Short-term remodeling of the host epithelial cell proteome by the listeriolysin O pore-forming toxin

Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria induce transcriptional changes, in order to modify the set of proteins synthetized by the host cell, or target pre-existing proteins by modulating their post-translational modifications or by triggering their degradation. Protein levels variations in host cells during infection integrates both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the bacterial pathogen Listeria monocytogenes, and more specifically the Listeria toxin Listeriolysin O (LLO). In order to characterize host proteome alterations induced by LLO, we performed a shotgun proteomics analysis on HeLa cells treated or not with the LLO toxin. To this end, in a first analysis we used SILAC labelling (stable isotope labelling by amino acids in cell culture) to compare in a quantitative manner the protein content from two differentially treated cell populations: one control population and one population incubated with a sublytic dose of LLO (3 nM). We decided to expose cells to LLO during only 20 min to limit protein level changes resulting from transcriptional changes. We performed two independent experiments (experiment #1 and experiment #2) with swapped SILAC labelling to rule out putative labelling-dependent effects. A total of 2,009 and 2,577 proteins was quantified in each independent experiment, respectively, and 1,834 proteins were quantified in both experiments. Among these 1834 proteins, we identified a total of 151 proteins for which protein levels were consistently decreased in cells treated with LLO (i.e. with a log2 control/LLO ratio >0.5 in both experiments). In addition to this preliminary study, we carried out a second experiment using label-free quantitative shotgun proteomics to compare protein abundance in cells treated or not with LLO. Four independent biological replicates were included in this second screen in order to perform a robust statistical analyses of downregulated proteins. Among the 2,973 proteins that were quantified in all independent replicates, we identified a total of 149 proteins (5.0 %) for which protein levels were significantly decreased in cells treated with LLO. In contrast to these decreases, only 16 proteins (0.5 %) showed significant increased levels in LLO-treated cells. This result confirmed that LLO remodels the cell proteome mainly by decreasing the level of host targets. To assess whether the host proteasome was involved in the decrease of these 149 proteins, we repeated our label-free proteomics analysis on HeLa cells pre-treated with proteasome inhibitors before exposure to LLO. Interestingly, we observed that the vast majority (i.e. 83%) of host proteins identified as strongly downregulated in response to LLO also displayed significant decreased levels in the presence of proteasome inhibitors, indicating that the majority of observed decreases in host protein levels induced by LLO are not due to proteasome-mediated degradation. In follow-up experiments, we validated that LLO decreases in particular the protein level of two E2 ubiquitin enzymes, UBE2K and UBE2N, leading to major changes in the host ubiquitylome. This toxin-induced proteome remodeling involves post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. These decreases in host protein levels were observed in different epithelial cell lines but not in macrophages. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data show that different bacterial pore-forming toxins induce deep proteome remodeling, that may impair host epithelial cell functions.

细菌病原体可通过多种策略干扰宿主细胞的正常功能。在这些策略中，细菌可诱导转录组改变，以修饰宿主细胞合成的蛋白质谱，或通过调控靶标蛋白的翻译后修饰、触发其降解来靶向预存在的蛋白质。感染过程中宿主细胞内的蛋白质水平变化，整合了病原体诱导的转录与转录后调控机制。

本研究聚焦于单核细胞增生李斯特菌（Listeria monocytogenes）这一细菌病原体诱导的宿主蛋白质组改变，更具体地针对其分泌的毒素——李斯特菌溶血素O（Listeriolysin O，LLO）展开研究。

为表征LLO诱导的宿主蛋白质组改变，我们对经LLO毒素处理与未处理的海拉细胞（HeLa cells）开展了鸟枪法蛋白质组学分析。首轮分析中，我们采用细胞培养氨基酸稳定同位素标记（stable isotope labelling by amino acids in cell culture, SILAC）技术，以定量方式比较两组经不同处理的细胞群体的蛋白质含量：一组为对照组细胞，另一组为经亚溶细胞剂量LLO（3 nM）孵育的细胞群体。为限制由转录变化导致的蛋白质水平改变，我们将细胞暴露于LLO的时长设定为仅20分钟。我们开展了两次独立实验（实验1与实验2），并采用反向SILAC标记策略以排除潜在的标记依赖效应。两次独立实验中分别定量到2009和2577种蛋白质，且有1834种蛋白质在两次实验中均被定量到。在这1834种蛋白质中，我们共鉴定出151种蛋白质，其在LLO处理组细胞中的水平持续下调（即两次实验中log₂(对照组/LLO处理组)比值均大于0.5）。

除上述预实验外，我们还开展了第二项实验，采用无标记定量鸟枪法蛋白质组学技术比较LLO处理与未处理细胞的蛋白质丰度。该第二轮筛选包含4个独立生物学重复，以对下调蛋白质开展稳健的统计学分析。在所有重复中均被定量到的2973种蛋白质中，我们共鉴定出149种蛋白质（占比5.0%），其在LLO处理组细胞中的水平显著下调。与这些下调现象形成对比的是，仅有16种蛋白质（占比0.5%）在LLO处理组细胞中表现出水平显著上调。该结果证实，LLO主要通过降低宿主靶标蛋白的水平来重塑细胞蛋白质组。

为探究宿主蛋白酶体是否参与这149种蛋白质的水平下调，我们对经蛋白酶体抑制剂预处理后再暴露于LLO的海拉细胞重复了无标记蛋白质组学分析。有趣的是，我们观察到，绝大多数（即83%）在LLO处理后被鉴定为显著下调的宿主蛋白质，在蛋白酶体抑制剂存在的情况下同样表现出水平显著下降，这表明LLO诱导的宿主蛋白质水平降低大多并非由蛋白酶体介导的降解所导致。

在后续实验中，我们验证了LLO可特异性降低两种E2泛素结合酶UBE2K与UBE2N的蛋白质水平，进而引发宿主泛素化修饰组（ubiquitylome）的显著改变。这种由毒素诱导的蛋白质组重塑仅涉及转录后调控，因为对应基因的转录水平未观察到任何变化。

上述宿主蛋白质水平降低现象在多种上皮细胞系中均有观测到，但在巨噬细胞中未出现。此外，我们证实，另一种与LLO结构相似的细菌孔形成毒素——产气荚膜梭菌溶血素O（Perfringolysin O）同样可诱导宿主蛋白质组改变。

综上，我们的研究数据表明，不同的细菌孔形成毒素均可诱导深度蛋白质组重塑，这可能会损害宿主上皮细胞的正常功能。