The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis

Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with Mycobacterium tuberculosis (Mtb) bacilli. We report dose- and time-dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2–24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that Mtb bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that Mtb strains vary in their ability to facilitate aggregation and utilization of LR.质膜中富含胆固醇的动态区域被称为脂筏（lipid rafts, LR），已有研究观察到其在多种病原体感染宿主细胞的过程中形成，并可能直接参与该感染进程。本研究聚焦于结核分枝杆菌（Mycobacterium tuberculosis, Mtb）感染肺泡上皮细胞过程中诱导的脂筏聚集现象。我们发现，在感染复数（multiplicity of infection, MOI）为1、10和100的条件下，使用3株不同菌株感染宿主细胞后，脂筏聚集程度呈剂量与时间依赖性升高，检测时间覆盖感染后2至24小时（hours post infection, hpi）。H37Rv、HN878与CDC1551三株菌株间存在特异性的菌株依赖性差异：在24小时感染周期中，MOI为100时，H37Rv诱导的脂筏聚集最为显著，每细胞聚集数（aggregates per cell, APC）从15个升高至27个。分别使用各菌株的培养滤液蛋白、总脂质以及γ辐照全菌体处理上皮细胞，均未能达到活菌株感染时诱导的脂筏聚集水平。然而，感染宿主细胞后的上皮细胞过滤上清液可诱导相似程度的脂筏聚集，这表明感染宿主细胞过程中分泌的分枝杆菌产物是导致脂筏聚集的关键因素。感染前阻断脂筏形成的实验表明，结核分枝杆菌可利用脂筏聚落在上皮细胞内实现内化与存活。使用脂筏阻断剂菲林III（Filipin III）处理宿主细胞后，H37Rv与HN878菌株的活菌数在感染6小时后下降近22%，CDC1551菌株则下降7%。本研究证实，结核分枝杆菌可显著诱导肺泡上皮细胞质膜发生改变，且不同Mtb菌株在促进脂筏聚集与利用方面的能力存在差异。