Effect of Neisserial Porin in Immune Cell Apoptosis

NeisSerial孔蛋白(淋球菌蛋白I[PIA或PIB]或脑膜炎球菌1类[PorA]、2或3[PorB]蛋白)是NeisSerial外膜的主要蛋白质成分。我们之前已经证明，NeisSerial porins可以激活B淋巴细胞，增加共刺激配体B7-2的表面表达，从而提高B细胞“共刺激”T淋巴细胞的能力。B7-2蛋白的诱导表达与其佐剂能力直接相关。在这项工作的过程中，我们发现当孔蛋白激活B细胞时，降低了这些B细胞对激活诱导的细胞死亡(AICD)或凋亡的敏感性。我们发现，抗凋亡作用是由于孔蛋白与线粒体(细胞凋亡的中央控制点)的直接相互作用。免疫沉淀实验表明，PorB与线粒体孔蛋白VDAC相互作用。我们推测，这种VDAC-NeisSerial Porin蛋白-蛋白相互作用类似于VDAC与抗凋亡分子Bcl-2的相互作用，是这些孔蛋白的抗凋亡作用的原因。这种相互作用导致细胞存活的增强和B细胞的持续激活，这将加强B细胞参与特定的免疫反应。因此，这种抗凋亡作用可能是孔蛋白免疫增强能力的一个潜在的额外机制。这项建议将研究NeisSerial孔蛋白对B细胞、上皮细胞和其他类型细胞对凋亡的易感性的影响。基本目的包括：1)检测NeisSeries porin与线粒体和线粒体VDAC的相互作用；2)确定细胞培养条件的改变(尤其是血清撤除)是否可以改变Porins对细胞凋亡的影响，以及这是否与细胞内ATP水平的变化有关；3)确定完整的NeisSeries生物对细胞凋亡的影响以及Porin是否在这一效应中发挥作用。这些研究将产生重要的发现，既可以阐明Por(以及潜在的其他细菌成分)的免疫刺激作用，也可以更好地将Por用作疫苗佐剂。此外，这些结果可能在NeisSeries发病机制中具有重要意义，特别是因为我们有新的初步数据表明，完整的活脑膜炎球菌也可以保护细胞免受凋亡。

Neisserial porins (gonococcal Protein I [PIA or PIB] or meningococcal class 1 [PorA], 2 or 3 [PorB] proteins) are the major protein constituents of the Neisserial outer membrane. We have previously demonstrated that the Neisserial porins activate B lymphocytes, increasing the surface expression of the costimulatory ligand B7-2, which improves the B cell's ability to "costimulate" T lymphocytes. The induction of B7-2 expression is directly related to the porins' adjuvant ability. During the course of this work, we have discovered that the porins, when activating B cells, decreased the susceptibility of these B cells to activation induced cell death (AICD) or apoptosis. We found that the anti-apoptotic effect is due to a direct interaction of the porin with mitochondria (a central control point for apoptotic cell death). Immunoprecipitation experiments revealed that PorB interacts with the mitochondrial porin, VDAC. We hypothesize that this VDAC-Neisserial porin protein-protein interaction, which is similar to that of VDAC with the anti- apoptotic molecule Bcl-2, is responsible for the anti-apoptotic effect of the porins. This interaction results in an enhancement of cell survival and continued activation of B cells, which would potentiate the B cells' involvement in specific immune responses. Therefore, it is possible that this anti-apoptotic effect is a potential additional mechanism of the porins' immunopotentiating ability. This proposal will investigate the effect of Neisserial porins on the susceptibility of B cells, epithelial cells and other cell types to apoptosis. The basic aims include 1) examining the interaction of Neisserial porin with mitochondria and mitochondrial VDAC, 2) determining if alterations in cell culture conditions (especially serum withdrawal) can alter the effect of porins on apoptosis and whether this could be related to changes in cellular ATP levels and 3) determining the effect of intact Neisserial organisms on apoptosis and whether porin might play a role in this effect. These studies will yield significant findings that could both elucidate the immune stimulatory effect of Por (and potentially other bacterial components), allowing for the better use of Por as a vaccine adjuvant. In addition, these results can have significant implications in Neisserial pathogenesis, especially since we have new preliminary data that intact live meningococci can also protect cells from apoptosis.