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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.

ABSTRACT: UNLABELLED: 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.

SUBMITTER: Sojka D

PROVIDER: S-EPMC2289814 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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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 Daniel D Franta Zdenek Z Horn Martin M Hajdusek Ondrej O Caffrey Conor R CR Mares Michael M Kopácek Petr P

Parasites & vectors 20080318 1

<h4>Unlabelled</h4><h4>Background</h4>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 prac ...[more]

PMID: 18348719

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Project description:The tick microbiota is a highly complex ensemble of interacting microorganisms. Keystone taxa, with a central role in the microbial networks, support the stability and fitness of the microbial communities. The keystoneness of taxa in the tick microbiota can be inferred from microbial co-occurrence networks. Microbes with high centrality indexes are highly connected with other taxa of the microbiota and are expected to provide important resources to the microbial community and/or the tick. We reasoned that disturbance of vector microbiota by removal of ubiquitous and abundant keystone bacteria may disrupt the tick-microbiota homeostasis causing harm to the tick host. These observations and reasoning prompted us to test the hypothesis that antibodies targeting keystone bacteria may harm the ticks during feeding on immunized hosts. To this aim, in silico analyses were conducted to identify keystone bacteria in the microbiota of Ixodes nymphs. The family Enterobacteriaceae was among the top keystone taxa identified in Ixodes microbiota. Immunization of α-1,3-galactosyltransferase-deficient-C57BL/6 (α1,3GT KO) mice with a live vaccine containing the Enterobacteriaceae bacterium Escherichia coli strain BL21 revealed that the production of anti-E. coli and anti-α-Gal IgM and IgG was associated with high mortality of I. ricinus nymphs during feeding. However, this effect was absent in two different strains of wild type mice, BALB/c and C57BL/6. This result concurred with a wide distribution of α-1,3-galactosyltransferase genes, and possibly α-Gal, in Enterobacteriaceae and other bacteria of tick microbiota. Interestingly, the weight of I. ricinus nymphs that fed on E. coli-immunized C57BL/6 was significantly higher than the weight of ticks that fed on C57BL/6 immunized with a mock vaccine. Our results suggest that anti-tick microbiota vaccines are a promising tool for the experimental manipulation of vector microbiota, and potentially the control of ticks and tick-borne pathogens.

Project description:BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Screening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for "Lipocalin from I. ricinus" and numbered from 1 to 14 (LIR1 to LIR14). According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50-70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4. CONCLUSIONS/SIGNIFICANCE: This work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C4.

Project description:BackgroundIxodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed.MethodsRNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV).ResultsDifferential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive controls, also resulted in increased LGTV replication and production in both cell lines, confirming their role in the antiviral RNAi pathway.ConclusionsThis systems biology approach identified several molecules that may be involved in the tick cell innate immune response against flaviviruses and highlighted that ticks, in common with other invertebrate species, have other antiviral responses in addition to RNAi.

Dataset Information

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

Publications

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

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