Intrinsic Degradation of the Type-II Antitoxin ParD from Pseudomonas aeruginosa
Type-II Toxin Antitoxin (TA) systems are regulated by differential half-lives of the resulting non-secreted proteins, such that the neutralizing antitoxin undergoes continual degradation and replenishment to maintain neutralization of its cognate toxin. Antitoxin proteins are widely reported as labile, including upon purification and in vitro storage. During the course of studies on a ParDE TA system we noted a prevalent in vitro degradation of the ParD antitoxin. In efforts to combat this for practical use in assays, we characterized parameters impacting the degradation as well as the resulting products. These revealed a mechanism likely mediated by a serine or metal-dependent protease. Using Direct Infusion Mass Spectrometry, the cleavage products were identified as an essentially intact DNA binding region of the antitoxin and with the toxin binding domain completely removed. No other species were identified in the solution, such as a contaminant that may mediate such cleavage. Therefore, while our studies revealed viable strategies to mitigate the in vitro degradation they did not identify any protease, leaving open the possibility of a potential auto-catalytic proteolytic activity of the antitoxin proteins.