Samantha J. Emery

Eimeria trichosuri: Phylogenetic position of a marsupial coccidium, based on 18S rDNA sequences

Phylogenetic analysis of the genus Eimeria suggests that parasite and host have coevolved over broad evolutionary timescales. Here we extend this analysis by determining the 18S rDNA gene sequence of the marsupial coccidium, Eimeria trichosuri, and assessing its phylogenetic position relative to Eimeria from birds, reptiles and placental mammals. This analysis placed E. trichosuri clones in a clade that diverged before the major clade comprising species from placental mammals. The position of E.trichosuri is consistent with host phylogeny where marsupials represent an ancient evolutionary line that predates the placental mammal line.

Into the Wild: Dissemination of Antibiotic Resistance Determinants via a Species Recovery Program

Management strategies associated with captive breeding of endangered species can establish opportunities for transfer of pathogens and genetic elements between human and animal microbiomes. The class 1 integron is a mobile genetic element associated with clinical antibiotic resistance in gram-negative bacteria. We examined the gut microbiota of endangered brush-tail rock wallabies Petrogale penicillata to determine if they carried class 1 integrons. No integrons were detected in 65 animals from five wild populations. In contrast, class 1 integrons were detected in 48% of fecal samples from captive wallabies. The integrons contained diverse cassette arrays that encoded resistance to streptomycin, spectinomycin, and trimethoprim. Evidence suggested that captive wallabies had acquired typical class 1 integrons on a number of independent occasions, and had done so in the absence of strong selection afforded by antibiotic therapy. Sufficient numbers of bacteria containing diverse class 1 integrons must have been present in the general environment occupied by the wallabies to account for this acquisition. The captive wallabies have now been released, in an attempt to bolster wild populations of the species. Consequently, they can potentially spread resistance integrons into wild wallabies and into new environments. This finding highlights the potential for genes and pathogens from human sources to be acquired during captive breeding and to be unwittingly spread to other populations.

Proteomic analysis in Giardia duodenalis yields insights into strain virulence and antigenic variation.

Giardia duodenalis is a protozoan parasite of the small intestine in vertebrates, including humans. Assemblage A of G. duodenalis is one of the two discrete subtypes that infects humans, and is considered a zoonotic assemblage. Two G. duodenalis Assemblage A strains BRIS/95/HEPU/2041 and BRIS/83/HEPU/106, constituting virulent and control strains respectively, were analyzed in one of the first comparative shotgun proteomic studies performed in this parasite. Protein extracts were prepared using a multiplatform approach with both an in‐gel and in‐solution sample preparation to enable us to assess the complementarity for future Giardia proteomic studies. Protein analysis revealed that BRIS/95/HEPU/2041 possessed a wider and more varied repertoire of variant surface proteins (VSPs), which are hypothesized to be involved in host adaptation, immune evasion, and virulence. A total of 35 VSPs were identified, with three common to both strains, six unique to BRIS/82/HEPU/106, and twenty‐six unique to BRIS/95/HEPU/2041. Additionally, up to 25.6% of all differentially expressed proteins in BRIS/95/HEPU/2041 belonged to the VSP family, a trend not seen in the control BRIS/83/HEPU/106. Greater antigen variation in BRIS/95/HEPU/2041 may explain aspects of virulence phenotypes in G. duodenalis, with a highly diverse population capable of evading host immune responses.

Induction of virulence factors in giardia duodenalis independent of host attachment

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response.

Quantitative proteomic analysis of Giardia duodenalis assemblage A: A baseline for host, assemblage, and isolate variation.

Giardia duodenalis is a gastrointestinal protozoan parasite of vertebrates and is a species complex comprised of eight assemblages, with the zoonotic assemblage A one of two subtypes infective for humans. With increasing genomic and transcriptomic data publicly available through the centralized giardiaDB.org, we have quantitatively analyzed the proteomes of eight G. duodenalis assemblage A strains (seven A1 and one A2) to provide a proteomic baseline to complement the available data. A nonredundant total of 1197 subassemblage A1 proteins and 719 subassemblage A2 proteins were identified with an average of 770 proteins in each strain. The eight strains were also searched against both assemblage A genome sequences (subassemblage A1 and A2 genomes) and demonstrated subassemblage specific differences in protein identifications, especially for variable gene families. Substantial differences were observed in the numbers and abundance in the variable surface protein family, and two different variable surface protein expression profiles that were independent of host origin, subassemblage, or geographic origin. We hypothesize that this variation in surface antigen switching events may be related to karotype and chromosomal variation, which would indicate an assemblage‐independent mechanism of diversity generation in G. duodenalis. All MS data have been deposited in the ProteomeXchange with identifier PXD001272 (http://proteomecentral.proteomexchange.org/dataset/PXD001272).

The generation gap: Proteome changes and strain variation during encystation in Giardia duodenalis

The prevalence of Giardia duodenalis in humans is partly owed to its direct and simple life cycle, as well as the formation of the environmentally resistant and infective cysts. Proteomic and transcriptomic studies have previously analysed the encystation process using the well-characterised laboratory genomic strain, WB C6. This study presents the first quantitative study of encystation using pathogenically relevant and alternative assemblage A strains: the human-derived BRIS/82/HEPU/106 (H-106)and avian-derived BRIS/95/HEPU/2041 (B-2041). We utilised tandem MS/MS with a label-free quantitative approach to compare cysts and trophozoite life stages for strain variation, as well as confirm universal encystation markers of assemblage A. A total of 1061 non-redundant proteins were identified from both strains, including trophozoite- and cyst-specific proteomes and life-stage differentially expressed proteins. Additionally, 24 proteins previously classified in the literature as encystation-specific were confirmed as strain-independent markers of encystation. Functional cluster analysis of differentially expressed proteins saw significant overlap between strains, including protein trafficking and localisation in cysts, NEK kinase function, and carbohydrate metabolism in trophozoites. Two significant points of strain specific adaptations in cysts were also identified. B-2041 possessed major up-regulation of the ankyrin repeat protein 21.1 family compared to H-106. Furthermore, cysts of B-2041 retained near-complete VSP variant diversity between cysts and trophozoites, while H-106 lost 45% of its VSP variant diversity between life cycle stages, a constriction previously observed in studies of WB C6. This is the first report of strain variation in the cyst stage in G. duodenalis, and highlights cyst variation and its impacts on reinfection and life cycle success.

Analysis of rice proteins using SDS-PAGE shotgun proteomics

In this chapter we describe the workflow used in our laboratory to analyze rice leaf samples using label-free shotgun proteomics based on SDS-PAGE fractionation of proteins. Rice proteomics has benefitted substantially from successful execution of shotgun proteomics techniques. We describe steps on how to proceed starting from rice protein extraction, SDS-PAGE, in-gel protein digestion with trypsin, nanoLC-MS/MS, and database searching using the GPM. Data from these experiments can be used for spectral counting, where simultaneous quantitation of several thousand proteins can be obtained.

Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines

Giardia duodenalis is an intestinal parasite that causes 200-300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID10, 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid α-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID10 diverged from those in 713-r and WB-r (r ≤ 0.2), which were more similar to each other (r = 0.47). In 106-2ID10, a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.

Data from a proteomic baseline study of Assemblage A in Giardia duodenalis.

Eight Assemblage A strains from the protozoan parasite Giardia duodenalis were analysed using label-free quantitative shotgun proteomics, to evaluate inter- and intra-assemblage variation and complement available genetic and transcriptomic data. Isolates were grown in biological triplicate in axenic culture, and protein extracts were subjected to in-solution digest and online fractionation using Gas Phase Fractionation (GPF). Recent reclassification of genome databases for subassemblages was evaluated for database-dependent loss of information, and proteome composition of different isolates was analysed for biologically relevant assemblage-independent variation. The data from this study are related to the research article “Quantitative proteomics analysis of Giardia duodenalis Assemblage A – a baseline for host, assemblage and isolate variation” published in Proteomics (Emery et al., 2015 [1]).

Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis

ABSTRACT Understanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and α-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control.