Culture conditions particularly temperature which affect the
Culture conditions (particularly temperature), which affect the pathogenicity of Y. enterocolitica (Bottone, 1997), may also impact its inhibitory properties against cysteine proteases. However, culturing the bacterial strains at 37 instead of 28 °C did not alter remarkably the inhibitory potential of their extracts (except for the strain 19), but had an apparent effect on the proton pump inhibitor of papain inhibitors by most of the tested strains, causing either induction or impairment of this process (Figs. 2A and B). Some authors have also investigated the effect of culture temperature on the production of cysteine protease inhibitors by several bacterial species. Sarafeddinov et al. (2011) showed that the actinomycete Streptomyces mobaraensis secreted the inhibitor of papain into the medium when cultured at temperature values ranging from 28 to 42 °C. Changing the culture temperature from 28 to 37 °C during the logarithmic growth phase resulted in the increased secretion of the inhibitor. Hoang et al. (2008a) cultured the marine strain of Pseudomonas sp. at 16–40 °C, but only at 28 °C the bacterium secreted large amount of the inhibitors of cathepsin B into the medium. Then, in our study we demonstrated that Y. enterocolitica strains produced the inhibitors of papain and cathepsin L regardless of the bacterial growth phase (i.e., time of culture). The research on S. mobaraensis (Sarafeddinov et al., 2011) and Pseudomonas sp. (Hoang et al., 2008a) also confirmed the synthesis of cysteine protease inhibitors (inhibitors of papain and cathepsin B, respectively) by these bacteria at different growth phases. Hoang et al. (2008a) suggested that the inhibitors of cathepsin B, secreted by Pseudomonas sp., might constitute primary metabolites of the coccobacillus as they were produced during the intensive bacterial growth, which was also observed for the inhibitors of papain and cathepsin L synthesized by Y. enterocolitica strains analyzed in the present work. Additionally, we showed that culturing Y. enterocolitica under nutrient limitation (in M9 minimal medium) caused the increased secretion of papain inhibitors by all tested strains (Fig. 2B). Pavlova et al. (2006) discovered that the cell surface of P. shigelloides exhibited the increased inhibitory activity against papain when the bacterium was grown under the other stress conditions (anaerobically). Culturing Pseudomonas sp. in the medium containing organic nitrogen, and glucose and maltose as carbon sources yielded the effective secretion of cathepsin B inhibitors (Hoang et al., 2008a). In the present study, three different assays were applied to confirm the reversible mode of papain and cathepsin L inhibition by the extract of the Y. enterocolitica strain 19 (4/O:3) (Fig. 4). The results suggested the proteinaceous nature of the bacterial inhibitors since the vast majority of proteinaceous inhibitors bind reversibly to their target cysteine proteases (Turk et al., 2012). Subsequently, we demonstrated that the inhibitors present in the bacterial extracts were thermolabile as the inhibitory potential of the extracts was completely lost upon incubation at 60 °C (Fig. 5A). The inhibitors were stable at temperature values below 50 °C as was the case for the proteinaceous noncompetitive inhibitor of papain, isolated from the plasma of chum salmon (Li et al., 2008). On the contrary, the periplasmic extract of P. shigelloides (Pavlova et al., 2006), as well as the substrate of transglutaminase from S. mobaraensis (Sarafeddinov et al., 2011) retained their inhibitory activity against papain after incubation at 100 °C. The cystatin-like inhibitor from C. albicans was also thermostable as it inhibited papain upon incubation at 40–90 °C (Tsushima et al., 1992). The observed thermolability of the inhibitors present in the extracts of Y. enterocolitica allowed us to assume that these inhibitors probably do not possess a cystatin-like fold (Katunuma and Kominami, 1995).