Search

Ascofuranone (AF), a meroterpenoid isolated from various filamentous fungi, including Acremonium egyptiacum, has been reported as a potential lead candidate for drug development against parasites and cancer. In this study, we demonstrated that AF and its derivatives are potent anthelminthic agents, particularly against Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis. We measured the inhibitory activities of AF and its derivatives on the mitochondrial aerobic and anaerobic respiratory systems of E. multilocularis larvae. Several derivatives inhibited complex II (succinate:quinone reductase [SQR]; IC50 = 0.037 to 0.135 mM) and also complex I to III (NADH:cytochrome c reductase; IC50 = 0.008 to 0.401 mM), but not complex I (NADH:quinone reductase), indicating that mitochondrial complexes II and III are the targets. In particular, complex II inhibition in the anaerobic pathway was notable because E. multilocularis employs NADH:fumarate reductase (fumarate respiration), in addition to NADH oxidase (oxygen respiration), resulting in complete shutdown of ATP synthesis by oxidative phosphorylation. A structure-activity relationship study of E. multilocularis complex II revealed that the functional groups of AF are essential for inhibition. Binding mode prediction of AF derivatives to complex II indicated potential hydrophobic and hydrogen bond interactions between AF derivatives and amino acid residues within the quinone binding site. Ex vivo culture assays revealed that AF derivatives progressively reduced the viability of protoscoleces under both aerobic and anaerobic conditions. These findings confirm that AF and its derivatives are the first dual inhibitors of fumarate and oxygen respiration in E. multilocularis and are potential lead compounds in the development of anti-echinococcal drugs., Antimicrobial Agents and Chemotherapy, art. no. e01428-22; 2023

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc1 complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC50). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug., Parasitology International, 75, art.no.102004; 2020

The resistance/susceptibility to Echinococcus multilocularis infection in mice is genetically controlled. However, genetic factors responsible for these differences remain unknown. Our previous study in genetic linkage analysis has revealed that there is a significant quantitative trait locus (QTL) for the establishment of cyst (Emcysl), and a highly significant QTL for the development of protoscolex of E. multilocularis larvae (Empscl), on mouse chromosomes 6 and 1, respectively. The current study aimed to confirm these QTLs and narrow down the critical genetic region that controls resistance/susceptibility to E. multilocularis infection by establishing congenic and subcongenic lines from C57BL/6 (B6) and DBA/2 (D2) mice. For protoscolex development phenotype, two congenic lines, B6.D2-Empscl and D2.B6-Empscl were developed, where responsible QTL, Empsc1 was introgressed from D2 into B6 background and vice versa. For cyst establishment phenotype, two congenic lines, B6.D2-Emcys1 and D2.B6-Emcys1 were developed, where responsible QTL, Emcys1 was introgressed from D2 into B6 background and vice versa. Because there was no significant difference in cyst establishment between B6.D2Emcysl and D2.B6-Emcys1 mice after challenge with E. multilocularis, it is suggested that the Emcysl does not solely control the cyst establishment in mouse liver. However, infection experiments with B6.D2-Empscl and D2.B6-Empsc1 mice showed a significant difference in protoscolex development in the cyst. It confirms that the Empsc1 controls phenotype of the protoscolex development in the cyst. Subsequently, two subcongenic lines, B6.D2-Empsc1.1 and B6.D2-Empsc1.2 from B6.D2-Emcysl and one subcongenic line, D2.B6-Empsc1.1 from D2.B6-Empscl were developed to narrow down the critical region responsible for protoscolex development. From the results of infection experiments with E. multilocularis in these subcongenic mice, it is concluded that a gene responsible for protoscolex development is loc

Background: We have previously evaluated the vaccine efficacies of seven tetraspanins of Echinococcus multilocularis (Em-TSP1-7) against alveolar echinococcosis (AE) by subcutaneous (s.c.) administration with Freund's adjuvant. Over 85% of liver cyst lesion number reductions (CLNR) were achieved by recombinant Em-TSP1 (rEm-TSP1) and -TSP3 (rEm-TSP3). However, to develop an efficient and safe human vaccine, the efficacy of TSP mucosal vaccines must be thoroughly evaluated. Methodology/Principal Findings: rEm-TSP1 and -TSP3 along with nontoxic CpG ODN (CpG oligodeoxynucleotides) adjuvant were intranasally (i.n.) immunized to BALB/c mice and their vaccine efficacies were evaluated by counting liver CLNR (experiment I). 37.1% (p<0.05) and 62.1% (p<0.001) of CLNR were achieved by these two proteins, respectively. To study the protection-associated immune responses induced by rEm-TSP3 via different immunization routes (i.n. administration with CpG or s. c. immunization with Freund's adjuvant), the systemic and mucosal antibody responses were detected by ELISA (experiment II). S. c. and i.n. administration of rEm-TSP3 achieved 81.9% (p<0.001) and 62.8% (p<0.01) CLNR in the liver, respectively. Both the immunization routes evoked strong serum IgG, IgG1 and IgG2a responses; i.n. immunization induced significantly higher IgA responses in nasal cavity and intestine compared with s. c. immunization (p<0.001). Both immunization routes induced extremely strong liver IgA antibody responses (p<0.001). The Th1 and Th2 cell responses were assessed by examining the IgG1/IgG2 alpha ratio at two and three weeks post-immunization. S. c. immunization resulted in a reduction in the IgG1/IgG2 alpha ratio (Th1 tendency), whereas i.n. immunization caused a shift from Th1 to Th2. Moreover, immunohistochemistry showed that Em-TSP1 and -TSP3 were extensively located on the surface of E. multilocularis cysts, protoscoleces and adult worms with additional expression of Em-TSP3 in

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6 × D2)F1 and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host–parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection.