Genetic Manipulation of Schistosoma haematobium, the Neglected Schistosome
ABSTRACT Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia.
Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium.
These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen.
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ABSTRACT: Infection with helminth parasites remains a persistent public health problem in developing countries. Three of these pathogens, the liver flukes Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, are of particular concern due to their classification as Group 1 carcinogens: infection with these worms is carcinogenic. Using liquid chromatography-mass spectrometry (LC-MS/MS) approaches, we identified steroid hormone like (e.g., oxysterol-like, catechol estrogen quinone-like, etc.) metabolites and related DNA-adducts, apparently of parasite origin, in developmental stages including eggs of S. haematobium, in urine of people with urogenital schistosomiasis, and in the adult stage of O. viverrini. Since these kinds of sterol derivatives are metabolized to active quinones that can modify DNA, which in other contexts can lead to breast and other cancers, helminth parasite associated sterols might induce tumor-like phenotypes in the target cells susceptible to helminth parasite associated cancers, i.e., urothelial cells of the bladder in the case of urogenital schistosomiasis and the bile duct epithelia or cholangiocytes, in the case of O. viverrini and C. sinensis. Indeed we postulate that helminth induced cancers originate from parasite estrogen-host epithelial/urothelial cell chromosomal DNA adducts, and here we review recent findings that support this conjecture.Frontiers in Genetics 12/2014; 5:444. DOI:10.3389/fgene.2014.00444
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ABSTRACT: Exploration of genetic changes during active Schistosoma infection is important for anticipation and prevention of chronic sequelae. This study aimed to explore the genomic instability in chromosomal and cellular kinetics in Egyptians suffering from uncomplicated active schistosomiasis haematobium infection in addition to chronic schistosomiasis haematobium cases complicated by bilharzial-associated bladder cancer (BAC). This study was conducted on 46 schistosomiasis haemotobium cases, 22 were active (Viable S. haematobium eggs in urine samples as detected by microscopy) and 24 were chronic complicated with bladder cancer. Three cytogenetic techniques were applied; the first was quantitative nuclear-morphocytometry by means of which the Feulgen-stained nuclei were analyzed for parameters including shape, size, integrated optical-density and nuclear area. The second was Fluorescent In-Situ Hybridization (FISH) for specific p53gene-locus of chromosome 17 and the third technique was karyotyping. Concerning chronic complicated cases, the mean ± SD of DNA-content in urinary bladder tissue sections was 3.18 ± 0.65. Five samples (20.83%) of bladder tissue sections of chronic complicated cases showed diploid nuclei, 6 urinary bladder tissue samples (25%) were tetraploid, while 13 bladder samples (54.16%) were aneuploid. Epithelial cells of urine samples demonstrated aneuploidy (mean ± SD = 3.74 ± 0.36).Nuclear contents showed high proliferative DNA index in all urinary epithelial cells. In the acute uncomplicated group, nuclear-DNA of urinary epithelial cells was found diploid with mean nuclear-DNA content of 2.2 ± 0.16SD. Half of these diploid smears had a high proliferation index. The difference between nuclear DNA-contents in acute and chronic cases was significant (P = 0.0001). FISH technique for specific p53gene-locus and karyotyping were done on urinary bladder tissue specimens and peripheral blood monocytes of 8 chronic cases respectively. Three samples (37.5%) with invasive BAC had a deletion of the p53 gene. Karyotyping showed three cases out of the 8 chronic schistosomiasis haematobium patients with chromosomal fragmentations. DNA morphometry was valuable in detection of gross genetic changes in urothelial tissues. It is an important prognostic factor in established schistosomiasis haematobium induced bladder malignancy. It has the great advantage of being applicable on urine cells making it suitable for the prediction of a tendency towards genetic instability in active schistosomiasis haematobium patients.Molecular Cytogenetics 01/2015; 8(1):1. DOI:10.1186/s13039-014-0104-5 · 2.66 Impact Factor
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ABSTRACT: Approximately 200,000,000 people have schistosomiasis (schistosome infection). Among the schistosomes, Schistosoma haematobium is responsible for the most infections- 110 million people globally, mostly in sub-Saharan Africa. This pathogen causes an astonishing breadth of sequelae- hematuria, anemia, dysuria, stunting, uremia, bladder cancer, urosepsis, and HIV co-infection. Refined estimates of the quality of life impact of schistosomiasis suggest it rivals malaria. Despite S. haematobium's importance, relevant research has lagged. Here, we review advances that will deepen knowledge of S. haematobium. Three sets of breakthroughs will accelerate discoveries in the pathogenesis of urogenital schistosomiasis (UGS): (1) comparative genomics; (2) the development of functional genomic tools; and (3) use of animal models to explore S. haematobium-host interactions. Comparative genomics for S. haematobium is feasible, given the sequencing of multiple schistosome genomes. Features of the S. haematobium genome that are conserved among platyhelminth species, and others that are unique to S. haematobium may provide novel diagnostic and drug targets for UGS. Though there are technical hurdles, the integrated use of these approaches can elucidate host-pathogen interactions during this infection, and inform development of techniques for investigating schistosomes in their human and snail hosts and therapeutics and vaccines for the control of UGS.The Journal of Infectious Diseases 09/2014; DOI:10.1093/infdis/jiu527 · 5.78 Impact Factor