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PLoS Negl Trop Dis
2014 Jun 05;86:e2870. doi: 10.1371/journal.pntd.0002870.
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Targeting Echinococcus multilocularis stem cells by inhibition of the Polo-like kinase EmPlk1.
Schubert A
,
Koziol U
,
Cailliau K
,
Vanderstraete M
,
Dissous C
,
Brehm K
.
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BACKGROUND: Alveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation.
METHODOLOGY/PRINCIPAL FINDINGS: We herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation.
CONCLUSIONS/SIGNIFICANCE: We conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy.
Figure 2. Expression of emplk1 in E. multilocularis larval stages.Total RNA was isolated from primary cell cultures (Pc) after 2, 5 and 11 days (d2, d5, d11) of development towards the metacestode stage, from dormant (Ps_non) and pepsin/low pH-activated protoscoleces (Ps_act), as well as from mature metacestode vesicles (Mc). The isolated RNA was reverse transcribed to cDNA and equal amounts of serial (10-fold) dilutions of cDNA were subjected to gene-specific RT-PCR for emplk1 and the constitutively expressed gene elp (control) as indicated to the right. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. gDNA indicates positive control lanes where PCR has been performed on genomic DNA. ‘RT-neg’ indicates the negative control where reverse transcriptase has been omitted. Marker sizes are indicated to the left.
Figure 3. WISH detection of emplk1 transcripts.(A) General view of a metacestode hybridized with the emplk1 antisense probe. Note the accumulation of emplk1 positive cells in the developing protoscoleces (arrows). (B) General view of a metacestode hybridized with the emplk1 sense control probe. No signal is detected. (C–F) Details of emplk1 WISH detection in different developmental stages. (C) Detail of the metacestode germinative layer, in which dispersed emplk1 positive cells can be distinguished (inset: higher magnification of a positive cell, combined with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining). (D) Accumulation of emplk1 positive cells in the early brood capsule bud (arrow). (E) Early protoscolex development, with abundant emplk1 positive cells in the interior. (F) Late protoscolex development. emplk1 positive cells accumulate at the base of the developing suckers (arrows). Bars represent 200 µm (A, B) and 50 µm (C–F).
Figure 4. BI 2536 inhibits the formation of metacestode vesicles from E. multilocularis primary cells.Primary cell cultures were established from metacestode vesicles and incubated under ideal growth conditions (A4 medium) in the presence of different concentrations of BI 2536 (5, 10, 25, 50, 100 nM) as indicated. After 14 days of incubation, fully formed (mature) metacestode vesicles were counted microscopically. ‘Control’ indicates cultures with A4 medium, ‘mock’ indicates cultures with DMSO (without inhibitor). All conditions were at least assessed in three different biological replicates with at least three technical replicates. *(p<0.05); **(p = 0.001–0.01) (Student's t-test).
Figure 5. Long-term BI 2536 effects on E. multilocularis metacestode vesicles.Metacestode vesicles were treated for 21 days with different concentrations (10, 25, 50 and 100 nM) of BI 2536, followed by three days of recovery without inhibitor. On day three of recovery, EdU labeling (5 h, 50 µM EdU) was carried out and labeled cells were microscopically counted. (A) Microscopic images of the GL of BI 2536 treated (10, 25, 50, 100 nM) and non-treated E. multilocularis metacestode vesicles, stained with EdU. Bar represents 25 µm. Blue, nuclear staining, DAPI; red, EdU staining. (B) EdU-positive cell counts per mm2 of GL. ‘Control’ indicates vesicles cultivated in A4 medium alone. ‘Mock’ indicates the DMSO control. ***(p<0.001) (Student's t-test). Please note that the specific examples shown in (A) display slightly different total cell numbers but that the overall cell numbers in metacestode vesicles were not altered by drug treatment, as already outlined in [14] for HU treatment.
Figure 6. Short-term treatment of metacestode vesicles with BI 2536.Metacestode vesicles were treated for 24, 48 or 72(as indicated) with 50 nM (light grey) or 100 nM (dark grey) of BI 2536, followed by one day of recovery without inhibitor. EdU labeling (5 h, 50 µM EdU) was carried out and labeled cells were microscopically counted. Shown are EdU-positive cell counts per mm2 of GL. ‘Control’ indicates vesicles cultivated in A4 medium alone. ‘Mock’ indicates the DMSO control. ***(p<0.001) (Student's t-test).
Figure 1. Amino acid sequence and structural analysis of EmPlk1.The EmPlk1 amino acid sequence was aligned to Plk1-like enzymes of S. mansoni (SmPlk1; GenBank accession number: AY747306), Homo sapiens (Plk1; P53350); D. melanogaster (Polo; P52304) and X. laevis (Plx1; P70032). The predicted kinase domain is boxed, predicted Polo-boxes (Polo-box 1; Polo-box 2) are indicated above the alignment. Residues identical in at least four of the sequences are shown in white on black background. The eleven sub-domains known to be highly conserved in kinases are indicated above (I–XI). Highly conserved amino acid residues and sequence motifs are indicated above the alignment. The highly conserved threonine residue which is critical for activation is marked by (T*) above the alignment. Residues important for phosphosubstrate binding of the C-terminal domain are marked by (#) below the alignment. Residues involved in binding of BI 2536 to Plk1 are indicated by grey dots below the alignment.
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