Histone deacetylase enzymes as potential drug targets of Neglected Tropical Diseases caused by cestodes

Abstract

Cestode parasites cause neglected diseases, such as echinococcosis and cysticercosis, which represent a significant problem in human and animal health. Benzimidazoles and praziquantel are the only available drugs for chemotherapy and it is therefore important to identify new alternative drugs against cestode parasites. Histone deacetylases (HDACs) are validated drug targets for the treatment of cancer and other diseases, including neglected diseases. However, knowledge of HDACs in cestodes is very scarce. In this work, we investigated cestode HDACs as potential drug targets to develop new therapies against neglected diseases caused by cestodes. Here we showed the full repertoire of HDAC coding genes in several members of the class Cestoda. Between 6 and 7 zinc-dependent HDAC coding genes were identified in the genomes of species from Echinococcus, Taenia, Mesocestoides and Hymenolepis genera. We classified them as Class I and II HDACs and analyzed their transcriptional expression levels throughout developmental stages of Echinococcus spp. We confirmed for the first time the complete HDAC8 nucleotide sequences from Echinococcus canadensis G7 and Mesocestoides corti. Homology models for these proteins showed particular structural features which differentiate them from HDAC8 from Homo sapiens. Furthermore, we showed that Trichostatin A (TSA), a pan-HDAC inhibitor, decreases the viability of M. corti, alters its tegument and morphology and produces an increment of the total amount of acetylated proteins, including acetylated histone H4. These results suggest that HDAC from cestodes are functional and might play important roles on survival and development. The particular structural features observed in cestode HDAC8 proteins suggest that these enzymes could be selectively targeted. This report provides the basis for further studies on cestode HDAC enzymes and for discovery of new HDAC inhibitors for the treatment of neglected diseases caused by cestode parasites.

Keywords: Cestode; Echinococcus; Histone deacetylases; Mesocestoides corti; Neglected Tropical Diseases; Trichostatin A.

Graphical abstract

Fig. 1

Phylogenetic tree of HDACs from Homo sapiens, Schistosoma mansoni and cestode parasites. Phylogenetic tree of the amino acid sequences of the HDAC catalytic domains among the following cestode parasites: Echinococcus canadensis G7 (Eca), Echinococcus granulosussensu stricto G1 (Egr), Echinococcus multilocularis (Emu), Hymenolepis diminuta (Hdi), Hymenolepis microstoma (HmN), Mesocestoides corti (Mco), Taenia asiatica (Tas), Taenia saginata (Tsa), Taenia solium (Tsm) and Homo sapiens (Hsa) and Schistosoma mansoni (Smp). Gene IDs are shown in brackets. Phylogenetic tree was obtained using the Maximum Likelihood method based on the JTT matrix-based model. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The analysis involved 90 amino acid sequences. All positions with less than 95% site coverage were eliminated. There were a total of 79 positions in the final dataset. Phylogenetic analysis was conducted in MEGA5.

Fig. 2

Comparison of HDAC catalytic domains amino acid residues. Percentage of identity of HDAC catalytic domains amino acid residues between the following cestodes: Echinococcus canadensis G7 (Eca), Echinococcus granulosussensu stricto G1 (Egr), Echinococcus multilocularis (Emu), Hymenolepis diminuta (Hdi), Hymenolepis microstoma (HmN), Mesocestoides corti (Mco), Taenia asiatica (Tas), Taenia saginata (Tsa), Taenia solium (Tsm) and Homo sapiens (Hsa) or Schistosoma mansoni (Smp). Each panel shows the percentage of identity for each HDAC gene. The values were taken from a percent identity matrix created by Clustal2.1.

Fig. 3

Transcriptional expression levels of HDAC genes in parasites of genus Echinococcus spp. HDAC transcriptional expression levels are shown as RPKM (Reads Per Kilobase Million). (A) Comparative HDAC transcriptional expression gene levels, determined by RNAseq, in several developmental stages of Echinococcus granulosussensu stricto G1: adult, oncospheres (Onc), cyst wall (CW) and protoscoleces (PSC) (Zheng et al., 2013). (B) Comparative HDAC transcriptional expression gene levels, determined by RNAseq, in several developmental stages of Echinococcus multilocularis: oncospheres (Onc), activated oncospheres (Act Onc) and 4-week metacestodes miniature vesicles (4wCW) (Huang et al., 2016).

Fig. 4

Comparative analysis of HDAC8 from Homo sapiens, Schistosoma mansoni, Echinococcus canadensis G7 and Mesocestoides corti. (A) Alignment of amino acid sequences of HDAC8 from H. sapiens (HsaHDAC8), S. mansoni (SmpHDAC8), E. canadensis G7 (EcaHDAC8) and M. corti (McoHDAC8). Sequence similarities are shown in green levels. Conserved residues indicated below the alignment are involved in coordinating the zinc ion (rhombus), catalysis and active site formation (quadrate).(B) Superposition of native HsaHDAC8 (red; PDB 1T64; H. sapiens), SmpHDAC8 (purple; PDB 4BZ5; S. mansoni.) and models of EcaHDAC8 (blue; E. canadensis G7) and McoHDAC8 (green; M. corti) structures represented as ribbons. All HDAC8 enzymes adopted the canonical HDAC fold. The yellow sphere represents the catalytic ion zinc (Zn). Close view of active sites of (C) HsaHDAC8, (D) SmpHDAC8, (E) EcaHDAC8 and (F) McoHDAC8. The residues involved in zinc binding, catalysis and active site formation are shown as sticks. Residues are conserved in parasite HDAC8s, only L31 and M274 are replaced by serine and histidine, respectively, EcaHDAC8 (S20; H286) and McoHDAC8 (S20; H286), as well as in SmpHDAC8 (S18; H292). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Amino acid residues surrounding the phenylalanine of HDAC8 from Homo sapiens, Schistosoma mansoni, Echinococcus canadensis G7 and Mesocestoides corti. Structures of HDAC8 from H. sapiens PDB 1T64 (HsaHDAC8 F152), S. mansoni PDB 4BZ5 (SmpHDAC8 F151), E. canadensis G7 (EcaHDAC8 F154) and M. corti (McoHDAC8 F154) are shown. The replacement of leucine in HsaHDAC8 (L31) by a smaller residue as serine in SmpHDAC8 (S18), EcaHDAC8 (S20) and McoHDAC8 (S20), together with several aromatic residues (“aromatic cage” is shown as mesh) in SmpHDAC8 (purple: F21, F104, Y110, W140 and Y153), EcaHDAC8 (blue: Y23, F107, Y113, W143 and Y156) and McoHDAC8 (green: Y23, F107, Y113, W143 and Y156), form a cavity behind the phenylalanine which can adopt a especial conformation know as “flipped-out”. Residues above mentioned are shown as sticks (red from HsaHDAC8 and white from SmpHDAC8, EcaHDAC8 and McoHDAC8). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Residues involved in Trichostatin A (TSA) binding in HDAC8 from Homo sapiens, Echinococcus canadensis G7 and Mesocestoides corti. Cartoon representations of (A) H. sapiens PDB 1T64 (HsaHDAC8; red) and model structures from (B) E. canadensis G7 (EcaHDAC8; blue) and M. corti (McoHDAC8; green). The yellow sphere represents the catalytic ion zinc (Zn). TSA and residues involved in TSA binding are shown as sticks. Most residues involved in TSA binding in HsaHDAC8 are conserved in EcaHDAC8 and McoHDAC8. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Effect of Trichostatin A (TSA) on viability of Mesocestoides corti tetrathyridium (TTy) in culture. (A) The AlamarBlue assay and (B) the motility assay by worm tracker device at 6 days of M. corti TTy treatment. TTy were incubated in 200 μL of culture medium with 1, 10 and 20 μM of TSA or the vehicle (DMSO 1%). Three independent biological replicates were used. Error bars represent the SD. Praziquantel (PZQ) and ethanol 70% (EtOH 70%) were used as positive controls. In all panels, asterisks indicate those values showing differences with statistical significance compared to control according to ANOVA tests (***, p < 0.001; ****, p < 0.0001). Inverted optical microscope images of M. corti TTy incubated with 40 μM TSA at (D) 3 and (F) 6 days or M. corti TTy incubated with DMSO 1% at (C) 3 and (D) 6 days. Scale bars represent 50 μm.

Fig. 8

Hiperacetylation of Mesocestoides corti tetrathyridium (TTy) proteins by Trichostatin A (TSA). (A) M. corti TTy were treated with 20 or 40 μM TSA or 1% DMSO (control) for 3 days, followed by preparation of protein lysates and Western Blot analyses using anti acetylated-histone H4 and anti pan-acetylated-protein antibodies. As negative controls, 20 μM praziquantel (PZQ) and albendazole (ABZ) were used. The panel below Western Blot represents the gel stained with Coomassie Blue. (B) Densitometry analyses from western blots were performed and normalized with the total amount of proteins of the same sample observed by Coomassie Blue staining. Results are expressed as the fold change in relative density compared to the control (1% DMSO), set to one. Error bars represent the SD.

Suppl Fig S2.png

Amino acid residues coordinating the catalytic ion zinc in HDAC8 from Homo sapiens, Schistosoma mansoni, Echinococcus canadensis G7 and Mesocestoides corti. Structures of HDAC8 from H. sapiens PDB 1T64 (HsaHDAC8; red), S. mansoni PDB 4BZ5 (SmpHDAC8; purple) and homology models of E. canadensis G7 (EcaHDAC8; blue) and M. corti (McoHDAC8; green) are shown. Surface of HsaHDAC8 is shown in gray. The catalytic ion zinc is shown as a yellow sphere.