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Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target.

ABSTRACT: BACKGROUND:Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown. OBJECTIVE:To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound. METHODS:We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a G. duodenalis aldose reductase (GdAldRed) homolog. RESULTS:KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that GdAldRed is a viable KPF target (?G = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog. CONCLUSIONS:KPF exerted a proapoptotic effect on G. duodenalis trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures. GdAldRed is a likely target underlying its antigiardial activity.

SUBMITTER: Arguello-Garcia R

PROVIDER: S-EPMC7577037 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target.

Argüello-García Raúl R Calzada Fernando F García-Hernández Normand N Chávez-Munguía Bibiana B Velázquez-Domínguez José Antonio JA

Memorias do Instituto Oswaldo Cruz 20201021

<h4>Background</h4>Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown.<h4>Objective</h4>To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound.<h4>Methods</h4>We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron m ...[more]

PMID: 33111756

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Project description:Fresh aqueous extracts (AGEs) and several thioallyl compounds (TACs) from garlic have an important antimicrobial activity that likely involves their interaction with exposed thiol groups at single aminoacids or target proteins. Since these groups are present in Giardia duodenalis trophozoites, in this work we evaluated the anti-giardial activity of AGE and several garlic's TACs. In vitro susceptibility assays showed that AGE affected trophozoite viability initially by a mechanism impairing cell integrity and oxidoreductase activities while diesterase activities were abrogated at higher AGE concentrations. The giardicidal activities of seven TACs were related to the molecular descriptor HOMO (Highest Occupied Molecular Orbital) energy and with their capacity to modify the -SH groups exposed in giardial proteins. Interestingly, the activity of several cysteine proteases in trophozoite lysates was inhibited by representative TACs as well as the cytopathic effect of the virulence factor giardipain-1. Of these, allicin showed the highest anti-giardial activity, the lower HOMO value, the highest thiol-modifying activity and the greatest inhibition of cysteine proteases. Allicin had a cytolytic mechanism in trophozoites with subsequent impairment of diesterase and oxidoreductase activities in a similar way to AGE. In addition, by electron microscopy a marked destruction of plasma membrane and endomembranes was observed in allicin-treated trophozoites while cytoskeletal elements were not affected. In further flow cytometry analyses pro-apoptotic effects of allicin concomitant to partial cell cycle arrest at G2 phase with the absence of oxidative stress were observed. In experimental infections of gerbils, the intragastric administration of AGE or allicin decreased parasite numbers and eliminated trophozoites in experimentally infected animals, respectively. These data suggest a potential use of TACs from garlic against G. duodenalis and in the treatment of giardiasis along with their additional benefits in the host's health.

Project description:Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.

Project description:BACKGROUND:Enterocytozoon bieneusi and Giardia duodenalis are common human and animal pathogens. Studies have increasingly shown that non-human primates (NHPs) are common hosts of these two zoonotic parasites. However, few studies have explored the genetic diversity and public health potential of these pathogens in laboratory monkeys. In this study, we examined the genetic diversity of the two pathogens in crab-eating macaques (Macaca fascicularis) in a commercial facility in Hainan, China. RESULTS:Enterocytozoon bieneusi and G. duodenalis were detected by PCR analysis in 461/1452 (31.7%) and 469/1452 (32.3%) fecal specimens from the animals, respectively. Significantly higher detection rates of E. bieneusi were detected in males (36.5%, 258/706) than in females (26.7%, 160/599; ?2?=?14.391, P?=?0.0001), in animals with loose stools (41.4%, 151/365) than those with normal stool (28.5%, 310/1087; ?2?=?20.83, P?<?0.0001), and in animals of over 3 years of age (38.6%, 135/350) than those of 1-3 years (29.6%, 326/1,102; ?2?=?9.90, P?=?0.0016). For G. duodenalis, the detection rate in males (33.4%, 236/706) was higher than in females but not statistically significant (30.2%, 181/599; ?2?=?1.54, P?=?0.2152), in monkeys with loose stools (41.1%, 150/365) than those with normal stools (29.3%, 319/1087; ?2?=?17.25, P?<?0.0001), and in monkeys of 1-3 years of age (36.6%, 403/1102) than those over 3 years (18.9%, 66/350; ?2?=?38.11, P?<?0.0001). Nine E. bieneusi genotypes were detected in this study by DNA sequence analysis of the internal transcribed spacer of the rRNA gene, namely Type IV (236/461), Peru8 (42/461), Pongo2 (27/461), Peru11 (12/461), D (4/461) and PigEbITS7 (1/461) previously seen in NHPs as well as humans, and CM1 (119/461), CM2 (17/461) and CM3 (3/461) that had been only detected in NHPs. DNA sequence analyses of the tpi, gdh and bg loci identified all G. duodenalis specimens as having assemblage B. Altogether, eight (4 known and 4 new), seven (6 known and 1 new) and seven (4 known and 3 new) subtypes were seen at the tpi, gdh and bg loci, leading to the detection of 53 multi-locus genotypes (MLG-B-hn01 to MLG-B-hn53). Most of them were genetically related to those previously seen in common Old-World monkeys. CONCLUSIONS:Data from this study indicate a common occurrence of zoonotic genotypes of E. bieneusi and assemblage B of G. duodenalis in farmed crab-eating macaques in Hainan, China.

Dataset Information

Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target.

Publications

Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target.

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