Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, United States of America.
Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, United States of America.
PLoS One. 2024 Feb 23;19(2):e0281564. doi: 10.1371/journal.pone.0281564. eCollection 2024.
Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-β secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the ΔsecA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the ΔsecA2 strain. Therefore, to better examine secreted IFN-β levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-β to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-β from macrophages infected with the ΔsecA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-β response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.
视黄酸诱导基因 I（Rig-I）是一种细胞溶质模式识别受体，其在识别病毒 RNA 方面的重要作用已得到广泛描述。越来越多的证据表明，源自细胞内细菌（如结核分枝杆菌）的细菌衍生 RNA 能够激活相同的宿主 Rig-I/线粒体抗病毒信号蛋白（MAVS）信号通路，从而引发 I 型干扰素反应，有助于体内细菌发病机制。在结核分枝杆菌中，这种反应是由蛋白分泌系统 SecA2 介导的，但人们对该过程是否在其他致病性分枝杆菌中保守，以及这些核酸如何进入宿主细胞质，知之甚少。由于结核分枝杆菌和海洋分枝杆菌的 SecA2 蛋白分泌系统在遗传和功能上具有高度的保守性，我们假设宿主巨噬细胞中 Rig-I/MAVS 的激活以及随后 IFN-β 的分泌也将在这两种分枝杆菌之间保守。为了验证这一点，我们构建了一个ΔsecA2 海洋分枝杆菌菌株以及表达海洋分枝杆菌或结核分枝杆菌 secA2 基因的互补菌株。我们的结果表明，ΔsecA2 菌株在体外具有生长缺陷，但在宿主巨噬细胞中没有。这些细胞内生长曲线还表明，在感染实验中，用于估计感染巨噬细胞单层时添加的细菌数量的计算方法低估了ΔsecA2 菌株的细菌输入量。因此，为了更好地检测感染水平相等时分泌 IFN-β 的水平，我们在感染后 2 小时进行了平板 CFU 计数，同时进行了 ELISA 基础感染实验。这使我们能够将分泌的 IFN-β 水平标准化到一个标准数量的细菌上。我们将该方法应用于 WT 和 MAVS-/-骨髓来源的巨噬细胞，与 WT 相比，感染ΔsecA2 海洋分枝杆菌菌株的巨噬细胞中 IFN-β 的分泌水平相等或更高。综上所述，在测试条件下，海洋分枝杆菌中 Rig-I/MAVS 胞质传感器的激活以及随后以 SecA2 依赖性方式诱导 IFN-β 反应并不保守。
