Understanding PinT, a noncoding RNA timer of virulence gene expression

细菌小RNA(SRNAs)调节大多数重要的生物学过程，但由于缺乏敏感的模型，对它们在毒力中的作用仍知之甚少。利用最近发展的双重RNA-SEQ方法同时研究宿主和病原体在感染过程中的转录，我们发现了一种在宿主细胞感染过程中高度诱导的沙门氏菌。初步研究表明，品脱的一个功能是作为沙门氏菌毒力程序的定时器。此外，这种sRNA的活性对宿主对沙门氏菌的反应具有前所未有的巨大影响。然而，PINT功能的全部范围仍不清楚，PINT介导的调节机制以及PINT在感染期间宿主-病原体串扰中作用的分子基础尚不清楚。在本项目中，我们将使用适体标记-PINT的脉冲表达和亲和纯化来编制感染相关条件下PINT靶标的完整目录。将使用适当的荧光报告构建体和定义的点突变体来验证这些候选基因。Pint介导的调节机制将使用结构探测和体外凝胶位移分析等生化方法相结合的方法来确定，以揭示Pint相关蛋白的作用。这些将与感染分析相补充，以了解pint在宿主-病原体相互作用中的作用。为了进一步剖析宿主和病原体在感染过程中的动态相互作用，将使用双重RNA-SEQ来研究宿主细胞环境对pint介导的调节的影响。通过评估PINT调节的单个mRNAs对DelapinT沙门氏菌感染中观察到的表型的贡献，将调查特定PINT靶标对宿主细胞反应的贡献。总之，这些方法将提高我们对品脱在沙门氏菌毒力和宿主细胞感染中的作用的理解。更广泛地说，该项目中开发的方法将提供新的策略来表征不同病原体中的sRNA，这些病原体的细胞功能不能使用基于细胞的感染分析或适当的动物模型中的标准遗传方法来观察。

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.