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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

Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases. Messenger RNA levels of individual enzymes were determined by semi-quantitative two-step RT PCR. Panel A: Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B: Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).
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Figure 8: Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases. Messenger RNA levels of individual enzymes were determined by semi-quantitative two-step RT PCR. Panel A: Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B: Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).

Mentions: Gene-specific PCR primer sets for the newly identified cDNAs and IrAE cDNA (Table 2) were used to amplify the relevant peptidase genes from different tick developmental stages and tissues. Semi-quantitative RT-PCR of whole-body homogenates revealed that IrCL, IrCC and IrAE are abundantly present in all developmental stages including eggs (Fig. 8A). In contrast, messages for IrCB and IrCD were absent from tick eggs. No apparent differences were observed for any enzyme expression between un-fed and freshly attached females suggesting that the enzyme messages are not changed in the initial feeding phase. Once partially engorged, it is possible to reliably dissect individual organs of females for RT-PCR tissue profiling (Fig. 8B) and the data demonstrate that all the peptidases of interest are co-expressed in the gut towards the end of the slow feeding period [7] what indicates their simultaneous action in a putative cascade or network. Moreover, IrCB, IrAE and IrCD seem to be strictly gut-specific, whereas messages for IrCL and IrCC were also found in other tick tissues. Negative controls in which the template cDNA was replaced by sterile distilled water gave no PCR products (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)

Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases. Messenger RNA levels of individual enzymes were determined by semi-quantitative two-step RT PCR. Panel A: Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B: Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases. Messenger RNA levels of individual enzymes were determined by semi-quantitative two-step RT PCR. Panel A: Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B: Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).
Mentions: Gene-specific PCR primer sets for the newly identified cDNAs and IrAE cDNA (Table 2) were used to amplify the relevant peptidase genes from different tick developmental stages and tissues. Semi-quantitative RT-PCR of whole-body homogenates revealed that IrCL, IrCC and IrAE are abundantly present in all developmental stages including eggs (Fig. 8A). In contrast, messages for IrCB and IrCD were absent from tick eggs. No apparent differences were observed for any enzyme expression between un-fed and freshly attached females suggesting that the enzyme messages are not changed in the initial feeding phase. Once partially engorged, it is possible to reliably dissect individual organs of females for RT-PCR tissue profiling (Fig. 8B) and the data demonstrate that all the peptidases of interest are co-expressed in the gut towards the end of the slow feeding period [7] what indicates their simultaneous action in a putative cascade or network. Moreover, IrCB, IrAE and IrCD seem to be strictly gut-specific, whereas messages for IrCL and IrCC were also found in other tick tissues. Negative controls in which the template cDNA was replaced by sterile distilled water gave no PCR products (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