WGS data for Salmonella enterica serovar Typhimurium deficient in cohorts of SPI-2 effector proteins. Salmonella multimutants reveal SPI-2 effector cohorts that promote gut inflammation and systemic colonisation

Salmonella enterica spp. rely on translocation of effector proteins through the SPI-2 encoded type III secretion system (T3SS) to achieve pathogenesis. More than 30 effectors contribute to manipulation of host cells through diverse mechanisms, but interdependency or redundancy between effectors complicates the discovery of effector phenotypes using single mutant strains. Here, we engineer six mutant strains to be deficient in cohorts of SPI-2 effector proteins, as defined by their reported function. Using various animal models of infection, we show that three principle phenotypes define the functional contribution of the SPI-2 T3SS to infection. Multimutant strains deficient for intracellular replication, for manipulation of host cell defences, or for expression of virulence plasmid effectors all showed strong attenuation in vivo, while mutants representing approximately half of the known effector complement showed phenotypes similar to the wild-type parent strain. By additionally removing the SPI-1 T3SS, we find cohorts of effector proteins that contribute to SPI-2 T3SS-driven enhancement of gut inflammation. Further, we provide an example of how iterative mutation can be used to find a minimal number of effector deletions required for attenuation, and thus establish that the SPI-2 effectors SopD2 and GtgE are critical for the promotion of gut inflammation. This strategy provides a powerful toolset for simultaneous parallel screening of all known SPI-2 effectors in a single experimental context, and further facilitates the identification of the responsible effectors, and thereby provides an efficient approach to study how individual effectors contribute to disease.

肠炎沙门氏菌（Salmonella enterica spp.）需通过沙门氏菌致病岛2（SPI-2，Salmonella Pathogenicity Island 2）编码的Ⅲ型分泌系统（T3SS，type III secretion system）转运效应蛋白，方可完成致病过程。目前已发现超过30种效应蛋白可通过多样机制操控宿主细胞，但效应蛋白间的相互依赖或功能冗余，使得利用单突变菌株探究效应蛋白表型的工作变得复杂。本研究中，我们依据已报道的功能分组，构建了6株缺失不同SPI-2效应蛋白组合的突变菌株。借助多种感染动物模型，我们发现三类核心表型可界定SPI-2 T3SS在感染过程中的功能贡献：缺失胞内增殖相关、宿主细胞防御操控相关或毒力质粒效应蛋白表达相关功能的多突变菌株，均在体内呈现显著的毒力减弱表型；而涵盖约半数已知效应蛋白组的突变菌株，其表型与野生型亲本菌株相近。进一步移除SPI-1 Ⅲ型分泌系统后，我们又鉴定出可介导SPI-2 T3SS驱动肠道炎症增强的效应蛋白组合。此外，本研究还展示了如何通过迭代突变筛选获得实现毒力减弱所需的最少效应蛋白缺失组合，并据此证实SPI-2效应蛋白SopD2与GtgE在肠道炎症增强过程中发挥关键作用。该策略构建了一套高效工具包，可在单一实验环境中同步平行筛选所有已知SPI-2效应蛋白，进一步助力致病效应蛋白的鉴定，从而为探究单个效应蛋白如何参与疾病致病过程提供了高效研究路径。