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Cholesterol-Rich Membrane Rafts and Lyn Are Involved in Phagocytosis during Pseudomonas aeruginosa Infection¹

Shibichakravarthy Kannan^*, Aaron Audet^*, Huang Huang^*, Li-juan Chen and Min Wu^2,

^* Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58203; and State Key Laboratory for Biotherapy, West China Hospital, Sichuan University, Chengdu, China

The mechanism of phagocytosis of pathogens remains to be fully characterized. We report a novel phagocytosis pathway for Pseudomonas aeruginosa, which is initiated by cholesterol-rich membrane rafts and is dependent on Lyn, primarily an immune regulator with both positive and negative roles. Blocking of Lyn or blocking of cholesterol synthesis significantly inhibited phagocytosis by alveolar macrophages. We found that Lyn, via Src homology 2 and 3 domains, bound to and then activated PI3K and Akt to regulate intracellular routing of the engulfed P. aeruginosa. Further analysis indicates that Lyn and raft components entered in phagosomes and late lysosomes. Finally, respiratory burst was dependent on Lyn and membrane rafts, as confirmed by small interfering RNA and dominant-negative strategies. Our investigations demonstrate that Lyn along with membrane rafts plays a fundamental role in phagocytosis by alveolar macrophages during infection.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants ES014690 and P20 RR017699 American Heart Association Scientist Development Grant (National Office). This work was also supported by National Key Basic Research Program of China (no. 2006CB504300).

² Address correspondence and reprint requests to Dr. Min Wu, 501 North Columbia Road, P.O. Box 9037, Grand Forks, ND 58203-9037. E-mail address: minwu{at}medicine.nodak.edu or miw100{at}hotmail.com

³ Abbreviations used in this paper: PA, Pseudomonas aeruginosa; AM, alveolar macrophage; CI, confidence interval; DN, dominant negative; Exo, exoenzyme; HMG, 3-hydroxy-3-methylglutaryl; LB, Luria-Bertani; MBS, MES buffered saline; MOI, multiplicity of infection; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4,d]pyrimidine; SH, Src homology; siRNA, small interfering RNA; TTSS, type III secretion system; wt, wild type; YFP, yellow fluorescent protein.