Vaccine and Infectious Disease Research Center, Translational Health Science and Technology Institute, Faridabad, Haryana, INDIA.
Drug Discovery Research Center, Translational Health Science and Technology Institute, Faridabad, Haryana, INDIA.
PLoS One. 2019 Apr 10;14(4):e0215123. doi: 10.1371/journal.pone.0215123. eCollection 2019.
Mycobacterium tuberculosis (Mtb) secretes proteases and peptidases to subjugate its host. Out of its sixty plus proteases, atleast three are reported to reach host macrophages. In this study, we show that Mtb also delivers a lysyl alanine aminopeptidase, PepN (Rv2467) into host macrophage cytosol. Our comparative in silico analysis shows PepNMtb highly conserved across all pathogenic mycobacteria. Non-pathogenic mycobacteria including M. smegmatis (Msm) also encode pepN. PepN protein levels in both Mtb (pathogenic) and Msm (non-pathogenic) remain uniform across all in vitro growth phases. Despite such tight maintenance of PepNs' steady state levels, upon supplementation, Mtb alone allows accumulation of any excessive PepN. In contrast, Msm does not. It not only proteolyzes, but also secretes out the excessive PepN, be it native or foreign. Interestingly, while PepNMtb is required for modulating virulence in vivo, PepNMsm is essential for Msm growth in vitro. Despite such essentiality difference, both PepNMtb and PepNMsm harbor almost identical N-terminal M1-type peptidase domains that significantly align in their amino acid sequences and overlap in their secondary structures. Their C-terminal ERAP1_C-like domains however align much more moderately. Our in vitro macrophage-based infection experiments with MtbΔpepN-expressing pepNMsm reveals PepNMsm also retaining the ability to reach host cytosol. Lastly, but notably, we determined the PepNMtb and PepNMsm interactomes and found them to barely coincide. While PepNMtb chiefly interacts with Mtb's secreted proteins, PepNMsm primarily coimmunoprecipitates with Msm's housekeeping proteins. Thus, despite high sequence homology and several common properties, our comparative analytical study reveals host-centric traits of pathogenic and bacterial-centric traits of non-pathogenic PepNs.
结核分枝杆菌（Mtb）分泌蛋白酶和肽酶来征服宿主。在其六十多种蛋白酶中，至少有三种被报道能到达宿主巨噬细胞。在这项研究中，我们表明 Mtb 还将赖氨酸丙氨酸氨肽酶 PepN（Rv2467）递送到宿主巨噬细胞质溶胶中。我们的比较计算机分析表明 PepN 在所有致病性分枝杆菌中高度保守。包括耻垢分枝杆菌（Msm）在内的非致病性分枝杆菌也编码 pepN。在 Mtb（致病性）和 Msm（非致病性）中，PepN 蛋白水平在所有体外生长阶段都保持均匀。尽管 PepN 的稳定状态水平保持得如此紧密，但在补充后，只有 Mtb 允许积累任何过量的 PepN。相比之下，Msm 则不能。它不仅能蛋白水解，而且还能分泌出过量的 PepN，无论是天然的还是外来的。有趣的是，虽然 PepNMtb 是调节体内毒力所必需的，但 PepNMsm 是 Msm 体外生长所必需的。尽管存在这种必要性差异，但 PepNMtb 和 PepNMsm 都拥有几乎相同的 N 端 M1 型肽酶结构域，在其氨基酸序列中显著对齐，并在其二级结构中重叠。然而，它们的 C 端 ERAP1_C 样结构域的对齐度要低得多。我们在体外基于巨噬细胞的感染实验中，用 MtbΔpepN 表达 pepNMsm 表明 PepNMsm 也保留了进入宿主细胞质的能力。最后，但值得注意的是，我们确定了 PepNMtb 和 PepNMsm 的互作组，并发现它们几乎没有重合。虽然 PepNMtb 主要与 Mtb 的分泌蛋白相互作用，但 PepNMsm 主要与 Msm 的管家蛋白共免疫沉淀。因此，尽管存在高度的序列同源性和几个共同的特性，但我们的比较分析研究揭示了致病性 PepN 的宿主中心特性和非致病性 PepN 的细菌中心特性。
