Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.

细胞内细菌病原体注入效应蛋白以劫持宿主细胞过程并促进其存活和增殖。为了系统地绘制感染过程中的效应-宿主蛋白-蛋白相互作用 (PPI)，我们生成了一个包含 32 种鼠伤寒沙门氏菌 (S Tm) 菌株的文库，这些菌株在巨噬细胞和上皮细胞中表达染色体编码的亲和标记效应子和量化的 PPI。我们确定了 446 种效应宿主 PPI，其中 25 种先前已描述，并通过相互免疫共沉淀验证了 13 种。虽然效应器集中在相同的宿主细胞过程上，但大多数都有多个目标，这些目标通常在细胞类型之间有所不同。我们证明 SseJ、SseL 和 SifA 在含沙门氏菌的液泡 (SCV) 部分通过胆固醇转运蛋白 Niemann-Pick C1 蛋白。PipB 将细胞器接触位点蛋白 PDZD8 招募到 SCV，SteC 通过磷酸化福尔明蛋白样蛋白来促进肌动蛋白捆绑。本研究提供了一种在感染过程中探测宿主病原体 PPI 的方法，以及一种研究S Tm 效应机制的资源。