Transcriptomic and proteomic analysis of Ascaris suum larvae during their hepato-tracheal migration

The gastro-intestinal nematodes Ascaris lumbricoides and Ascaris suum are amongst the most prevalent parasites of humans and pigs, respectively. Ascaris infections cause serious public health problems and significant economic losses in the pig industry. Traditionally ascariasis is controlled by mass treatment with anthelmintics. However, due to the short activity of the anthelmintics and an environment often highly contaminated with Ascaris eggs, reinfections can occur rapidly. In addition, the development of anthelmintic resistance which has been observed in other nematodes, suggests that current repeated doses of massive chemotherapy treatment will probably also lead to drug resistance in Ascaris spp. eventually. Therefore, investigation of the alternative means of ascariasis control such as vaccination is worthwhile for pursuing. To promote the rational development of an effective vaccine, a better understanding of the molecular biology and host-parasite relationships of Ascaris is required. In chapter two, a transcriptome dataset was generated and analyzed in order to identify both the highest transcribed and stage-specific transcripts for each larval stage, the metabolic changes and chemosensation pathways active in the larvae and, finally, the expression of potential molecular mimicry candidates. Illumina RNA sequencing of the A. suum infective-stage, liver-stage, lung-stage and intestinal-stage larvae resulted in 95,463,423 sequences yielding 18,543 contigs. Within the top 250 most highly transcribed contigs per stage, accounting for approximately 60% of the total transcription in each stage, cuticle collagens were by far the most abundant gene family. The analysis also identified a set of interesting stage-specific genes, such as venom allergen, chitinase, thioredoxin and cecropin, with a potentially important role in the host-parasite interaction processes. Analysis of the metabolic pathways showed that the degradation of complex carbohydrates likely forms an essential part of the energy metabolism of this parasite, as almost 10% of the total transcriptome of the L4 stage encodes for enzymes involved in this pathway. In comparison to Caenorhabditis elegans, a reduced number of olfactory molecules were identified in A. suum, in particular the aerotaxis molecules seem to absent, suggesting that they are less important for this parasite. Finally, 12 transcript sequences were identified as potential molecular mimicry candidates, including a suppressor of cytokine signaling family. Overall, the outcome of this transcriptomic analysis provides novel and valuable information regarding the biology of A. suum larvae, which can be used as a foundation for further research. In chapter three, we identified the excretory-secretory proteins of the migratory stages of A. suum utilizing LC-MS/MS. In total 106 proteins were identified, some of which are known as important players in the parasite-host interface. Interestingly, an abundance of glycosyl hydrolases was observed in the ES material of the intestinal L4 stage larvae. By combining the proteomic analysis with in-depth genomic, transcriptomic and enzymatic analyses we could show that the glycosyl hydrolase protein family has undergone a massive expansion in A. suum and that most of the glycolytic activity is present in the intestinal tissue of the adult parasites. Again, as already indicated by the transcriptomic analysis, this could suggest that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine. In chapter four, we employed two different approaches, i.e. biotin labeling and enzymatic shaving, combined with LC-MS/MS to study the cuticle surface associated proteins of the infective stage larvae of A. suum. In total, 17 proteins were identified as surface associated proteins by the labeling approach. Many of these molecules had been previously reported as surface exposed proteins in other helminth species. On the other hand, the MS/MS spectra for the shaving approach only resulted in the identification of 3 genuine surface attached proteins, i.e. a cuticle collagen, a tubulin and a protein with unknown function. These results extended the knowledge on the biology of Ascaris larvae and their cuticle proteins, which play important roles in host-parasite interactions and hopefully it will benefit the development of novel intervention strategies. In conclusion, the transcriptomic and proteomic investigations performed in this thesis have led to a significant progress in our understanding of Ascaris biology. The most important observations were the potentially important role of carbohydrate metabolism in the larvae during their hepato-tracheal migration and the identification of the molecules essential to parasite survival and development. The study provides a basis for further molecular investigations aimed at exploring the biological role of the proteins identified and their potential as vaccine and/or therapeutic targets.

Grantor dc:publisher

Ghent University. Faculty of Veterinary Medicine

Year dc:date

2014

Author dc:creator

• Wang, Tao

Contributors dc:contributor

• Geldhof, Peter

• Biology and Life Sciences
• Proteome
• Excretory-secretory products
• Transcriptome
• Cuticle surface
• Genome
• Ascaris

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://biblio.ugent.be/publication/4254816
urn:isbn:9789058643698
https://biblio.ugent.be/publication/4254816/file/4254817

OAI identifier oai:identifier

oai:archive.ugent.be:4254816