M. avium GPLs in macrophage activation and virulence

描述(申请人提供)：分枝杆菌感染导致巨噬细胞信号通路的激活，巨噬细胞信号通路是刺激宿主免疫反应的关键。然而，我们对分枝杆菌侵袭巨噬细胞的信号转导途径以及致病分枝杆菌是否调节这些信号的了解有限。 在我们的实验中，为了确定分枝杆菌感染过程中启动的巨噬细胞信号通路，我们确定了分枝杆菌感染后小鼠巨噬细胞中的丝裂原活化蛋白激酶(MAPK)p38和ERK1/2被激活。然而，与感染非致病性分枝杆菌的细胞相比，感染致病的禽类分枝杆菌感染的巨噬细胞的MAPK活性有限。在这些感染了鸟型支原体的细胞中也观察到了有限的肿瘤坏死因子-α的产生。抑制剂研究表明，MAPK是分枝杆菌介导的诱导肿瘤坏死因子-α所必需的。此外，与感染含有GPL的等基因2151株相比，感染GPL缺乏的M.avium 2151的巨噬细胞也显示出更多的MAPK激活和TNF-α产生。因此，我们假设MAPK是在分枝杆菌感染过程中启动的巨噬细胞信号通路中的关键成分，并且鸟分支杆菌已经进化出部分通过产生GPLs来限制其活性的机制。进一步的分析表明，通过缺失甲基转移酶D基因来改变GPL结构，导致了一株毒力减弱的禽类分支杆菌104菌株。因此，我们提出了以下的具体目标：1)建立一组缺乏参与GPL生物合成的特定基因的突变株724和104；2)对突变株的GPL结构和细胞壁成分进行生化表征；3)表征GPL对巨噬细胞激活和分枝杆菌毒力的影响。我们的长期目标是更好地了解分枝杆菌入侵过程中启动的巨噬细胞信号通路。我们相信，对这些巨噬细胞反应以及分枝杆菌如何修改它们进行更仔细的分析，将导致对分枝杆菌发病机制的新见解。

DESCRIPTION (PROVIDED BY APPLICANT): Mycobacterial infections result in the activation of macrophage signaling pathways that are essential for stimulating a host immune response. However our understanding is limited as to which signaling pathways are transduced in macrophages upon mycobacterial invasion and whether pathogenic mycobacteria modulate these signals. In our experiments to define the macrophage signaling pathways initiated during a mycobacterial infection we determined that the mitogen activated protein kinases (MAPK) p38 and ERK 1/2 were activated in murine macrophages upon mycobacterial infection. However, the MAPK activity was limited in macrophages infected with pathogenic M. avium strains relative to cells infected with non-pathogenic mycobacteria. A limited production of TNF-alpha was also observed in these M. avium infected cells. Inhibitor studies indicated that the MAPKs were required for the mycobacteria-mediated induction of TNF-alpha. Moreover, macrophages infected with a glycopeptidolipid (GPL) deficient M. avium 2151 also showed increased MAPK activation and TNF-alpha production compared to cells infected with a isogenic 2151 strain containing GPLs. Therefore, we hypothesize that the MAPKs are key components in the macrophage signaling pathways initiated during a mycobacterial infection and that M. avium has evolved mechanisms to limit their activity in part through production of GPLs. Additional analysis showed that modifying the GPL structure through deletion of the methyl transferase D gene resulted in a M. avium 104 strain with attenuated virulence. Thus, we propose the following specific aims: 1) produce a panel of M. avium 724 and 104 mutants that lack specific genes involved in GPL biosynthesis 2) Biochemically characterize the M. avium mutants for GPL structure and cell wall composition 3) Characterize GPLs for their affect on macrophage activation and mycobacterial virulence. Our long-term goal is to better understand the macrophage signaling pathways initiated during a mycobacterial invasion. We believe a more careful analysis of these macrophage responses and how mycobacteria may modify them will lead to novel insights into mycobacterial pathogenesis.