Dataset Information

Proteomics of Metronidazole Resistance in three seminal lines of Giardia duodenalis

ABSTRACT: Metronidazole (Mtz) is the frontline chemotherapeutic for a variety of anaerobic pathogens, including the gastroenterinal parasitic protist, Giardia duodenalis. However, treatment failure and clinical resistance is common and increasing. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in in vitro, isogenic cultures. These mechanisms centrally involve regulation of glycolytic and antioxidant oxidoreducatases, however this is inconsistent across isolate lines, likely incomplete, and further complicated by phenotypic plasticity in Giardia, which regains Mtz susceptibility after cessation of drug selection, or via passage through the life cycle. Global, quantitative approaches are required to reconstruct complex Mtz resistance phenotypes, resolve isolate variability, and explore unresolved hypotheses regarding post-translational modifications as the regulators of this phenotype. Our study addresses these gaps by comprehensively characterising proteomic changes in three seminal Mtz resistant lines compared to their isogenic, Mtz-susceptible, parental lines. Using quantitative proteomics, we have calculated differentially expressed proteins (DEPs) in MtzR lines, and probed via Western blot changes in protein acetylation, methylation, ubiquitination and phosphorylation post-translational modifications (PTMs). Finally, we have also performed the first controlled, longitudinal study

INSTRUMENT(S): Q Exactive

ORGANISM(S): Giardia Lamblia Atcc 50803

TISSUE(S): Trophozoite

DISEASE(S): Giardiasis

SUBMITTER: Samantha Emery

LAB HEAD: Aaron Jex

PROVIDER: PXD007183 | Pride | 2018-02-21

REPOSITORIES: Pride

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Publications

Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis.

Emery Samantha J SJ Baker Louise L Ansell Brendan R E BRE Mirzaei Mehdi M Haynes Paul A PA McConville Malcom J MJ Svärd Staffan G SG Jex Aaron R AR

GigaScience 20180401 4

<h4>Background</h4>Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarel ...[more]

PMID: 29688452

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Project description:Giardia causes more episodes of illness worldwide than any other parasite. It is a flagellated cyst-forming enteric pathogen that inhabits the lumen of the small intestine and although it is clear that more and less virulent strains do occur, pathogenesis and virulence of Giardia remains poorly understood. Identification of Giardia virulence factors is highly desirable since their expression would be the best indicator for determining disease outcome. The advent of quantitative proteomics has been timely in investigating which proteins are likely to be secreted virulence factors. Here we successfully apply such analyses to the whole parasite and to the supernatants derived from the parasite, in order to ascertain a repertoire of secreted proteins. In our initial analysis we compared replicates of Giardia cells and culture supernatants from the two major lineages which cause human disease – assemblage A and assemblage B. The genome of Giardia is believed to contain open reading frames which could encode as many as 6000 proteins. In our study we confirm expression of ~1600 proteins from each assemblage, the vast majority of which being common to both lineages. To look for actual enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet. This simple method defines a small group of enriched proteins, putatively secreted at a steady state by cultured growing Giardia trophozoites of both assemblages. This secretome contains a high proportion annotated to have N’ terminus signal peptides, with some showing evidence of having recently evolved under strong positive selective pressure. The most abundant secreted proteins include known virulence factors such as the Cathepsin B cysteine proteases and members of a Giardia superfamily of Cysteine Rich Proteins which comprises VSPs, HCMPs and a new class of virulence factors, the Giardia Tenascins. Since we also find that physiological function of human enteric epithelial cells can be disrupted by soluble factors even in the absence of the trophozoites we are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia derived soluble mediators.

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.

Dataset Information

Proteomics of Metronidazole Resistance in three seminal lines of Giardia duodenalis

Publications

Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis.

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