| Summary: | The effect of salmon gastrointestinal mucus on protease activity in Vibrio anguillarum M93Sm was investigated. Cells grown to stationary phase in nine salts solution plus mucus (NSSM) exhibited ninefold-greater protease activity than cells grown in Luria-Bertani broth plus 2% NaCl (LB20). Induction of protease activity in stationary phase cells occurred 60 min after addition of mucus and was ≥70-fold greater than protease activity measured in cells incubated in any other condition. Southern analysis and PCR confirmed that V. anguillarum M93 contained empA. An empA mutant of V. anguillarum did not exhibit protease activity after exposure to mucus, but did grow in mucus. Atlantic salmon challenged with V. anguillarum M93Sm and NB10 resulted in mortality when administered by intraperitoneal injection (IP) or anal intubation (AIB). NB12 (empA mutant) was avirulent via either route of infection. M99 (empA mutant) virulence was attenuated by AIB, but fully virulent by IP. It is hypothesized that differences in virulence are due to differences in regulation of empA. Northern analysis of empA and quantification of protease activity from LB20 or NSSM incubated cells or cells incubated in conditioned media demonstrate that expression of empA is differentially regulated in V. anguillarum strains NB10 and M93Sm and requires σ S, quorum sensing molecules, and gastrointestinal mucus. Autoinducer-2 (AI-2) is a non-AHL quorum sensing signal molecule that regulates bioluminescence in Vibrio harveyi. In an assay that measures luminescence, the V. harveyi sensor strain BB170 detected AI-2 activity in V. anguillarum culture supernatants during late-exponential growth phase and stationary phase. Southern analysis demonstrated the presence of a V. harveyi luxS (AI-2 synthase) homolog in V. anguillarum. M93Sm cells incubated in conditioned LB20 supernatants from a V. anguillarum luxS mutant, M01, which did not contain AI-2 activity, stimulated an earlier and greater level of protease activity than wild-type conditioned LB20. Wild-type V. anguillarum survived better during starvation in NSS than the luxS mutant. Infection studies in Atlantic salmon revealed no decline in virulence of M01 compared to M93Sm. It does not appear that the V. anguillarum LuxS quorum sensing system is important for growth or virulence, but may be important during starvation-survival.
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