resultsmentioning

confidence: 99%

Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi

et al. 2019

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Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.

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“…Trypanosoma cruzi wild type Y epimastigotes were cultured in medium containing liver and infusion tryptose (LIT) supplemented with 10% fetal bovine serum at 28 • C. The parasites expressing Cas9 were generated by transfection with plasmid pTREX-Cas9-Neo (Lander et al, 2015), selected and maintained in the same medium supplemented with 200 µg/mL Geneticin G418. For transfection, 4 × 10 7 exponentially growing Y-Cas9 epimastigotes were collected and centrifuged at 2,000 g for 5 min, parasites were washed in 1 mL of electroporation buffer (5 mM KCl, 0.15 mM CaCl 2 , 90 mM Na 2 HPO 4 , 50 mM HEPES, 50 mM Mannitol) (Pacheco-Lugo et al, 2017) and resuspended in 100 µL of electroporation buffer, along with 50 µL containing 2 µg of purified sgRNA and 25 µg of purified PCR fragments containing the BSD sequence. Parasites were transfected in 2 mm cuvettes using AMAXA Nucleofactor II apparatus, with two pulses of X-014 program.…”

Section: Generation Of Mutated Parasitesmentioning

confidence: 99%

GCN2-Like Kinase Modulates Stress Granule Formation During Nutritional Stress in Trypanosoma cruzi

Malvezzi

Aricó

Souza-Melo

et al. 2020

Front. Cell. Infect. Microbiol.

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The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (Tck1, Tck2, and Tck3) are found in the Trypanosoma cruzi genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several T. cruzi strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of T. cruzi devoid of TcK1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative T. cruzi epimastigotes depleted of TcK1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in TcK1 depleted line. However, eIF2α is still phosphorylated in TcK1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of Tck1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the TcK1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in T. cruzi affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.

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Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection

Cited by 23 publications

References 19 publications

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi

GCN2-Like Kinase Modulates Stress Granule Formation During Nutritional Stress in Trypanosoma cruzi

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