Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, 70101, Taiwan.
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, 70101, Taiwan.
J Biomed Sci. 2017 Aug 19;24(1):58. doi: 10.1186/s12929-017-0368-2.
Vibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans. We have previously shown that the multifunctional autoprocessing repeats in toxin (MARTX) of a biotype 1 V. vulnificus strain promotes survival of this organism in the host by preventing it from engulfment by the phagocytes. The purpose of this study was to further explore how MARTX inhibits phagocytosis of this microorganism by the macrophage.
We compared between a wild-type V. vulnificus strain and its MARTX-deficient mutant for a variety of phagocytosis-related responses, including morphological change and activation of signaling molecules, they induced in the macrophage. We also characterized a set of MARTX domain-deletion mutants to define the regions associated with antiphagocytosis activity.
The RAW 264.7 cells and mouse peritoneal exudate macrophages underwent cell rounding accompanied by F-actin disorganization in the presence of MARTX. In addition, phosphorylation of some F-actin rearrangement-associated signaling molecules, including Lyn, Fgr and Hck of the Src family kinases (SFKs), focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), phosphoinositide 3-kinase (PI3K) and Akt, but not p38, was decreased. By using specific inhibitors, we found that these kinases were all involved in the phagocytosis of MARTX-deficient mutant in an order of SFKs-FAK/Pyk2-PI3K-Akt. Deletion of the effector domains in the central region of MARTX could lead to reduced cytotoxicity, depending on the region and size of deletion, but did not affect the antiphagocytosis activity and ability to cause rounding of macrophage. Reduced phosphorylation of Akt was closely associated with inhibition of phagocytosis by the wild-type strain and MARTX domain-deletion mutants, and expression of the constitutively active Akt, myr-Akt, enhanced the engulfment of these strains by macrophage.
MARTX could inactivate the SFKs-FAK/Pyk2-PI3K-Akt signaling pathway in the macrophages. This might lead to impaired phagocytosis of the V. vulnificus-infected macrophage. The majority of the central region of MARTX is not associated with the antiphagocytosis activity.
创伤弧菌是一种海洋细菌，会引起机会性感染，在人类中表现为严重的皮肤损伤和暴发性败血症。我们之前已经表明，生物型 1 创伤弧菌菌株的多功能自加工重复毒素（MARTX）通过防止吞噬细胞吞噬它，促进该生物体在宿主中的存活。本研究的目的是进一步探讨 MARTX 如何抑制巨噬细胞吞噬这种微生物。
我们比较了野生型创伤弧菌菌株及其 MARTX 缺陷突变体在诱导巨噬细胞发生各种吞噬作用相关反应方面的差异，包括形态变化和信号分子的激活。我们还对一组 MARTX 结构域缺失突变体进行了特征描述，以确定与抗吞噬作用活性相关的区域。
在存在 MARTX 的情况下，RAW 264.7 细胞和小鼠腹腔渗出巨噬细胞发生细胞圆化，并伴有 F-肌动蛋白解聚。此外，一些与 F-肌动蛋白重排相关的信号分子的磷酸化，包括 Src 家族激酶（SFKs）中的 Lyn、Fgr 和 Hck、粘着斑激酶（FAK）、富含脯氨酸的酪氨酸激酶 2（Pyk2）、磷酸肌醇 3-激酶（PI3K）和 Akt，但不包括 p38，均降低。通过使用特异性抑制剂，我们发现这些激酶都按照 SFKs-FAK/Pyk2-PI3K-Akt 的顺序参与了 MARTX 缺陷突变体的吞噬作用。MARTX 中心区域效应结构域的缺失会导致细胞毒性降低，这取决于缺失的区域和大小，但不影响抗吞噬作用活性和引起巨噬细胞圆化的能力。Akt 的磷酸化减少与野生型菌株和 MARTX 结构域缺失突变体抑制吞噬作用密切相关，而组成型活性 Akt 的表达，myr-Akt，增强了这些菌株被巨噬细胞吞噬。
MARTX 可以使巨噬细胞中的 SFKs-FAK/Pyk2-PI3K-Akt 信号通路失活。这可能导致感染创伤弧菌的巨噬细胞吞噬作用受损。MARTX 的大部分中心区域与抗吞噬作用活性无关。
