Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila [exp1]. Mus musculus

The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires’ Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis. Overall design: Four-condition experiment: macrophages left uninfected (negative control), or infected with wildtype Legionella pneumophila, the flagellin-deficient mutant ΔflaA, or the secretion-deficient mutant ΔdotA (three experimental conditions). Biological replicates: two, independently infected, harvested, and hybridized to arrays. One to two technical replicates per array, as indicated in file titles.

胞内致病菌嗜肺军团菌（Legionella pneumophila）可引发一种名为军团病的炎症性肺炎。其毒力依赖于一套Dot/Icm IV型分泌系统（type IV secretion system），该系统可将细菌效应蛋白转运至宿主胞质溶胶中。缺失Dot/Icm系统的嗜肺军团菌会被Toll样受体（Toll-like receptors, TLRs）识别，进而激活依赖于NF-κB的经典转录应答。此外，表达功能性Dot/Icm系统的嗜肺军团菌可强效诱导独特的转录靶标，包括Il23a、Csf2这类促炎基因。
本研究证实，这种依赖于Dot/Icm系统的应答（我们将其命名为效应蛋白触发应答（effector-triggered response, ETR））需要五种可抑制宿主蛋白质合成的转运型细菌效应蛋白。在巨噬细胞感染强毒株嗜肺军团菌后，这五种效应蛋白会导致宿主翻译过程整体下调，从而阻断NF-κB转录因子的抑制蛋白IκB的合成。因此，感染野生型嗜肺军团菌的巨噬细胞会出现NF-κB的持续激活，这与Il23a、Csf2等ETR靶基因的转录密切相关。
缺失这五种效应蛋白的嗜肺军团菌突变株仍可激活TLRs和NF-κB，但由于该突变株可正常合成IκB，NF-κB的激活更为短暂，且不足以完全诱导ETR。表达酶学失活效应蛋白的嗜肺军团菌突变株同样无法完全诱导ETR；而多种通过不同机制抑制宿主蛋白质合成的化合物或细菌毒素，在与TLR配体联合使用时可重现ETR。
既往研究表明，宿主对细菌感染的应答主要由激活Toll样受体或胞质模式识别受体的特定微生物分子所诱导。本研究结果为该模型补充了关键证据：宿主对强毒株病原体的免疫应答同样可由病原体编码的活性（如抑制宿主蛋白质合成）所塑造。
整体实验设计：四组对照实验：未感染的巨噬细胞（阴性对照），或感染野生型嗜肺军团菌、鞭毛蛋白缺陷型突变株ΔflaA，或是分泌缺陷型突变株ΔdotA（共三种实验组）。生物学重复：共2次独立感染、收获样本并进行芯片杂交。每张芯片设置1~2次技术重复，详见文件标题标注。