Understanding PinT, a noncoding RNA timer of virulence gene expression

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Bacterial small RNAs (sRNAs) regulate most important biological processes but their roles in virulence remain poorly understood for a lack of sensitive models. Using a recently developed Dual RNA-seq approach to simultaneously study host and pathogen transcriptomes during infection, we have discovered a Salmonella sRNA, PinT, that is highly induced during the infection of host cells. Initial studies suggest that one function of PinT is to act as a timer of Salmonella virulence programs. In addition, the activity of this sRNA has an unprecedentedly massive effect on the host response to Salmonella. However, the full scope of PinT function remains unclear as do the mechanisms involved in PinT-mediated regulation, and the molecular basis for PinTs role in host-pathogen cross-talk during infections.In this project we will use pulse-expression and affinity purification of aptamer-tagged-PinT to compile a complete catalogue of PinT targets under infection-relevant conditions. These candidates will be validated using appropriate fluorescent reporter constructs and defined point mutants. The mechanism of PinT-mediated regulation will be determined using a combination of biochemical approaches such as structure-probing together with in vitro gel shift assays to reveal the role of PinT associated proteins. These will be complemented with infection assays to understand the role of PinT in host-pathogen interactions. To further dissect the dynamic interplay between host and pathogen during infection Dual RNA-seq will be used to study the impact of host cell environment in HeLa and porcine macrophages on PinT-mediated regulation. The contribution of specific PinT targets to the host cell response will be investigated by evaluating the contribution of individual PinT-regulated mRNAs to the phenotypes observed in infections using deltapinT Salmonella. Altogether, these approaches will improve our understanding of the role of PinT in Salmonella virulence and host cell infection. More generally, the approaches developed in this project will provide new strategies to characterise sRNAs in disparate pathogens whose cellular functions cannot be observed using standard genetic approaches in cell based infection assays or appropriate animal models.