The relationship between the infection of Toxoplasma gondii and Alzheimer’s disease

Toxoplasma gondii is a zoonotic parasite that commonly causes infections in a variety of warm-blooded animals including humans. This parasite causes a disease referred to as toxoplasmosis. T. gondii infections are known to have a significant impact on the host brain since the parasite can penetrate the blood-brain barrier and extensively colonize the central nervous system (CNS) for an extended period. Attacks of the brain tissue by the parasite can cause both neurological and structural damage. The parasite infects the brain, forms a cyst and affects several parts of the brain, including the amygdala, hippocampus, other cortical regions, and cerebellum; this can result in alterations in the hosts' behaviour. Studies have proposed that infections caused by T. gondii pose a risk in the development of several neuropsychiatric disorders like Alzheimer’s disease, Schizophrenia, and Parkinson's disease. The aims of this study were to conduct a pilot study on the association between T. gondii infections and Alzheimer's disease using tissue samples from the Manchester Brain Bank (MBB) and to investigate the effect of the parasite on oxidative stress in cultured human brain cellsAs a pilot study, previously prepared slides from 124 brain sections from Alzheimer’s patients and controls were examined for the presence of Toxoplasma gondii cysts. Despite a 10% prevalence of T. gondii in humans in the UK, no cysts were detected in any of the sections. It was concluded that unsustainably large tissue samples would be required from MBB to pursue an investigation using this approach.Instead, a line of investigation was pursued that explored the interactions between T. gondii infections and oxidative stress (a feature of neurological diseases such as Alzheimer's) using the development of a model system. SH-SY5Y cells were used as culture models for neuronal cells and flow cytometry and MTT assays were conducted to determine cell viability. To evaluate the effects of oxidative stress, differentiated and undifferentiated SH-SY5Y cells, were examined for cell viability and the effects of infection by T. gondii type I and II were investigated. When high doses of H2O2 [250 and 500uM] were used for 24 hours inundifferentiated SH-SY5Y cells, there was a reduction in the cell viability to 8.66% and 4.66%respectively. Conversely, when SH-SY5Y cells (differentiated) were subjected to high dosesof H2O2 (250 and 50

SPARC 2017 retrospect & prospects : Salford postgraduate annual research conference book of abstracts

Welcome to the Book of Abstracts for the 2017 SPARC conference. This year we not only celebrate the work of our PGRs but also the 50th anniversary of Salford as a University, which makes this year’s conference extra special. Once again we have received a tremendous contribution from our postgraduate research community; with over 130 presenters, the conference truly showcases a vibrant PGR community at Salford. These abstracts provide a taster of the research strengths of their works, and provide delegates with a reference point for networking and initiating critical debate. With such wide-ranging topics being showcased, we encourage you to exploit this great opportunity to engage with researchers working in different subject areas to your own. To meet global challenges, high impact research inevitably requires interdisciplinary collaboration. This is recognised by all major research funders. Therefore engaging with the work of others and forging collaborations across subject areas is an essential skill for the next generation of researchers

Biphasic zinc compartmentalisation in a human fungal pathogen

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2 Delta growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrtl/Zrt2-cellular import, followed by Zrcl-dependent intracellular compartmentalisation

Zincosome formation is Zrc1 dependent.

<p>(A) Zincosome screen. Wild type, ZnT deletion mutants and <i>zrc1</i>Δ+<i>ZRC1</i> strains were pulsed with 25 μM zinc for 20 minutes and zincosome fluorescence determined by staining with zinquin. Prepulsed cells were also stained as control. Experiment was performed at least twice in duplicates and all data normalised to the post-pulse value of wild type. ANOVA was first performed on initial (pre-normalised data). Asterisks indicate statistical significance compared to wild type and to relevant deletion mutant ** P <0.01. (B) As panel A, except zinquin fluorescence kinetics was determined by flow cytometry. Experiment performed three times. <i>zrc1</i>Δ exhibits significantly reduced zinquin fluorescence compared to wild type and revertant at 20 minutes <i>P</i> < 0.001, ANOVA.</p

Growth of <i>zrt2</i>Δ strains is specifically rescued by excess zinc.

<p>Indicated strains were cultured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007013#ppat.1007013.g001" target="_blank">Fig 1</a> with zinc, iron, manganese (100 μM) or copper (10 μM) and growth kinetics measured over 36 h in a microtitre plate. Experiment performed twice in triplicate. Iron had a moderate inhibitory effect on <i>C</i>. <i>albicans</i> growth. Note that only zinc rescued growth of <i>zrt2</i>Δ strains.</p

pH-dependent functionality and regulation of Zrt1 and Zrt2 in <i>C</i>. <i>albicans</i>.

<p>(<b>A</b>) Zrt2 is essential in acidic medium. Indicated strains, precultured in YPD, were washed and cultured in SD (YNB+glucose) medium alone, or supplemented with 100 μM ZnSO4 or with 50 mM HEPES pH 7.4. Asterisks indicate statistical significance compared to the wild type; # indicates statistical significance compare to the <i>zrt2</i>Δ in SD; P <0.05. (<b>B</b>) <i>ZRT1</i> promoter activity is pH regulated and <i>ZRT2</i> is constitutively expressed under zinc limitation. (<i>P</i>_<i>ZRT1</i>-GFP and <i>P</i>_<i>ZRT2</i>-GFP reporter strains in LZM buffered to indicated pH values). LZM was used due to lower green autofluorescence. Experiment performed three times. (<b>C</b>) Double deletion of <i>ZRT1</i> and <i>ZRT2</i> precludes growth at both acidic and neutral alkaline pH. Strains were cultured as in (A) and growth kinetics measured over 48 h in a microtitre plate. Experiment performed twice in triplicate.</p

Relationship between Zrc1, zincosomes and zinc tolerance.

<p>(A) Cells were challenged with potentially toxic zinc (1 mM), stained with zinquin and fluorescence determined. <i>P</i> < 0.0001 compared to wild type and revertant. (B) Micrographs of cells treated as in A. Note that <i>zrc1</i>Δ is highly defective for zincosome formation in response to 1 mM ZnSO₄ –a condition under which wild type, but not <i>zrc1</i>Δ cells can grow (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007013#ppat.1007013.s008" target="_blank">S7 Fig</a>).</p

<i>C</i>. <i>albicans</i> Zrt2 is required for kidney colonisation in the presence of functional calprotectin.

<p>Indicated mice strains were infected with indicated fungal strains and kidney colonisation determined by plating CFUs on day one and day three post-infection. At day three post-infection, <i>C</i>. <i>albicans</i> wild type kidney fungal burden had increased significantly by 6.5-fold (<i>P</i> = 0.034), Deletion of <i>ZRT2</i> precluded an increase in kidney fungal burden between day one and day three post-infection (<i>P</i> = 0.597), asterisk. Complementation of <i>zrt2</i>Δ with a single copy of <i>ZRT2</i> restored kidney colonisation at day three (4.5-fold higher than at day one, <i>P</i> = 0.004).</p

Kinetics of zincosome formation in <i>C</i>. <i>albicans</i>.

<p>Cells were incubated overnight in YNB-zinc-dropout medium (SD0) to deplete zincosomes and pulsed with 25 μM ZnSO₄ for indicated time points. Cells were then stained with zinquin to probe for zincosomal zinc and the cell wall stained with Concanavalin A conjugated to Alexa-647. Left hand column shows false colour overlay of cell wall (cyan) and zincosomes (magenta). Right hand column shows DIC; Experiment performed three times and representative images shown.</p