University of Technology, Sydney. Faculty of Science.Dientamoeba fragilis is a parasite of the human gastrointestinal tract that is often described as a cause of illness. Compared to other enteric protozoa of humans, information on D. fragilis is limited. Molecular studies on D. fragilis are particularly scarce and only three protein coding gene sequences are available for D. fragilis in Genbank. There is no published genome sequence for D. fragilis and knowledge on its metabolism, life cycle and mechanisms of pathogenicity is also lacking. Further research into these aspects of D. fragilis is warranted. Development of new diagnostic tests for D. fragilis has been a growing trend in recent years, though with a distinct focus on molecular diagnostics. Less attention has been paid to the development of antibody-based diagnostic tests for D. fragilis. While molecular assays are extremely sensitive and specific, antibody based assays are more applicable to routine diagnostic settings, mostly due to issues of cost and feasibility. Given the increasing importance of D. fragilis to human health, further research into the development of antibody-based diagnostic tests for D. fragilis is also warranted.
This project was designed to address the shortcomings described previously. One major objective of this project was to sequence the transcriptome of D. fragilis (aim 2 of this project). This would dramatically increase the volume of molecular data available for D. fragilis and encourage future research on this organism. Another major objective was to develop polyclonal antibodies against a specific D. fragilis antigen which could be implemented in a diagnostic test (aim 3 of this project). To fulfil aims 2 and 3 of this project, long term cultures of D. fragilis would also be required to provide the necessary starting materials; D. fragilis RNA’s for sequencing and D. fragilis cells for the evaluation polyclonal antibodies. Therefore, the first objective was to improve current techniques for the cultivation of D. fragilis by evaluating various xenic culture systems for their ability to support D. fragilis growth (aim 1 of this project).
Several media formulations were evaluated for their ability to support D. fragilis growth at various temperatures and atmospheric conditions. Different temperatures (from ambient room temperature to 42̊C) and various atmospheric conditions (aerobic, anaerobic and microaerophilic conditions) were also evaluated. Trophozoites were inoculated into fresh media under varying conditions and manual cell counts were performed daily to determine the media and conditions which supported optimal growth. Once optimal culture conditions were established, total RNA was extracted from culture sediments using TriSure reagent. As the culture systems employed were xenic (also containing a mixture of bacterial support flora), eukaryotic mRNA’s were enriched from total RNA extracts using oligo-(dT) cellulose chromatography. Purified eukaryotic mRNA’s were then subjected to next-generation sequencing. Upon assembly of the D. fragilis transcriptome, a suitable target protein was selected for polyclonal antibody development based on information obtained from the scientific literature. A homologue for this protein was identified in the D. fragilis transcriptome using blast searches. This nucleotide sequence then was translated into its amino acid sequence and immunogenic peptides were designed using this sequence as a template. Peptides were synthesised and conjugated to keyhole limpet hemocyanin for immunisation of rabbits. Anti-peptide antibodies were then purified from the resulting rabbit antisera by immunoaffinity chromatography. The resulting antibodies were evaluated by indirect fluorescent antibody testing (IFAT).
Loeffler’s slope medium incubated at between 37̊C and 42̊C under microaerophilic conditions, supported significantly better growth of D. fragilis trophozoites compared to other media formulations and conditions. Next-generation sequencing of D. fragilis mRNA's generated 6,946 unique contigs. These contigs had an average length of 710 nucleotides with an average GC content of 34%. For the development of polyclonal antibodies, a protein from the enolase family was selected as a suitable target. Multiple enolase homologues were detected in the D. fragilis transcriptome and one of these was used as a template for the design of immunogenic peptides. Two of seven immunogenic peptides designed from this protein gave rise to two respective antibody preparations which reacted strongly to the surface of fixed D. fragilis trophozoites, as determined by the strong fluorescent signal observable in IFAT's.
Each of the project aims was addressed succinctly and succesfully, to generate new knowledge on D. fragilis. Various culture systems were evaluated for their ability to support the growth of D. fragilis, and the optimal conditions (among those conditions tested) were determined. This knowledge will assist future investigators in the study of D. fragilis, as culture systems are an essential tool in the study of microorganisms. With the completion of the first D. fragilis transcriptome, thousands of unique nucleotide sequence can now be made available to future investigators. This work currently represents the largest scientific contribution to our understanding of D. fragilis molecular biology. Polyclonal antibodies against a surface antigen of D. fragilis were developed as a result of this project. These antibodies show great promise for implementation in an antibody-based diagnostic test for D. fragilis. Overall, this PhD project has contributed significantly to current knowledge on D. fragilis. It is hoped that this work will stimulate future research on D. fragilis so that our understanding of this organism continues to improve
