Identification of critical molecules involved in host-parasite interactions of Tritrichomonas foetus

Abstract

Trichomonads have attracted considerable attention over the years as parasites of human and veterinary importance. Unlike the human infecting parasite Trichomonas vaginalis, Tritrichomonas foetus can be found in a range of distantly related mammals. Initially identified as a commensal organism of the nasal cavity and digestive tract of pigs, T. foetus later became notorious as a parasite of high veterinary importance, parasitising the urogenital tract of cattle and thus contribute to infertility. Within the last decade T. foetus was also identified as the causative agent of large bowel diarrhoea in domestic cats. Current prevalence has seen feline trichomoniasis as a global veterinary problem and bovine trichomoniasis appears to be re-emerging as a serious disease burden in cattle. Although it is not unusual for a parasite to have a wide host range, the diverse tissue tropism, routes of transmission and pathogenicity of T. foetus isolates within different hosts is confounding. This in part, could be attributed to the limited knowledge of molecules governing T. foetus host-specificity and biological variation. Recent comparative genotypic and transcriptomic analysis of T. foetus have revealed a high degree of similarity exists between T. foetus isolates. However, they have been unable to define the dynamic molecules involved in driving this host-specificity. Quantitative proteomic analysis could address this and may shed light on the molecules governing T. foetus host-specificity. Therefore, this thesis aimed to provide an in-depth quantitative proteomic analysis of T. foetus baseline proteomes from three distinct hosts to enhance the understanding of T. foetus. To achieve this, experimental investigations were performed into aspects of parasite genotypic variation and virulence, all of which have generated quantitative proteomic data. Firstly, quantitative proteomic analysis using gel-based, two-dimensional gel electrophoresis (2DE) was undertaken using whole cell lysates from two parasitic bovine and feline T. foetus isolates. Both isolates exhibited largely similar proteomes with only 24 spots identified as having a four-fold or greater change. However, deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes, which may help account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies and potentially highlights the importance of CPs as major determinants of parasite virulence. Following this initial investigation, the proteomes of a commensal porcine T. foetus isolate, as well as parasitic bovine and feline T. foetus isolates were characterised using a gel-free, label free 'bottom-up' approach. This constituted the first comprehensive baseline proteome of a commensal porcine T. foetus isolate. T. foetus isolates, from diverse hosts and unique host niches, were analysed using mass spectrometry to characterise both the commonality of their proteomes and to identify isolate-specific variations. A high level of similarity between the proteomes of T. foetus from unique hosts was confirmed. Of note, a greater degree of similarity was identified between the bovine and porcine isolates that constitutes the 'bovine genotype' with 67% of proteins shared (only ~60% of proteins were shared between the isolates from the 'bovine genotype' and 'feline genotype'). Interestingly, both quantitative proteomics and fluorogenic analysis identified a unique CP profile amongst all three T. foetus isolates, with markedly reduced CP activity in commensal porcine T. foetus whole cell lysates when compared with the parasitic T. foetus whole cell lysates, suggesting these CPs may influence the isolates' virulent phenotype. In addition, comparison of this gel-free method with the in-gel quantitative analysis highlighted both techniques were necessary to attain a complete picture of complex proteomes of T. foetus isolates. The third study investigated secretomes isolated from parasitic bovine and feline T. foetus. This fraction is directly involved in host-parasite interactions and contains proteins integral to the infection process. In-gel 2DE coupled with glycoprotein and phosphoprotein specific stains were applied to analyse the secretomes. This constitutes the first proteomic analysis of the secretome of T. foetus from any host origin. A range of unique virulent protein profiles were resolved between isolates including those of the multigene CP family. In particular CP8 was more abundant in the secretome of the bovine isolate, while CP7 was more abundant in the feline isolate. Furthermore, a number of proteins involved in facilitating isolate-specific adaptations that may impact reinfection success in subsequent generations were identified. These included proteins involved in combating host defence systems, such as thioredoxin peroxidase and dual functioning proteins, such as fructose-1,6-bisphosphate with roles in adhesion. Furthermore, the dynamic post-translational modifications, glycosylation and phosphorylation, were identified on approximately 64% (glycosylation) and 30% (phosphorylation) of bovine secretomes and 41% (glycosylation) and 30% (phosphorylation) of feline secretomes and are suggested to contribute to the unique secretome profiles of bovine and feline T. foetus. Overall, this thesis has created a platform to advance future T. foetus research in the fields of epidemiology and drug target discovery. The three proteomes generated for two parasitic T. foetus isolates and a newly isolated commensal isolate, cultured in identical environmental conditions, provide a comprehensive baseline towards understanding host-specific differences between isolates originating from the three unique hosts. This work demonstrates that a range of quantitative proteomic approaches are suitable for analysis of T. foetus, all of which provide important insights into key aspects of parasite biology. These studies provide an important proteomic complement for transcriptomic data currently available in the literature, which is necessary for undertaking a systems biology approach to understanding T. foetus.

Date of Award	2017
Original language	English