Understanding how pathogens subvert the host cell cytoskeleton to induce their own internalization is of great interest, opening new avenues to develop treatments to control antibiotic-resistant infections as well as furthering our understanding of fundamental aspects of cell biology. In the experiments reported here, we used RNAi-mediated gene inactivation in Drosophila S2 cells to carry out an unbiased forward genetic screen to identify host molecules crucial to entry of P. aeruginosa
. As S2 cells are phagocytic in origin, our screen had the potential to identify genes involved in phagocytosis or in pathogen-directed uptake into non-phagocytic cells. We identified the tyrosine kinase Abl, the adaptor protein Crk, the Rho family GTPases Rac1 and Cdc42, and Pak as components of a host signaling pathway which has not previously been demonstrated to be required for P. aeruginosa
entry. Using comprehensive and complementary approaches, we validated the role of the Abl kinase pathway in P. aeruginosa
entry into mammalian epithelial cells. Remarkably, three of its components, Crk, Rac1 and Cdc42, are known targets of ExoS and/or ExoT, T3SS effector proteins of P. aeruginosa
that have been shown to modulate P. aeruginosa
internalization into mammalian cells 
. Our results further reveal new complexities in the regulation of bacterial entry by ExoS and ExoT.
Through the use of a chemical inhibitor of Abl kinase, an Abl/Arg deficient cell line, and RNAi-mediated depletion of Abl, we demonstrate that this cytoplasmic tyrosine kinase is essential for efficient internalization of P. aeruginosa
by mammalian cells (). Abl kinase has been shown to be a key component of various steps in the infection of several pathogens, including actin motility in poxvirus infection, pedestal formation in enterophathogenic E. coli
, and the entry of Coxsackievirus, and Shigella flexneri
into non-phagocytic cells 
. However, the requirement for Abl in P. aeruginosa
internalization does not simply reflect utilization of a general phagocytic pathway, as Abl is not required for the phagocytosis of dead bacteria 
. Likewise, Abl depletion apparently does not affect the uptake of several other pathogens, including S. typhimurium
), Listeria monocytogenes
, Mycobacterium fortuitum
, and Candida albicans 
. Taken together, these results suggest that a subset of microbial pathogens subvert Abl-dependent pathways during pathogenesis.
Our data also provide new evidence that Crk plays a role in P. aeruginosa
internalization (), that CrkII is phosphorylated by Abl upon P. aeruginosa
infection (), and that the phosphorylation of CrkII contributes to the internalization of P. aeruginosa
(). The phosphorylation of CrkII at tyrosine 221, which is required for its membrane localization, has been shown to modulate the ability of this adaptor protein to interact with other signaling molecules and to regulate the localization of Rac1 and Rac1-dependent signaling 
. Phosphorylation of CrkII has also been demonstrated to be essential for Rac1 and Cdc42 activation upon Shigella
. Based on these findings, we postulate that infection with P. aeruginosa
leads to phosphorylation of CrkII, facilitating its transport to the plasma membrane, where it interacts with other signaling molecules such as the small GTPases, eventually leading to bacterial internalization. The role of Crk in P. aeruginosa
internalization is even more intriguing as this adaptor protein has been identified as the substrate for the T3SS effector ExoT 
. ExoT has been shown to ADP ribosylate Crk on arginine 20 of its SH2 domain, disrupting its ability to interact with Paxillin and p130Cas 
. Our data also suggest that ExoT inhibits CrkII phosphorylation (). Thus, upon translocation of its effector protein ExoT, P. aeruginosa
can downregulate its internalization, at least in part by disruption of CrkII phosphorylation and function.
This study further reveals that invasion of PAK into epithelial cells is at least in part a Cdc42 and Rac1 dependent process () that is subject to complex regulation. Using isogenic mutants in ExoS and/or ExoT, we examined the state of Rac1 and Cdc42 activation, the effect of depletion of Rac1 or Cdc42, and the kinetics of entry to formulate the following model. The effector deficient strain, PAKΔSΔT, likely locally activates Rac1 and Cdc42 to enhance entry into non-phagocytic cells, possibly through the insertion of the T3SS complex. PAKΔT, which translocates ExoS, is even more invasive than PAKΔSΔT, likely because of enhanced activation of Rac1 by the ADPRT activity of ExoS. Our finding that depletion of Rac1 or Cdc42 diminished entry suggests that there may be local activation of Cdc42 in addition to the observed ExoS-dependent activation of Rac1. PAKΔS is the least invasive of the four strains, and is least affected by depletion of Rac1 or Cdc42. This finding suggests that ExoT is able to effective abrogate Rac1 and Cdc42 activation. Finally, the phenotype of PAK may be explained as a complex combination of the synergistic and antagonistic effects of ExoS and ExoT. It is less invasive than PAKΔSΔT and PAKΔT, likely because the GAP activity of ExoT partially counteracts the activation of Rac1 by ExoS.
Previously, our lab reported that ectopic expression of ExoS in PA103ΔUΔT, a P. aeruginosa
strain that does not normally express ExoS, inhibited internalization into macrophages but variably inhibited internalization into MDCK cells 
. These disparate results are readily explained by reports showing that the ability of the ADPRT domain of ExoS to activate Rac1 are cell type specific; Rac1 activation was observed in fibroblasts and epithelial cells, but not macrophages 
. Our finding that ExoS has a slight stimulating effect on bacterial internalization into epithelial cells is particularly remarkable as it might represent a mechanism that explains why ExoS-expressing strains of P. aeruginosa
are more invasive than strains that do not express ExoS. Furthermore, it implies that the effect of ExoS on invasion is context (i.e. cell type) specific. The exact physiological consequence of this remains to be determined, but it is striking that the vast majority of P. aeruginosa
strains produce both ExoS and ExoT 
. It is interesting to speculate that this imparts a flexibility that allows PAK to enter epithelial cells, such as those that line the mucosal barrier, while avoiding uptake by macrophages. Alternatively or in addition, ExoS/ExoT producing strains may exhibit enhanced fitness in the environment.
Our work further demonstrates a role for Pak1 in P. aeruginosa
invasion. Pak1 belongs to a family of highly conserved serine/threonine kinases that are implicated in cytoskeletal rearrangements induced by GTP-bound forms of Rac1 and Cdc42 
(). Interestingly, expression of a constitutively active mutant as well as a kinase-dead mutant of Pak1 inhibited bacterial internalization (). These findings corroborate that cycling of this kinase between an active and inactive state is required for its function 
Pak1 may facilitate P. aeruginosa
invasion by regulating Arp2/3-dependent actin polymerization. Indeed, the Arp2/3 complex is also required in P. aeruginosa
invasion (Table S1
). Pak1 has been shown to interact both in vivo
and in vitro
with p41-Arc, a putative regulatory component of the human Arp2/3 complex 
. Pak1 phosphorylation of p41-Arc regulates its localization with the Arp2/3 complex in the cortical nucleation regions of cells 
. This interaction may represent a mechanism by which the signaling cascade triggered by P. aeruginosa
influences the function of the Arp2/3-complex, leading to the formation of new actin filaments and lamellipodia, and eventually to bacterial uptake.
The activation of the Arp2/3 complex is also mediated by the Wiscott-Aldrich syndrome proteins WASP and WAVE, which are known effectors of Cdc42 and Rac1, respectively 
. As RNAi mediated depletion of WASP and WAVE decreased internalization of P. aeruginosa
into S2 cells (Table S1
), these proteins may also be involved in processes leading to bacterial uptake. This is furthermore supported by the finding that depletion of Abi, Sra-1 and Kette, which form a complex that regulates the function of WASP and WAVE in coordinating the formation of F-actin 
, also affected bacterial internalization (Table S1
). Whether these observations are relevant to non-phagocytic cells remains to be determined.
Our RNAi screen also identified Phosphatidylinositol 3-kinase (PI3K) and Protein kinase B/Akt as host molecules that contribute to efficient P. aeruginosa
internalization. Indeed, recent work in our laboratory demonstrated that PI3K and its downstream effector Protein kinase B/Akt are required for internalization of PAK in MDCK cells 
. It will be important to determine if the PI3K/Akt pathway intersects with the Abl kinase internalization pathway. Preliminary results using the pharmacological Abl inhibitor Gleevec and the PI3K inhibitor LY294002 suggest that these signaling pathways may be separate (Pielage and Engel, unpublished data). It is also possible that the interaction between these two pathways occurs further downstream, such as at the level of the Rho family GTPases. Alternatively, they may share a mutual receptor, though further work will be required to elucidate the details.
As clinically important antibiotic resistance of P. aeruginosa
continues to increase, the identification of host genes essential for the pathogenesis of P. aeruginosa
infections may lead to new drug targets. The Abl inhibitor Gleevec, a well tolerated drug which has become a mainstay for the treatment for chronic myelogenous leukemia and stromal tumors with few side effects 
, has been shown to protect against vaccinia virus infection in mice 
and may prove to be effective against P. aeruginosa
and other pathogens that subvert Abl kinase-dependent pathways. As drugs such as Gleevec affect host instead of bacterial proteins, they are much less likely to engender resistance compared to conventional antimicrobial treatments, and may be applicable to a wide range of pathogens. Future studies will be directed towards assessing these host cell targets as candidates for new therapies.