Crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata

Abstract

Synopsis The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help overcome the host’s defense during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease-vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å and used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the proinflammatory cytokines TNF-α and IL-12, and proliferation of antigen-specific CD4+ T cells. This suggests that OmC2 may suppress the host’s adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.

Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

“…It is able to inhibit cathepsin B, cathepsin L and cathepsin C (L. F. Parizi, personal communication). As these enzymes are important in some immunologic processes, these cystatins in R. microplus saliva could act as immunomodulators during the slow feeding phase of cattle tick parasitism, as previously shown for other tick cystatins, facilitating blood feeding and pathogen transmission [126]–[130]. The importance of these inhibitors in blood feeding was underscored in studies that showed that neutralization of cystatins (through gene silencing in ticks or vaccines) significantly reduces tick feeding ability [128], [131], [132].…”

Salivary Tick Cystatin OmC2 Targets Lysosomal Cathepsins S and C in Human Dendritic Cells

“…However, the inhibition of the exoprotease activity of cathepsins B, C, H by cystatin OmC2 is much more effective than that of sialostatin L (Salát et al, 2010); cystatin OmC2 inhibits cathepsin C with K i = 0.19 nM (Grunclová et al, 2006). Our results demonstrated that substantial amounts of the analyzed cathepsins C, B, and H were present in the pull-down fractions from treated cells, which indicated that all of them, in addition to cathepsin S, bind to the internalized cystatin OmC2 (Figure 6C).…”

Bioinformatics and expression analyses of the Ixodes scapularis tick cystatin family

“…Studies based on recombinant tick cystatins have provided insight that native tick-encoded cystatins are functional inhibitors of cathepsin-like cysteine proteases (Kotsyfakis et al 2006; Lima et al 2006; Zhou et al 2006, 2009, 2010; Grunclová et al 2006a, b; Yamaji et al 2009). In other studies, recombinant cystatins affected the function of immune cell functions (Salát et al 2010; Sá-Nunes et al 2009). In this study we have used bioinformatics analyses to identify cystatins that are conserved in most ticks and RT-PCR expression analyses to describe the relationship of I. scapularis cystatins to the tick feeding cycle.…”

“…Several lines of research point to the importance of cystatins in tick physiology (Schwarz et al 2012; Horka et al 2012). RNAi silencing of cystatins in I. scapularis (Kotsyfakis et al 2007) and A. americanum (Karim et al 2005) or feeding I. scapularis ticks or Guinea pigs (Kotsyfakis et al 2008) or Ornithodoros moubata (Salát et al 2010) that were immunized with a recombinant tick salivary gland cystatin caused significant reductions in tick feeding efficiency. In a recent study an I. scapularis tick salivary gland cystatin that retained the consensus cystatin secondary structure fold was shown involved in B. burgdorferi transmission (Kotsyfakis et al 2010a, b).…”