The inhibitory potential of Y

The inhibitory potential of Y. enterocolitica strains from pigs with reproductive disorders (Platt-Samoraj et al., 2006, Platt-Samoraj et al., 2009) was tested against papain family cysteine proteases in a few steps. Initially, we investigated the effect of Y. enterocolitica cell-free extracts, conditioned culture media and cellular surface on the activity of papain and cathepsins B and L. Then, the impact of different culture conditions on the activity of cysteine protease bacterial inhibitors was assessed. The inhibitory potential of the coccobacillus was also compared with the antiproteolytic activity of two other widespread and often pathogenic Gram-negative species, Escherichia coli and Pseudomonas aeruginosa, which were included in our study as the reference bacteria since their genomes have already been shown to encode the fbpase affecting the activity of cysteine proteases (Garcia-Ferrer et al., 2015, Sanderson et al., 2003). Finally, the biochemical and molecular characterization of the inhibitors synthesized by Y. enterocolitica was performed and concluded with the identification of a proteinaceous putative inhibitor of papain and cathepsin L in both Y. enterocolitica and E. coli.
Material and methods
Results
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 genes (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.