Translocation of Pseudomonas aeruginosa from the Intestinal Tract Is Mediated by the Binding of ExoS to an Na,K-ATPase Regulator, FXYD3

Abstract

The intestinal tract is considered the most important reservoir of Pseudomonas aeruginosa in intensive care units (ICUs). Gut colonization by P. aeruginosa underlies the development of invasive infections such as gut-derived sepsis. Intestinal colonization by P. aeruginosa is associated with higher ICU mortality rates. The translocation of endogenous P. aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Here we identify bacterial and host proteins associated with bacterial penetration through the intestinal epithelial barrier. We first show by comparative genomic hybridization analysis that the exoS gene, encoding the type III effector protein, ExoS, was specifically detected in a clinical isolate that showed higher virulence in silkworms following midgut injection. We further show using a silkworm oral infection model that exoS is required both for virulence and for bacterial translocation from the midgut to the hemolymph. Using a bacterial two-hybrid screen, we show that the mammalian factor FXYD3, which colocalizes with and regulates the function of Na,K-ATPase, directly binds ExoS. A pulldown assay revealed that ExoS binds to the transmembrane domain of FXYD3, which also interacts with Na,K-ATPase. Na,K-ATPase controls the structure and barrier function of tight junctions in epithelial cells. Collectively, our results suggest that ExoS facilitates P. aeruginosa penetration through the intestinal epithelial barrier by binding to FXYD3 and thereby impairing the defense function of tight junctions against bacterial penetration.Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of infection-related mortality among individuals with compromised immune systems. Fatality rates among patients infected with P. aeruginosa are higher than those among patients infected with any other opportunistic Gram-negative bacterium (48, 51). The lungs are a major site of P. aeruginosa infection in ill patients; however, a considerable number of such infections occur through direct contamination of the lungs by gastrointestinal flora or through hematogenous spread from the intestine to the lungs (51). In particular, the presence of highly virulent strains of P. aeruginosa within the intestinal tract alone is the main source of sepsis and death among immunocompromised patients, even in the absence of established extraintestinal infection and bacteremia (34,41,51). Furthermore, the lethal effects of intestinal P. aeruginosa are dependent upon its ability to adhere to and disrupt the intestinal epithelial barrier (1).The intestinal tract is considered to be the most important reservoir of P. aeruginosa (2). The rate of mortality of patients in intensive care units (ICUs) suffering from intestinal colonization by P. aeruginosa is significantly higher than that of patients without such colonization (34). The notion that gut colonization by P. aeruginosa sets the stage for the underlying development of invasive infection is supported by reports demonstrating a reduction in rates of...

Degradation of interleukin 8 by the serine protease MucD of Pseudomonas aeruginosa

“…The wild-type (PAO1), DexoS, and DexoS/exoS strains are our laboratory stock strains of P. aeruginosa [20]. The DmucD and DPA3535 strains were constructed using the suicide vector pEX18Tc, as described previously [20].…”

“…The DmucD and DPA3535 strains were constructed using the suicide vector pEX18Tc, as described previously [20]. The 502-bp upstream sequence of the mucD gene was amplified with primers PA0766FF (5 0 -GAGGAAGCTTCC CAGAAATGATCGAGGAGCAGGG-3 0 ) and PA0766FR (5 0 -GCCAGCCGGCCGACTACAGCTCCCGTGATGTTC G-3 0 ).…”

“…In addition, the ADPRT activity of ExoS inhibits IL-1b and IL-18 secretion by repressing the activation of caspase-1 in the host cell [14]. Very recently, we have shown that ExoS disrupts intact TJs in Caco-2 cells by altering the distribution of the TJ proteins, ZO-1 and occludin, by binding to the mammalian factor, FXYD3, which co-localizes with and regulates the function of Na, K-ATPase [20].…”

“…Four effector proteins (ExoS, ExoT, ExoU, and ExoY) of the P. aeruginosa type III secretion system have been identified. ExoS is a multifunctional toxin possessing an N-terminal Rho GTPase activating protein (RhoGAP) activity that targets small GTPases [13][14][15][16], a highly promiscuous C-terminally encoded ADP-ribosylation activity (ADPRT) towards small GTP-binding proteins [14,[17][18][19], and an activity to disrupt intact tight junctions (TJs) through its binding to the mammalian factor, FXYD3 [20]. The RhoGAP activity of ExoS causes disruption of the host cell actin cytoskeleton by modulating the switch between the active GTP-bound form and the inactive GDP-bound form of the GTPase.…”

Klebsiella pneumoniae Translocates across the Intestinal Epithelium via Rho GTPase- and Phosphatidylinositol 3-Kinase/Akt-Dependent Cell Invasion

“…In the transcellular pathway, well-studied enteropathogens such as Salmonella, Shigella, Listeria, and Yersinia species invade nonphagocytic epithelial cells by subverting host cytoskeleton dynamics (20). In the paracellular pathway, bacteria such as Vibrio cholerae (21), Campylobacter jejuni (22), and Pseudomonas aeruginosa (23) perturb epithelial integrity to facilitate bacterial translocation by disrupting the cell tight junctions (TJs), the structures between epithelial cells that control paracellular permeability.…”