In this paper, we identify what we believe to be a novel role for the E3 ubiquitin ligase and adaptor molecule Cbl-b in the early defense response and in bacterial pathogenesis. We specifically show that ExoT, a type III–secreted effector and critical virulence factor of P. aeruginosa
, is ubiquitinated and undergoes rapid proteasomal degradation in a Cbl-b–dependent manner whereas the closely related effector ExoS is not rapidly degraded. We further demonstrate that the E3 ubiquitin ligase activity of Cbl-b is required for ExoT ubiquitination and degradation and that the rate of degradation is regulated by ExoT ADPRT activity. We find that Cbl-b is tyrosine phosphorylated upon addition of P. aeruginosa
and is found in a complex with ExoT and its substrate Crk. Crk appears to be required for the interaction of ExoT and Cbl-b that leads to rapid degradation of ExoT. Our data demonstrate that, in vitro, ExoT has a prolonged half-life in the absence of Cbl-b or Cbl-b ubiquitin ligase activity. These observations highlight Cbl-b as one of the few identified examples of a mammalian gene product that is specifically required to degrade a type III effector (22
Using 2 different mouse models, acute pneumonia and acute peritonitis, we demonstrate the in vivo relevance of these findings. In both models, Cbl-b was specifically required to limit the dissemination of ExoT-producing bacteria whereas c-Cbl had no effect. We observed higher bacterial loads as well as increased dissemination to distant organs and increased ascites fluid in cbl-b–/– mice infected with PA103ΔexoU compared with wild-type mice. Our findings reinforce the role for ExoT in dissemination of P. aeruginosa in a mouse model of acute pneumonia and demonstrate, for what we believe is the first time, a role for ExoT in the i.p. infection model. The loss of Cbl-b did not appear to have an impact on colonization or spread of ExoT-deficient P. aeruginosa, eliminating the possibility that cbl-b–/– mice are simply more susceptible to microbial infections.
mice, we provide the first evidence, to our knowledge, that this multifaceted protein is involved in the early defense response against infectious agents: the defect in limiting dissemination of ExoT-producing bacteria was observed as early as 12–18 hpi. These findings are distinct from previous studies that implicated a role for Cbl-b in humoral immunity, specifically in limiting T cell receptor stimulation in response to antigen (25
). As Cbl-b is widely expressed (25
), it might be predicted to play multiple roles in host defense. For example, by enhancing the degradation of a bacterial toxin, it may help to preserve the integrity of the epithelial or endothelial barrier. Alternatively, it may be required in neutrophil- or macrophage-mediated responses. Experiments to test these possibilities are in progress.
Our data support a model in which ExoT, Crk, and Cbl-b form a complex that leads to ubiquitination of ExoT by Cbl-b. Phosphorylation of Cbl-b occurs rapidly upon addition of P. aeruginosa
, within 5 minutes, prior to the translocation of ExoT (which is not detectable under the conditions of our experiments prior to 15 minutes; unpublished data). Phosphorylated Cbl-b binds to Crk SH2 domain. Once translocated, ExoT binds to the Crk–Cbl-b complex. Cbl-b is not a known substrate of ExoT (9
) and therefore would not be predicted to bind ExoT directly. As Crk itself does not have any enzymatic activity, we propose that Crk, functioning as a molecular scaffold, recruits Cbl-b to ExoT. Indeed, siRNA depletion of Crk alone is sufficient to increase ExoT stability. In the absence of Cbl-b or Cbl-b ubiquitin ligase activity, ExoT has a longer half-life, thus leading to increased ExoT-mediated damage to host cells and presumably increased bacterial dissemination in vivo.
The formation and activity of the ExoT–Crk–Cbl-b complex may be subject to extensive regulation, at least in response to P. aeruginosa
infection. We found that addition of P. aeruginosa
increased the phosphorylation of both Crk and Cbl-b, which may have complicated and opposing biological consequences (unpublished data). Additionally, we found that ExoT-ADPRT activity enhances degradation. Changes in the amount of tyrosine phosphorylated Cbl-b or Crk may modulate the interaction of Cbl-b and ExoT. We have preliminary data indicating that ADP-ribosylation by ExoT decreases tyrosine phosphorylation of CrkII (unpublished data). ADP ribosylation of Crk itself also alters the affinity of Crk for some SH2-binding proteins (10
). Whether this affects Cbl-b binding to Crk is currently unknown. It is also possible that ExoT(G-A+) and ExoT(G-A-) bind to Crk and/or Cbl-b with different affinities, resulting in their differential susceptibility to degradation.
Our findings that cbl-b–/–
mice but not c-cbl–/–
mice are uniquely susceptible to ExoT-producing P. aeruginosa
, that only Cbl-b is phosphorylated by P. aeruginosa
, and that Cbl-b is uniquely involved in ExoT degradation provides extensive new information to support the notion that c-Cbl and Cbl-b, though highly homologous, can have functionally distinct roles. These findings are consistent with reports that c-Cbl and Cbl-b perform different roles in regulating T cell signaling (25
). c-Cbl primarily regulates the T cell receptor complex whereas Cbl-b controls T cell signaling through the Rho family guanine exchange factor VAV and Wiskott-Aldrich syndrome protein (WASP), which eventually leads to cytoskeletal remodeling. Understanding how ExoT discriminates between these 2 proteins may shed fundamental insights into their function. One possibility is that bacterial binding and entry lead to Crk and specifically Cbl-b recruitment, consistent with its proposed involvement in regulating components of the actin cytoskeleton during T cell signaling. Indeed, we have preliminary evidence that Cbl-b plays a role in P. aeruginosa
entry into cultured epithelial cells (unpublished observations).
Alternatively, the interaction between ExoT and Cbl-b may be a mechanism by which P. aeruginosa
temporally regulates the expression of the toxin in the host cell. Indeed, previous studies have demonstrated that, while type III–secreted effectors are necessary for the establishment of an initial acute infection, these functions are lost under conditions of chronic persistence (3
). Thus, modulation of ExoT stability by Cbl-b–mediated degradation may facilitate a transition from an acute infection to a chronic or persistent infection.
The differential susceptibility of the closely related toxins ExoT and ExoS to proteasomal degradation adds them to the list of bacterial toxins whose activity is modulated by host-mediated proteasomal degradation (21
). Interestingly, unlike the Salmonella enterica
proteins SopE and SptP, whose N terminal secretion and translocation domains modulate the temporal regulation of their activities through ubiquitination and differential proteasomal degradation (21
), the rate of degradation of ExoS and ExoT is regulated by their ADPRT-domain binding partners.
The results described in this paper describe a novel role for Cbl-b in bacterial pathogenesis and suggest that Cbl-b is important in host response to infection. By identifying a host protein that specifically limits bacterial dissemination mediated by a single effector, they highlight how the host develops a fine-tuned mechanism to downregulate or inactivate bacterial toxins. This new information could help in developing strategies directed toward enhancing host defense and/or limiting excessive host response to improve outcome in P. aeruginosa lung infections.