RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.
Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.
Pielage
Julia F
JF
Program in Microbial Pathogenesis and Host Defense, University of California San Francisco, California, USA.
Powell
Kimberly R
KR
Kalman
Daniel
D
Engel
Joanne N
JN
D036002:ADP Ribose Transferases / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism; D002460:Cell Line; D003599:Cytoskeleton / Q000382:microbiology / Q000502:physiology; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000166:cytology; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020868:Gene Silencing; D054884:Host-Pathogen Interactions / Q000502:physiology*; D008264:Macrophages / Q000201:enzymology / Q000382:microbiology*; D051379:Mice; D018345:Mice, Knockout; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000472:pathogenicity*; D034622:RNA Interference; D034741:RNA, Small Interfering
D048868:Adaptor Proteins, Signal Transducing; D001427:Bacterial Toxins; D029721:Drosophila Proteins; C477521:ExoT protein, Pseudomonas aeruginosa; D020690:GTPase-Activating Proteins; D034741:RNA, Small Interfering; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D016315:Proto-Oncogene Proteins c-abl