Please can you give a brief introduction to the Asian liver fluke, Opisthorchis viverrini? How does this parasite infect humans?
Opisthorchis viverrini is a parasitic flatworm (or liver fluke) endemic throughout Thailand, the Lao People’s Democratic Republic, Vietnam and Cambodia. Humans are infected with this parasite when they eat a fluke encysted in inadequately cooked/preserved freshwater fish.
Once eaten, the larval stage enters the human bile duct where they develop into an adult that resides in the bile duct for several years. Adults produce eggs that pass into the stool, enter freshwater and are ingested by an aquatic snail host.
Inside the snail, these parasites develop into a free-living stage, which leaves the snail and encysts in the muscle of the freshwater fish, completing their complex lifecycle.
Current estimates suggest almost 10 million people are infected with these parasites across South East Asia.
In the human bile duct, O. viverrini cause opisthorchiasis, a disease associated with an enlarged liver, bile duct inflammation and bile duct cancer (cholangiocarcinoma; CCA), a deadly cancer of the biliary tree, with a very poor prognosis.
Does this parasite contribute to bile duct cancer?
Opisthorchis viverrini has been classified as a Class I carcinogen. We still don’t know the exact mechanisms involved, although researchers believe that several factors contribute to cancer development.
For example, host inflammation caused by cell damage or cell death, linked to parasite feeding and/or attachment, likely contributes to carcinogenesis.
In addition, recent evidence suggests that O. viverrini secrete proteins that directly promote cell growth and tumour development.
Another factor that is thought to contribute to tumorigenesis is DNA damage caused by a diet rich in nitrosamines, a potent human Class I carcinogen. Together, these factors create a “perfect storm” in the human bile duct that results in a high incidence of bile duct cancer.
It was recently announced that the genome of the Asian live fluke had been sequenced. What did this involve?
An International team, including parasitologists and genomics experts from the University of Melbourne (Australia), the Genome Institute of Singapore (Singapore), Khon Kaen University (Thailand) and the Beijing Genomics Institute (China) was involved in sequencing and annotating the genome.
First, we extracted O. viverrini genomic DNA and used high-throughput sequencing machines to produce more than 60–fold coverage of the genome as small sequence fragments. These small fragments were then assembled into genome scaffolds. In these large genome scaffolds, each gene region was identified and annotated.
A range of tools were then used to predict the function of each O. viverrini gene, including key molecules associated with metabolism and their adaptation to life in the human bile duct.
What were the main challenges in characterising the largest parasitic worm genome studied to date?
The biggest challenge was the presence of large numbers of repetitive elements found throughout the genomic DNA of O. viverrini.
For example, only 3.4% of the genome contained genetic information encoding proteins. In contrast more than 30% of the genome contained repetitive elements present in more than one location in the genome. This meant assembly was challenging.
Luckily, we were able to use new software (called Opera) that was developed by our collaborators at the Genomics Institute of Singapore.
Using Opera, we produced a high quality draft genome that we used to functionally annotate the gene set and obtain vital insights into the fundamental molecular biology of this parasite, including the identification of essential pathways linked to worm–host interactions.
What insight does this study provide into how the fluke survives within the human bile duct?
Our study showed that these parasites are highly adapted to a life in the bile duct, leading to their establishment within the bile duct for up to 10 years.
For instance, O. viverrini is adapted to survive in a low oxygen environment and are experts at detoxifying harmful compounds, such as free oxygen radicals released in bile during lipid peroxidation.
The metabolic profiles of these parasites also suggest an adaptation to feeding on epithelial cells and/or bile constituents that are rich in complex lipids and proteins. Evasion of the host immune response is also important for these parasites, particularly if inflammation ensues from cellular damage caused by feeding and/or attachment. Our data indicate that a number of secreted molecules can modulate the host immune response.
Did you find evidence that the proteins these parasites release alter human tissue?
We identified genes encoding proteins other researchers have experimentally shown to alter human tissues. These molecules (called granulin-like molecules) are thought to mimic human growth factors that promote cell growth.
In our study, we found another previously undescribed granulin-like molecule that may also be involved in regulating cell growth. The role of these molecules in promoting cancer is now being investigated.
What treatments are currently available for infection with Asian liver fluke, and are your results likely to lead to improved treatment options?
The simplest intervention strategy would be to ensure people thoroughly cook/preserve fish used for human consumption. Unfortunately, the cultural practice of eating raw fish remains common in many areas, despite concerted efforts to teach communities about the risks associated with O. viverrini infection.
Presently, there is no vaccine to prevent infection, and chemotherapy relies on the use of a single drug, praziquantel.
Recent studies suggest the excessive use of praziquantel can reduce treatment efficacy and induce inflammation of the biliary system. Moreover, even after successful treatment, reinfection with O. viverrini is common. Alternative strategies to intervene with O. viverrini infection are thus urgently needed.
A genome is an important resource that can be used to understand how a drug or vaccine works. It can also be used to identify and prioritise new drugs.
Given that these parasites are hard to maintain in the laboratory, a list of drug targets should be first prioritised before undertaking a costly drug screening program.
What impact will your new genome resource have on the understanding of other cancer-causing parasites?
In our study, we included a comparison of the O. viverrini genome to a closely related liver fluke, Clonorchis sinensis, a species endemic in Vietnam, China and South Korea and another parasite classified as a Class I carcinogen.
Genomic comparisons revealed similarities and differences between these parasites, including differences in the secreted granulin-like molecules that are thought to contribute to the development of cancer.
Understanding the biology of both liver flukes will improve our understanding of cancer development and will help consolidate efforts to control these worms throughout South East Asia.
We also study another important cancer-causing parasite, Schistosoma haematobium, a blood fluke living in the urogenital blood vessels and that infects hundreds of millions of people across sub-Saharan Africa. These parasites are very different to O. viverrini, and our analyses didn’t find any common genes linked with the development of cancer.
What are the next steps in your research?
We are now screening the genomes of all flatworms and prioritising genes to target using our drug discovery pipeline.
We hope to find genes/proteins that are common to all parasites and that can be targeted using existing or novel drugs. One of our major aims is to provide medical practitioners with a broader range of drugs to treat infection.
In addition, we will explore novel protein families identified in O. viverrini to improve our understanding of how they can live within the human bile duct for several years without being removed by the immune system.
To date, when we characterise a new parasite genome we find an array of novel proteins that give us a glimpse of their unique adaptations to the human host. As biologists, we are continuously surprised at what we find, and excited about the unravelling the story.
Where can readers find more information?
We recently published the O. viverrini genome and all of our findings in Nature Communications (DOI: 10.1038/ncomms5378). You can also find out more information about O. viverrini at the Centers of Disease Control and Prevention website (http://www.cdc.gov/parasites/opisthorchis/).
Neil Young is an National Health and Medical Research Council (NHMRC) Early Career Research Fellow and our research is funded by the Australian Research Council, the NHMRC of Australia and BGI-Shenzhen. Our research is also supported by a Victorian Life Sciences Computation Initiative Grant (grant number VR0007) on its Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Government.
About Dr Neil Young
Neil Young BSc PhD is a National Health and Medical Research Council Early Career Fellow, undertaking genomic research on helminth parasites within the Pathogen Genomics and Genetics Programme (PGGP) in The University of Melbourne.
He uses a multidisciplinary approach to undertake genomic and molecular research to characterise the biology of parasitic flatworms (class Trematoda). By studying these parasites, he hopes to better understand their evolution with the human host and contribute to their control and elimination.
Since undertaking his post-doctoral training he has undertaken collaborative research analysing genomic and transcriptomic sequence data to characterise the biology of a wide range of socioeconomically important parasites.
He has published >40 articles or books in high quality international peer-reviewed literature. Current projects led by Dr Young relate to sequencing the genomes of cancer-causing parasites as well as improving the annotation and current gene sets using a range of complementary bioinformatic approaches.
Liver cells effectively serve as immune checkpoint regulating anti-cancer immunity