Identification and characterization of Epp, the secreted processing protease for the Vibrio anguillarum EmpA metalloprotease.

Vibrio anguillarum, a marine bacterium, is the causative agent of vibriosis, a systemic disease of both wild and cultured marine fish characterized by hemorrhagic septicemia (2). Outbreaks of vibriosis result in high mortalities among infected fish, and this disease continues to be a major obstacle for the aquaculture industry (2). V. anguillarum enters its fish host through the gastrointestinal (GI) tract and quickly colonizes this nutrient-rich environment (21, 22). Garcia et al. (9) have shown that V. anguillarum grows extremely well in salmon intestinal mucus and that mucus-grown cells specifically express a number of different proteins, including several outer membrane proteins (9) and the extracellular metalloprotease EmpA (7). The zinc metalloprotease EmpA has been identified as a virulence factor for V. anguillarum and is important for virulence during infection of the GI tract of Atlantic salmon (Salmo salar) (7, 16, 19). In V. anguillarum wild-type strain M93Sm, EmpA is expressed during stationary phase when cells are incubated in Atlantic salmon GI mucus (6, 7). The creation of an empA null mutant (M99) by insertional mutagenesis showed that EmpA is responsible for the protease activity observed for wild-type strain M93Sm (7). However, EmpA activity is dependent on successful secretion and processing of the nascent protein to an active mature protease (16, 25). According to Milton et al. (16), EmpA is synthesized as a 66.7-kDa preproenzyme. Sequence analysis predicts that the removal of both pre- and propeptides during secretion would result in a mature protein with a molecular mass of 44.6 kDa. However, EmpA protease activity was repeatedly associated with a 36-kDa protein (16). This suggests that the 44.6-kDa protein undergoes further processing to a 36-kDa active form. Recently, Staroscik et al. (25) used Western blot analysis to study EmpA secretion in V. anguillarum culture supernatants. Using anti-LasB antibodies for detection, Western blot analysis revealed an ∼46-kDa band in all culture supernatants as well as an ∼36-kDa band that could be detected only in culture supernatants possessing protease activity (25). The ∼46- and ∼36-kDa bands correspond to the sizes of the predicted secreted proenzyme and the mature protein, respectively (16). Staroscik et al. (25) also confirmed the presence of the cytoplasmic preproenzyme. It has been suggested that the activation of the proenzyme occurs after secretion by the removal of an ∼10-kDa peptide. The protease responsible for the posttranslational modification of pro-EmpA to the mature enzyme has yet to be identified. Multiple processing steps have been proposed for other metalloproteases, such as the hemagglutinin/protease (HA/protease) of V. cholerae (12), the Vvp protease of V. vulnificus (17), and the LasB elastase of Pseudomonas aeruginosa (16). In this study, we sought to further examine the processing of pro-EmpA to mature EmpA metalloprotease. Minitransposon mutagenesis (mini-Tn10Km) was used to create and screen for protease mutants. One mutant that exhibited a reduction in protease activity compared to wild-type protease activity was detected. The region surrounding this mutation in V. anguillarum was cloned and sequenced. One open reading frame was identified and mutated by insertional mutagenesis. The resulting strain was designated M101. Protease assays and Western blots were performed to determine changes in protease activity and processing of EmpA from what was seen for wild-type strain M93Sm. These experiments led to the identification of a putative EmpA-processing protease gene (epp). Our results strongly suggest that epp is the protease responsible for the activation of pro-EmpA to the mature EmpA protease.