Given the importance of cysteine cathepsin inhibitors
Given the importance of cysteine cathepsin inhibitors as putative therapeutic molecules and microbial virulence factors, we chose to investigate whether Yersinia enterocolitica may be a potent producer of such inhibitors. Y. enterocolitica is the most heterogeneous species within the genus Yersinia. It comprises Gram-negative coccobacilli, which cause yersiniosis – the third most common zoonotic bacterial disease of gastrointestinal tract in the European Union and other countries in temperate climate zones (Rahman et al., 2011). Swine are the primary reservoir of both presumably nonpathogenic (biotype 1A) and pathogenic (biotypes 1B, 2–5) Y. enterocolitica strains, which can be transmitted to humans via contaminated food, such as raw or undercooked pork, raw or pasteurized milk, liquid egg products and vegetables (Bottone, 1997, Rahman et al., 2011). The bacterium may then colonize the distal small intestine and proximal colon, where it penetrates the epithelium and, subsequently, is internalized by phagocytes and transported within them to the mesenteric lymph nodes, liver and spleen (Bottone, 1997, Fàbrega and Vila, 2012). The virulence factors of Y. enterocolitica, which are encoded by the genes located on the bacterial chromosome and the 70 kb virulence plasmid pYV, include: Ail (attachment-invasion locus) enabling adhesion to and invasion of mammalian cells, as well as conferring resistance to the bactericidal activity of serum; Yst (Yersinia stable toxin) damaging the intestinal mucus layer; Yops (Yersinia outer proteins) limiting immune and inflammatory responses (Fàbrega and Vila, 2012, Skorek et al., 2013). There are several reasons for selecting Y. enterocolitica to analyze its inhibitory potential on cysteine proteases. Firstly, the bacterium secretes virulent cysteine proteases – the ARL 67156 trisodium salt proteins YopP and YopT (the latter being homologous to papain) (Fàbrega and Vila, 2012, Rawlings et al., 2012), but it has not been investigated whether the activity of these enzymes is controlled intracellularly by any endogenous inhibitors as is the case for cysteine proteases synthesized by other pathogenic bacteria (Kędzior et al., 2016). Secondly, diverse bacterial species secrete the homologues of α2-macroglobulin, a homotetrameric glycoprotein capable of inhibiting proteases of any catalytic type. These homologues prevent the degradation of bacterial cells by the host\'s proteolytic defense system (Wong and Dessen, 2014). The homologue of α2-macroglobulin is also encoded in the genome of Y. enterocolitica (Rawlings et al., 2012), but it remains unclear whether this protein is produced by the bacterium as a functional protease inhibitor. Finally, Y. enterocolitica may replicate effectively within phagocytes (Mills et al., 1997) and induce the apoptosis of antigen-presenting cells (Erfurth et al., 2004) by involving the secretory machinery named the type III secretion system (T3SS) (Fàbrega and Vila, 2012), thereby restraining innate and adaptive immune responses, respectively. Several pathogenic microbes impair these responses through the action of their cysteine cathepsin inhibitors (Kędzior et al., 2016), which have not yet been shown to be synthesized by Y. enterocolitica. 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 proteins 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.