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 1

ExoT is degraded by proteasomes and is polyubiquitinated.

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ExoT is degraded by proteasomes and is polyubiquitinated. (A–C) HeLa cel...
(A–C) HeLa cells were cocultivated with PA103ΔexoU/exoT(G-A+) for 1.5 hours in the (A) presence or (B) absence of 10 μM lactacystin. Extracellular bacteria were then killed by the addition of amikacin. Protein synthesis of internalized bacteria was inhibited by the addition of chloramphenicol. At the indicated times, cytoplasmic extracts were prepared, and translocated ExoT was assayed by immunoblotting with anti-ExoT antiserum (upper panels, A and B). To control for loading efficiency, the blots were probed with anti-GAPDH mAbs (lower panels, A and B). (C) The rate of degradation of ExoT was quantified by normalizing the amount of ExoT at each time to the corresponding amount of GAPDH. This ratio was plotted as a percentage of the ratio at time 0 (2 hpi). Shown are the means ± SD. In some cases, the error bars are too small to be seen. (D and E) HeLa cells were transfected with the indicated plasmids for 18 hours; 10 μM lactacystin was added, and the cells were further incubated for 4 hours. (D) To assess overall levels of transfected proteins, whole-cell lysates were immunoblotted with anti-HA (upper panel) or anti-Myc (for ExoT) (lower panel). (E) ExoT was immunoprecipitated using anti-ExoT antiserum and immunoblotted with anti-HA (upper panel, ubiquitinated ExoT) or anti-Myc (lower panel, total ExoT). Polyubiquitinated ExoT, indicated by the slower migrating forms of ExoT, is only visible in cells cotransfected with the ExoT and HA-Ub vectors (lane 4, upper portion of E).