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  • br Material and methods br Results br Discussion In the


    Material and methods
    Discussion In the present study, twenty strains of Y. enterocolitica were analyzed for their inhibitory potential on cysteine proteases. Five of these strains belonged to bioserotype 4/O:3, which is distinguished for its pathogenicity towards humans. The rest of strains represented biotype 1A, which comprises presumably nonpathogenic coccobacilli, predominantly lacking virulence markers, such as ail and ystA p450 inhibitors (Platt-Samoraj et al., 2006, Platt-Samoraj et al., 2009). However, our results (Table A.1) remain contradictory to the notion that ail confers Y. enterocolitica resistance to the bactericidal activity of serum (Fàbrega and Vila, 2012), thus questioning the contribution of this gene to bacterial virulence. Besides, the tested strains of biotype 1A contained the ystB gene (Table 1), which encodes a thermostable enterotoxin homologous to the product of ystA. Many strains of biotype 1A were isolated from the organs of humans suffering from digestive tract disorders, while the toxin YstB proved to be the major cause of diarrhea (Singh and Virdi, 2004). Moreover, several strains of biotype 1A may induce the clinical symptoms similar to those caused by the pathogenic strains of other biotypes (Bottone, 1997). Therefore, all Y. enterocolitica strains tested in this study could be regarded as potential pathogens. The same strains had been previously isolated from the organs colonized typically by pathogenic Y. enterocolitica, and were then suggested to cause the reproductive disorders in pigs (Platt-Samoraj et al., 2009). We hypothesized that these strains were likely to develop the effective defense mechanisms against their host's immune system and, hence, to produce the inhibitors of cysteine proteases involved in immune responses. The inhibitory potential of Y. enterocolitica on cysteine proteases was compared with that of other Gram-negative bacteria – E. coli and P. aeruginosa. It remains unclear whether E. coli synthesizes cysteine cathepsin inhibitors to restrain either phagocytosis or antigen presentation to survive within its host; however, the E. coli genome encodes the homologue of α2-macroglobulin synthesized as a functional inhibitor of endopeptidases of different catalytic types (Garcia-Ferrer et al., 2015). P. aeruginosa has not yet been shown to produce any cysteine cathepsin inhibitors involved in virulence, but its genome encodes the homologue of cysteine protease inhibitor chagasin. A recombinant form of P. aeruginosa chagasin homologue inhibited the activity of mammalian cathepsin L (Sanderson et al., 2003). The marine strain of Pseudomonas sp. also proved to secrete two small-molecule inhibitors of cathepsin B (Hoang et al., 2008b). The inhibitory activity of bacteria was tested against cysteine proteases of the p450 inhibitors papain family (C1): papain, cathepsin B and cathepsin L. Papain is an archetype of all other enzymes of the family C1. It was applied as a model peptidase in the course of designing new potent and selective inhibitors of cysteine cathepsins (LaLonde et al., 1998). Bovine cathepsin B and human cathepsin L, 28 and 39% homologous to papain (respectively), are greatly involved in phagocytosis by macrophages and other phagocytic cells in mammals (Flannagan et al., 2009), and – especially cathepsin L – in antigen presentation through the major histocompatibility complex (MHC) class II molecules (Watts, 1997). We have discovered the presence of papain inhibitors in the extracts, conditioned culture media and cell suspensions of E. coli, P. aeruginosa and several Y. enterocolitica strains (Figs. 1, 2 and A.2). All tested extracts and cell suspensions inhibited additionally the activity of cathepsin L (Figs. 2 and A.2), but only the extracts of E. coli and P. aeruginosa affected the activity of cathepsin B (Fig. A.2). Having regard to the limited access of high-molecular-weight substrates and inhibitors to the active site of cathepsin B (Yamamoto et al., 2000), we could assume that Y. enterocolitica strains, in contrast to other tested bacteria, produced exclusively the high-molecular-weight inhibitors of cysteine proteases. These inhibitors could be differently distributed in bacterial cells as the cellular surface, unlike the cell-free extracts, exhibited predominantly higher inhibitory activity against cathepsin L than against papain. Other authors have also confirmed the inhibitory potential of bacterial extracts, post-culture media and intact cells on the analyzed peptidases. Pavlova et al. (2006) demonstrated that the periplasmic extract of Plesiomonas shigelloides inhibited papain and mammalian cathepsins B and L, and the cell surface of this bacterium influenced the activity of papain. In another study, the conditioned medium obtained from the culture of the marine strain of Pseudomonas sp. was shown to inhibit cathepsin B (Hoang et al., 2008a).