Project description:Giardia lamblia is one of most common agents causing persistent abdominal symptoms in developed and developing countries. There are several diagnostic methods for Giardia infection, but none are optimal. In this study our aim was to find a new method based on Giardia microRNA (miRNA) that would contribute to the currently available diagnostic methods of giardiasis. Profiling Giardia small RNAs by deep sequencing revealed that the previously reported putative miR5 and miR6 are expressed in several G. lamblia isolates. These miRNAs were later tested by PCR in duodenal biopsies from 8 patients with positive pathology for giardiasis, while gastric biopsies served as matched negative controls. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. All 8 duodenal samples of patients with histologically proven G. lamblia infection were positive for Giardia miR5 with a mean Ct of 23.7. These results were superior to Ct levels of G. lamblia DNA, which were 26.3 (p=0.004). The miR6 results were close to negative. All 10 gastric biopsies were negative for miR5. Stool studies showed 90% specificity but only 50% sensitivity in diagnosing giardiasis using miR6. The results of miR5 in stool were even less accurate. In conclusion, miR5 testing for Giardia infection in duodenal biopsies, may be a breakthrough method for diagnosis of giardiasis. It seems to be superior to G. lamblia DNA in duodenal biopsies. It would be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies and duodenal aspirates from patients with persistent abdominal symptoms.
Project description:Background: Cyst formation in parasitic protist Giardia duodenalis is critical to its transmission. Proteomic data is essential for the system-level understanding of the molecular mechanisms managing stage transition from a flagellated, binucleate trophozoite to a tetra-nucleate cyst. The current encystation proteome quantifies 16% of predicted protein coding genes and lacks complete coverage of the developmental transition. Given recent gains in mass spectrometry instrumentation, we have generated significantly improved proteomes for Giardia across encystation, providing framework for transcriptome-proteome correlation drawing insights into molecular networks modulating encystation in Giardia. Results: We have reproducibly identified 3,796 (64.6% of Giardia proteins) and quantified 3,402 proteins (56.12 % of Giardia proteins), tripling the currently published encystation proteome coverage. This includes timepoints from standard trophozoite growth (TY), during low-bile encystation priming (LB), mid-encystation (EC), and enriched, mature cysts (C), making this the first proteome study across the entire encystation process. This is also the first proteome analysis of trophozoites during low bile priming prior to in vitro induction of encystation, and analysis of mature cyst proteomes. Almost 28% (1,102 proteins) of the proteome is differentially changed during encystation including within proteasomal machinery, metabolic pathways and lipid metabolism, indicating a multimodal regulation in encystation. To further understand novel observations in lipid metabolism, we used hidden markov model (HMM) based domain prediction strategies and i-Tasser based structural modelling to provide evidence for the presence of three families of lipid transporters (LTP’s) StARkin (steroidogenic acute regulatory protein–related lipid transfer), ORP/Osh (Oxysterol Binding protein related protein), GLTP (Glycosphingolipid transfer protein) in Giardia. Finally, we performed functional interaction studies using in silico docking of newly identified LTP’s with corresponding lipid ligands to validate their interaction. This identified a shift in lipid species dependency in Giardia as the parasite progress towards cyst formation.