ABSTRACT: Whole genome sequencing of dog specific assemblages C and D of Giardia duodenalis from single and pooled cysts indicates host associated genes
Project description:Despite of Giardia duodenalis being one of the most commonly found intestinal pathogens in humans and animals, little is known of the host-parasite interactions in natural hosts. Therefore, the objective of this study was to investigate the intestinal response in calves following a G. duodenalis infection, using a bovine high-density oligo microarray to analyze global gene expression in the small intestine. The resulting microarray data suggested a decrease in inflammation, immune response and immune cell migration in infected animals, which was examined in more detail by quantitative real-time PCR on a panel of cytokines combined with histological analyses. The cytokine transcription levels showed a trend of down regulated expression in infected animals compared to the negative controls, best seen in jejunum for IL-6 and IL-8 and statistically significant for IL-17, IL-13 and IFN-?. No increased immune cell recruitment could be seen after infection, as well as no intestinal pathologies, such as villus shortening or increased levels of apoptosis. Key regulators in this intestinal response seem to be the nuclear peroxisome proliferator-activated receptors alpha (PPARA) and gamma (PPARG), for which an up-regulated expression was seen on microarray and qRT-PCR data. The activation of PPARs can exert an anti-inflammatory effect with inhibition of pro-inflammatory cytokines and a decrease in cell recruitment. . How the PPARs are activated during a Giardia infection still needs to be further elucidated. Eight male Holstein calves aged two to four weeks old were used for the trial. Prior to arrival, all animals were screened for the presence of Giardia cysts in their faecal samples. After confirming their negative status for all these pathogens, four of the animals were randomly chosen and placed in a G. duodenalis contaminated environment, whereas the four remaining animals were kept as negative controls in separate G. duodenalis-free stables. All calves in the study received the same commercial milk replacer. After three weeks, the presence or absence of a G. duodenalis infection was confirmed by IFA on faecal samples after which the animals were euthanized. Changes in gene expression profiles induced by Giardia duodenalis infection were compared using a high-density 60mer bovine oligo microarray.
Project description:Giardia duodenalis is a protozoan parasite of a wide range of vertebrates and one of the leading causes of gastroenteritis worldwide. G. duodenalis is a species complex of 8 assemblages with the zoonotic assemblage A as one of two discrete subtypes that is infective for humans. With increasing genomic and transcriptomic data now publicly available through the centralised giardiaDB.org, we have quantitatively analysed the proteomes of 8 G. duodenalis assemblage A strains (7 A1 and 1 A2) to provide a comprehensive proteomic baseline to complement these studies. Protein analysis identified a non-redundant total of 1220 proteins with an average of 764 proteins in each strain. At least 10% of all proteins identified were from the 4 protein families in the G. duodenalis variable genome, and substantial differences in number and abundance profiles in the Variable Surface Protein (VSP) family was observed. We also searched the 8 strains against both assemblage A genomes (subassemblage A1 and A2 genomes) and showed losses in protein identifications, especially for protein identifications associated with Giardia variable gene families which are sub-assemblage specific. We observed two expression profiles of VSPs within Giardia, which was independent to host origin, subassemblage, geographic origin and introduction to axenic culture and may indicate variation in surface antigen switching events and population heterogeneity. We hypothesise this variation may be related to karotype and chromosomal variation, which would indicate an assemblage-independent mechanism of variation in G. duodenalis.
Project description:Homeostatic interactions between the host and its resident microbiota is important for normal physiological functions and if altered, it could lead to dysbiosis, a change in the structure and function of the microbiota, and as a result to various pathophysiologies. Altered structure in bacterial community is associated with various pathophysiologies, but we are just beginning to understand how these structural changes translate into functional changes. Environmental factors including pathogenic infections can lead to altered interactions between the host and its resident microbiota. We used microarray analysis and a C. elegans model system to gain insights on the mechanisms of functional changes in host-commensal bacteria interaction in the presence or absence of G. duodenalis and identified expression pattern in commensal bacteria that are characteristic of homeostatic and dysbiotic interactions. E. coli HB101 exposed to C. elegans in the presence or absence of G. duodenalis conditioned S-basal complete media for 24 hours were used for RNA extraction and hybridization on Affymetrix microarrays. We collected expression data for E. coli HB101, E. coli HB101 exposed to C. elegans, E. coli HB101 exposed to Giardia conditioned media, and E. coli HB101 exposed to both C. elegans and Giardia conditioned media.
Project description:Giardia duodenalis is a protozoan parasite responsible for gastroenteritis in vertebrates, including humans. The prevalence of G. duodenalis is partly owed to its direct and simple life cycle, as well as the formation of the environmentally resistant and infective cysts. Several proteomic and transcriptomic studies have previously analysed global changes during the encystation process using the well-characterised laboratory isolate and genome strain, WBC6. To expand current comparative analyses, this study presents the first quantitative global study of encystation using pathogenically relevant and alternative assemblage A strains: the human-derived BRIS/82/HEPU/106 and avian-derived BRIS/95/HEPU/2041. We have utilised tandem MS/MS with a label-free quantitative approach to compare cysts and trophozoite life stages between strains for variation, as well as confirm universal encystation markers of Assemblage A.
Project description:Giardia gene expression is being examined using Serial Analysis of Gene Expression (SAGE) to monitor genome-wide levels of messenger RNA (mRNA) expression throughout Giardia's life cycle. Examination of genome-wide gene expression patterns will provide a coherent picture of activation and inactivation of biological pathways. This research will provide a comprehensive understanding of changes in giardial gene expression in response to important host physiological signals and will serve as a valuable model for study of other parasites and complex eukaryotes, such as yeast and animals. It will provide a dynamic framework, in the context of the life cycle, to the annotation of the Giardia genome, including the detection of unpredicted genes via detection of their tags. Keywords: Giardia, SAGE, trophozoites, encystation, excystation, cysts
Project description:To investigate the magnitude of the transcriptional changes occurring during the life cycle of Giardia lamblia we compared the transcriptome of trophozoites and cysts. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. In cysts which have lost infectivity the transcriptome is further depleted. In contrast, exposure of cysts to conditions which promote excystation induced transcription. Six cysts and three trophozoites life cycle stages were analyzed.
Project description:To investigate the magnitude of the transcriptional changes occurring during the life cycle of Giardia lamblia we compared the transcriptome of trophozoites and cysts. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. In cysts which have lost infectivity the transcriptome is further depleted. In contrast, exposure of cysts to conditions which promote excystation induced transcription.
Project description:In this study we compare at the transcriptomic level, how human gene expresion changes over time when Caco-2 cells co-incubated with Giardia cysts.
Project description:Giardia duodenalis is the protozoan agent responsible for the majority of parasitic gastroenteritis in humans worldwide. Disease pathology includes malabsorption and maldigestion, cell apoptosis and small intestinal barrier dysfunction, which occurs in absence of known toxins, cell invasion and overt inflammation. To understand pathogenesis, host-parasite in vitro interaction models provide global insights into disease induction and virulence. Hence, we performed the first proteomic analysis of G. duodenalis trophozoites interacted with intestinal epithelial cells (IECs, HT-29) for 6 hours, and compared it to trophozoites exposed to cell-free fractions of host-soluble signals. This has allowed us to demonstrate that distinct and independent protein cascades are induced by host attachment compared to host soluble signals. We utilised a tandem mass tag (TMT) approach and evaluated it as quantitative proteomics for the first time in Giardia.