Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

Abstract

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.

Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors against Trypanosoma cruzi: implications for Chagas disease drug discovery and development

“…U2OS cells grow as a monolayer and present a large cytoplasm that allow for improved quantification of T. cruzi amastigotes in high content analysis46. Infected cultures were exposed to compounds for 4 days, except for Dm28c, which were exposed to compounds for 3 days.…”

A Genome-Wide siRNA Screen to Identify Host Factors Necessary for Growth of the Parasite Toxoplasma gondii

“…Host cell molecules responsible for Mycobacterium tuberculosis intracellular survival and regulation of bacterial load in macrophages were identified using large-scale siRNA screens [10], [11]. A genome-wide siRNA screen identified 162 genes important for growth and persistence of the parasite Trypanosoma cruzi [12]. siRNA silencing of the human hepatoma cell kinome during Plasmodium berghei sporozoite infection identified five proteins involved in parasite replication [13].…”

High-Content Assay for Measuring Intracellular Growth ofLeishmaniain Human Macrophages

“…High-throughput assays are also increasingly being used to undertake genome-wide siRNA screens to identify host genes that impact on the virulence of intracellular pathogens. [30][31][32][33][34] In this study, we have established a novel vital staining protocol for tracking the proliferation of live amastigotes in THP-1 macrophages.…”

“…After 24 h, the medium was aspirated using a BioTek EL 406 liquid handling robot and replaced with pre-stained amastigotes at an MOI of 1, 3, 5, 10, 30, 50 (well volume made up to 50 mL with RPMI supplemented with 10% iFCS); higher MOIs (30,50) were dispensed at a five-fold greater cell concentration. Following a 24-h incubation (33°C, 5% CO 2 , 95% humidity), wells were washed with PBS and fresh medium was supplied ( Table 2, steps 5-7).…”