Transcriptomic analysis of reduced sensitivity to praziquantel in Schistosoma mansoni

Abstract

Schistosomiasis is an intravascular parasitic infection estimated to affect over 206 million people, the majority of whom live in Africa where the trematode worms Schistosoma mansoni and Schistosoma haematobium are the major causative agents. While a number of drugs have been used to treat schistosomiasis, praziquantel (PZQ) is the only one that is widely available, relatively cheap, and easy to use. The reliance on a single drug for the treatment of such a prevalent disease is a cause for concern due to the potential for resistance to render PZQ ineffective. In this study, we examine the transcriptome of three generations of a laboratory strain of S. mansoni (PR1) whose susceptibility to PZQ has been diminished across 9 passages through exposure to increasing sub-lethal doses of the drug. Miracidial susceptibility was significantly reduced after exposure to 2 × 50 mg/Kg PZQ during the first passage. Susceptibility of worms in vivo was first assessed during passage 5 when mice infected with PZQ-selected schistosomes were dosed with a lethal dose of 3 × 300 mg/kg PZQ resulting in only a 10% reduction in worm number compared to control treatment. The emergence of reduced sensitivity was marked by a shift in sex ratio from a predominantly male to a female population, a reduction in the length of females and ultimately the loss of the PZQ-selected line after passage 9. Analysis of differentially regulated transcripts did not suggest that any particular gene product or pathway was associated with drug resistance suggesting either a loss of function mutation to a single gene or an epistatic interaction of multiple gene products as the underlying cause of reduced susceptibility.

Keywords: Drug resistance; Helminth; Praziquantel; Schistosoma; Schistosomiasis; Transcriptome.

Fig 1.. Changes in S. mansoni length and width during PZQ-selection.

The length and width of male and female schistosomes isolated during passages (P) 1–9 are shown. (A) Length of male parasites. (B) Width of male parasites. (C) Length of female parasites. (D) Width of female parasites. Significance of observed differences between control and PZQ-selected schistosomes were assessed with 2-way ANOVA with Sidak’s multiple comparisons test. Error bars represent mean with standard deviation, n = 10 sex/treatment/passage. * = p< 0.05, and *** = p< 0.001.

Fig 2.. In vitro assay of S. mansoni miracidia survival after exposure to PZQ.

Miracidia were hatched from control and PZQ-selected groups and exposed to 10⁻⁵ M PZQ for 20 min and the number surviving counted. (A) P1, (B) P3, (C) P4, (D) P5, (E) P6, (F) P7, and (G) P8. Passage 2 and P9 data are not included due to low miracidia yield. Statistical significance determined between control and PZQ-selected parasites using the Holm-Sidak multiple comparisons method with alpha = 0.05. Error bars represent mean with standard deviation. * = p< 0.05, ** = p< 0.01, and *** = p< 0.001.

Fig 3.. Venn diagrams of differentially expressed genes.

Indicated in the Venn diagrams are the numbers of (A) up-regulated and (B) down-regulated differentially expressed genes shared between or exclusive to S. mansoni derived from P6, P7 and P8. The diameter of each circle is proportional to the number of transcripts it represents.

Fig 4.. Expression of selected S. mansoni genes during passages (P) 6, 7 and 8.

Heat map of the 50 most differentially expressed genes (25 up- and 25 down-regulated) in PZQ-selected parasites relative to controls. Genes not differentially expressed are colored gray and increased and decreased gene expression are teal and pink respectively. The color scale indicates log₂ fold change for the average of four biological replicates.