Inventory and Evolution of Mitochondrion-localized Family A DNA Polymerases in Euglenozoa

The order Trypanosomatida has been well studied due to its pathogenicity and the unique biology of the mitochondrion. In Trypanosoma brucei, four DNA polymerases, namely PolIA, PolIB, PolIC, and PolID, related to bacterial DNA polymerase I (PolI), were shown to be localized in mitochondria experimentally. These mitochondrion-localized DNA polymerases are phylogenetically distinct from other family A DNA polymerases, such as bacterial PolI, DNA polymerase gamma (Polγ) in human and yeasts, “plant and protist organellar DNA polymerase (POP)” in diverse eukaryotes. However, the diversity of mitochondrion-localized DNA polymerases in Euglenozoa other than Trypanosomatida is poorly understood. In this study, we discovered putative mitochondrion-localized DNA polymerases in broad members of three major classes of Euglenozoa—Kinetoplastea, Diplonemea, and Euglenida—to explore the origin and evolution of trypanosomatid PolIA-D. We unveiled distinct inventories of mitochondrion-localized DNA polymerases in the three classes: (1) PolIA is ubiquitous across the three euglenozoan classes, (2) PolIB, C, and D are restricted in kinetoplastids, (3) new types of mitochondrion-localized DNA polymerases were identified in a prokinetoplastid and diplonemids, and (4) evolutionarily distinct types of POP were found in euglenids. We finally propose scenarios to explain the inventories of mitochondrion-localized DNA polymerases in Kinetoplastea, Diplonemea, and Euglenida

The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasis

The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). Arobust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.Charles University [GAUK 1642314/2014]; European Union (EU) grant [FP7-261504]; EU's Horizon research and innovation programme under the Marie Sklodowska-Curie grant [642609]; Fundacao para a Ciencia e a Tecnologia [SFRH/BPD/44082/2008]; Ministerio da Educacao e Ciencia (Foundation for Science and Technology, Ministry of Education and Science), Portuga

Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles

<div><p>Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in <i>Trypanosoma brucei</i>. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds.</p></div

2-furyl(phenyl)methanol isolated from Atractilis gummifera rhizome exhibits anti-leishmanial activity

The file attached to this record is the Publisher's final version. Crown copyright.We report for the first time the isolation of 2-furyl(phenyl)methanol (5) from the chloroform extracts of the Atractylis gummifera roots. A. gummifera is a thistle belonging to the Asteraceae family that produces the ent-kaurane diterpenoid glycoside atractyloside (ATR). ATR (1) was isolated and chemically modified to obtain its aglycone atractyligenin (2) and the methylated derivatives ATR-OMe (3) and genine-OMe (4). The compounds 1-5 were structurally characterised and evaluated against the intracellular amastigote, cultured within macrophages, and the extracellular promastigote of Leishmania donovani, the protozoan parasite responsible for the highly infective disease visceral leishmaniasis, which is fatal if untreated. The 2-furyl(phenyl)methanol 5 exhibited notable activity against the promastigote

Nosema bombi (Microsporidia: Nosematidae) and trypanosomatid prevalence in spring bumble bee queens (Hymenoptera: Apidae: Bombus) in Kansas

Citation: Tribodi, A., Cibils-Stewart, X., McCornack, B., & Szlanski, A. (2014). Nosema bombi (Microsporidia: Nosematidae) and Trypanosomatid Prevalence in Spring Bumble Bee Queens (Hymenoptera: Apidae: Bombus) in Kansas. Journal of the Entomological Society, 87(2), 225-233. https://doi.org/10.2317/JKES130730.1Several species of bumble bees are declining in the United States; these declining populations often show higher prevalence of Nosema bombi, a microsporidian pathogen. To date, surveys of bumble bee pathogens in the United States have only been conducted on workers and males, yet the health of a population is ultimately dependent on the success of colony-founding queens. We conducted a molecular-diagnostic survey of the prevalence of N. bombi and trypanosomatids, such as Crithidia bombi, in six species of spring queens (n  =  142) collected in 2011 and 2013 at three sites in central Kansas. Nosema bombi was found in 27% of Bombus pensylvanicus and 13% of B. auricomus but was not found in the other species sampled. Trypanosomatids were only found in B. pensylvanicus (9%) during the May 2013 sampling period. The high prevalence of N. bombi in B. pensylvanicus is consistent with other surveys for this pathogen in other castes, but the high prevalence of N. bombi in B. auricomus is a novel finding. Although the conservation status of B. auricomus has not been thoroughly assessed, two recently published surveys showed that B. auricomus were less common in portions of the species' range. Based on those findings and an oft-cited link between N. bombi prevalence and bumble bee species' decline (e.g., B. pensylvanicus) in other studies, our findings suggest B. auricomus populations in Kansas may warrant further scrutiny

NommPred: Prediction of Mitochondrial and Mitochondrion-Related Organelle Proteins of Nonmodel Organisms

To estimate the functions of mitochondria of diverse eukaryotic nonmodel organisms in which the mitochondrial proteomes are not available, it is necessary to predict the protein sequence features of the mitochondrial proteins computationally. Various prediction methods that are trained using the proteins of model organisms belonging particularly to animals, plants, and fungi exist. However, such methods may not be suitable for predicting the proteins derived from nonmodel organisms because the sequence features of the mitochondrial proteins of diversified nonmodel organisms can differ from those of model organisms that are present only in restricted parts of the tree of eukaryotes. Here, we proposed NommPred, which predicts the mitochondrial proteins of nonmodel organisms that are widely distributed over eukaryotes. We used a gradient boosting machine to develop 2 predictors—one for predicting the proteins of mitochondria and the other for predicting the proteins of mitochondrion-related organelles that are highly reduced mitochondria. The performance of both predictors was found to be better than that of the best method available

Establishment of a tRNA over-expression system in Trypanosoma brucei to study the role of post-transcriptional modifications on function.

Undergraduate Research ScholarshipMayers Summer InternshipDean's Undergraduate Research FundTrypanosomatids are parasitic protozoa which infect millions of humans and animals across the globe every year. The two primary types of these unicellular flagellates are Trypanosoma and Leishmania, and the World Health Organization estimates that approximately 400 million people worldwide are at substantial risk to develop infections caused by these pathogens. Trypanosomes also possess many significant biological and biochemical properties, and in particular the tRNAs of trypanosomatids undergo substantial in vivo chemical modifications. We focus on understanding how one particular set of modifications, i.e. adenosine to inosine changes in the anticodon, impact tRNA function. We are also interested on how other modifications like methylations may affect inosine formation. The goals of this project are two-fold: first, to construct a vector capable of successfully over-expressing tRNA in Trypanosoma brucei, and second, to successfully introduce two non-endogenous, tagged tRNAs into T. brucei in order to further analyze in vivo modifications. To this end, we have engineered two distinct tRNA variants with mutations in the anticodon arm (Tag 22) and anticodon arm and loop (Tag 23). These mutations serve as tags to differentiate them from endogenous tRNAs naturally found in T. brucei cells. Prior to testing our overexpression system, the two "tagged" variants were analyzed for their ability to support known modification reactions in vitro. Since these variants are derived from a tRNA that undergoes adenosine to inosine changes in their anticodon in vivo, we tested and further showed that the mutations used to generate the variants do not interfere with this reaction in vitro. In the next phase of the project, the two variants will be cloned in a plasmid vector and expressed under the control of an endogenous T7 promoter and tetracycline (TET) operator, followed by transfection into Trypanosoma brucei cells. Total RNA isolated from the transfected cells will thus provide a measure of overexpression of our two tRNA variants. Successful completion of this phase of the project will allow further exploration into the in vivo posttranscriptional modifications of tRNAs through analysis of the tagged tRNA constructs. By comparing the tagged tRNA construct to endogenous tRNAs, important modification reactions such as adenosine to inosine and cytidine to uridine editing can be further studied. As mentioned earlier, since these modifications play a vital role in the function of tRNA, their identification could be useful for future design of anti-trypanosomal treatments.American Heart AssociationNational Science Foundatio

Bee trypanosomatids and their interactions with pollutants

Honeybees (Apis mellifera) are highly valued worldwide for their products, but also as pollinators of crops and wild plants. Due to the close proximity of bees in the beehive and their foraging habits, a large number of pathogens that weaken the bee colonies are spread both inside and between colonies. In recent decades, high annual losses of bees have been recorded, which still do not have a clear explanation. The sudden loss of bees (known as colony collapse disorder) is likely due to the interaction of multiple factors at once. A large part of the problem is attributed to viral diseases and some parasitic organisms; parasites of the Trypanosomatida group play an uncertain role. A member of this group, Crithidia bombi, is considered a problematic inhabitant of the digestive tract of bumblebees, which can cause serious complications. Close relatives, C. mellificae and Lotmaria passim, parasites of honeybees, are sometimes associated with their higher mortality. Pesticides, whose effects can be very toxic, have also come to the forefront in assessing bee losses, and most pesticides have therefore been banned in the EU. This thesis examines the prevalence, infection development, and host specificity of monoxenous trypanosomatids in bees and their pesticide interactions. A total of 26 species of...Včely medonosné (Apis mellifera) jsou celosvětově vysoce ceněné díky svým produktům, ale také jako opylovači hospodářských plodin a planě rostoucích rostlin. Vzhledem k těsné blízkosti včel a jejich sociálnímu uspořádání se jak uvnitř včelstva, tak i mezi včelstvy šíří velké množství patogenů způsobující následně oslabení včelstva. V posledních desetiletích byly zaznamenány vysoké meziroční ztráty včel, které nemají doposud jednoznačné vysvětlení. Příčina náhlých ztrát včel (označováno jako syndrom zhroucení včelstev) bude pravděpodobně ve vzájemném působení více faktorů najednou. Velký podíl je přisuzován virovým onemocněním i některým parazitickým organismům; nejistou úlohu pak hrají paraziti ze skupiny Trypanosomatida. Zástupce této skupiny, Crithidia bombi, je považován za problematického obyvatele trávicího traktu čmeláků, kterým může působit vážné komplikace. Blízce příbuzné druhy, C. mellificae a Lotmaria passim, jsou pak parazity zejména včel medonosných a někdy bývají spojováni s jejich vyšší úmrtností. Do popředí při hodnocení ztrát včel se dostávají také pesticidy, jejichž účinky mohou být velmi toxické a většina pesticidů byla proto v EU zakázána. Tato diplomová práce je zaměřena na zkoumání prevalence, rozvoje infekce a hostitelské specifity včelích jednohostitelských trypanosomatid a...Katedra parazitologieDepartment of ParasitologyFaculty of SciencePřírodovědecká fakult

Monoxenous trypanosomatids of invertebrates

T ída Kinetoplastea zahrnuje voln žijící i parazitické zástupce. Jednou z nejznám jších skupin je ád Trypanosomatida, do kterého pat í obligátní parazité (Trypanosoma, Leishmania) širokého spektra hostitel . Mnohé druhy jsou velmi nebezpe nými patogeny lov ka, jiné p sobí velké ekonomické ztráty v zem d lství. Nicmén p evážná ást trypanosomatid pat í mezi jednohostitelské parazity hmyzu, kte í jsou pro své hostitele v tšinou nepatogenní. Jednohostitelská trypanosomatida parazitují p evážn v ádech polok ídlých a dvouk ídlých. P edložená diplomová práce se v nuje detekci nákaz jednohostitelskými trypanosomatidy u šváb odchycených na území eské republiky a šváb pocházejících z n kolika r zných chov . Švábi jsou vhodní mechani tí p enaše i r zných patogen (v etn parazit ), kte í ohrožují zdraví lov ka i zví at. První popis trypanosomatid ve švábech byl zdokumentován již na po átku 20. století, ale žádná studie se t mto nález m d kladn ji nev novala. Cílem této práce byla i podrobná morfologická a ultramikroskopická analýza a studium hostitelské specifity nedávno popsaného druhu švábí herpetomonády Herpetomonas tarakana. Tato diplomová práce byla áste n podkladem již publikovaného lánku "Diversity of trypanosomatids in cockroaches and the description of Herpetomonas tarakana sp. n.", na který navazuje a...The class Kinetoplastea contains free-living and parasitic species. One of the most dominant group within the class is the order Trypanosomatida which includes obligate parasites (Trypanosoma, Leishmania) infecting a wide range of hosts. Some species are serious pathogens of humans and domestic animals and cause considerable losses. However, the majority of trypanosomatids belongs to monoxenous parasites of insect which are usually harmless to their hosts. Monoxenous trypanosomatids predominantly infect Hemiptera and Diptera. This diploma thesis is focused on the detection of monoxenous trypanosomatids in cockroaches captured in the Czech Republic and cockroaches from different breedings. Cockroaches are very suitable mechanical vectors of many different pathogens (including parasites) and are significant health threat for humans and animals. First trypanosomatids in cockroaches were documented at the beginning of the 20th century, but there is no study focused on this topic specifically. Another aim of this thesis is morphological and ultramicroscopic analysis and the study of the host specificity of the recently described species Herpetomonas tarakana, isolated from a cockroach. My findings were partly used in the already published study "Diversity of trypanosomatids in cockroaches and the...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc