Ghosh Moumita, Roy Koushik, Das Mukherjee Dipanwita, Chakrabarti Gopal, Roy Choudhury Kingshuk, Roy Syamal
Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
Department of Biotechnology, University of Calcutta, Kolkata, India.
PLoS Negl Trop Dis. 2014 Dec 4;8(12):e3367. doi: 10.1371/journal.pntd.0003367. eCollection 2014 Dec.
The protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.
METHODOLOGY/PRINCIPAL FINDINGS: We determined the lateral mobility of a membrane protein in normal, LD infected and liposome treated LD infected cells using GFP-tagged PLCδ1 as a probe. The mobility of PLCδ1 was computationally analyzed from the time lapse experiment using boundary distance plot and radial profile movement. Our results showed that the lateral mobility of the membrane protein, which is increased in infection, is restored to normal upon liposomal cholesterol treatment. The results of FRAP experiment lent further credence to the above notion. The membrane proteins are intimately linked with cellular actin and alteration of cellular actin may influence lateral mobility. We found that F-actin is decreased in infection but is restored to normal upon liposomal cholesterol treatment as evident from phalloidin staining and also from biochemical analysis by immunoblotting.
CONCLUSIONS/SIGNIFICANCES: To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.
原生动物寄生虫杜氏利什曼原虫（LD）可能为自身利益而降低宿主细胞的胆固醇水平。胆固醇主要存在于质膜的特殊区域。膜蛋白的流动性与膜胆固醇消耗之间的关系仍然是一个重要问题。利什曼原虫感染会改变膜蛋白流动性这一观点源于我们之前的研究，在该研究中我们发现，LD感染细胞表面的IFNγ受体（R1和R2）亚基之间的距离增加，但通过脂质体胆固醇处理可恢复正常。
方法/主要发现：我们使用绿色荧光蛋白标记的PLCδ1作为探针，测定了正常细胞、LD感染细胞以及脂质体处理的LD感染细胞中膜蛋白的侧向流动性。通过使用边界距离图和径向轮廓运动对延时实验进行计算分析，得出PLCδ1的流动性。我们的结果表明，感染时增加的膜蛋白侧向流动性在脂质体胆固醇处理后恢复正常。FRAP实验的结果进一步证实了上述观点。膜蛋白与细胞肌动蛋白密切相关，细胞肌动蛋白的改变可能会影响侧向流动性。我们发现，从鬼笔环肽染色以及免疫印迹的生化分析中可以明显看出，感染时F - 肌动蛋白减少，但脂质体胆固醇处理后恢复正常。
结论/意义：据我们所知，这是首次直接证明LD寄生虫在其细胞内生命周期中增加了感染巨噬细胞中膜蛋白的侧向流动性并降低了F - 肌动蛋白水平。这些缺陷可能导致细胞内信号传导无效以及其他细胞功能异常。
