Salmonella systemic infections claim thousands of lives worldwide even today. Certain cases lead to an infection in the brain culminating in meningitis and associated neurological abnormalities. Multiple reports have indicated neurological manifestations in patients suffering from typhoid fever during the course of infection and afterwards. While the meanderings of Salmonella systemic infections are fairly well studied, the flow of events in the brain is very poorly understood. We investigated the colonization of various brain parts by Salmonella in mice. It was observed that the bacterium is frequently able to invade various brain parts in mice. Selected mutants namely deletion mutants of key proteins encoded by the Salmonella pathogenicity islands (SPIs) 1 and 2 and ompA gene were also used to decipher the roles of specific genes in establishing an infection in the brain. Our results suggest roles for the Salmonella pathogenicity island (SPI) 1 and outer membrane protein A gene in enabling blood-brain barrier penetration by the pathogen. We further investigated behavioral abnormalities in infected mice and used an antibiotic treatment regime in an attempt to reverse the same. Results show some mice still display behavioral abnormalities and a high bacterial burden in brain despite clearance from spleen and liver. Overall, our study provides novel insights into S. Typhimurium's capacity to invade the mouse brain and the effectiveness of antibiotic treatment on behavioral manifestations due to infection. These observations could have important implications in understanding reported neurological manifestations in typhoid patients.

Targeting the translation initiation complex eIF4F, which binds the 5′ cap of mRNAs, is a promising anti-cancer approach. Silvestrol, a small molecule inhibitor of eIF4A, the RNA helicase component of eIF4F, inhibits the translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor, which, in turn, reduces the transcription of the gene encoding one of the major immune checkpoint proteins, i.e., programmed death ligand-1 (PD-L1) in melanoma cells. A large proportion of human genes produce multiple mRNAs differing in their 3′-ends through the use of alternative polyadenylation (APA) sites, which, when located in alternative last exons, can generate protein isoforms, as in the STAT1 gene. Here, we provide evidence that the STAT1α, but not STAT1β protein isoform generated by APA, is required for silvestrol-dependent inhibition of PD-L1 expression in interferon-γ-treated melanoma cells. Using polysome profiling in activated T cells we find that, beyond STAT1, eIF4A inhibition downregulates the translation of some important immune-related mRNAs, such as the ones encoding TIM-3, LAG-3, IDO1, CD27 or CD137, but with little effect on the ones for BTLA and ADAR-1 and no effect on the ones encoding CTLA-4, PD-1 and CD40-L. We next apply RT-qPCR and 3′-seq (RNA-seq focused on mRNA 3′ ends) on polysomal RNAs to analyze in a high throughput manner the effect of eIF4A inhibition on the translation of APA isoforms. We identify about 150 genes, including TIM-3, LAG-3, AHNAK and SEMA4D, for which silvestrol differentially inhibits the translation of APA isoforms in T cells. It is therefore crucial to consider 3′-end mRNA heterogeneity in the understanding of the anti-tumor activities of eIF4A inhibitors.

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The recognition of polyadenylation signals (PAS) in eukaryotic pre-mRNAs is usually coupled to transcription termination, occurring while pre-mRNA is chromatin-bound. However, for some pre-mRNAs, this 3 0 -end processing occurs post-transcriptionally, i.e., through a co-transcriptional cleavage (CoTC) event downstream of the PAS, leading to chromatin release and subsequent PAS cleavage in the nucleoplasm. While DNA-damaging agents trigger the shutdown of co-transcriptional chromatin-associated 3 0 -end processing, specific compensatory mechanisms exist to ensure efficient 3 0 -end processing for certain pre-mRNAs, including those that encode proteins involved in the DNA damage response, such as the tumor suppressor p53. We show that cleavage at the p53 polyadenylation site occurs in part post-transcriptionally following a cotranscriptional cleavage event. Cells with an engineered deletion of the p53 CoTC site exhibit impaired p53 3 0 -end processing, decreased mRNA and protein levels of p53 and its transcriptional target p21, and altered cell cycle progression upon UV-induced DNA damage. Using a transcriptome-wide analysis of PAS cleavage, we identify additional pre-mRNAs whose PAS cleavage is maintained in response to UV irradiation and occurring posttranscriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows certain pre-mRNAs to escape 3 0 -end processing inhibition in response to UV-induced DNA damage.

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Background: Eukaryotic initiation factor 4F (eIF4F) complex plays a pivotal role in the selective translational control of protein expression in cancer. We previously showed that the inhibition of eIF4F had both a direct antitumor effect that could overcome resistance to BRAF inhibition in BRAFV600 melanoma and an indirect immune-mediated antitumor effect via PD-L1 down regulation resulting from STAT1 decreased translation. We now explore further the translational control of immune checkpoints (ICs) on T cell and its link with lymphocyte exhaustion. Methods: We performed polysome profiling (PP) on peripheral blood-derived activated and resting T lymphocytes as well as tumor-infiltrating lymphocytes (TILs) isolated from melanoma specimens in the presence or absence of silvestrol as an eIF4A (the helicase component of eIF4F) inhibitor. We studied the expression of ICs and exhaustion-associated transcription factors on lymphocytes at the various stages of exhaustion. The tumor cell lysing capacity of PBMC was evaluated with and without silvestrol. We also assessed the effect of silvestrol on tumor microenvironment in the BP syngeneic melanoma tumor model. Results: Silvestrol decreased the translation of multiple ICs in activated CD4+ and CD8+ T cells, including TOX, NFAT1, NFAT2, LAG3, and TIM3 (the two latter being also transcriptionally regulated). FACS analysis confirmed the dose-dependent decrease of protein expression of exhaustion-associated transcription factors TOX, NFATc1, TCF1 and T-bet in activated lymphocytes. This effect was more prominent in more exhausted T cells. Silvestrol decreased T cell proliferation but, in co-culture experiments, it increased immune cell killing of melanoma cells by T-lymphocytes. In the BP murine model, the antitumor effect of silvestrol was associated with denser T cell infiltration and less exhausted TILs than control tumors. Conclusion: Our findings demonstrate the role of eIF4F in the orchestrated regulation of functionally related proteins involved in T cell exhaustion. They highlight the drugability of eIF4A for the cancer treatment and suggest that eIF4A inhibitors could be useful with IC inhibitors in combination or sequentially. Citation Format: Samad Muhammadnejad, Biswendu Biswas, Ramdane Guemiri, Naima Benannoune, Jérémy Lavigne, Virginie Quidville, Jean-Yves Scoazec, Stephan Vagner, Caroline Robert. Translational control of T cell exhaustion in melanoma, perspectives in therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4167.

Pre-mRNA 3'-end processing by cleavage and polyadenylation (CPA) is a nuclear process in which RNA polymerase II (Pol II) transcripts are cleaved at the polyadenylation site (PAS cleavage) before addition of a poly(A) tail. While PAS cleavage is usually coupled to transcription termination, for some pre-mRNAs it occurs post-transcriptionally, i.e. after pre-mRNA release from chromatin to nucleoplasm through a downstream co-transcriptional cleavage (CoTC) event. DNA-damaging agents such as ultraviolet-C (UV) irradiation trigger rapid shutdown of pre-mRNA 3'-end processing. However, specific compensatory mechanisms exist to ensure efficient 3'-end processing for some pre-mRNAs encoding proteins involved in the DNA damage response (DDR), such as the p53 tumor suppressor protein. Here, we show that PAS cleavage of the p53 pre-mRNA occurs in part post-transcriptionally, in a PCF11-independent manner, in the nucleoplasm, following a CoTC-type event. Upon UV-irradiation, cells with an engineered deletion of the p53 CoTC site exhibit impaired 3'-end processing of the p53 pre-mRNA, decreased mRNA and protein levels of p53 and its transcriptional target, p21, and altered cell cycle progression. Finally, using a transcriptome-wide analysis of PAS cleavage, we identified additional -including DDR related- pre-mRNAs whose PAS cleavage is maintained in response to UV and occurs post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows specific pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.