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

Sojka D, Franta Z, Horn M, Hajdusek O, Caffrey CR, Mares M, Kopácek P - Parasit Vectors (2008)

Bottom Line: Overall, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Ceské Budejovice, CZ-370 05, The Czech Republic. dsojka@seznam.cz.

ABSTRACT

Background: Ticks 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.

Results: Using 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.

Conclusion: Overall, 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.

No MeSH data available.


Related in: MedlinePlus

The pH profile of hemoglobinolytic activity in the I. ricinus gut tissue extracts. Gut tissue was dissected from partially engorged tick females (the 5-th day of feeding), washed from the gut contents and extracted. Fluorescently labeled AMC-hemoglobin was digested in vitro with gut extract at various pH values. The relative rate of degradation of the substrate was determined using the measurement of fluorescence in a continuous hemoglobinolytic assay. The error bars indicate standard deviations of the mean of triplicates.
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Figure 1: The pH profile of hemoglobinolytic activity in the I. ricinus gut tissue extracts. Gut tissue was dissected from partially engorged tick females (the 5-th day of feeding), washed from the gut contents and extracted. Fluorescently labeled AMC-hemoglobin was digested in vitro with gut extract at various pH values. The relative rate of degradation of the substrate was determined using the measurement of fluorescence in a continuous hemoglobinolytic assay. The error bars indicate standard deviations of the mean of triplicates.

Mentions: Gut tissue extract prepared from partially engorged I. ricinus females (5th day of feeding) was tested on degradation of the biologically relevant substrate hemoglobin. Proteolysis was analyzed using a fluorescence assay incorporating AMC-hemoglobin. Optimal proteolysis was measured at acidic pH between 3.0 and 4.5 (Fig. 1). No substantial degradation occurred above pH 6.0. At optimum pH (~4.0), hemoglobinolytic activity was inhibited by the small molecule inhibitors E64 and pepstatin that selectively target cysteine and aspartic peptidases, respectively. A combined application of both compounds resulted in nearly complete blockage of the activity (~97% inhibition), the individual treatment showed about 80% inhibition (~83% and ~78% for E64 and pepstatin, respectively). In contrast, inhibitors of serine peptidases and metallopeptidases Pefabloc and EDTA, respectively, were ineffective (data not shown).


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

Sojka D, Franta Z, Horn M, Hajdusek O, Caffrey CR, Mares M, Kopácek P - Parasit Vectors (2008)

The pH profile of hemoglobinolytic activity in the I. ricinus gut tissue extracts. Gut tissue was dissected from partially engorged tick females (the 5-th day of feeding), washed from the gut contents and extracted. Fluorescently labeled AMC-hemoglobin was digested in vitro with gut extract at various pH values. The relative rate of degradation of the substrate was determined using the measurement of fluorescence in a continuous hemoglobinolytic assay. The error bars indicate standard deviations of the mean of triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2289814&req=5

Figure 1: The pH profile of hemoglobinolytic activity in the I. ricinus gut tissue extracts. Gut tissue was dissected from partially engorged tick females (the 5-th day of feeding), washed from the gut contents and extracted. Fluorescently labeled AMC-hemoglobin was digested in vitro with gut extract at various pH values. The relative rate of degradation of the substrate was determined using the measurement of fluorescence in a continuous hemoglobinolytic assay. The error bars indicate standard deviations of the mean of triplicates.
Mentions: Gut tissue extract prepared from partially engorged I. ricinus females (5th day of feeding) was tested on degradation of the biologically relevant substrate hemoglobin. Proteolysis was analyzed using a fluorescence assay incorporating AMC-hemoglobin. Optimal proteolysis was measured at acidic pH between 3.0 and 4.5 (Fig. 1). No substantial degradation occurred above pH 6.0. At optimum pH (~4.0), hemoglobinolytic activity was inhibited by the small molecule inhibitors E64 and pepstatin that selectively target cysteine and aspartic peptidases, respectively. A combined application of both compounds resulted in nearly complete blockage of the activity (~97% inhibition), the individual treatment showed about 80% inhibition (~83% and ~78% for E64 and pepstatin, respectively). In contrast, inhibitors of serine peptidases and metallopeptidases Pefabloc and EDTA, respectively, were ineffective (data not shown).

Bottom Line: Overall, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Ceské Budejovice, CZ-370 05, The Czech Republic. dsojka@seznam.cz.

ABSTRACT

Background: Ticks 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.

Results: Using 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.

Conclusion: Overall, 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.

No MeSH data available.


Related in: MedlinePlus