On the development of novel molecular and microscopy methodologies for the interrogation of host-pathogen interactions at the single-cell level

ended

Over the course of host-pathogen evolution, both the players in this game have set out strategies to overcome each other, by adapting to challenging environments and camouflaging on the one side, and by detecting and eradicating the enemy on the other. In this context, the identification of the pathogenic potential, that is, the genetic elements conferring virulence and how they subvert the host’s defences, is fundamental. With a multiplexed approach, I aim at developing a pooled genetic screen using CRISPR interference to produce pathogen knocked-down libraries impaired in a curated subset of virulence factors of Salmonella Typhi. This pathogen is of particular interest because of the severity of its systemic infection combined with its human host-restriction, which left research lagging behind. Several cellular systems (macrophages, PBMCs, organoids) will be probed upon infection of S. typhi mutants, and the host transcriptome retrieved by single-cell RNA-seq. This analysis will provide a systematic and thorough map of the cause and effect network of the pathogen virulence factors and host responses. Interestingly, in this pathogen-host “war”, each battle can end with a different winner, even when the teams are comprised of the same players, meaning that the heterogeneity in the cellular state of both the pathogen and the host previous to the infection, can lead to the occurrence of diverse outcomes within a single population. The pathogen can either replicate, persist or be cleared by the host. This intrinsic cell-to-cell variability posits the importance of profiling simultaneously the host and the pathogen at the level of single cells. To achieve this goal, I will profile the transcriptomes of the host and the pathogen at the same time, by using multiplexed smFISH. This analysis will quantitatively untangle the contribution of heterogeneity to disease outcomes.