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 5

Cbl-b limits P. aeruginosa infection in an ExoT-dependent manner.

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Cbl-b limits P. aeruginosa infection in an ExoT-dependent manner. ...
(A–E) C57BL/6 (cbl-b+/+) or C57BL/6 (cbl-b–/–) were infected i.n. with 2.5 × 108 of PA103ΔexoUΔexoT (A and B) or 1 × 108 of PA103ΔexoU (C and D). A higher dose of PA103ΔexoUΔexoT compared with PA103ΔexoU was necessary in order to quantify lung or liver CFUs at 18 hours. At 18 hpi, mice were sacrificed and bacterial CFUs were enumerated from the lung (A and C) and liver (B and D) tissues by plating serial dilutions of tissue homogenates on LB agar plates. (E) The average ± SEM of the liver/lung ratio of bacterial counts in cbl-b+/+ and cbl-b–/–mice was determined for each mouse. (F–G) cbl-b+/+ or cbl-b–/– mice were infected i.n. with 1 × 108 CFUs of PA103ΔexoU. Mice were sacrificed at the indicated times after infection, and bacterial CFUs in the lungs (F) and liver (G) were enumerated. Liver/lung ratio of bacterial counts was determined for each mouse and plotted as an average for cbl-b+/+ and cbl-b–/– mice (H). (I–M) cbl-b+/+ or cbl-b–/– were infected i.p. with 1 × 107 of PA103ΔexoUΔexoT or PA103ΔexoU. At 18 hpi, mice were sacrificed and bacterial CFUs were enumerated from the lungs (I), liver (J), and spleen (K). The lung/liver (L) or lung/spleen (M) ratio of bacterial counts was determined for each mouse and plotted as an average ± SEM for cbl-b+/+ and cbl-b–/–mice. Statistical analyses were performed by the 2-tailed Mann-Whitney U test. *P < 0.05; **P < 0.005; ***P < 0.001.