Developmental Sensitivity in Schistosoma mansoni to Puromycin To Establish Drug Selection of Transgenic Schistosomes

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.

Keywords: antibiotic susceptibility; drug selection; functional genomics; puromycin; schistosomiasis.

FIG 1

Schistosomula of Schistosoma mansoni cultured in the presence of puromycin. (A) Schistosomula were cultured for 10 days in the presence of increasing concentrations of antibiotic. Culture media and puromycin were replaced when indicated (P). CT, cercarial transformation; P, puromycin. (B) Representative micrographs of schistosomula cultured for 8 days in 0 μg/ml (top), 2.5 μg/ml (center), or 12.5 μg/ml (bottom) puromycin and costained with propidium iodide (PI) and fluorescein diacetate (FDA) to label dead (red) and live (green) parasites, respectively. Selected concentrations induced minimal harm from no treatment, substantial harm from 2.5 μg/ml, and complete killing from 12.5 μg/ml. Bar, 200 μm. (C) Survival of schistosomula over time displayed as means ± standard errors of the means (SEM). For clarity, selected curves only are presented. (D) Dose-response curves (DRC) of schistosomula exposed to puromycin (means ± SEM). (E) Fifty percent lethal concentration (LC₅₀) over time indicating 95% confidence intervals.

FIG 2

Schistosoma mansoni sporocysts cultured in the presence of puromycin. (A) Primary sporocysts were cultured for 21 days in increasing concentrations of the antibiotic. Culture media and puromycin were replaced when indicated (P). ST, sporocyst transformation; P, puromycin. (B) Representative micrographs of primary sporocysts cultured in 0 μg/ml (top), 250 μg/ml (center), or 1,000 μg/ml (bottom) puromycin and costained with propidium iodide (PI) and fluorescein diacetate (FDA) to stain dead (red) and live (green) parasites, respectively. Bar, 200 μm. (C) Sporocyst survival over time displayed as means ± SEM. (D) Dose-response curves (DRC) of sporocysts exposed to puromycin (means ± SEM). (E) Fifty percent lethal concentration (LC₅₀) over time indicating 95% confidence interval error bars.

FIG 3

Puromycin impairs the egg maturation. (A) Schematic showing the experimental design. Eggs laid in vitro (in vitro laid eggs [IVLEs]) by female adult schistosomes cultured for 3 days after perfusion were collected and exposed to the indicated concentrations of puromycin or vehicle control for 4 days. The culture media and puromycin were replaced on day 5 after perfusion, and 2 days later eggs were induced to hatch by transfer into water and exposure to bright light. (B) Percentages of eggs at different maturation stages as categorized using the staging system of Vogel and Prata (35) for the indicated days after perfusion and puromycin concentrations. (C) Representative pictures of IVLEs cultured in the presence of 0, 1, and 100 μg/ml of puromycin at 7 days after perfusion. Bar, 200 μm. (D) Dose-response curves (DRC) of IVLEs exposed to puromycin (means ± SEM). (E) Fifty percent lethal concentration (LC₅₀) over time; 95% confidence intervals are indicated.

FIG 4

Toxicity of puromycin for schistosome eggs isolated from mouse livers. (A) Representative micrographs of eggs cultured in the presence of 0, 125, and 1,000 μg/ml puromycin for 5 days, hatched under bright light for 2 h, and fixed. Bar, 200 μm. (B) Eggs isolated from liver were cultured for 5 days in increasing concentrations of puromycin or controls (untreated and vehicle control, i.e., 1 mM HEPES). Thereafter, the eggs were washed, transferred to water, induced to hatch under bright light for 2 h, fixed, and counted. Eight to 10 nonoverlapping field pictures showing an average of 12 eggs per field were inspected in each group, i.e., 96 to 120 eggs per treatment group. Each dot within the groups indicates the percentage of hatched eggs in each nonoverlapping field, and the means of hatched eggs for treatments group are presented. One-way ANOVA among the groups: P ≤ 0.05; t test between each group and HEPES control: *, P ≤ 0.05; **, P ≤ 0.01. (C) Dose-response curves (DRC) of relative hatched eggs exposed to increasing concentrations of puromycin (means ± SE). Fifty percent lethal concentration (LC₅₀) is shown.

FIG 5

Puromycin kills adult schistosomes in vitro. (A) Experimental design: 1 day after collection (C) the adult worms were separated into males and females and exposed to increasing concentrations of puromycin (P). The medium and antibiotic were changed every other day. (B) Representative micrographs of adult female worms cultured in the presence of 1,000 μg/ml of antibiotic (left) or HEPES-treated control group (right). Bar, 250 μm. (C) Adult motility measured by xWORM assay of female worms exposed to the indicated concentrations of puromycin over time. (D) Dose-response curves (DRC) of survival relative to control cultured in the absence of antibiotic at three different time points as indicated (means ± SE). (E) Fifty percent lethal concentration (LC₅₀) over time indicating 95% confidence intervals for female and male adult schistosomes 48 h after exposure to the antibiotic.

FIG 6

Rescue from puromycin toxicity of PuroR-expressing schistosomula. (A) Micrographs of control (top) or MLV-transduced parasites expressing puroR (bottom) cultured in the presence of puromycin for 6 days. Bar, 100 μm. (B) Percentage of live control or MLV-transduced schistosomula expressing PuroR cultured in the presence of 2.5 μg/ml of puromycin for 6 days (means ± SD; SD_control = 0.09, SD_{MLV_puroR} = 0.14; n = 5 micrographs showing nonoverlapping fields containing ≥50 parasites). Student's t test between groups: *, P ≤ 0.05. (C) Relative PuroR transgene expression in control or MLV-transduced schistosomula cultured in 2.5 μg/ml of puromycin for 6 days. The experiment was repeated at least twice with different batches of virions.