RNAi Screen Reveals an Abl Kinase-Dependent Host Cell Pathway Involved in Pseudomonas aeruginosa Internalization

Abstract

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 ∼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.

Multifunctional Abl kinases in health and disease

“…Subsequent reports showed that Abl kinases are also required for infection by several bacteria (e.g. Helicobacter pylori, EPEC, Salmonella enterica, Anaplasma phagocytophilum, Pseudomonas aeruginosa, Chlamydia trachomatis, Mycobacterium tuberculosis) (Bauer et al, 2009;Bruns et al, 2012;Elwell et al, 2008;Jayaswal et al, 2010;Lin et al, 2007;Ly and Casanova, 2009;Muller, 2012;Napier et al, 2011;Pielage et al, 2008;Poppe et al, 2007;Swimm et al, 2004;Tammer et al, 2007) and viruses [vaccinia virus,coxsackievirus,enterovirus 71 (EV71), HIV, hepatitis C virus (HCV) and Ebola virus] (Chen et al, 2007;Coyne and Bergelson, 2006;Garcia et al, 2012;Harmon et al, 2010;Newsome et al, 2006;Reeves et al, 2005;Yamauchi et al, 2015). Microbial pathogens exploit the ability of Abl kinases to regulate cytoskeletal processes in order to achieve efficient internalization, intracellular motility, pedestal formation, cell-to-cell spread, membrane modeling and release (egress).…”

A lipid zipper triggers bacterial invasion

“…The opportunistic bacterium Pseudomonas aeruginosa can also invade and survive in diverse epithelial and endothelial cells (8)(9)(10)(11)(12)(13)(14), which significantly contributes to its pathogenicity (8,10,15). Numerous reports suggested various host cell factors, including cell surface receptors and signaling components, including α 5 β 1 integrin, cystic fibrosis transmembrane conductance regulator, and Abelson tyrosine-protein kinase 1-dependent pathway (16)(17)(18), that are involved in the cellular uptake of P. aeruginosa. Host-cell GSLs have been identified as important molecules contributing to host specificity and adhesion of P. aeruginosa (19,20).…”

Pathogen and Toxin Entry - How Pathogens and Toxins Induce and Harness Endocytotic Mechanisms

“…Both functions redundantly disrupt the actin cytoskeleton [120]. Surprisingly, ExoS and ExoT act as anti-internalization factors [121], probably by interfering with components of the Abl pathway [122]: Rac1, Cdc42 and Crk were demonstrated to be activated by, and necessary for, the cellular uptake of P. aeruginosa [123]. Interestingly, additional experiments performed with ΔExoS and ΔExoT deletion mutants of P. aeruginosa revealed that ExoT abrogated Rac1 and Cdc42 activation, whereas ExoS activates these GTPases in order to promote efficient uptake [123].…”