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Quantitative proteomic analyses of isolate variation and virulence in Giardia duodenalis

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posted on 28.03.2022, 13:38 by Samantha Jane Emery
Giardia duodenalis is a parasitic protozoan with a global human infection burden of 250 million, and is therefore the largest parasitic cause of diarrheal disease worldwide. Though some cases are asymptomatic, giardiasis can be acute and chronic, with post-infection sequellae including irritable bowel syndrome, chronic fatigue, obesity and type II diabetes.Importantly, Giardia is problematic in children under the age of five, causing ill-thrift and failure-to-thrive. In addition, diarrheal diseases including Giardia constitute the second leading cause of mortality for this age category. Giardia has a direct life cycle, where infective, tetranucleated cysts are transmitted via the faeco-oral route, and then excyst in the duodenum into virulent, flagellated trophozoites. The prevalence of the parasite is also due to its wide host range, with zoonotic transfer from wild, livestock and domestic animal species to humans. Efforts continue to define the mechanisms of virulence and pathophysiology, as more research is needed to elucidate the relationship between host and parasite factors. Advances in genetic epidemiology have defined clear assemblages that segregate phylogenetically according to host range, and multiple assemblage and subassemblage genome sequences are now available. These genome sequences have provided the databases necessary for bottom-up, or shotgun, proteomics, and as such have expanded possibilities for quantitative analyses in this parasite. This thesis aimed to provide a thorough quantitative proteomic foundation to enhance the Giardia research field both biologically and technically. To achieve this, the thesis consists of four experimental investigations into aspects of parasite variation and virulence, all of which have generated quantitative proteomic data. Firstly, two different protein sample preparation and fractionation methods were compared for label-free quantitative proteomics. These were applied to two G. duodenalis assemblage A1 isolates with different phenotypes, in order to investigate possible sources of isolate variation. The optimised protocol generated from this initial investigation was applied in later studies, which are also contained within this thesis. In addition, phenotypes associated with pathogenicity correlated with up-regulation of known virulence factors in Giardia.Following this initial investigation, quantitative data was generated using the same label free approach for eight assemblage A isolates, which constituted the first comprehensive proteomic baseline for this taxonomic group. Isolates of diverse host, geographic and subassemblage origins were analysed using mass spectrometry to characterise their common proteomes and isolate-specific variations. In addition, both the A1 and A2 subassemblage genome databases were evaluated for peptide to spectrum matching, which demonstrated the importance of subassemblage databases to improve identifications from the Giardia variable genome. The third study investigated isolate variation in the biological context of the process of differentiation in G. duodenalis. Label-free quantitative proteomics was used to analyse the proteomes of cysts and trophozoites from two genome-alternate subassemblage A1 isolates. This is the first post-genomic analysis of the life cycle beyond the genome isolate,WB. A range of isolate-independent, universal encystation markers were identified, as well as several indications of isolate-specific life-cycle adaptations which may impact reinfection success in subsequent generations. Finally, the last experiment in this thesis investigated disease induction using in vitro host parasite interaction models between intestinal epithelial cell (IEC) lines and trophozoites.We used isobaric Tandem Mass Tags (TMT) to sensitively quantitate changes introphozoites which were either allowed to attach to host-cell monolayers, or were exposed to host-cell secretions alone. This is the first use of TMT label technologies for quantitative proteomics in Giardia. This has demonstrated that distinct protein cascades are induced by both levels of host-signals, and also that induction of virulence factors is not dependent on parasite attachment to host cells. Through these experiments, this thesis demonstrates that a range of quantitative proteomic approaches are suitable for G. duodenalis, all of which are capable of providing important insight into key aspects of parasite biology. These studies provide an important proteomic complement for genomic and transcriptomic data currently available in the literature, which is necessary for undertaking a systems biology approach to understanding Giardia.


Table of Contents

1. Quantitative proteomics in Giardia duodenalis -achievements and challenges -- 2. Proteomic analysis in Giardia duodenalis yields insights into strain virulence and antigenic variation -- 3. Quantitative proteomics analysis of Giardia duodenalis Assemblage A - a baseline for host, assemblage and isolate variation -- 4. The generation gap: proteome changes and strain variation during encystation in Giardia duodenalis -- 5. Differential stimulation of Giardia duodenalis trophozoites between host soluble signals and host cell attachment during in vitro interactions -- 6. General discussion.


Includes bibliographical references Thesis by publication.

Awarding Institution

Macquarie University

Degree Type

Thesis PhD


PhD, Macquarie University, Faculty of Science and Engineering, Department of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award


Principal Supervisor

Paul Haynes


Copyright Samantha Jane Emery 2015. Copyright disclaimer: http://mq.edu.au/library/copyright




1 online resource (183 pages) illustrations

Former Identifiers

mq:68947 http://hdl.handle.net/1959.14/1249052