Translational profiling of macrophages infected with Leishmania donovani identifies mTOR- and eIF4A-sensitive immune-related transcripts

Visnu Chaparro; Louis-Philippe Leroux; Laia Masvidal; Julie Lorent; Tyson E. Graber; Aude Zimmermann; Guillermo Arango Duque; Albert Descoteaux; Tommy Alain; Ola Larsson; Maritza Jaramillo

256 donovani-infected BMDMs (Fig 5D) without affecting Tgfb1 mRNA levels (Fig 5E) . Of note, . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2019.12.20.884338 doi: bioRxiv preprint 14 257 no acute toxicity was detected in BMDMs and extracellular parasites exposed to silvestrol (S4 273 Interestingly, parasite numbers increased in presence of rapamycin at 24 h post-infection (~92% 274 increase compared to DMSO control) (Fig 6A) whereas the opposite effect was observed upon 275 cell exposure to silvestrol (~57% reduction compared to DMSO control) (Fig 6B) . These data 276 indicate that mTOR limits L. donovani persistence within the host cell while eIF4A promotes it. Table. 425 426 Quantitative RT-PCR 427 Purified RNA (500 ng) was reverse transcribed using the Superscript IV VILO Master Mix 428 (Invitrogen). Quantitative PCR was performed with PowerUp™ SYBR ® Green Master Mix 429 (Applied Biosystems). Relative quantification was calculated using the comparative Ct method 430 (Ct) (61) and relative expression was normalized to mouse Actb. Experiments were performed 431 in independent biological replicates (n=3); each sample was analyzed in a technical triplicate, the 432 average of which was plotted against the respective conditions used. Primers were designed 433 using NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (S4 Table) . The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2019.12.20.884338 doi: bioRxiv preprint 26 507 Where applicable, data are presented as the mean ± standard deviation (SD) of the mean. 508 Statistical significance was determined by using one-way ANOVA followed by a Tukey post-509 hoc test; calculations were performed by using Prism 7 software package (GraphPad). 510 Differences were considered significant when *p < 0.05, ** p < 0.01, *** p < 0.001.