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.
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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

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Figure 4

The stability of ExoT in the host cytosol is modulated by the activity of its ADPRT domain.

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The stability of ExoT in the host cytosol is modulated by the activity o...
(A) HeLa cells were cocultivated with PA103ΔexoU/exoT(G-A+) or PA103ΔexoU/exoT(G-A-) for 1.5 hours. Translocation assays were performed as described for Figure 1. Translocated ExoT was quantified by immunoblot analyses using antiserum against ExoT (upper panels). As a control, blots were probed with anti-GAPDH mAbs (lower panels). (B) The rates of degradation were quantified as described for Figure 1C. The ratio obtained at each time point was plotted as a percentage of the ratio at time 0 (2 hpi). Shown are means ± SD. In some cases, error bars are too small to be seen. (C) HeLa cells were cocultivated with PA103ΔexoUΔexoT + pUCP20ExoS(G-A+) or PA103ΔexoU/ΔexoT + pUCP20ExoS(G-A-), and translocation assays were performed as previously described. (D) HeLa cells were transfected with p-IRES–ExoT or the indicated ExoT mutants. Eighteen hours after transfection, cells were lysed and steady-state levels of ExoT quantified by immunoblotting with anti-ExoT (upper panel). Gels were immunoblotted with anti-GAPDH as a control (lower panel). As reported earlier, ADPRT-deficient variants of ExoT migrate with a slower mobility than ADPRT+ forms of ExoT (24).