Publications by authors named "Andreas J Stroehlein"

25 Publications

  • Page 1 of 1

High-quality reference genome for Clonorchis sinensis.

Genomics 2021 May 4;113(3):1605-1615. Epub 2021 Mar 4.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia.

The Chinese liver fluke, Clonorchis sinensis, causes the disease clonorchiasis, affecting ~35 million people in regions of China, Vietnam, Korea and the Russian Far East. Chronic clonorchiasis causes cholangitis and can induce a malignant cancer, called cholangiocarcinoma, in the biliary system. Control in endemic regions is challenging, and often relies largely on chemotherapy with one anthelmintic, called praziquantel. Routine treatment carries a significant risk of inducing resistance to this anthelmintic in the fluke, such that the discovery of new interventions is considered important. It is hoped that the use of molecular technologies will assist this endeavour by enabling the identification of drug or vaccine targets involved in crucial biological processes and/or pathways in the parasite. Although draft genomes of C. sinensis have been published, their assemblies are fragmented. In the present study, we tackle this genome fragmentation issue by utilising, in an integrated way, advanced (second- and third-generation) DNA sequencing and informatic approaches to build a high-quality reference genome for C. sinensis, with chromosome-level contiguity and curated gene models. This substantially-enhanced genome provides a resource that could accelerate fundamental and applied molecular investigations of C. sinensis, clonorchiasis and/or cholangiocarcinoma, and assist in the discovery of new interventions against what is a highly significant, but neglected disease-complex.
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http://dx.doi.org/10.1016/j.ygeno.2021.03.001DOI Listing
May 2021

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Int J Mol Sci 2021 Feb 11;22(4). Epub 2021 Feb 11.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, , and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.
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http://dx.doi.org/10.3390/ijms22041811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918261PMC
February 2021

Major SCP/TAPS protein expansion in Lucilia cuprina is associated with novel tandem array organisation and domain architecture.

Parasit Vectors 2020 Nov 27;13(1):598. Epub 2020 Nov 27.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, VIC, 3010, Australia.

Background: Larvae of the Australian sheep blowfly, Lucilia cuprina, parasitise sheep by feeding on skin excretions, dermal tissue and blood, causing severe damage known as flystrike or myiasis. Recent advances in -omic technologies and bioinformatic data analyses have led to a greater understanding of blowfly biology and should allow the identification of protein families involved in host-parasite interactions and disease. Current literature suggests that proteins of the SCP (Sperm-Coating Protein)/TAPS (Tpx-1/Ag5/PR-1/Sc7) (SCP/TAPS) superfamily play key roles in immune modulation, cross-talk between parasite and host as well as developmental and reproductive processes in parasites.

Methods: Here, we employed a bioinformatics workflow to curate the SCP/TAPS protein gene family in L. cuprina. Protein sequence, the presence and number of conserved CAP-domains and phylogeny were used to group identified SCP/TAPS proteins; these were compared to those found in Drosophila melanogaster to make functional predictions. In addition, transcription levels of SCP/TAPS protein-encoding genes were explored in different developmental stages.

Results: A total of 27 genes were identified as belonging to the SCP/TAPS gene family: encoding 26 single-domain proteins each with a single CAP domain and a solitary double-domain protein containing two conserved cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domains. Surprisingly, 16 SCP/TAPS predicted proteins formed an extended tandem array spanning a 53 kb region of one genomic region, which was confirmed by MinION long-read sequencing. RNA-seq data indicated that these 16 genes are highly transcribed in all developmental stages (excluding the embryo).

Conclusions: Future work should assess the potential of selected SCP/TAPS proteins as novel targets for the control of L. cuprina and related parasitic flies of major socioeconomic importance.
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http://dx.doi.org/10.1186/s13071-020-04476-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694928PMC
November 2020

High-quality nuclear genome for Sarcoptes scabiei-A critical resource for a neglected parasite.

PLoS Negl Trop Dis 2020 10 1;14(10):e0008720. Epub 2020 Oct 1.

Cell and Molecular Biology Department, Infectious Diseases Program, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

The parasitic mite Sarcoptes scabiei is an economically highly significant parasite of the skin of humans and animals worldwide. In humans, this mite causes a neglected tropical disease (NTD), called scabies. This disease results in major morbidity, disability, stigma and poverty globally and is often associated with secondary bacterial infections. Currently, anti-scabies treatments are not sufficiently effective, resistance to them is emerging and no vaccine is available. Here, we report the first high-quality genome and transcriptomic data for S. scabiei. The genome is 56.6 Mb in size, has a a repeat content of 10.6% and codes for 9,174 proteins. We explored key molecules involved in development, reproduction, host-parasite interactions, immunity and disease. The enhanced 'omic data sets for S. scabiei represent comprehensive and critical resources for genetic, functional genomic, metabolomic, phylogenetic, ecological and/or epidemiological investigations, and will underpin the design and development of new treatments, vaccines and/or diagnostic tests.
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http://dx.doi.org/10.1371/journal.pntd.0008720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591027PMC
October 2020

First record of a tandem-repeat region within the mitochondrial genome of Clonorchis sinensis using a long-read sequencing approach.

PLoS Negl Trop Dis 2020 08 26;14(8):e0008552. Epub 2020 Aug 26.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

Background: Mitochondrial genomes provide useful genetic markers for systematic and population genetic studies of parasitic helminths. Although many such genome sequences have been published and deposited in public databases, there is evidence that some of them are incomplete relating to an inability of conventional techniques to reliably sequence non-coding (repetitive) regions. In the present study, we characterise the complete mitochondrial genome-including the long, non-coding region-of the carcinogenic Chinese liver fluke, Clonorchis sinensis, using long-read sequencing.

Methods: The mitochondrial genome was sequenced from total high molecular-weight genomic DNA isolated from a pool of 100 adult worms of C. sinensis using the MinION sequencing platform (Oxford Nanopore Technologies), and assembled and annotated using an informatic approach.

Results: From > 93,500 long-reads, we assembled a 18,304 bp-mitochondrial genome for C. sinensis. Within this genome we identified a novel non-coding region of 4,549 bp containing six tandem-repetitive units of 719-809 bp each. Given that genomic DNA from pooled worms was used for sequencing, some variability in length/sequence in this tandem-repetitive region was detectable, reflecting population variation.

Conclusions: For C. sinensis, we report the complete mitochondrial genome, which includes a long (> 4.5 kb) tandem-repetitive region. The discovery of this non-coding region using a nanopore-sequencing/informatic approach now paves the way to investigating the nature and extent of length/sequence variation in this region within and among individual worms, both within and among C. sinensis populations, and to exploring whether this region has a functional role in the regulation of replication and transcription, akin to the mitochondrial control region in mammals. Although applied to C. sinensis, the technological approach established here should be broadly applicable to characterise complex tandem-repetitive or homo-polymeric regions in the mitochondrial genomes of a wide range of taxa.
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http://dx.doi.org/10.1371/journal.pntd.0008552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449408PMC
August 2020

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Mol Biol Evol 2020 12;37(12):3525-3549

Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.
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http://dx.doi.org/10.1093/molbev/msaa186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743719PMC
December 2020

Expanded complement of Niemann-Pick type C2-like protein genes in Clonorchis sinensis suggests functions beyond sterol binding and transport.

Parasit Vectors 2020 Jan 23;13(1):38. Epub 2020 Jan 23.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia.

Background: The parasitic flatworm Clonorchis sinensis inhabits the biliary tree of humans and other piscivorous mammals. This parasite can survive and thrive in the bile duct, despite exposure to bile constituents and host immune attack. Although the precise biological mechanisms underlying this adaptation are unknown, previous work indicated that Niemann-pick type C2 (NPC2)-like sterol-binding proteins might be integral in the host-parasite interplay. Expansions of this family in some invertebrates, such as arthropods, have shown functional diversification, including novel forms of chemoreception. Thus, here we curated the NPC2-like protein gene complement in C. sinensis, and predicted their conserved and/or divergent functional roles.

Methods: We used an established comparative genomic-bioinformatic approach to curate NPC2-like proteins encoded in published genomes of Korean and Chinese isolates of C. sinensis. Protein sequence and structural homology, presence of conserved domains and phylogeny were used to group and functionally classify NPC2-like proteins. Furthermore, transcription levels of NPC2-like protein-encoding genes were explored in different developmental stages and tissues.

Results: Totals of 35 and 32 C. sinensis NPC2-like proteins were predicted to be encoded in the genomes of the Korean and Chinese isolates, respectively. Overall, these proteins had low sequence homology and high variability of sequence alignment coverage when compared with curated NPC2s. Most C. sinensis proteins were predicted to retain a conserved ML domain and a conserved fold conformation, with a large cavity within the protein. Only one protein sequence retained the conserved amino acid residues required in bovine NPC2 to bind cholesterol. Non-canonical C. sinensis NPC2-like protein-coding domains clustered into four distinct phylogenetic groups with members of a group frequently encoded on the same genome scaffolds. Interestingly, NPC2-like protein-encoding genes were predicted to be variably transcribed in different developmental stages and adult tissues, with most being transcribed in the metacercarial stage.

Conclusions: The results of the present investigation confirms an expansion of NPC2-like proteins in C. sinensis, suggesting a diverse array of functions beyond sterol binding and transport. Functional explorations of this protein family should elucidate the mechanisms enabling the establishment and survival of C. sinensis and related flukes in the biliary systems of mammalian hosts.
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http://dx.doi.org/10.1186/s13071-020-3910-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6979364PMC
January 2020

The developmental phosphoproteome of Haemonchus contortus.

J Proteomics 2020 02 14;213:103615. Epub 2019 Dec 14.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Protein phosphorylation plays essential roles in many cellular processes. Despite recent progress in the genomics, transcriptomics and proteomics of socioeconomically important parasitic nematodes, there is scant phosphoproteomic data to underpin molecular biological discovery. Here, using the phosphopeptide enrichment-based LC-MS/MS and data-independent acquisition (DIA) quantitation, we characterised the first developmental phosphoproteome of the parasitic nematode Haemonchus contortus - one of the most pathogenic parasites of ruminant livestock. Totally, 1804 phosphorylated proteins with 4406 phosphorylation sites ('phosphosites') from different developmental stages/sexes were identified. Bioinformatic analyses of quantified 'phosphosites' exhibited distinctive stage- and sex-specific patterns during development, and identified a subset of phosphoproteins proposed to play crucial roles in processes such as spindle positioning, signal transduction and kinase activity. A sequence-based comparison of the phosphoproteome of H. contortus with those of two free-living nematode species (Caenorhabditis elegans and Pristionchus pacificus) suggested a limited number of common protein phosphorylation events among these species. Our findings infer active roles for protein phosphorylation in the adaptation of a parasitic nematode to a constantly changing external environment. The phosphoproteomic data set for H. contortus provides a basis to better understand phosphorylation and associated biological processes (e.g., regulation of signal transduction), and might enable the discovery of novel anthelmintic targets. SIGNIFICANCE: Here, we report the first phosphoproteome for a socioeconomically parasitic nematode (Haemonchus contortus). This phosphoproteome exhibits distinctive patterns during development, suggesting active roles of post-translational modification in the parasite's adaptation to changing environments within and outside of the host animal. This work sheds a light on the developmental phosphorylation in a parasitic nematode, and could enable the discovery of novel interventions against major pathogens.
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http://dx.doi.org/10.1016/j.jprot.2019.103615DOI Listing
February 2020

High-quality Schistosoma haematobium genome achieved by single-molecule and long-range sequencing.

Gigascience 2019 09;8(9)

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Corner Flemington Road and Park Drive, Parkville, VIC 3010, Australia.

Background: Schistosoma haematobium causes urogenital schistosomiasis, a neglected tropical disease affecting >100 million people worldwide. Chronic infection with this parasitic trematode can lead to urogenital conditions including female genital schistosomiasis and bladder cancer. At the molecular level, little is known about this blood fluke and the pathogenesis of the disease that it causes. To support molecular studies of this carcinogenic worm, we reported a draft genome for S. haematobium in 2012. Although a useful resource, its utility has been somewhat limited by its fragmentation.

Findings: Here, we systematically enhanced the draft genome of S. haematobium using a single-molecule and long-range DNA-sequencing approach. We achieved a major improvement in the accuracy and contiguity of the genome assembly, making it superior or comparable to assemblies for other schistosome species. We transferred curated gene models to this assembly and, using enhanced gene annotation pipelines, inferred a gene set with as many or more complete gene models as those of other well-studied schistosomes. Using conserved, single-copy orthologs, we assessed the phylogenetic position of S. haematobium in relation to other parasitic flatworms for which draft genomes were available.

Conclusions: We report a substantially enhanced genomic resource that represents a solid foundation for molecular research on S. haematobium and is poised to better underpin population and functional genomic investigations and to accelerate the search for new disease interventions.
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http://dx.doi.org/10.1093/gigascience/giz108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736295PMC
September 2019

Dauer signalling pathway model for Haemonchus contortus.

Parasit Vectors 2019 Apr 29;12(1):187. Epub 2019 Apr 29.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, VIC, 3010, Australia.

Background: Signalling pathways have been extensively investigated in the free-living nematode Caenorhabditis elegans, but very little is known about these pathways in parasitic nematodes. Here, we constructed a model for the dauer-associated signalling pathways in an economically highly significant parasitic worm, Haemonchus contortus.

Methods: Guided by data and information available for C. elegans, we used extensive genomic and transcriptomic datasets to infer gene homologues in the dauer-associated pathways, explore developmental transcriptomic, proteomic and phosphoproteomic profiles in H. contortus and study selected molecular structures.

Results: The canonical cyclic guanosine monophosphate (cGMP), transforming growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1) and steroid hormone signalling pathways of H. contortus were inferred to represent a total of 61 gene homologues. Compared with C. elegans, H. contortus has a reduced set of genes encoding insulin-like peptides, implying evolutionary and biological divergences between the parasitic and free-living nematodes. Similar transcription profiles were found for all gene homologues between the infective stage of H. contortus and dauer stage of C. elegans. High transcriptional levels for genes encoding G protein-coupled receptors (GPCRs), TGF-β, insulin-like ligands (e.g. ins-1, ins-17 and ins-18) and transcriptional factors (e.g. daf-16) in the infective L3 stage of H. contortus were suggestive of critical functional roles in this stage. Conspicuous protein expression patterns and extensive phosphorylation of some components of these pathways suggested marked post-translational modifications also in the L3 stage. The high structural similarity in the DAF-12 ligand binding domain among nematodes indicated functional conservation in steroid (i.e. dafachronic acid) signalling linked to worm development.

Conclusions: Taken together, this pathway model provides a basis to explore hypotheses regarding biological processes and regulatory mechanisms (via particular microRNAs, phosphorylation events and/or lipids) associated with the development of H. contortus and related nematodes as well as parasite-host cross talk, which could aid the discovery of new therapeutic targets.
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http://dx.doi.org/10.1186/s13071-019-3419-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489264PMC
April 2019

Comparative bioinformatic analysis suggests that specific dauer-like signalling pathway components regulate Toxocara canis development and migration in the mammalian host.

Parasit Vectors 2019 Jan 14;12(1):32. Epub 2019 Jan 14.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, 3010, Australia.

Background: Toxocara canis is quite closely related to Ascaris suum but its biology is more complex, involving a phase of arrested development (diapause or hypobiosis) in tissues as well as transplacental and transmammary transmission routes. In the present study, we explored and compared dauer-like signalling pathways of T. canis and A. suum to infer which components in these pathways might associate with, or regulate, this added complexity in T. canis.

Methods: Guided by information for Caenorhabditis elegans, we bioinformatically inferred and compared components of dauer-like signalling pathways in T. canis and A. suum using genomic and transcriptomic data sets. In these two ascaridoids, we also explored endogenous dafachronic acids (DAs), which are known to be critical in regulating larval developmental processes in C. elegans and other nematodes, by liquid chromatography-mass spectrometry (LC-MS).

Results: Orthologues of C. elegans dauer signalling genes were identified in T. canis (n = 55) and A. suum (n = 51), inferring the presence of a dauer-like signalling pathway in both species. Comparisons showed clear differences between C. elegans and these ascaridoids as well as between T. canis and A. suum, particularly in the transforming growth factor-β (TGF-β) and insulin-like signalling pathways. Specifically, in both A. suum and T. canis, there was a paucity of genes encoding SMAD transcription factor-related protein (daf-3, daf-5, daf-8 and daf-14) and insulin/insulin-like peptide (daf-28, ins-4, ins-6 and ins-7) homologues, suggesting an evolution and adaptation of the signalling pathway in these parasites. In T. canis, there were more orthologues coding for homologues of antagonist insulin-like peptides (Tc-ins-1 and Tc-ins-18), an insulin receptor substrate (Tc-ist-1) and a serine/threonine kinase (Tc-akt-1) than in A. suum, suggesting potentiated functional roles for these molecules in regulating larval diapause and reactivation. A relatively conserved machinery was proposed for DA synthesis in the two ascaridoids, and endogenous Δ4- and Δ7-DAs were detected in them by LC-MS analysis. Differential transcription analysis between T. canis and A. suum suggests that ins-17 and ins-18 homologues are specifically involved in regulating development and migration in T. canis larvae in host tissues.

Conclusion: The findings of this study provide a basis for functional explorations of insulin-like peptides, signalling hormones (i.e. DAs) and related nuclear receptors, proposed to link to development and/or parasite-host interactions in T. canis. Elucidating the functional roles of these molecules might contribute to the discovery of novel anthelmintic targets in ascaridoids.
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http://dx.doi.org/10.1186/s13071-018-3265-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332619PMC
January 2019

Interactive online application for the prediction, ranking and prioritisation of drug targets in Schistosoma haematobium.

Parasit Vectors 2018 Nov 27;11(1):605. Epub 2018 Nov 27.

Melbourne Veterinary School, Department of Veterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, 3010, Australia.

Background: Human schistosomiasis is a neglected tropical disease caused by parasitic worms of the genus Schistosoma that still affects some 200 million people. The mainstay of schistosomiasis control is a single drug, praziquantel. The reliance on this drug carries a risk of resistance emerging to this anthelmintic, such that research towards alternative anti-schistosomal drugs is warranted. In this context, a number of studies have employed computational approaches to prioritise proteins for investigation as drug targets, based on extensive genomic, transcriptomic and small-molecule data now available.

Methods: Here, we established a customisable, online application for the prioritisation of drug targets and applied it, for the first time, to the entire inferred proteome of S. haematobium. This application enables selection of weighted and ranked proteins representing potential drug targets, and integrates transcriptional data, orthology and gene essentiality information as well as drug-drug target associations and chemical properties of predicted ligands.

Results: Using this application, we defined 25 potential drug targets in S. haematobium that associated with approved drugs, and 3402 targets that (although they could not be linked to any compounds) are conserved among a range of socioeconomically important flatworm species and might represent targets for new trematocides.

Conclusions: The online application developed here represents an interactive, customisable, expandable and reproducible drug target ranking and prioritisation approach that should be useful for the prediction of drug targets in schistosomes and other species of parasitic worms in the future. We have demonstrated the utility of this online application by predicting potential drug targets in S. haematobium that can now be evaluated using functional genomics tools and/or small molecules, to establish whether they are indeed essential for parasite survival, and to assist in the discovery of novel anti-schistosomal compounds.
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http://dx.doi.org/10.1186/s13071-018-3197-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6257948PMC
November 2018

The small RNA complement of adult Schistosoma haematobium.

PLoS Negl Trop Dis 2018 05 29;12(5):e0006535. Epub 2018 May 29.

Melbourne Veterinary School, Department of Veterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

Background: Blood flukes of the genus Schistosoma cause schistosomiasis-a neglected tropical disease (NTD) that affects more than 200 million people worldwide. Studies of schistosome genomes have improved our understanding of the molecular biology of flatworms, but most of them have focused largely on protein-coding genes. Small non-coding RNAs (sncRNAs) have been explored in selected schistosome species and are suggested to play essential roles in the post-transcriptional regulation of genes, and in modulating flatworm-host interactions. However, genome-wide small RNA data are currently lacking for key schistosomes including Schistosoma haematobium-the causative agent of urogenital schistosomiasis of humans.

Methodology: MicroRNAs (miRNAs) and other sncRNAs of male and female adults of S. haematobium and small RNA transcription levels were explored by deep sequencing, genome mapping and detailed bioinformatic analyses.

Principal Findings: In total, 89 transcribed miRNAs were identified in S. haematobium-a similar complement to those reported for the congeners S. mansoni and S. japonicum. Of these miRNAs, 34 were novel, with no homologs in other schistosomes. Most miRNAs (n = 64) exhibited sex-biased transcription, suggestive of roles in sexual differentiation, pairing of adult worms and reproductive processes. Of the sncRNAs that were not miRNAs, some related to the spliceosome (n = 21), biogenesis of other RNAs (n = 3) or ribozyme functions (n = 16), whereas most others (n = 3798) were novel ('orphans') with unknown functions.

Conclusions: This study provides the first genome-wide sncRNA resource for S. haematobium, extending earlier studies of schistosomes. The present work should facilitate the future curation and experimental validation of sncRNA functions in schistosomes to enhance our understanding of post-transcriptional gene regulation and of the roles that sncRNAs play in schistosome reproduction, development and parasite-host cross-talk.
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http://dx.doi.org/10.1371/journal.pntd.0006535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993326PMC
May 2018

Molecular alterations during larval development of Haemonchus contortus in vitro are under tight post-transcriptional control.

Int J Parasitol 2018 08 22;48(9-10):763-772. Epub 2018 May 22.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

In this study, we explored the molecular alterations in the developmental switch from the L3 to the exsheathed L3 (xL3) and to the L4 stage of Haemonchus contortus in vitro using an integrated transcriptomic, proteomic and bioinformatic approach. Totals of 9,754 mRNAs, 88 microRNAs (miRNAs) and 1,591 proteins were identified, and 6,686 miRNA-mRNA pairs inferred in all larval stages studied. Approximately 16% of transcripts in the combined transcriptome (representing all three larval stages) were expressed as proteins, and there were positive correlations (r = 0.39-0.44) between mRNA transcription and protein expression in the three distinct developmental stages of the parasite. Of the predicted targets, 1,019 (27.0%) mRNA transcripts were expressed as proteins, and there was a negative correlation (r = -0.60 to -0.50) in the differential mRNA transcription and protein expression between developmental stages upon pairwise comparison. The changes in transcription (mRNA and miRNA) and protein expression from the free-living to the parasitic life cycle phase of H. contortus related to enrichments in biological pathways associated with metabolism (e.g., carbohydrate and lipid degradation, and amino acid metabolism), environmental information processing (e.g., signal transduction, signalling molecules and interactions) and/or genetic information processing (e.g., transcription and translation). Specifically, fatty acid degradation, steroid hormone biosynthesis and the Rap1 signalling pathway were suppressed, whereas transcription, translation and protein processing in the endoplasmic reticulum were upregulated during the transition from the free-living L3 to the parasitic xL3 and L4 stages of the nematode in vitro. Dominant post-transcriptional regulation was inferred to elicit these changes, and particular miRNAs (e.g., hco-miR-34 and hco-miR-252) appear to play roles in stress responses and/or environmental adaptations during developmental transitions of H. contortus. Taken together, these integrated results provide a comprehensive insight into the developmental biology of this important parasite at the molecular level in vitro. The approach applied here to H. contortus can be readily applied to other parasitic nematodes.
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http://dx.doi.org/10.1016/j.ijpara.2018.03.008DOI Listing
August 2018

Improved strategy for the curation and classification of kinases, with broad applicability to other eukaryotic protein groups.

Sci Rep 2018 05 1;8(1):6808. Epub 2018 May 1.

Melbourne Veterinary School, Department of Veterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, 3010, Australia.

Despite the substantial amount of genomic and transcriptomic data available for a wide range of eukaryotic organisms, most genomes are still in a draft state and can have inaccurate gene predictions. To gain a sound understanding of the biology of an organism, it is crucial that inferred protein sequences are accurately identified and annotated. However, this can be challenging to achieve, particularly for organisms such as parasitic worms (helminths), as most gene prediction approaches do not account for substantial phylogenetic divergence from model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, whose genomes are well-curated. In this paper, we describe a bioinformatic strategy for the curation of gene families and subsequent annotation of encoded proteins. This strategy relies on pairwise gene curation between at least two closely related species using genomic and transcriptomic data sets, and is built on recent work on kinase complements of parasitic worms. Here, we discuss salient technical aspects of this strategy and its implications for the curation of protein families more generally.
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http://dx.doi.org/10.1038/s41598-018-25020-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931623PMC
May 2018

Advances in kinome research of parasitic worms - implications for fundamental research and applied biotechnological outcomes.

Biotechnol Adv 2018 Jul - Aug;36(4):915-934. Epub 2018 Mar 19.

Melbourne Veterinary School, Department of Veterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Protein kinases are enzymes that play essential roles in the regulation of many cellular processes. Despite expansions in the fields of genomics, transcriptomics and bioinformatics, there is limited information on the kinase complements (kinomes) of most eukaryotic organisms, including parasitic worms that cause serious diseases of humans and animals. The biological uniqueness of these worms and the draft status of their genomes pose challenges for the identification and classification of protein kinases using established tools. In this article, we provide an account of kinase biology, the roles of kinases in diseases and their importance as drug targets, and drug discovery efforts in key socioeconomically important parasitic worms. In this context, we summarise methods and resources commonly used for the curation, identification, classification and functional annotation of protein kinase sequences from draft genomes; review recent advances made in the characterisation of the worm kinomes; and discuss the implications of these advances for investigating kinase signalling and developing small-molecule inhibitors as new anti-parasitic drugs.
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http://dx.doi.org/10.1016/j.biotechadv.2018.02.013DOI Listing
February 2019

Screening of the 'Stasis Box' identifies two kinase inhibitors under pharmaceutical development with activity against Haemonchus contortus.

Parasit Vectors 2017 Jul 5;10(1):323. Epub 2017 Jul 5.

Faculty of Veterinary and Agricultural Sciences, Melbourne Veterinary School, The University of Melbourne, Parkville, VIC, 3010, Australia.

Background: In partnership with the Medicines for Malaria Venture (MMV), we screened a collection ('Stasis Box') of 400 compounds (which have been in clinical development but have not been approved for illnesses other than neglected infectious diseases) for inhibitory activity against Haemonchus contortus, in order to attempt to repurpose some of the compounds to parasitic nematodes.

Methods: We assessed the inhibition of compounds on the motility and/or development of exsheathed third-stage (xL3s) and fourth-stage (L4) larvae of H. contortus using a whole-organism screening assay.

Results: In the primary screen, we identified compound MMV690767 (also known as SNS-032) that inhibited xL3 motility by ~70% at a concentration of 20 μM after 72 h as well as compound MMV079840 (also known as AG-1295), which induced a coiled xL3 phenotype, with ~50% inhibition on xL3 motility. Subsequently, we showed that SNS-032 (IC = 12.4 μM) and AG-1295 (IC = 9.92 ± 1.86 μM) had a similar potency to inhibit xL3 motility. Although neither SNS-032 nor AG-1295 had a detectable inhibitory activity on L4 motility, both compounds inhibited L4 development (IC values = 41.24 μM and 7.75 ± 0.94 μM for SNS-032 and AG-1295, respectively). The assessment of the two compounds for toxic effects on normal human breast epithelial (MCF10A) cells revealed that AG-1295 had limited cytotoxicity (IC > 100 μM), whereas SNS-032 was quite toxic to the epithelial cells (IC = 1.27 μM).

Conclusions: Although the two kinase inhibitors, SNS-032 and AG-1295, had moderate inhibitory activity on the motility or development of xL3s or L4s of H. contortus in vitro, further work needs to be undertaken to chemically alter these entities to achieve the potency and selectivity required for them to become nematocidal or nematostatic candidates.
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http://dx.doi.org/10.1186/s13071-017-2246-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499055PMC
July 2017

Whipworm kinomes reflect a unique biology and adaptation to the host animal.

Int J Parasitol 2017 11 10;47(13):857-866. Epub 2017 Jun 10.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia. Electronic address:

Roundworms belong to a diverse phylum (Nematoda) which is comprised of many parasitic species including whipworms (genus Trichuris). These worms have adapted to a biological niche within the host and exhibit unique morphological characteristics compared with other nematodes. Although these adaptations are known, the underlying molecular mechanisms remain elusive. The availability of genomes and transcriptomes of some whipworms now enables detailed studies of their molecular biology. Here, we defined and curated the full complement of an important class of enzymes, the protein kinases (kinomes) of two species of Trichuris, using an advanced and integrated bioinformatic pipeline. We investigated the transcription of Trichuris suis kinase genes across developmental stages, sexes and tissues, and reveal that selectively transcribed genes can be linked to central roles in developmental and reproductive processes. We also classified and functionally annotated the curated kinomes by integrating evidence from structural modelling and pathway analyses, and compared them with other curated kinomes of phylogenetically diverse nematode species. Our findings suggest unique adaptations in signalling processes governing worm morphology and biology, and provide an important resource that should facilitate experimental investigations of kinases and the biology of signalling pathways in nematodes.
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http://dx.doi.org/10.1016/j.ijpara.2017.04.005DOI Listing
November 2017

Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain.

G3 (Bethesda) 2016 09 8;6(9):2847-56. Epub 2016 Sep 8.

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia

Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation.
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http://dx.doi.org/10.1534/g3.116.032961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015942PMC
September 2016

CAP protein superfamily members in Toxocara canis.

Parasit Vectors 2016 06 24;9(1):360. Epub 2016 Jun 24.

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia.

Background: Proteins of the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 (CAP) superfamily are recognized or proposed to play roles in parasite development and reproduction, and in modulating host immune attack and infection processes. However, little is known about these proteins for most parasites.

Results: In the present study, we explored CAP proteins of Toxocara canis, a socioeconomically important zoonotic roundworm. To do this, we mined and curated transcriptomic and genomic data, predicted and curated full-length protein sequences (n = 28), conducted analyses of these data and studied the transcription of respective genes in different developmental stages of T. canis. In addition, based on information available for Caenorhabditis elegans, we inferred that selected genes (including lon-1, vap-1, vap-2, scl-1, scl-8 and scl-11 orthologs) of T. canis and their interaction partners likely play central roles in this parasite's development and/or reproduction via TGF-beta and/or insulin-like signaling pathways, or via host interactions.

Conclusion: In conclusion, this study could provide a foundation to guide future studies of CAP proteins of T. canis and related parasites, and might assist in finding new interventions against diseases caused by these parasites.
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http://dx.doi.org/10.1186/s13071-016-1642-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921028PMC
June 2016

Reconstruction of the insulin-like signalling pathway of Haemonchus contortus.

Parasit Vectors 2016 Feb 3;9:64. Epub 2016 Feb 3.

The University of Melbourne, Faculty of Veterinary and Agricultural Sciences, Parkville, VIC, Australia.

Background: In the present study, we reconstructed the insulin/insulin-like growth factor 1 signalling (IIS) pathway for Haemonchus contortus, which is one of the most important eukaryotic pathogens of livestock worldwide and is related to the free-living nematode Caenorhabditis elegans.

Methods: We curated full-length open-reading frames from assembled transcripts, defined the complement of genes that encode proteins involved in this pathway and then investigated the transcription profiles of these genes for all key developmental stages of H. contortus.

Results: The core components of the IIS pathway are similar to their respective homologs in C. elegans. However, there is considerable variation in the numbers of isoforms between H. contortus and C. elegans and an absence of AKT-2 and DDL-2 homologs from H. contortus. Interestingly, DAF-16 has a single isoform in H. contortus compared with 12 in C. elegans, suggesting novel functional roles in the parasitic nematode. Some IIS proteins, such as DAF-18 and SGK-1, vary in their functional domains, indicating distinct roles from their homologs in C. elegans.

Conclusions: This study paves the way for the further characterization of key signalling pathways in other socioeconomically important parasites and should help understand the complex mechanisms involved in developmental processes.
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http://dx.doi.org/10.1186/s13071-016-1341-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741068PMC
February 2016

The Haemonchus contortus kinome--a resource for fundamental molecular investigations and drug discovery.

Parasit Vectors 2015 Dec 8;8:623. Epub 2015 Dec 8.

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia.

Background: Protein kinases regulate a plethora of essential signalling and other biological pathways in all eukaryotic organisms, but very little is known about them in most parasitic nematodes.

Methods: Here, we defined, for the first time, the entire complement of protein kinases (kinome) encoded in the barber's pole worm (Haemonchus contortus) through an integrated analysis of transcriptomic and genomic datasets using an advanced bioinformatic workflow.

Results: We identified, curated and classified 432 kinases representing ten groups, 103 distinct families and 98 subfamilies. A comparison of the kinomes of H. contortus and Caenorhabditis elegans (a related, free-living nematode) revealed considerable variation in the numbers of casein kinases, tyrosine kinases and Ca(2+)/calmodulin-dependent protein kinases, which likely relate to differences in biology, habitat and life cycle between these worms. Moreover, a suite of kinase genes was selectively transcribed in particular developmental stages of H. contortus, indicating central roles in developmental and reproductive processes. In addition, using a ranking system, drug targets (n = 13) and associated small-molecule effectors (n = 1517) were inferred.

Conclusions: The H. contortus kinome will provide a useful resource for fundamental investigations of kinases and signalling pathways in this nematode, and should assist future anthelmintic discovery efforts; this is particularly important, given current drug resistance problems in parasitic nematodes.
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http://dx.doi.org/10.1186/s13071-015-1231-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672506PMC
December 2015

Defining the Schistosoma haematobium kinome enables the prediction of essential kinases as anti-schistosome drug targets.

Sci Rep 2015 Dec 4;5:17759. Epub 2015 Dec 4.

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

The blood fluke Schistosoma haematobium causes urogenital schistosomiasis, a neglected tropical disease (NTD) that affects more than 110 million people. Treating this disease by targeted or mass administration with a single chemical, praziquantel, carries the risk that drug resistance will develop in this pathogen. Therefore, there is an imperative to search for new drug targets in S. haematobium and other schistosomes. In this regard, protein kinases have potential, given their essential roles in biological processes and as targets for drugs already approved by the US Food and Drug Administration (FDA) for use in humans. In this context, we defined here the kinome of S. haematobium using a refined bioinformatic pipeline. We classified, curated and annotated predicted kinases, and assessed the developmental transcription profiles of kinase genes. Then, we prioritised a panel of kinases as potential drug targets and inferred chemicals that bind to them using an integrated bioinformatic pipeline. Most kinases of S. haematobium are very similar to those of its congener, S. mansoni, offering the prospect of designing chemicals that kill both species. Overall, this study provides a global insight into the kinome of S. haematobium and should assist the repurposing or discovery of drugs against schistosomiasis.
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http://dx.doi.org/10.1038/srep17759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669435PMC
December 2015

Lucilia cuprina genome unlocks parasitic fly biology to underpin future interventions.

Nat Commun 2015 Jun 25;6:7344. Epub 2015 Jun 25.

Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.

Lucilia cuprina is a parasitic fly of major economic importance worldwide. Larvae of this fly invade their animal host, feed on tissues and excretions and progressively cause severe skin disease (myiasis). Here we report the sequence and annotation of the 458-megabase draft genome of Lucilia cuprina. Analyses of this genome and the 14,544 predicted protein-encoding genes provide unique insights into the fly's molecular biology, interactions with the host animal and insecticide resistance. These insights have broad implications for designing new methods for the prevention and control of myiasis.
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http://dx.doi.org/10.1038/ncomms8344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491171PMC
June 2015

Flatworms have lost the right open reading frame kinase 3 gene during evolution.

Sci Rep 2015 May 15;5:9417. Epub 2015 May 15.

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

All multicellular organisms studied to date have three right open reading frame kinase genes (designated riok-1, riok-2 and riok-3). Current evidence indicates that riok-1 and riok-2 have essential roles in ribosome biosynthesis, and that the riok-3 gene assists this process. In the present study, we conducted a detailed bioinformatic analysis of the riok gene family in 25 parasitic flatworms (platyhelminths) for which extensive genomic and transcriptomic data sets are available. We found that none of the flatworms studied have a riok-3 gene, which is unprecedented for multicellular organisms. We propose that, unlike in other eukaryotes, the loss of RIOK-3 from flatworms does not result in an evolutionary disadvantage due to the unique biology and physiology of this phylum. We show that the loss of RIOK-3 coincides with a loss of particular proteins associated with essential cellular pathways linked to cell growth and apoptosis. These findings indicate multiple, key regulatory functions of RIOK-3 in other metazoan species. Taking advantage of a known partial crystal structure of human RIOK-1, molecular modelling revealed variability in nucleotide binding sites between flatworm and human RIOK proteins.
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http://dx.doi.org/10.1038/srep09417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894443PMC
May 2015
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