Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

ABSTRACT The minute virus of mice, an autonomous parvovirus, requires entry of host cells into the S phase of the cell cycle for its DNA to be amplified and its genes expressed. This work focuses on the P4 promoter of this parvovirus, which directs expression of the transcription unit encoding the parvoviral nonstructural polypeptides. These notably include protein NS1, necessary for the S-phase-dependent burst of parvoviral DNA amplification and gene expression. The activity of the P4 promoter is shown to be regulated in a cell cycle-dependent manner. At the G1/S-phase transition, the promoter is activated via a cis-acting DNA element which interacts with phase-specific complexes containing the cellular transcription factor E2F. It is inhibited, on the other hand, in cells arrested in G1 due to contact inhibition. This inhibitory effect is not observed in serum-starved cells. It is mediated in cis by cyclic AMP response elements (CREs). Unlike serum-starved cells, confluent cells accumulate the cyclin-dependent kinase inhibitor p27, suggesting that the switch from CRE-mediated activation to CRE-mediated repression involves the p27 protein. Accordingly, plasmid-driven overexpression of p27 causes down-modulation of promoter P4 in growing cells, depending on the presence of at least two functional CREs. No such effect is observed with two other cyclin-dependent kinase inhibitors, p16 and p21. Given the importance of P4-driven synthesis of protein NS1 in parvoviral DNA amplification and gene expression, the stringent S-phase dependency of promoter P4 is likely a major determinant of the absolute requirement of the minute virus of mice for host cell proliferation.

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An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009994 ◽

2021 ◽

Vol 15 (11) ◽

pp. e0009994

Author(s):

Roberto I. Cuevas-Hernández ◽

Richard M. B. M. Girard ◽

Luka Krstulović ◽

Miroslav Bajić ◽

Ariel Mariano Silber

Keyword(s):

Cell Cycle ◽

Trypanosoma Cruzi ◽

Host Cells ◽

Mammalian Host ◽

Cycle Arrest ◽

Mitochondrial Inner Membrane ◽

Intracellular Ca ◽

Low Toxicity ◽

Plasma Membrane Permeabilization ◽

Chimeric Molecules

Trypanosoma cruzi is a hemoflagellated parasite causing Chagas disease, which affects 6–8 million people in the Americas. More than one hundred years after the description of this disease, the available drugs for treating the T. cruzi infection remain largely unsatisfactory. Chloroquinoline and arylamidine moieties are separately found in various compounds reported for their anti-trypanosoma activities. In this work we evaluate the anti-T. cruzi activity of a collection of 26 “chimeric” molecules combining choroquinoline and amidine structures. In a first screening using epimastigote forms of the parasite as a proxy for the clinically relevant stages, we selected the compound 7-chloro-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]quinoline (named here as A6) that performed better as an anti-T. cruzi compound (IC50 of 2.2 ± 0.3 μM) and showed a low toxicity for the mammalian cell CHO-K1 (CC50 of 137.9 ± 17.3 μM). We initially investigated the mechanism of death associated to the selected compound. The A6 did not trigger phosphatidylserine exposure or plasma membrane permeabilization. Further investigation led us to observe that under short-term incubations (until 6 hours), no alterations of mitochondrial function were observed. However, at longer incubation times (4 days), A6 was able to decrease the intracellular Ca2+, to diminish the intracellular ATP levels, and to collapse mitochondrial inner membrane potential. After analysing the cell cycle, we found as well that A6 produced an arrest in the S phase that impairs the parasite proliferation. Finally, A6 was effective against the infective forms of the parasite during the infection of the mammalian host cells at a nanomolar concentration (IC50(tryps) = 26.7 ± 3.7 nM), exhibiting a selectivity index (SI) of 5,170. Our data suggest that A6 is a promising hit against T. cruzi.

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Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

Virology Journal ◽

10.1186/1743-422x-4-52 ◽

2007 ◽

Vol 4 (1) ◽

pp. 52 ◽

Cited By ~ 9

Author(s):

Susan D Wharam ◽

Matthew J Hall ◽

William H Wilson

Keyword(s):

Gene Expression ◽

Nucleic Acid ◽

Nucleic Acid Amplification ◽

Host Cells ◽

Infected Host ◽

Isothermal Nucleic Acid Amplification ◽

Nucleic Acid Amplification Assay ◽

Amplification Assay ◽

Synechococcus Sp

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An Aromatic Diamidine That Targets Kinetoplast DNA, Impairs the Cell Cycle in Trypanosoma cruzi, and Diminishes Trypomastigote Release from Infected Mammalian Host Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01595-15 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5867-5877 ◽

Cited By ~ 10

Author(s):

Richard M. B. M. Girard ◽

Marcell Crispim ◽

Ivana Stolić ◽

Flávia Silva Damasceno ◽

Marcelo Santos da Silva ◽

...

Keyword(s):

Cell Cycle ◽

Trypanosoma Cruzi ◽

Nuclear Dna ◽

Host Cells ◽

Mammalian Host ◽

Kinetoplast Dna ◽

Content Type ◽

Parasite Cell ◽

Infection Index ◽

Dependent Inhibition

ABSTRACTTrypanosoma cruziis the etiological agent of Chagas disease, affecting approximately 10 million people in the Americas and with some 40 million people at risk. The objective of this study was to evaluate the anti-T. cruziactivity of three new diamidines that have a 3,4-ethylenedioxy extension of the thiophene core, designated MB17, MB19, and MB38. All three diamidines exhibited dose-dependent inhibition of epimastigote replication. The mechanisms of action of these diamidines were investigated. Unlike MB17 and MB19, MB38 exhibited a significant increase in the number of annexin-propidium iodide double-labeled cells compared to levels in control parasites. As MB17 had shown a lower 50% inhibitory concentration (IC50) against epimastigote growth, the mechanism of action of this drug was studied in more detail. MB17 triggered a decrease in the intracellular ATP levels. As a consequence, MB17 affected the genomic DNA and kinetoplast DNA (kDNA) and impaired the parasite cell cycle. Moreover, MB17 caused DNA fragmentation, with a more severe effect on kDNA than on nuclear DNA, resulting in dyskinetoplastic cells. MB17 was tested for toxicity and effectiveness for the treatment of infected CHO-K1cells, exhibiting a 50% cytotoxic concentration (CC50) of 13.47 ± 0.37 μM and an IC50of 0.14 ± 0.12 μM against trypomastigote release. MB17 also diminished the infection index by 60% at 0.5 μM. In conclusion, despite belonging to the same family, these diamidines have different efficiencies. To summarize, MB17 was the most potent of these diamidines against epimastigotes, producing DNA damage preferentially in kDNA, impairing the parasite cell cycle, and decreasing the infection index and trypomastigote release from infected mammalian host cells, with a high selectivity index (SI) (<90). These data suggest that MB17 could be an interesting lead compound againstT. cruzi.

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