Publications by authors named "David Leitsch"

43 Publications

A reassessment of the role of oxygen scavenging enzymes in the emergence of metronidazole resistance in trichomonads.

Int J Parasitol Drugs Drug Resist 2021 Apr 30;16:38-44. Epub 2021 Apr 30.

Institute for Specific Prophylaxis and Tropical Medicine Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Kinderspitalgasse 15, A-1090 Vienna, Austria. Electronic address:

Trichomonads are an order of parasitic protists which infect a wide range of hosts. The human parasite Trichomonas vaginalis and the bovine parasite Tritrichomonas foetus which also infects cats and swine are of considerable medical and veterinary importance, respectively. Since trichomonads are microaerophiles/anaerobes they are susceptible to 5-nitroimidazoles such as metronidazole. 5-nitroimidazoles are exclusively toxic to microaerophilic/anaerobic organisms because reduction, i.e. activation, of the drug can only occur in a highly reductive environment. 5-nitroimidazoles have remained a reliable treatment option throughout the last decades but drug resistance can be a problem. Clinical resistance to 5-nitroimidazoles has been studied in more detail in T. vaginalis and has been ascribed to defective oxygen scavenging mechanisms which lead to higher intracellular oxygen concentrations and, consequently, to less drug being reduced. Two enzymes, flavin reductase (FR) and NADH oxidase have been suggested to be the major oxygen scavenging enzymes in T. vaginalis. The loss, or at least an impairment of FR which reduces oxygen to hydrogen peroxide, has been proposed as the central mechanism that enables the emergence of 5-nitroimidazole resistance. In this study we explored if T. foetus also encodes a homolog of FR and if it is, likewise, involved in resistance. T. foetus was indeed found to express a FR but it was only weakly active as compared to the T. vaginalis homolog. Further, activity of FR in T. foetus was unchanged in metronidazole-resistant cell lines, ruling out that it has a role in metronidazole resistance. Finally, we measured oxygen scavenging rates in metronidazole-sensitive and -resistant cell lines and found that NADH oxidase and FR are not the major oxygen scavenging enzymes in trichomonads and that oxygen scavenging is possibly a consequence, rather than a cause of metronidazole resistance.
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http://dx.doi.org/10.1016/j.ijpddr.2021.04.004DOI Listing
April 2021

Recent advances in the molecular biology of the protist parasite .

Authors:
David Leitsch

Fac Rev 2021 4;10:26. Epub 2021 Mar 4.

Department of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria.

is an anaerobic/microaerophilic protist parasite which causes trichomoniasis, one of the most prevalent sexually transmitted diseases worldwide. . not only is important as a human pathogen but also is of great biological interest because of its peculiar cell biology and metabolism, in earlier times fostering the erroneous notion that this microorganism is at the root of eukaryotic evolution. This review summarizes the major advances in the last five years in the . field with regard to genetics, molecular biology, ecology, and pathogenicity of the parasite.
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http://dx.doi.org/10.12703/r/10-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946386PMC
March 2021

Molecular characterization of metronidazole resistant Bacteroides strains from Kuwait.

Anaerobe 2021 Mar 11;69:102357. Epub 2021 Mar 11.

Institute of Clinical Microbiology, Faculty of Medicine, University of Szeged, Szeged, Hungary. Electronic address:

Eleven metronidazole resistant Bacteroides and one newly classified Phocaeicola dorei strain from Kuwait were investigated for their resistance mechanisms and the emergence of their resistant plasmids. All but one strain harbored nimE genes on differently sized plasmids. Of the 11 nimE genes, 9 were preceded by full copies of the prototype ISBf6 insertion sequence element, one carried a truncated ISBf6 and one was activated by an additional copy of IS612B. Nucleotide sequencing results showed that the nimE ISBf6 distances were constant and all five different plasmids shared a common region, suggesting that (i) the nimE-ISBf6 configuration was inserted into an undisclosed common genetic element, (ii) over time, this common element was mutated by insertions and deletions, spreading the resultant plasmids. Of the 10 B. fragilis strains in this collection, 6 were also cfiA-positive, one with full imipenem resistance, indicating a tendency for multidrug resistance (MDR) among such isolates. The significant number of metronidazole resistant Bacteroides spp. and P. dorei strains with the MDR phenotype warns of difficulties in treatment and suggests promoting adherence to antibiotic stewardship recommendations in Kuwait.
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http://dx.doi.org/10.1016/j.anaerobe.2021.102357DOI Listing
March 2021

A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation.

Malar J 2021 Feb 28;20(1):121. Epub 2021 Feb 28.

Malaria Research Initiative Bandarban, Vienna, Austria.

Background: The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease.

Methods: A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum.

Results: Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1-2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe.

Conclusions: Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.
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http://dx.doi.org/10.1186/s12936-021-03662-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916309PMC
February 2021

An unusual thioredoxin system in the facultative parasite Acanthamoeba castellanii.

Cell Mol Life Sci 2021 Apr 18;78(7):3673-3689. Epub 2021 Feb 18.

Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Kinderspitalgasse 15, 1090, Vienna, Austria.

The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii's response to oxidative stress. The role of the large TrxR, however, remains elusive.
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http://dx.doi.org/10.1007/s00018-021-03786-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038987PMC
April 2021

Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp.

Sci Rep 2020 06 25;10(1):10362. Epub 2020 Jun 25.

Institute of Specific Prophylaxis und Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Many open questions can be addressed by gene expression studies, however, for Acanthamoeba reliable standards have not yet been established. In this study, suitable reference genes (RGs) for RT-qPCR in Acanthamoeba were comprehensively evaluated, comparing different Acanthamoeba strains and employing four different algorithms (NormFinder, GeNorm, BestKeeper and RefFinder). Expression stability was assessed under various conditions and the potentials of the most promising RGs for accurate normalization of target genes were evaluated. Expression stability of RGs varied depending on conditions and employed algorithms, however, the genes for the 18S rRNA and the hypoxanthine phosphoribosyl transferase seem to be widely suitable RGs. Normalization with a combination of two carefully chosen RGs resulted in reliable expression data for target genes, while normalization with unsuitable RGs led to significant misinterpretation of expression profiles. Thus, a careful evaluation of RGs prior to expression studies is essential.
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http://dx.doi.org/10.1038/s41598-020-67035-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316857PMC
June 2020

A novel 5-Plex qPCR-HRM assay detecting human diarrheal parasites.

Gut Pathog 2020 29;12:27. Epub 2020 May 29.

Institute for Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria.

Background: Intestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges. spp., (and, arguably, and spp.) are among the most important and common parasitic protozoans causing diarrhea. Several multiplex real-time PCR assays have been developed for the synchronous detection of these parasites. However, most assays include the use of hydrolysis probes, increasing the cost of stool examination. In this study, we designed and evaluated a real-time PCR protocol, based on high-resolution melting (HRM) curve analysis, to simultaneously detect and differentiate five gastrointestinal parasites.

Results: Using a blinded panel of 143 clinical samples with laboratory diagnostic data to evaluate the method, we obtained a 95.8% concordance with conventional methods. Moreover, 4.2% of the samples were positive for and 2.8% additional infections were found with our multiplex assay. Our method is sensitive and specific for the selected parasites with the additional possibility of being run in single-plex as a backup control for mixed infections.

Conclusions: The assay is a convenient and cost-effective method that could contribute to a quicker and accurate diagnosis as well as to more targeted therapies of parasite-derived diarrhea. Finally, this new multiplex PCR assay could also be instrumental in epidemiology studies on these parasites.
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http://dx.doi.org/10.1186/s13099-020-00365-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7257150PMC
May 2020

Drug resistance in Giardia: Mechanisms and alternative treatments for Giardiasis.

Adv Parasitol 2020 17;107:201-282. Epub 2020 Jan 17.

Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México, Mexico. Electronic address:

The use of chemotherapeutic drugs is the main resource against clinical giardiasis due to the lack of approved vaccines. Resistance of G. duodenalis to the most used drugs to treat giardiasis, metronidazole and albendazole, is a clinical issue of growing concern and yet unknown impact, respectively. In the search of new drugs, the completion of the Giardia genome project and the use of biochemical, molecular and bioinformatics tools allowed the identification of ligands/inhibitors for about one tenth of ≈150 potential drug targets in this parasite. Further, the synthesis of second generation nitroimidazoles and benzimidazoles along with high-throughput technologies have allowed not only to define overall mechanisms of resistance to metronidazole but to screen libraries of repurposed drugs and new pharmacophores, thereby increasing the known arsenal of anti-giardial compounds to some hundreds, with most demonstrating activity against metronidazole or albendazole-resistant Giardia. In particular, cysteine-modifying agents which include omeprazole, disulfiram, allicin and auranofin outstand due to their pleiotropic activity based on the extensive repertoire of thiol-containing proteins and the microaerophilic metabolism of this parasite. Other promising agents derived from higher organisms including phytochemicals, lactoferrin and propolis as well as probiotic bacteria/fungi have also demonstrated significant potential for therapeutic and prophylactic purposes in giardiasis. In this context the present chapter offers a comprehensive review of the current knowledge, including commonly prescribed drugs, causes of therapeutic failures, drug resistance mechanisms, strategies for the discovery of new agents and alternative drug therapies.
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http://dx.doi.org/10.1016/bs.apar.2019.11.003DOI Listing
July 2020

Metabolomic Profiling of Wildtype and Transgenic Strains by H HR-MAS NMR Spectroscopy.

Metabolites 2020 Jan 30;10(2). Epub 2020 Jan 30.

Institute of Parasitology, Vetsuisse Faculty, University of Bern, Länggass-Strasse 122, CH-3012 Bern, Switzerland.

, a causative agent of persistent diarrhea in humans, domestic animals, and cattle, is usually treated with nitro compounds. Consequently, enzymes involved in anaerobic nitro reduction have been investigated in detail as potential targets. Their role within the normal metabolic context is, however, not understood. Using H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, we analyzed the metabolomes of trophozoites overexpressing three nitroreductases (NR1-NR3) and thioredoxin reductase (TrxR), most likely a scavenger of reactive oxygen species, as suggested by the results published in this study. We compared the patterns to convenient controls and to the situation in the nitro drug resistant strain C4 where NR1 is downregulated. We identified 27 metabolites in trophozoites. Excluding metabolites of high variability among different wildtype populations, only trophozoites overexpressing NR1 presented a distinct pattern of nine metabolites, in particular arginine catabolites, differing from the respective controls. This pattern matched a differential pattern between wildtype and strain C4. This suggests that NR1 interferes with arginine and thus energy metabolism. The exact metabolic function of NR1 (and the other nitroreductases) remains to be elucidated.
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http://dx.doi.org/10.3390/metabo10020053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073884PMC
January 2020

Identification of the NADH-oxidase gene in Trichomonas vaginalis.

Parasitol Res 2020 Feb 18;119(2):683-686. Epub 2019 Dec 18.

Institute for Specific Prophylaxis and Tropical Medicine Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Kinderspitalgasse 15, A-1090, Vienna, Austria.

The microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide. Due to its anaerobic metabolism, it has to quickly remove intracellular oxygen in order to avoid deactivation of essential metabolic enzymes such as oxygen-sensitive pyruvate:ferredoxin oxidoreductase (PFOR). Two major enzyme activities which are responsible for the removal, i.e. reduction, of molecular oxygen have been identified in T. vaginalis flavin reductase, formerly designated NADPH oxidase, which indirectly reduces oxygen to hydrogen peroxide via flavin mononucleotide (FMN), and NADH oxidase which reduces oxygen to water. Flavin reductase has been identified and characterized at the gene level as well as enzymatically, but NADH oxidase has so far only been characterized enzymatically with enzyme isolated from T. vaginalis cell extracts. In this study, we identified NADH oxidase by mass spectrometry after isolation of the enzyme from gel bands positively staining for NADH oxidase activity. In strain C1 (ATCC 30001) which is known to lack NADH oxidase activity completely, the NADH oxidase gene has a deletion at position 1540 of the open reading frame leading to a frame shift and, as a consequence, to premature termination of the encoded polypeptide.
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http://dx.doi.org/10.1007/s00436-019-06572-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985181PMC
February 2020

Activity of methylgerambullin from Glycosmis species (Rutaceae) against Entamoeba histolytica and Giardia duodenalis in vitro.

Int J Parasitol Drugs Drug Resist 2019 08 10;10:109-117. Epub 2019 Aug 10.

Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, A-1090, Vienna, Austria. Electronic address:

Entamoeba histolytica and Giardia duodenalis are widespread intestinal protozoan parasites which both spread via cysts that have to be ingested to infect a new host. Their environment, the small intestine for G. duodenalis and the colon for E. histolytica, contains only very limited amounts of oxygen, so both parasites generate energy by fermentation and substrate level phosphorylation rather than by oxidative phosphorylation. They both contain reducing agents able to reduce and activate nitroimidazole drugs such as metronidazole which is the gold standard drug to treat Entamoeba or Giardia infections. Although metronidazole works well in the majority of cases, it has a number of drawbacks. In animal models, the drug has carcinogenic activity, and concerns about a possible teratogenic activity remain. In addition, the treatment of G. duodenalis infections is hampered by emerging metronidazole resistance. Plant-derived drugs play a dominant role in human medicine, therefore we tested the activity of 14 isolated plant compounds belonging to seven different classes in vitro against both parasites. The tests were performed in a new setting in microtiter plates under anaerobic conditions. The compound with the highest activity was methylgerambullin, a sulphur-containing amide found in Glycosmis species of the family Rutaceae with an EC of 14.5 μM (6.08 μg/ml) after 24 h treatment for E. histolytica and 14.6 μM (6.14 μg/ml) for G. duodenalis. The compound was successfully synthesised in the laboratory which opens the door for the generation of new derivatives with higher activity.
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http://dx.doi.org/10.1016/j.ijpddr.2019.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722286PMC
August 2019

Redox Pathways as Drug Targets in Microaerophilic Parasites.

Trends Parasitol 2018 07 26;34(7):576-589. Epub 2018 May 26.

Department of Parasitology, Charles University, Faculty of Science, Prague, Czech Republic.

The microaerophilic parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia lamblia jointly cause hundreds of millions of infections in humans every year. Other microaerophilic parasites such as Tritrichomonas foetus and Spironucleus spp. pose a relevant health problem in veterinary medicine. Unfortunately, vaccines against these pathogens are unavailable, but their microaerophilic lifestyle opens opportunities for specifically developed chemotherapeutics. In particular, their high sensitivity towards oxygen can be exploited by targeting redox enzymes. This review focusses on the redox pathways of microaerophilic parasites and on drugs, either already in use or currently in the state of development, which target these pathways.
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http://dx.doi.org/10.1016/j.pt.2018.04.007DOI Listing
July 2018

A review on metronidazole: an old warhorse in antimicrobial chemotherapy.

Authors:
David Leitsch

Parasitology 2019 08 23;146(9):1167-1178. Epub 2017 Nov 23.

Institute of Specific Prophylaxis and Tropical Medicine,Medical University of Vienna,Kinderspitalgasse 15,Vienna A-1090,Austria.

The 5-nitroimidazole drug metronidazole has remained the drug of choice in the treatment of anaerobic infections, parasitic as well as bacterial, ever since its development in 1959. In contrast to most other antimicrobials, it has a pleiotropic mode of action and reacts with a large number of molecules. Importantly, metronidazole, which is strictly speaking a prodrug, needs to be reduced at its nitro group in order to become toxic. Reduction of metronidazole, however, only takes place under very low concentrations of oxygen, explaining why metronidazole is exclusively toxic to microaerophilic and anaerobic microorganisms. In general, resistance rates amongst the pathogens treated with metronidazole have remained low until the present day. Nevertheless, metronidazole resistance does occur, and for the treatment of some pathogens, especially Helicobacter pylori, metronidazole has become almost useless in some parts of the world. This review will give an account on the current status of research on metronidazole's mode of action, metronidazole resistance in eukaryotes and prokaryotes, and on other 5-nitroimidazoles in use.
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http://dx.doi.org/10.1017/S0031182017002025DOI Listing
August 2019

Drug susceptibility testing in microaerophilic parasites: Cysteine strongly affects the effectivities of metronidazole and auranofin, a novel and promising antimicrobial.

Authors:
David Leitsch

Int J Parasitol Drugs Drug Resist 2017 12 5;7(3):321-327. Epub 2017 Sep 5.

Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Electronic address:

The microaerophilic parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia lamblia annually cause hundreds of millions of human infections which are treated with antiparasitic drugs. Metronidazole is the most often prescribed drug but also other drugs are in use, and novel drugs with improved characteristics are constantly being developed. One of these novel drugs is auranofin, originally an antirheumatic which has been relabelled for the treatment of parasitic infections. Drug effectivity is arguably the most important criterion for its applicability and is commonly assessed in susceptibility assays using in vitro cultures of a given pathogen. However, drug susceptibility assays can be strongly affected by certain compounds in the growth media. In the case of microaerophilic parasites, cysteine which is added in large amounts as an antioxidant is an obvious candidate because it is highly reactive and known to modulate the toxicity of metronidazole in several microaerophilic parasites. In this study, it was attempted to reduce cysteine concentrations as far as possible without affecting parasite viability by performing drug susceptibility assays under strictly anaerobic conditions in an anaerobic cabinet. Indeed, T. vaginalis and E. histolytica could be grown without any cysteine added and the cysteine concentration necessary to maintain G. lamblia could be reduced to 20%. Susceptibilities to metronidazole were found to be clearly reduced in the presence of cysteine. With auranofin the protective effect of cysteine was extreme, providing protection to concentrations up to 100-fold higher as observed in the absence of cysteine. With three other drugs tested, albendazole, furazolidone and nitazoxanide, all in use against G. lamblia, the effect of cysteine was less pronounced. Oxygen was found to have a less marked impact on metronidazole and auranofin than cysteine but bovine bile which is standardly used in growth media for G. lamblia, displayed a marked synergistic effect with metronidazole.
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http://dx.doi.org/10.1016/j.ijpddr.2017.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595233PMC
December 2017

Evaluation of Giardia lamblia thioredoxin reductase as drug activating enzyme and as drug target.

Int J Parasitol Drugs Drug Resist 2016 12 22;6(3):148-153. Epub 2016 Jul 22.

Institute of Parasitology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, CH-3012, Bern, Switzerland.

The antioxidative enzyme thioredoxin reductase (TrxR) has been suggested to be a drug target in several pathogens, including the protist parasite Giardia lamblia. TrxR is also believed to catalyse the reduction of nitro drugs, e.g. metronidazole and furazolidone, a reaction required to render these compounds toxic to G. lamblia and other microaerophiles/anaerobes. It was the objective of this study to assess the potential of TrxR as a drug target in G. lamblia and to find direct evidence for the role of this enzyme in the activation of metronidazole and other nitro drugs. TrxR was overexpressed approximately 10-fold in G. lamblia WB C6 cells by placing the trxR gene behind the arginine deiminase (ADI) promoter on a plasmid. Likewise, a mutant TrxR with a defective disulphide reductase catalytic site was strongly expressed in another G. lamblia WB C6 cell line. Susceptibilities to five antigiardial drugs, i.e. metronidazole, furazolidone, nitazoxanide, albendazole and auranofin were determined in both transfectant cell lines and compared to wildtype. Further, the impact of all five drugs on TrxR activity in vivo was measured. Overexpression of TrxR rendered G. lamblia WB C6 more susceptible to metronidazole and furazolidone but not to nitazoxanide, albendazole, and auranofin. Of all five drugs tested, only auranofin had an appreciably negative effect on TrxR activity in vivo, albeit to a much smaller extent than expected. Overexpression of TrxR and mutant TrxR had hardly any impact on growth of G. lamblia WB C6, although the enzyme also exerts a strong NADPH oxidase activity which is a source of oxidative stress. Our results constitute first direct evidence for the notion that TrxR is an activator of metronidazole and furazolidone but rather question that it is a relevant drug target of presently used antigiardial drugs.
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http://dx.doi.org/10.1016/j.ijpddr.2016.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971154PMC
December 2016

Editorial.

Authors:
David Leitsch

Mol Biochem Parasitol 2016 Mar-Apr;206(1-2)

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http://dx.doi.org/10.1016/j.molbiopara.2016.05.001DOI Listing
January 2018

Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds.

Int J Parasitol Drugs Drug Resist 2015 Aug 25;5(2):37-43. Epub 2015 Mar 25.

Institute of Parasitology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland.

Giardia lamblia is a protozoan parasite that causes giardiasis, a diarrhoeal disease affecting humans and various animal species. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for treatment of giardiasis. Nitroreductases such as GlNR1 and GlNR2 may play a role in activation or inactivation of these drugs. The aim of this work is to characterise these two enyzmes using functional assays. For respective analyses recombinant analogues from GlNR1 and GlNR2 were produced in Escherichia coli. E. coli expressing GlNR1 and GlNR2 alone or together were grown in the presence of nitro compounds. Furthermore, pull-down assays were performed using HA-tagged GlNR1 and GlNR2 as baits. As expected, E. coli expressing GlNR1 were more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions whereas E. coli expressing GlNR2 were susceptible to neither drug. Interestingly, expression of both nitroreductases gave the same results as expression of GlNR2 alone. In functional assays, both nitroreductases had their strongest activities on the quinone menadione (vitamin K3) and FAD, but reduction of nitro compounds including the nitro drugs metronidazole and nitazoxanide was clearly detected. Full reduction of 7-nitrocoumarin to 7-aminocoumarin was preferentially achieved with GlNR2. Pull-down assays revealed that GlNR1 and GlNR2 interacted in vivo forming a multienzyme complex. These findings suggest that both nitroreductases are multifunctional. Their main biological role may reside in the reduction of vitamin K analogues and FAD. Activation by GlNR1 or inactivation by GlNR2 of nitro drugs may be the consequence of a secondary enzymatic activity either yielding (GlNR1) or eliminating (GlNR2) toxic intermediates after reduction of these compounds.
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http://dx.doi.org/10.1016/j.ijpddr.2015.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813764PMC
August 2015

Recent Advances in the Trichomonas vaginalis Field.

Authors:
David Leitsch

F1000Res 2016 11;5. Epub 2016 Feb 11.

Institute of Parasitology, Vetsuisse Faculty of the University of Bern, University of Bern, Längassstrasse, Bern, 3012, Switzerland.

The microaerophilic protist parasite Trichomonas vaginalis is occurring globally and causes infections in the urogenital tract in humans, a condition termed trichomoniasis. In fact, trichomoniasis is the most prevalent non-viral sexually transmitted disease with more than 250 million people infected every year. Although trichomoniasis is not life threatening in itself, it can be debilitating and increases the risk of adverse pregnancy outcomes, HIV infection, and, possibly, neoplasias in the prostate and the cervix. Apart from its role as a pathogen, T. vaginalis is also a fascinating organism with a surprisingly large genome for a parasite, i. e. larger than 160 Mb, and a physiology adapted to its microaerophilic lifestyle. In particular, the hydrogenosome, a mitochondria-derived organelle that produces hydrogen, has attracted much interest in the last few decades and rendered T. vaginalis a model organism for eukaryotic evolution. This review will give a succinct overview of the major advances in the T. vaginalis field in the last few years.
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http://dx.doi.org/10.12688/f1000research.7594.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755396PMC
February 2016

Drug Resistance in the Microaerophilic Parasite .

Authors:
David Leitsch

Curr Trop Med Rep 2015;2(3):128-135

Institute of Parasitology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland.

The microaerophilic parasite is a causative agent of dysentery affecting hundreds of millions of people around the globe every year. The symptoms of the disease, commonly referred to as giardiasis, are diarrhea, nausea, and malabsorption. Treatment of giardiasis is exclusively based on chemotherapy with antigiardial drugs, including metronidazole, albendazole, and nitazoxanide. In this review, all drugs currently used in the treatment of infections are discussed with a special emphasis on treatment failure and drug resistance.
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http://dx.doi.org/10.1007/s40475-015-0051-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523694PMC
January 2015

Pentamycin shows high efficacy against Trichomonas vaginalis.

Int J Antimicrob Agents 2015 Apr 2;45(4):434-7. Epub 2015 Feb 2.

Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria. Electronic address:

Trichomonas vaginalis is the causative agent of the venereal disease trichomoniasis, which is the most frequent non-viral sexually transmitted disease worldwide. Since the 1960s, metronidazole has been the standard treatment, however an increasing number of cases with metronidazole-resistant strains is being reported. In this study, pentamycin, a polyene antibiotic, was tested for its in vitro efficacy against T. vaginalis using four strains with different metronidazole susceptibilities. It was shown that pentamycin is highly active against T. vaginalis and that the effect is prompt and independent of underlying metronidazole resistance. The effective concentrations (EC values) after 1 h of treatment were in the range 1.74-2.62 μg/mL (EC50) and 4.91-6.51 μg/mL (EC90). Total eradication of trichomonads (EC100) was achieved in all strains by treatment with 15 μg/mL (22 μM) for 1 h or with ≥1 μg/mL (≥1.5 μM) for 24 h. Long-term cultivation (12 months) under permanent drug pressure did not induce stable resistance against pentamycin in any of the strains tested. Pentamycin has been approved for intravaginal use and is a promising candidate for the topical treatment of trichomoniasis.
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http://dx.doi.org/10.1016/j.ijantimicag.2014.12.024DOI Listing
April 2015

Protein profiles and immunoreactivities of Acanthamoeba morphological groups and genotypes.

Exp Parasitol 2014 Nov 20;145 Suppl:S50-6. Epub 2014 May 20.

Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria. Electronic address:

Acanthamoeba is a free-living protozoan found in a wide variety of habitats. A classification of Acanthamoeba into currently eighteen genotypes (T1-T18) has been established, however, data on differences between genotypes on the protein level are scarce. The aim of this study was to compare protein and immunoreactivity profiles of Acanthamoeba genotypes. Thirteen strains, both clinical and non-clinical, from genotypes T4, T5, T6, T7, T9, T11 and T12, representing three morphological groups, were investigated for their protein profiles and IgG, IgM and IgA immunoreactivities. It was shown that protein and immunoreactivity profiles of Acanthamoeba genotypes T4, T5, T6, T7, T9, T11 and T12 are clearly distinct from each other, but the banding patterns correlate to the morphological groups. Normal human sera revealed anti-Acanthamoeba antibodies against isolates of all investigated genotypes, interestingly, however only very weak IgM and virtually no IgA immunoreactivity with T7 and T9, both representing morphological group I. The strongest IgG, IgM and IgA immunoreactivities were observed for genotypes T4, T5 and T6. Differences of both, protein and immunological patterns, between cytopathic and non-cytopathic strains, particularly within genotype T4, were not at the level of banding patterns, but rather in expression levels.
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http://dx.doi.org/10.1016/j.exppara.2014.05.006DOI Listing
November 2014

A study on Nim expression in Bacteroides fragilis.

Microbiology (Reading) 2014 Mar 21;160(Pt 3):616-622. Epub 2014 Jan 21.

Institute of Clinical Microbiology, Faculty of Medicine, University of Szeged, Szeged, Hungary.

Members of the genus Bacteroides, mainly Bacteroides fragilis, can cause severe disease in man, especially after intestinal perforation in the course of abdominal surgery. Treatment is based on a small number of antibiotics, including metronidazole, which has proved to be highly reliable throughout the last 40 to 50 years. Nevertheless, metronidazole resistance does occur in Bacteroides and has been mainly attributed to Nim proteins, a class of proteins with a suggested nitroreductase function. Despite the potentially high importance of Nim proteins for human health, information on the expression of nim genes in B. fragilis is still lacking. It was the aim of this study to demonstrate expression of nim genes in B. fragilis at the protein level and, furthermore, to correlate Nim levels with the magnitude of metronidazole resistance. By the application of 2D gel electrophoresis, Nim proteins could be readily identified in nim-positive strains, but their levels were not elevated to a relevant extent after induction of resistance with high doses of metronidazole. Thus, the data herein do not provide evidence for Nim proteins acting as nitroreductases using metronidazole as a substrate, because no correlation between Nim levels and levels of metronidazole resistance could be observed. Furthermore, no evidence was found that Nim proteins protect B. fragilis from metronidazole by sequestering the activated antibiotic.
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http://dx.doi.org/10.1099/mic.0.074807-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215050PMC
March 2014

Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance.

Mol Microbiol 2014 Jan 20;91(1):198-208. Epub 2013 Nov 20.

Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.
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http://dx.doi.org/10.1111/mmi.12455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437529PMC
January 2014

Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites.

Exp Parasitol 2013 Jul 8;134(3):374-80. Epub 2013 Apr 8.

Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension.
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http://dx.doi.org/10.1016/j.exppara.2013.03.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682184PMC
July 2013

A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia lamblia.

Antimicrob Agents Chemother 2013 May 12;57(5):2029-35. Epub 2013 Feb 12.

Department of Medicine, University of California, San Diego, La Jolla, California, USA.

Giardiasis is one of the most common causes of diarrheal disease worldwide. Treatment is primarily with 5-nitro antimicrobials, particularly metronidazole. Resistance to metronidazole has been described, and treatment failures can occur in up to 20% of cases, making development of alternative antigiardials an important goal. To this end, we have screened a chemical library of 746 approved human drugs and 164 additional bioactive compounds for activity against Giardia lamblia. We identified 56 compounds that caused significant inhibition of G. lamblia growth and attachment. Of these, 15 were previously reported to have antigiardial activity, 20 were bioactive but not approved for human use, and 21 were drugs approved for human use for other indications. One notable compound of the last group was the antirheumatic drug auranofin. Further testing revealed that auranofin was active in the low (4 to 6)-micromolar range against a range of divergent G. lamblia isolates representing both human-pathogenic assemblages A and B. Most importantly, auranofin was active against multiple metronidazole-resistant strains. Mechanistically, auranofin blocked the activity of giardial thioredoxin oxidoreductase, a critical enzyme involved in maintaining normal protein function and combating oxidative damage, suggesting that this inhibition contributes to the antigiardial activity. Furthermore, auranofin was efficacious in vivo, as it eradicated infection with different G. lamblia isolates in different rodent models. These results indicate that the approved human drug auranofin could be developed as a novel agent in the armamentarium of antigiardial drugs, particularly against metronidazole-resistant strains.
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http://dx.doi.org/10.1128/AAC.01675-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632933PMC
May 2013

Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example.

Biochem J 2013 Apr;451(2):277-88

Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, A-1090 Vienna, Austria.

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.
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http://dx.doi.org/10.1042/BJ20121798DOI Listing
April 2013

Nitroimidazole drugs vary in their mode of action in the human parasite Giardia lamblia.

Int J Parasitol Drugs Drug Resist 2012 Dec 12;2:166-70. Epub 2012 May 12.

Institute of Specific Prophylaxis and Tropical Medicine at the Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Giardia lamblia (syn. duodenalis, intestinalis) is a globally occurring micro-aerophilic human parasite that causes gastrointestinal disease. Standard treatment of G. lamblia infections is based on the 5-nitroimidazole drugs metronidazole and tinidazole. In two other micro-aerophilic parasites, Entamoeba histolytica and Trichomonas vaginalis, 5-nitroimidazole drugs bind to proteins involved in the thioredoxin-mediated redox network and disrupt the redox equilibrium by inhibiting thioredoxin reductase and depleting intracellular thiol pools. The major aim of this study was to assess whether nitroimidazoles exert a similar toxic effect on G. lamblia physiology. The 5-nitroimidazoles metronidazole and tinidazole were found to bind to the same subset of proteins including thioredoxin reductase. However, in contrast to E. histolytica and T. vaginalis, none of the other proteins bound are candidates for being involved in the thioredoxin-mediated redox network. Translation elongation factor EF-1γ, an essential factor in protein synthesis, was widely degraded upon treatment with 5-nitroimidazoles. 2-Nitroimidazole (azomycin) and the 5-nitroimidazole ronidazole did not bind to any G. lamblia proteins, which is in contrast to previous findings in E. histolytica and T. vaginalis. All nitroimidazoles tested reduced intracellular thiol pools in G. lamblia, but metronidazole, also in contrast to the situation in the other two parasites, had the slightest effect. Taken together, our results suggest that nitroimidazole drugs affect G. lamblia in a fundamentally different way than E. histolytica and T. vaginalis.
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http://dx.doi.org/10.1016/j.ijpddr.2012.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3862438PMC
December 2012

Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis.

Mol Biochem Parasitol 2012 Jun 17;183(2):177-83. Epub 2012 Mar 17.

Institute of Specific Prophylaxis and Tropical Medicine at the Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria.

The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem. In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE). Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde.
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http://dx.doi.org/10.1016/j.molbiopara.2012.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3341570PMC
June 2012

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1α.

Vet Parasitol 2012 Apr 29;185(2-4):138-44. Epub 2011 Sep 29.

Institute of Parasitology, Vetsuisse Faculty, University of Berne, Länggassstrasse 122, 3012 Berne, Switzerland.

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1α) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1α was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.
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http://dx.doi.org/10.1016/j.vetpar.2011.09.032DOI Listing
April 2012

The N-glycans of Trichomonas vaginalis contain variable core and antennal modifications.

Glycobiology 2012 Feb 7;22(2):300-13. Epub 2011 Oct 7.

Department für Chemie, Universität für Bodenkultur, Vienna, Austria.

Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data have been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and high-performance liquid chromatography data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man(5)GlcNAc(2)) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species.
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http://dx.doi.org/10.1093/glycob/cwr149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272381PMC
February 2012