| Vibrio vulnificus is a Gram-negative, motile marine bacterium found in many coastal areas around the world that can cause human infection if ingested with undercooked seafood or through wound exposure to contaminated seawater. Upon exposure V. vulnificus spreads throughout the body rapidly and this spread is mediated by the toxins it secretes once inside the human host. Two toxins cause most of the damage: hemolysin and the multifunctional autoprocessing repeats-in-toxin toxin, or MARTX Vv. MARTXVv is of particular interest because it is composed of several effector domains, which have distinct and unknown functions. One of these effector domains, domain of unknown function in the 5th position (DUF5), has been shown to be important in the mouse model of infection. When deleted from the MARTX holotoxin the LD50 after intragastric infection increases by 10-50 fold. DUF5 is an effector domain of unknown function with limited sequence identity to Pasteurella multocida toxin. Genetic, molecular and cell biology techniques were applied to identifying the host target of DUF5, its function, and residues essential for activity. In this work, DUF5 is shown to be an endopeptidase capable of cleaving mammalian Ras and Rap1A proteins. The effect of Ras and Rap1A cleavage is complete dephosphorylation of ERK1/2 followed by cell rounding and inhibition of cell division. The same effect is elicited by DUF5 in the context of a bacterial challenge, indicating that Ras and Rap1A proteolysis may be important for V. vulnificus pathogenesis in vivo. Thus, DUF5 has been renamed RRSP, for its ability to cleave Ras and Rap1A. One key implication of this work is that RRSP is the first known protein toxin that cleaves Ras and Rap1A in the switch I region, and this makes RRSP a toxin with a novel biochemical activity but also a potential immunotoxin that may be developed further for the treatment of cancer. |
