Developmental sensitivity in Schistosoma mansoni to puromycin to establish drug selection of transgenic schistosomes

Yan, Hong-Bin, Smout, Michael J., Ju, Chuan, Folley, Anne E., Skinner, Danielle E., Mann, Victoria H., Loukas, Alex, Hu, Wei, Brindley, Paul J., and Rinaldi, Gabriel (2018) Developmental sensitivity in Schistosoma mansoni to puromycin to establish drug selection of transgenic schistosomes. Antimicrobial Agents and Chemotherapy, 62 (8). e02568-17.

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Abstract

Schistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and loss-of-function approaches can be expected to precipitate the discovery of novel interventions, and drug selection of transgenic schistosomes would facilitate the establishment of stable lines of engineered parasites. Sensitivity of developmental stages of schistosomes to the aminonucleoside antibiotic puromycin was investigated. For the schistosomulum and sporocyst stages, viability was quantified by fluorescence microscopy following dual staining with fluorescein diacetate and propidium iodine. By 6 days in culture, the 50% lethal concentration (LC50) for schistosomula was 19 μg/ml whereas the sporocysts were 45-fold more resilient. Puromycin potently inhibited the development of in vitro-laid eggs (LC50, 68 ng/ml) but was less effective against liver eggs (LC50, 387 μg/ml). Toxicity for adult stages was evaluated using the xCELLigence-based, real-time motility assay (xWORM), which revealed LC50s after 48 h of 4.9 and 17.3 μg/ml for male and female schistosomes, respectively. Also, schistosomula transduced with pseudotyped retrovirus encoding the puromycin resistance marker were partially rescued when cultured in the presence of the antibiotic. Together, these findings will facilitate selection on puromycin of transgenic schistosomes and the enrichment of cultures of transgenic eggs and sporocysts to facilitate the establishment of schistosome transgenic lines. Streamlining schistosome transgenesis with drug selection will open new avenues to understand parasite biology and hopefully lead to new interventions for this neglected tropical disease.

Item ID: 55020
Item Type: Article (Research - C1)
ISSN: 1098-6596
Keywords: antibiotic susceptibility, drug selection, functional genomics, puromycin, schistosomiasis
Copyright Information: Copyright © 2018 American Society for Microbiology. All Rights Reserved.
Funders: National Institute of Allergy and Infectious Diseases (NIH NIAID), Wellcome Trust
Projects and Grants: NIH NIAID Award R01AI072773, NIH NIAID Award R21AI109532, Wellcome Trust Strategic Award number 107475/Z/15/Z, Wellcome Trust Wellcome Sanger Institute award number 206194
Date Deposited: 15 Aug 2018 07:31
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110899 Medical Microbiology not elsewhere classified @ 100%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 100%
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