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

9 142 changes in protein levels imposed by altered transcription or mRNA stability (24). Interestingly, 143 a large subset of transcripts whose abundance changed upon infection with L. donovani 144 amastigotes or promastigotes was buffered at the level of translation (21% out of 1,051 and 29% 145 out of 1,604 mRNAs, respectively) (Fig 1D and S1 Table) . In contrast, only a small number of 146 transcripts (71 mRNAs) whose levels differed between L. donovani promastigote-and 147 amastigote-infected BMDMs (S1B-C Figs) were translationally buffered. These data indicate (Fig 2A and S2 Table) . Enriched categories, among 157 proteins encoded by translationally activated transcripts in BMDMs infected by either parasite 158 life stage, included chromatin remodeling, regulation of mRNA metabolism (i.e. splicing, export 159 from the nucleus, stability and translation), regulation of type I IFN production and protein 160 deubiquitination (Fig 2A, 228 Translation of eIF4A-sensitive mRNAs is activated upon L. donovani infection 229 As mentioned above, the RNA helicase eIF4A facilitates translation of transcripts harboring long 230 and highly structured 5' UTR sequences. Some such encoded proteins are involved in tumor 231 immune evasion (36) and progression of viral (37) and protozoan parasitic infections (12), 232 suggesting that eIF4A-dependent translation may contribute to herein observed changes in 233 translational efficiencies (Fig 1) . In addition to eIF4F-complex formation, the unwinding activity . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 13 234 of eIF4A is enhanced by the translation initiation factor eIF4B (14). Consistent with eIF4B-235 dependent modulation of eIF4A activity, levels of phosphorylated and total eIF4B protein were 236 increased in BMDMs infected with L. donovani amastigotes or promastigotes (Fig 5A) . To test 237 whether this may contribute to selective regulation of mRNA translation following parasite 238 infection, we assessed translational efficiencies of a compilation of previously described eIF4A-239 sensitive transcripts (36, 38-40). Indeed, following infection independently of parasite stage, the 240 translational efficiencies of such mRNAs were elevated as compared to background transcripts 241 (p <0.001) (Fig 5B) . From a total of 1198 previously described eIF4A-sensitive mRNAs, 149 242 were translationally activated whereas 80 were translationally suppressed upon infection with 243 promastigotes or amastigotes of L. donovani (S3B Table) . The presence of a 5' UTR G-244 quadruplex-forming guanine quartet (CGG) 4 motif is an indirect approach to assess whether 245 transcripts are expected to be more dependent of eIF4A for their translation (41). Indeed, 246 analysis of Motif Enrichment (AME) revealed a significant enrichment of the (CGG) 4 motif in 5' 247 UTRs of transcripts with highly activated translation (≥4-fold increase in translational efficiency 248 upon infection) as compared to 5' UTRs from transcripts with unaltered translational efficiency 249 (p = 0.0036) (Fig 5C) . TGF-β is a key cytokine implicated in the distinctive immune suppression 250 that follows L. donovani infection in vivo (42, 43) . Upon infection with L. donovani amastigotes 251 or promastigotes, anota2seq analysis identified augmented translational efficiency of the