Funkční analýza SUF dráhy v buňce Monocercomonoides exilis a Paratrimastix pyriformis

Syntéza železo-sirných center je nepostradatelný buněčný proces, který je závislý na složitých biosyntetických drahách. U modelových eukaryot jsou těmito drahami ISC v mitochondrii a CIA v cytosolu. Nedávná analýza genomu a transkriptomu amitochondriálního prvoka Monocercomonoides exilis prokázala, že tento organismus postrádá ISC dráhu, která byla nahrazena bakteriální SUF dráhou. Blízký volně žijící příbuzný M. exilis, Paratrimastix pyriformis, obsahuje organely příbuzné mitochondrii, přesto zde byla ISC dráha také nahrazena dráhou SUF. Získání SUF dráhy je považováno za podmínku pro následnou ztrátu mitochondrie u M. exilis, který je prvním zdokumentovaným eukaryotickým organismem zcela bez této organely. SUF dráha byla doposud předmětem mnoha studií u bakterií, avšak její role hlavního poskytovatele železo-sirných center pro eukaryotické buňky byla popsána pouze u několika případů, a to především na základě genomických dat. Tato práce se zabývá domnělou ATPázou SufC a domnělým proteinovým lešením SufB. Oba proteiny byly úspěšně izolovány v rekombinantní formě. U SufC byla naměřena ATPázová aktivita, která by zvýšená za přítomnosti SufB. Podmínky pro ATPázovou aktivity SufC byly standardizovány. Rekombinantní forma SufC byla použita k přípravě protilátky, která byla použita k lokalizaci SUF...The synthesis of iron-sulfur clusters is an essential cellular process, which depends on complex biosynthetic pathways. In model eukaryotes, these pathways are the ISC pathway in the mitochondria and the CIA pathway in the cytosol. A recent genome and transcriptome analysis showed, that an amitochondriate protist Monocercomonoides exilis lacks the canonical ISC pathway, which has been replaced by a bacterial SUF pathway. A close free-living relative of M. exilis, Paratrimastix pyriformis possesses a mitochondrion-related organelle, yet also possesses a SUF pathway instead of ISC. The acquisition of the SUF pathway has been suggested as the primordial cause for mitochondrial loss in M. exilis, which is the first documented eukaryotic organism without a mitochondrion. The SUF pathway has been the subject of numerous studies in bacteria, however, its role as the core provider of iron-sulfur clusters for eukaryotic cells has been reported in merely a handful of eukaryotes and was based predominantly on genomic data. This thesis focuses on the putative ATPase SufC and the putative scaffold protein SufB. Both proteins were successfully produced in recombinant forms. SufC has been found to possess ATPase activity in vitro, which was increased upon interaction with SufB. The conditions for theATPase...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

New advances in the in-vitro culture of Dientamoeba fragilis

Dientamoeba fragilis is an intestinal protozoan in humans that is commonly associated with diarrhoea and other gastrointestinal complaints. Studies conducted to investigate the biology of this parasite are limited by methods for in vitro cultivation. The objective of this study was to improve a biphasic culture medium, based on the Loeffler's slope, by further supplementation in order to increase the yield of trophozoites in culture. The current in vitro culture of D. fragilis is a xenic culture with a mix of bacteria. Three different liquid overlays were evaluated including Earle's balanced salt solution (EBSS), PBS and Dulbecco's modified PBS (DPBS), for their ability to support the in vitro growth of D. fragilis trophozoites. Out of these 3 overlays EBSS gave the highest increase in the trophozoite numbers. The effect of supplementation was analysed by supplementing EBSS with ascorbic acid, ferric ammonium citrate, L-cysteine, cholesterol and alpha-lipoic acid and quantification of in vitro growth by cell counts. A new liquid overlay is here described based upon EBSS supplemented with cholesterol and ferric ammonium citrate that, in conjunction with the Loeffler's slope, supports the growth of D. fragilis trophozoites in vitro. This modified overlay supported a 2-fold increase in the numbers of trophozoite in culture from all 4 D. fragilis isolates tested, when compared to a PBS overlay. These advances enable the harvest of a larger number of trophozoites needed for further studies on this parasite. © Cambridge University Press 2012

Analýzy velikosti genomu, ploidie a karyotypu u kmenů Monocercomonoides

Oxymonads are a group of flagellate protists living in low oxygen environments - mainly the guts of insects and vertebrates. In this study, we focus on the analysis of ploidy and karyotype of various species of oxymonads using Fluorescence In Situ Hybridization (FISH) with probes against single copy genes and telomeric repeats as well as estimating the DNA content in the nuclei of these oxymonads using flow cytometry. Using specific FISH probes against SufDSU gene, which is present in a single copy in the haploid genome, we showed that all studied strains are probably haploid. From the genome of Monocercomonoides exilis strain PA203 we know that oxymonads have the ancestral type of telomeric repeat (TTAGGG). Using a probe against these repeats we tried to label chromosome ends and estimate the number of chromosomes for seven strains (five species) of Monocercomonoides. With a single exception, the average number of signals per nucleus was below 20 indicating number of chromosomes below 10. In the strains of M. mercovicensis, we observed much higher number of signals suggesting that the cells have much higher number of chromosomes. Finally, we established the DNA content for several strains using flow cytometry. We used as a standard M. exilis strain PA203 knowing that the haploid genome size is...Oxymonády jsou skupinou bičíkatých prvoků, žijících v prostředí s nízkou koncentrací kyslíku. Obývají především střeva hmyzu a obratlovců. V této studii se zaměřujeme na analýzu ploidie a karyotypu různých druhů oxymonád pomocí metody fluorescence in situ hybridizace (FISH) s použitím prób proti jednokopiovým genům a telomerickým repeticím. Také jsme se pokusili odhadnout velikost genomu těchto druhů oxymonád pomocí průtokové cytometrie. S použitím specifických FISH prób proti SufDSU genu, který je pravděpodobně přítomenv jedné kopii v genomu, ukázali, že všechny studované kmeny jsou haploidní. Z genomu Monocercomonoides exilis víme, že oxymonády mají původní typ telomerické repetice (TTAGGG). Použitím próby proti těmto telomerickým repeticím jsme se pokusili odhadnout počet chromozomů u sedmi kmenů (pěti druhů) Monocercomonoides. Kromě jedné vyjímky byl průměrný počet signálů pod 20, což naznačuje počet chromozomů v řádu jednotek. V kmenech M. mercovicensis jsme ovšem zaznamenali mnohem vyšší počet signálů naznačujících, že buňky mají mnohem vyšší počty chromozomů. Nakonec jsme stanovili obsah DNA v jádreh těchto kmenů pomocí průtokové cytometrie se standardem M. exilis PA203, jehož velikost genomu je známa (82Mbp). Výsledky ukazují, že většina kmenů má menší velikost genomu podobnou nebo menší,...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Blastocystis: Isolation, Xenic Cultivation, and Cryopreservation.

Blastocystis is an intestinal parasite that is very easily isolated in culture from fresh stool samples. In fact, the parasite grows so readily in culture that short-term in vitro culture is sometimes used as a diagnostic tool in the absence of DNA-based methods. While axenizing Blastocystis cultures remains a significant challenge, the parasite can be propagated for several months in the presence of metabolically active bacteria (xenic culture). Hence, culture can be used for maintaining live Blastocystis strain libraries. This enables the production of a stable resource of reference material, which for instance can be used for DNA-based assays and research. Blastocystis isolates can also be cryopreserved with a view to reestablishing them in culture. Here, we provide protocols for xenic in vitro culture and cryopreservation of Blastocystis. © 2016 by John Wiley & Sons, Inc

Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase

The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Although analysis of residues inside the nucleotide-binding site suggests that Blastocystis SCS is GTP-specific, we demonstrate that it is ATP-specific. Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis SCS active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity. It has been proposed that residues in direct contact with the substrate determine nucleotide specificity in SCS. However, our results indicate that, in Blastocystis, an electrostatic gatekeeper controls which ligands can enter the binding site

Newly defined conditions for the in vitro cultivation and cryopreservation of Dientamoeba fragilis: New techniques set to fast track molecular studies on this organism

Dientamoeba fragilis is a pathogen of the human gastrointestinal tract that is a common cause of diarrhoea. A paucity of knowledge on the in vitro cultivation and cryopreservation of Dientamoeba has meant that few studies have been conducted to investigate its biology. The objective of this study was to define, for the first time, in vitro culture conditions able to support the long-term in vitro growth of Dientamoeba. Also, we aimed to define a suitable method for cryopreserving viable Dientamoeba trophozoites. A modified BD medium, TYGM-9, Loeffler's slope medium, Robinson's medium, Medium 199, Trichosel and a Tritrichomonas fetus medium were compared, using cell counts, for their ability to support the growth of D. fragilis at various temperatures and atmospheric conditions. Loeffler's slope medium supported significantly better growth compared to other media. A temperature of 42C and a microaerophilic atmosphere were also optimum for Dientamoeba growth. To our knowledge, this is the first study to describe and compare different culture media and conditions for the growth of clinical isolates of D. fragilis. This new technology will aid the development of diagnostics for dientamoebiasis as well as facilitate large-scale sequencing projects that will fast track molecular studies on D. fragilis. © Cambridge University Press 2010

Phylogenetic analyses of Chilomastix and Retortamonas species using in vitro excysted flagellates

Abstract In vitro excystation of cysts of microscopically identified Chilomastix mesnili and Retortamonas sp. isolated from Japanese macaques and Retortamonas sp. isolated from small Indian mongooses could be induced using an established protocol for Giardia intestinalis and subsequently by culturing with H2S-rich Robinson’s medium supplemented with Desulfovibrio desulfuricans. Excystation usually began 2 h after incubation in Robinson’s medium. DNA was isolated from excysted flagellates after 4 h of incubation or from cultured excysted flagellates. Phylogenetic analysis based on their 18S rRNA genes revealed that two isolates of C. mesnili from Japanese macaques belonged to the same cluster as a C. mesnili isolate from humans, whereas a mammalian Retortamonas sp. isolate from a small Indian mongoose belonged to the same cluster as that of an amphibian Retortamonas spp. isolate from a ‘poison arrow frog’ [sequence identity to AF439347 (94.9%)]. These results suggest that the sequence homology of the 18S rRNA gene of the two C. mesnili isolates from Japanese macaques was similar to that of humans, in addition to the morphological similarity, and Retortamonas sp. infection of the amphibian type in the small Indian mongoose highlighted the possibility of the effect of host feeding habitats

Current status of Blastocystis: A personal view.

Despite Blastocystis being one of the most widespread and prevalent intestinal eukaryotes, its role in health and disease remains elusive. DNA-based detection methods have led to a recognition that the organism is much more common than previously thought, at least in some geographic regions and some groups of individuals. Molecular methods have also enabled us to start categorizing the vast genetic heterogeneity that exists among Blastocystis isolates, wherein the key to potential differences in the clinical outcome of Blastocystis carriage may lie. In this review we summarize some of the recent developments and advances in Blastocystis research, including updates on diagnostic methods, molecular epidemiology, genetic diversity, host specificity, clinical significance, taxonomy, and genomics. As we are now in the microbiome era, we also review some of the steps taken towards understanding the place of Blastocystis in the intestinal microbiota

Comparison of the efficiencies of customized CSA-and rlectin-elisas for detection of anti-amoebic antibody based on selected aborigines serum samples

Orang Asli, the Malaysian aborigines live in remote jungles, are endemic for amoebiasis. Understanding the disease prevalence and distribution is important for the control and preventive measures. A major indicator that reflects on the disease incidence is the presence of highly elevated anti-amoebic antibody. However, continuous screening of the antibody using commercial kits is impractical due to the high cost. Cheaper but valid in-house customized assay should be employed instead to attain cost-effective and sustainable outcome. Hence, the presence study aimed to compare the efficiencies of customized crude soluble antigen (CSA)-ELISA and recombinant lectin (rLectin)- ELISA for detection of anti-amoebic antibody in selected Orang Asli serum samples, which were prior defined by commercial indirect haemagglutination assay (IHA). CSA was produced from axenically grown Entamoeba histolytica trophozoites, while rLectin was overexpressed and purified from Escherichia coli harbouring the corresponding recombinant gene. For the development of the two customized ELISAs, antigen concentration, serum and conjugated antibody dilutions were optimized. The efficiencies of both customised ELISAs were determined by 30 positive and negative serum samples pre-determined by commercial IHA. According to receiver operating characteristics (ROC) curve analysis, both customized ELISAs showed excellent diagnostic performances. As compared to the reference assay, the positive percentage agreement of CSA-ELISA (91%) was higher than that of rLectin-ELISA (61%), whileboth ELISAs showed the same negative percentage agreement, which was 97%. According to analysis of ELISA ODs, the positive delta value for CSA-ELISA (0.4472) was higher than that of rLectin-ELISA (0.2673), while the negative delta value of rLectin-ELISA (-1.209) was lower than that of CSA-ELISA (-1.175). In conclusion, this study demonstrated that CSA-ELISA was more sensitive in detecting the presence of anti-amoebic antibody, while rLectin-ELISA was more specific in ruling out negative cases