UCSF

The opportunistic human pathogen Pseudomonas aeruginosa utilizes a type III secretion system to deliver virulence factors that disrupt host cell processes and host innate immunity. Four type III-secreted toxins have been identified in this bacterium: ExoS, ExoT, ExoU and ExoY. ExoT is a bifunctional enzyme whose N-terminus encodes a GTPase activating protein with activity towards Rho, Rac, and Cdc42 while the C-terminus encodes an ADP ribosyl transferase with activity towards Crk. ExoT inhibits bacterial internalization into host cells, disrupts the actin cytoskeleton, and blocks wound repair. We previously showed that ExoT is degraded in a proteasomal dependent manner and that in vivo resistance against this toxin is Cbl-b-dependent. Though Cbl-b is known to influence actin cytoskeleton and neutrophil infiltration into LPS treated host tissues, we show that Cbl-b is not necessary for phagocytosis of P. aeruginosa by macrophages or neutrophil infiltration into P. aeruginosa infected host tissues. In addition, we demostrate that ExoT lysine residues 49 and 74 control its proteasomal dependent degradation in the host cytoplasm. We further demonstrate by host cell rounding assays and an acute pneumonia mouse model that mutating these lysine residues to arginine increases the virulence of P. aeruginosa. Interestingly, mutating lysine 49 and 74 did not alter the virulence of P. aeruginosa in cbl-b-/- mice, which suggests that Cbl-b-mediated protection of the host against P. aeruginosa must depend on the targeting of these two lysine residues in the toxin. Although mutation of lysine 49 to arginine is able to increase toxin secretion rate into the medium, we found that mutations in both lysine 49 and 74 are necessary for increased levels of translocated toxin in the host cytoplasm. Collectively, these data demonstrate that ExoT lysine residues at positions 49 and 74 play a key role in toxin degradation by the host proteasome, which leads to an alteration of ExoT-mediated virulence of P. aeruginosa. Our findings also suggest that Cbl-b targets these residues through either ubiquitination or another yet unidentified molecular process. Experiments are underway to determine whether Cbl-b is able to polyubiquitinate the mutant toxin as it does on wild type ExoT.