The ubiquitin ligase Cbl-b limits Pseudomonas aeruginosa exotoxin T–mediated virulence
Priya Balachandran, … , Arthur Weiss, Joanne Engel
Priya Balachandran, … , Arthur Weiss, Joanne Engel
Published February 1, 2007
Citation Information: J Clin Invest. 2007;117(2):419-427. https://doi.org/10.1172/JCI28792.
View: Text | PDF
Research Article Microbiology

The ubiquitin ligase Cbl-b limits Pseudomonas aeruginosa exotoxin T–mediated virulence

Abstract

Pseudomonas aeruginosa, an important cause of opportunistic infections in humans, delivers bacterial cytotoxins by type III secretion directly into the host cell cytoplasm, resulting in disruption of host cell signaling and host innate immunity. However, little is known about the fate of the toxins themselves following injection into the host cytosol. Here, we show by both in vitro and in vivo studies that the host ubiquitin ligase Cbl-b interacts with the type III–secreted effector exotoxin T (ExoT) and plays a key role in vivo in limiting bacterial dissemination mediated by ExoT. We demonstrate that, following polyubiquitination, ExoT undergoes regulated proteasomal degradation in the host cell cytosol. ExoT interacts with the E3 ubiquitin ligase Cbl-b and Crk, the substrate for the ExoT ADP ribosyltransferase (ADPRT) domain. The efficiency of degradation is dependent upon the activity of the ADPRT domain. In mouse models of acute pneumonia and systemic infection, Cbl-b is specifically required to limit the dissemination of ExoT-producing bacteria whereas c-Cbl plays no detectable role. To the best of our knowledge, this represents the first identification of a mammalian gene product that is specifically required for in vivo resistance to disease mediated by a type III–secreted effector.

Authors

Priya Balachandran, Leonard Dragone, Lynne Garrity-Ryan, Armando Lemus, Arthur Weiss, Joanne Engel

×

Figure 3

Role of Crk and Cbl-b in the ubiquitination and degradation of ExoT.

Options: View larger image (or click on image) Download as PowerPoint
Role of Crk and Cbl-b in the ubiquitination and degradation of ExoT. (A–...
(A–E) HeLa cells were pretreated with (A) no siRNA, (B and E) control siRNA, (C) Cbl-b siRNA, or (F) Crk siRNA for 30 hours prior to infection with PA103ΔexoU/exoT(G-A+). Translocation assays were performed as described for Figure 1. Cytoplasmic extracts were analyzed by immunoblotting using Abs against ExoT (A–C, E, and F; top panels) and loading control GAPDH (A–C, E, and F; middle panels). siRNA knockdown efficiency was determined by immunoblotting against Cbl-b (A–C; bottom panels) or Crk (E and F; bottom panels). (D) Rates of degradation in the presence or absence of Cbl-b siRNA were quantified as described for Figure 1C and plotted as means ± SD. Error bars are too small to be seen at some points. (G–H) HeLa cells were pretreated with (G) vector control pCEFL or (H) pCEFL–HA–Cbl-b (C373A) for 24 hours. Translocation assays were performed as described for Figure 1. Cytoplasmic extracts were analyzed by immunoblotting using Abs against ExoT (top panels) and GAPDH (middle panels). Blots were probed with anti-HA Abs (bottom panels) to verify expression of transfected protein. (I) HeLa cells were transfected with pCruzMycB-ExoT(G-A+), pcDNA3-(HA-Ub)4 and pCEFL, pCEFL–HA–Cbl-b, or pCEFL–HA–Cbl-b (C373A) for 18 hours. Ubiquitination of ExoT was assessed as described for Figure 1D. ExoT was immunoprecipitated using anti-ExoT antiserum under conditions in which ExoT and Cbl-b do not coimmunoprecipitate (in the presence of 0.5% SDS) and immunoblotted with anti-HA (top panel, ubiquitinated ExoT) or anti-Myc (bottom panel, total ExoT).