Iron in parasitic protists - from uptake to storage and where we can interfere.

It is well known that iron is a crucial micronutrient for all living organisms. Due to its chemical properties, iron is an irreplaceable cofactor of many essential enzymes but is also potentially toxic when present in excess. The acquisition of iron from the environment can be challenging for organisms, especially for parasitic protists that rely solely on the host for available nutrients. One of the host defense mechanisms is to starve parasites by detaining the crucial iron in a form unreachable for pathogens. In this review, we summarize current information about iron homeostasis-related pathways of important human parasites, such as Plasmodium, trypanosomes, Leishmania, pathogenic amoebas and Trichomonas. We focus on the parasites' strategies of iron acquisition, storage/detoxification, trafficking, and iron-regulated protein expression and address the questions of iron-influenced virulence and anti-parasitic chemotherapeutics targeted to iron metabolism. Finally, we outline the potential of understudied and somewhat neglected iron chelating agents as safe chemotherapeutics against protozoan parasites.

An Alliance of Gel-Based and Gel-Free Proteomic Techniques Displays Substantial Insight Into the Proteome of a Virulent and an Attenuated Histomonas meleagridis Strain.

The unicellular protozoan Histomonas meleagridis is notorious for being the causative agent of histomonosis, which can cause high mortality in turkeys and substantial production losses in chickens. The complete absence of commercially available curative strategies against the disease renders the devising of novel approaches a necessity. A fundamental step toward this objective is to understand the flagellate's virulence and attenuation mechanisms. For this purpose we have previously conducted a comparative proteomic analysis of an in vitro cultivated virulent and attenuated histomonad parasite using two-dimensional electrophoresis and MALDI-TOF/TOF. The current work aimed to substantially extend the knowledge of the flagellate's proteome by applying 2D-DIGE and sequential window acquisition of all theoretical mass spectra (SWATH) MS as tools on the two well-defined strains. In the gel-based experiments, 49 identified protein spots were found to be differentially expressed, of which 37 belonged to the in vitro cultivated virulent strain and 12 to the attenuated one. The most frequently identified proteins in the virulent strain take part in cytoskeleton formation, carbohydrate metabolism and adaptation to stress. However, post-translationally modified or truncated ubiquitous cellular proteins such as actin and GAPDH were identified as upregulated in multiple gel positions. This indicated their contribution to processes not related to cytoskeleton and carbohydrate metabolism, such as fibronectin or plasminogen binding. Proteins involved in cell division and cytoskeleton organization were frequently observed in the attenuated strain. The findings of the gel-based studies were supplemented by the gel-free SWATH MS analysis, which identified and quantified 42 significantly differentially regulated proteins. In this case proteins with peptidase activity, metabolic proteins and actin-regulating proteins were the most frequent findings in the virulent strain, while proteins involved in hydrogenosomal carbohydrate metabolism dominated the results in the attenuated one.

Cholesterol supplementation improves growth rates of Histomonas meleagridis in vitro.

Research on the energy metabolism of various protozoan parasites showed the essentiality and benefits of cholesterol in the cultivation of these organisms. However, not much is known about the energy metabolism of Histomonas meleagridis, although such information is of high importance to improve cultivation of the parasite for advancements in diagnostics, research and vaccine development. By supplementing a serum enriched cultivation medium with cholesterol, numbers of parasites could be doubled in comparison to unsupplemented negative controls. This effect was demonstrated for two different strains of the parasite, at different levels of in vitro-passages and for histomonads under xenic or monoxenic settings. Supplementing medium free of serum with cholesterol, resulted in significant growth of the parasite over 72 h. However, there were differences in growth behaviour in serum free medium between the different histomonad cultures and continuous passaging of the cultures without serum was not possible. Monitoring the bacterial growth of two different co-cultivated E. coli strains in monoxenic histomonad cultures during these experiments showed that there was no significant impact of cholesterol on the bacteria. Therefore, a direct effect of cholesterol on the parasite itself could be demonstrated. The results of these experiments supply new insights into the metabolism of H. meleagridis and it can be concluded that cholesterol is an important component to enhance parasite growth in vitro.

Unravelling the differences: comparative proteomic analysis of a clonal virulent and an attenuated Histomonas meleagridis strain.

The current study focused on Histomonas meleagridis, a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P < 0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis-specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation.

Development of a Dry Medium for Isolation of Histomonas meleagridis in the Field.

Blackhead disease is caused by Histomonas meleagridis, an anaerobic protozoan parasite, and results in mortality rates of up to 100% in turkeys and 30% in chickens. Outbreaks of blackhead disease are unpredictable, and the harvesting of H. meleagridis strains from the field would be a great resource for researchers to study its epidemiology. Therefore, the objective of this study was to develop a dry medium that would allow storage at ambient temperatures until needed. Fifty milliliters of horse serum was dried and then mixed with dry medium M199 with Hanks balanced salts (10.6 g), sodium bicarbonate (0.35 g), and rice powder (0.8 g). To test the ability of reconstituted medium to support growth of H. meleagridis, groups of 10 flasks containing 0.2 g of dry medium were stored for 24 hr at 25 and 60 C before testing. Other groups of flasks containing dry medium were stored at 25, 37, and 42 C for 1, 3, or 6 mo. At each test period, the flasks were reconstituted with 10 ml of water, inoculated with 100 000 H. meleagridis cells, and incubated at 40 C for 48 hr. Fresh liquid medium was used as a control. There were no differences in cell counts in medium stored at 25 or 60 C for 24 hr. After 1 mo, cell counts in reconstituted medium were about half that of fresh liquid medium after 48 hr of incubation. But after 3 and 6 mo, the cell counts were not significantly different in all groups (P < 0.05) after 72 hr of incubation. These results show that dried Dwyer medium can be stored at ambient temperatures for extended times and would be an effective tool for obtaining isolates of H. meleagridis from the field.

The costa of trichomonads: A complex macromolecular cytoskeleton structure made of uncommon proteins.

BACKGROUND INFORMATION: The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. RESULTS: Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. CONCLUSIONS: The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. SIGNIFICANCE: Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms.

Responding to a Zoonotic Emergency with Multi-omics Research: Pentatrichomonas hominis Hydrogenosomal Protein Characterization with Use of RNA Sequencing and Proteomics.

Pentatrichomonas hominis is an anaerobic flagellated protist that colonizes the large intestine of a number of mammals, including cats, dogs, nonhuman primates, and humans. The wide host range of this organism is alarming and suggests a rising zoonotic emergency. However, knowledge on in-depth biology of this protist is still limited. Similar to the human pathogen, Trichomonas vaginalis, P. hominis possesses hydrogenosomes instead of mitochondria. Studies in T. vaginalis indicated that hydrogenosome is essential for cell survival and associated with numerous pivotal biological functions, including drug resistance. To further decipher the biology of this important organelle, we undertook proteomic research in P. hominis hydrogenosomes. Lacking a decoded P. hominis genome, we utilized an RNA sequencing (RNA-seq) data set generated from P. hominis axenic culture as the reference for proteome analysis. Using this in-house reference data set and mass spectrometry (MS), we identified 442 putative hydrogenosomal proteins. Interestingly, the composition of the P. hominis hydrogenosomal proteins is very similar to that of T. vaginalis, but proteins such as Hmp36, Pam16, Pam18, and Isd11 are absent based on both MS and the RNA-seq. Our data underscore that P. hominis expresses different homologs of multiple gene families from T. vaginalis. To the best of our knowledge, we present here the first hydrogenosome proteome in a protist other than T. vaginalis that offers crucial new scholarship for global health, therapeutics, diagnostics, and veterinary medicine research. In addition, the research strategy used here using RNA sequencing and proteomics might inform future multi-omics research in other understudied organisms without decoded genomes.

The Cytoskeleton of Parabasalian Parasites Comprises Proteins that Share Properties Common to Intermediate Filament Proteins.

Certain protist lineages bear cytoskeletal structures that are germane to them and define their individual group. Trichomonadida are excavate parasites united by a unique cytoskeletal framework, which includes tubulin-based structures such as the pelta and axostyle, but also other filaments such as the striated costa whose protein composition remains unknown. We determined the proteome of the detergent-resistant cytoskeleton of Tetratrichomonas gallinarum. 203 proteins with homology to Trichomonas vaginalis were identified, which contain significantly more long coiled-coil regions than control protein sets. Five candidates were shown to associate with previously described cytoskeletal structures including the costa and the expression of a single T. vaginalis protein in T. gallinarum induced the formation of accumulated, striated filaments. Our data suggests that filament-forming proteins of protists other than actin and tubulin share common structural properties with metazoan intermediate filament proteins, while not being homologous. These filament-forming proteins might have evolved many times independently in eukaryotes, or simultaneously in a common ancestor but with different evolutionary trajectories downstream in different phyla. The broad variety of filament-forming proteins uncovered, and with no homologs outside of the Trichomonadida, once more highlights the diverse nature of eukaryotic proteins with the ability to form unique cytoskeletal filaments.

Feline Tritrichomonas foetus adhere to intestinal epithelium by receptor-ligand-dependent mechanisms.

Tritrichomonas foetus (TF) is a protozoan that infects the feline ileum and colon resulting in chronic diarrhea. Up to 30% of young purebred cats are infected with TF and the infection is recognized as pandemic. Only a single drug, characterized by a narrow margin of safety and emerging development of resistance, is effective for treatment. While the venereal pathogenicity of bovine TF is attributed to adherence to uterovaginal epithelium, the pathogenesis of diarrhea in feline TF infection is unknown. The aim of this study was to establish an in vitro model of feline TF adhesion to intestinal epithelium. Confluent monolayers of porcine intestinal epithelial cells (IPEC-J2) were infected with axenic cultures of feline TF that were labeled with [(3)H] thymidine or CFSE and harvested at log-phase. The effect of multiplicity and duration of infection, viability of TF, binding competition, formalin fixation and cytoskeletal inhibitors on adherence of feline TF to IPEC-J2 monolayers was quantified by liquid scintillation counting and immunofluorescence. [(3)H] thymidine and CFSE-labeled TF reproducibly adhered to IPEC-J2 monolayers. Clinical isolates of feline TF adhered to the intestinal epithelium in significantly greater numbers than Pentatrichomonas hominis, the latter of which is a presumably nonpathogenic trichomonad. Adhesion of TF required viable trophozoites but was independent of cytoskeletal activity. Based on saturation and competition binding experiments, adherence of feline TF to the epithelium occurred via specific receptor-ligand interactions. The developed model provides a valuable resource for assessing pathogenic mechanisms of feline TF and developing novel pharmacologic therapies for blocking the adhesion of feline TF to the intestinal epithelium.

Identification of gene expression elements in Histomonas meleagridis using splinkerette PCR, a variation of ligated adaptor PCR.

Histomonas meleagridis is the causative agent of blackhead disease in gallinaceous birds. Limited genetic information exists for this organism, with the majority of sequence information coming from the coding regions of genes. No information is available for intergenic regions that contain DNA elements required for the regulation of gene expression. In this study, we demonstrate that splinkerette PCR, a variation of ligated adaptor PCR, can be used to identify regions of unknown sequence that lie upstream and downstream of known genomic sequences. Using this technique, we identified upstream sequences of 2 ß-tubulin genes. Sequence analysis identified the 5' coding portions of the ß-tubulin genes, the intergenic regions, and 2 different open reading frames encoding for a putative serine/threonine phosphatase and a putative ras-related protein, racG. We predict that these intergenic regions contain polyadenylation and cleavage signals for the 2 open reading frames and initiator elements for the ß-tubulin genes. Our research demonstrates the use of splinkerette PCR as a valuable tool to identify unknown DNA sequences. In addition, the identification of the regulatory elements necessary for gene transcription in H. meleagridis will provide tools for future studies on its gene expression.

Acetate and succinate production in amoebae, helminths, diplomonads, trichomonads and trypanosomatids: common and diverse metabolic strategies used by parasitic lower eukaryotes.

Parasites that often grow anaerobically in their hosts have adopted a fermentative strategy relying on the production of partially oxidized end products, including lactate, glycerol, ethanol, succinate and acetate. This review focuses on recent progress in understanding acetate production in protist parasites, such as amoebae, diplomonads, trichomonads, trypanosomatids and in the metazoan parasites helminths, as well as the succinate production pathway(s) present in some of them. We also describe the unconventional organisation of the tricarboxylic acid cycle associated with the fermentative strategy adopted by the procyclic trypanosomes, which may resemble the probable structure of the primordial TCA cycle in prokaryotes.

Identification and molecular characterization of numerous Histomonas meleagridis proteins using a cDNA library.

SUMMARYHistomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or 'blackhead disease'. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed from a mono-eukaryotic culture of H. meleagridis. The library was screened with polyclonal rabbit serum raised against purified H. meleagridis trophozoites. Polyclonal rabbit serum specifically recognized the same major H. meleagridis antigens as chicken and turkey sera originating from animal trials, but displayed a significantly lower bacteria-dependent background signal. After 2 rounds of screening, a total of 95 positive clones were sequenced. Bioinformatics analyses were performed on nucleotide and deduced amino acid sequences, identifying 37 unique clones. Based on the homology to other protozoan parasites, mostly Trichomonas vaginalis, the clones were grouped according to functional aspects: structural proteins, possible surface proteins, oxygen reducing proteins, ribosomal proteins, protein kinases and various other intracellular proteins.

DNA fluorescent stain accumulates in the Golgi but not in the kinetosomes of amitochondriate protists.

Hindgut symbiotic trichomonads (uninucleate Caduceia versatilis, and multinucleate Stephanonympha sp. and Snyderella tabogae) from the dry-wood-eating termite Cryptotermes cavifrons (Kalotermitidae) accumulate DAPI (4,6diamidino-2-phenylindole) in the membranous sacs of the Golgi complex. This form of Golgi complex, typical of protists in the class Parabasalia, is called a parabasal body. Trichomonads contain organellar systems, mastigonts, that consist of four undulipodia (e.g. eukaryotic flagella and cilia), axostylar microtubules, a parabasal body and other structures. These cells bear from one (in the case of Caduceia) to hundreds (in the case of Snyderella) of mastigonts. These features are characteristic of their protist class (Parabasalia). The nuclei of all three species stained with DNA-specific stains: DAPI, SYTOX, acridine orange, propidium iodide, ethidium bromide and Feulgen, at optimal concentrations, but kinetosomes failed to stain at all. The nuclei, parabasal bodies and symbiotic bacteria (but no microtubular structures) fluoresced in glutaraldehyde-fixed cells stained with 1.45 microM DAPI. Parabasal bodies of Snyderella and Caduceia treated to remove lipids with Triton X-100, or treated with 5% trichloroacetic acid, lacked DAPI-fluorescence. I conclude that DNA, present as expected in nuclei and bacterial symbionts, is absent from and not associated with calonymphid kinetosomes. The reason for DNA-RNA stain accumulation in the Golgi cistemae is not clear.

Hemolytic activity of Monocercomonas spp.

The hemolytic activity of an isolate of Monocercomonas spp. from Tropidophis melanurus (snake: Boidae) was investigated. The isolate was tested against human erythrocytes of groups A, B, AB and O and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results show that Monocercomonas spp. exerted an hemolytic activity against all erythrocytes tested.