Elucidating virulence factors of Histomonas meleagridis

The flagellated protozoon Histomonas meleagridis is the aetiological agent of an enterohepatitis termed histomonosis or "blackhead disease". Untreated, histomonosis causes high rates of mortality in turkeys, whereas clinical signs in chickens vary considerably. For decades, the disease was well controlled by the use of chemotherapeutics as preventative and curative drugs. However, due to potential consumer health risks, effective drugs have been withdrawn from markets in the EU and USA, leading to the re-emergence of histomonosis. Lack of treatment with chemotherapeutics and the growing popularity of free-range housing expose poultry to the pathogen, contributing to the increased occurrence of the disease. Histomonosis is of special importance today, as the lack of effective treatment allows ist progression and causes considerable losses for the poultry industry. Limited molecular data on H. meleagridis have been accumulating over the past few years and most studies have related to the parasite`s phylogenetic position. There is still nothing known about the molecular basis of pathogenesis or about the parasite`s virulence. The aim of the present study is to identify and characterize putative virulence factors of H. meleagridis. Comparative proteome and secretome analyses of virulent and avirulent monoxenic cultures of H. meleagridis will be undertaken. Such cultures, displaying the same genetic background, have been generated in our laboratory and are well established. Proteins that are strongly up-regulated or present only in the virulent strain of the parasite will be analysed by mass spectroscopy. To complement the data gained by proteome and secretome analyses, differences in the transcriptomes of virulent and avirulent strains will also be investigated. The high-priority hits, consisting of proteins with significant homology to known virulence factors as well as unknown proteins displaying striking up-regulation in the virulent strain, will be further characterized with respect to the organization of their genomic locus, transcription start site, transcript size and abundance. Furthermore, in vivo experiments will explore the potential of selected proteins to elicit an immune response in the host. The role of selected secreted proteins in the virulence of H. meleagridis will be investigated and ultimately the value of selected proteins as markers in diagnostic tests will be evaluated.

The aim of the present project was to identify and characterize putative virulence factors of Histomonas meleagridis and by that contribute to better understanding of the mechanisms this important poultry parasite uses to conquer the host. The flagellated protozoon, H. meleagridis, is the aetiological agent of histomonosis or blackhead disease. Untreated, histomonosis can lead to the loss of entire turkey flocks, whereas in chickens the disease is less severe. This globally distributed disease was controlled for decades by the use of chemotherapeutics for prevention and treatment. In recent years, changes in drug legislation in European Union and the USA have led to reappearance of the histomonosis. The growing popularity of free-range housing means that poultry is increasingly exposed to the pathogen, contributing to the reoccurrence of the disease as well. In vivo experiments at the Clinic for Poultry and Fish Medicine, Veterinary University Vienna, have shown that fatal histomonosis can be protected by vaccination with an attenuated strain of the parasite; however commercially available prevention and curative strategies are still not available. Considering the state of emergency with numerous new outbreaks, devising novel approaches for protection becomes a necessity. A fundamental step towards this objective is to understand the flagellates virulence and attenuation mechanisms. For that purpose the current project focused on the comparative analyses of the entire set of soluble cellular and excreted proteins (proteomes and exoproteomes) from virulent and attenuated H. meleagridis strains. Considering the fact that the genome of this protozoan parasite is still not sequenced, de novo transcriptome analysis of both strains was undertaken to gain substantial genomic information on this parasite and facilitate the proteome analysis. The analyses employed in vitro cultures of virulent and attenuated strains of H. meleagridis that were derived from a single parasitic cell and grown in the background of a single bacterial strain. The proteome study identified a number of proteins differentially regulated between virulent and attenuated H. meleagridis parasites. The expression profiles often mirrored the adaptation process of the parasite to the in vitro conditions. Aside to proteins related to the adaptation to the in vitro growth, upregulation of several cysteine peptidases and post-translationally modified ubiquitous cellular proteins in the virulent strain, suggests their possible functional contribution towards the virulence. In addition to differences in the protozoan protein expression, differences in the expression of bacterial proteins, in regard to parasites phenotype, were demonstrated. This was especially prominent in the exoproteome analysis. The de novo transcriptome analysis of virulent and attenuated H. meleagridis resulted in the establishment of the reference transcriptome for H. meleagridis. The gene ontology analysis and data mining provided novel molecular insights into different aspect of cell biology, environmental adaptation and potential pathogenic mechanisms of H. meleagridis. The transcriptome data were used for a in sillico drug screen to identify potential anti-histomonal drugs. In conclusion, proteome and transcriptome data recruited from virulent and attenuated parasites facilitate better understanding of the parasites` molecular biology aiding the development of novel diagnostics and future research. Furthermore, the finding of bacterial differential protein regulation in respect to parasites phenotype supports the view of interdependence between H. meleagridis and the co-cultivating bacteria rather than a pure predator-prey relationship, whose understanding will be crucial to implement robust prevention strategies.