Michael Mareš

Hemoglobin Digestion in Blood-Feeding Ticks: Mapping a Multipeptidase Pathway by Functional Proteomics

Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.

New insights into the machinery of blood digestion by ticks.

Blood-protein digestion is a key physiological process providing essential nutrients for ticks and is a prerequisite for the transmission of tick-borne pathogens. Recently, substantial progress has been made in determining the proteolytic machinery in tick gut tissue, which is based on a dynamic multienzyme network capable of processing a vast amount of host blood. In this article we summarize our current knowledge of the molecular mechanisms of tick hematophagy and their similarities to those of Platyhelminthes, nematodes, and Plasmodium. Future research perspectives, including the potential for rational control of ticks and transmitted diseases, are also discussed.

Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

BackgroundTicks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets.ResultsUsing the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood.ConclusionOverall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

Two secreted cystatins of the soft tick Ornithodoros moubata: differential expression pattern and inhibitory specificity.

Abstract Two genes coding for cysteine peptidase inhibitors of the cystatin family (Om-cystatin 1 and 2) were isolated from a gut-specific cDNA library of the soft tick Ornithodoros moubata. Both cystatins were clearly down-regulated after a blood meal. Om-cystatin 1 is mainly expressed in the tick gut, while Om-cystatin 2 mRNA was also found in other tick tissues. Authentic Om-cystatin 2 was significantly more abundant than Om-cystatin 1 in the gut contents of fasting ticks and was associated with hemosome-derived residual bodies accumulated in the gut lumen. Om-cystatin 2 was also expressed by type 2 secretory cells in the salivary glands of unfed ticks. The inhibitory specificity of recombinant Om-cystatins 1 and 2 was tested with mammalian cysteine peptidases, as well as endogenous cysteine peptidases present in the tick gut. Both cystatins efficiently inhibited papain-like peptidases, including cathepsin B and H, but differed significantly in their affinity towards cathepsin C and failed to block asparaginyl endopeptidase. Our results suggest that the secreted cystatin isoinhibitors are involved in the regulation of multiple proteolytic targets in the tick digestive system and tick-host interaction.

Structural Basis for Inhibition of Cathepsin B Drug Target from the Human Blood Fluke, Schistosoma mansoni.

Schistosomiasis caused by a parasitic blood fluke of the genus Schistosoma afflicts over 200 million people worldwide. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated peptidase that digests host blood proteins as a source of nutrients. It is under investigation as a drug target. To further this goal, we report three crystal structures of SmCB1 complexed with peptidomimetic inhibitors as follows: the epoxide CA074 at 1.3 Å resolution and the vinyl sulfones K11017 and K11777 at 1.8 and 2.5 Å resolutions, respectively. Interactions of the inhibitors with the subsites of the active-site cleft were evaluated by quantum chemical calculations. These data and inhibition profiling with a panel of vinyl sulfone derivatives identify key binding interactions and provide insight into the specificity of SmCB1 inhibition. Furthermore, hydrolysis profiling of SmCB1 using synthetic peptides and the natural substrate hemoglobin revealed that carboxydipeptidase activity predominates over endopeptidolysis, thereby demonstrating the contribution of the occluding loop that restricts access to the active-site cleft. Critically, the severity of phenotypes induced in the parasite by vinyl sulfone inhibitors correlated with enzyme inhibition, providing support that SmCB1 is a valuable drug target. The present structure and inhibitor interaction data provide a footing for the rational design of anti-schistosomal inhibitors.

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

The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the hosts defence 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 in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was 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 A (1 A=0.1 nm) and was 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 pro-inflammatory cytokines tumour necrosis factor alpha and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the hosts 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.

Digestive α‐amylases of the flour moth Ephestia kuehniella– adaptation to alkaline environment and plant inhibitors

The digestive tract of lepidopteran insects is extremely alkaline. In the present work, molecular adaptation of amylolytic enzymes to this environment was investigated in the flour moth Ephestia kuehniella, an important stored‐product pest. Three digestive α‐amylases [Ephestia kuehniellaα‐amylase isoenzymes 1–3 (EkAmy1–3)] with an alkaline pH optimum were purified from larvae and biochemically characterized. These isoenzymes differ significantly in their sensitivity to α‐amylase inhibitors of plant origin that are directed against herbivores as antifeedants. Such functional variability renders the amylolytic system less vulnerable to suppression by plant defensive molecules. Moreover, we found that expression of α‐amylases is upregulated in larvae feeding on a diet enriched with an α‐amylase inhibitor. The α‐amylases are secreted into the larval midgut by an exocytotic mechanism, as revealed by immunogold microscopy. The cDNA sequence of EkAmy3 was determined, and a homology model of EkAmy3 was built in order to analyze the structural features responsible for adaptation to alkaline pH. First, the overall fold was found to be stabilized by remodeling of ion pairs. Second, molecular simulations supported by activity measurements showed that EkAmy3 does not bind a Cl–, owing to an Arg‐to‐Gln mutation in a conserved binding site. The Cl–‐binding residues are in contact with the catalytic residues, and this change might help to fine‐tune the catalytic pKa values to an alkaline pH optimum. We conclude that lepidopteran α‐amylases are evolutionarily adapted in terms of structure and expression dynamics for effective functioning in the digestive system.

Explosive Backpacks in Old Termite Workers

As Neocapritermes taracua termites age, they develop a suicidal toxic apparatus that bursts during aggressive encounters. By nature, defensive behavior is risky. In social insects, such behavior is more likely to occur in individuals whose potential for other tasks is diminished. We show that workers of the termite Neocapritermes taracua develop an exceptional two-component suicidal apparatus consisting of copper-containing protein crystals, stored in external pouches, and internal salivary glands. During aggressive encounters, their bodies rupture, and the crystals react with the salivary gland secretion to produce a toxic droplet. Both the amount of defensive substances and the readiness to explode increase with workers’ age, as their food-collecting ability declines.

IrCL1 – The haemoglobinolytic cathepsin L of the hard tick, Ixodes ricinus

Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In our model tick, Ixodes ricinus, cathepsin L (IrCL1) is part of a gut-associated multi-peptidase complex; its endopeptidase activity is important in the initial phase of haemoglobinolysis. We present the functional and biochemical characterisation of this enzyme. We show, by RNA interference (RNAi), that cathepsin L-like activity that peaks during the slow feeding period of females is associated with IrCL1. Recombinant IrCL1 was expressed in bacteria and yeast. Activity profiling with both peptidyl and physiological protein substrates (haemoglobin and albumin) revealed that IrCL1 is an acidic peptidase with a very low optimum pH (3-4) being unstable above pH 5. This suggests an endo/lysosomal localisation that was confirmed by indirect fluorescence microscopy that immunolocalised IrCL1 inside the vesicles of digestive gut cells. Cleavage specificity determined by a positional scanning synthetic combinatorial library and inhibition profile indicated that IrCL1 has the ligand-binding characteristics of the cathepsin L subfamily of cysteine peptidases. A non-redundant proteolytic function was demonstrated when IrCL1-silenced ticks had a decreased ability to feed compared with controls. The data suggest that IrCL1 may be a promising target against ticks and tick-borne pathogens.

Characterization of Gut-associated Cathepsin D Hemoglobinase from Tick Ixodes ricinus (IrCD1)

Background: Aspartic peptidase activity initiates a multienzyme hemoglobinolysis inside tick guts. Results: IrCD1 is a structurally unique hemoglobinolytic cathepsin D that is up-regulated in tick gut cells during feeding. Conclusion: IrCD1 is the major intestinal aspartic peptidase of I. ricinus. Significance: Biochemical and functional characterization of IrCD1 completes our knowledge on initial host hemoglobin degradation inside tick gut cells. To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1′ position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1′ regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2′. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.