Dagmar S. Urgast

Fungal Iron Availability during Deep Seated Candidiasis Is Defined by a Complex Interplay Involving Systemic and Local Events

Nutritional immunity – the withholding of nutrients by the host – has long been recognised as an important factor that shapes bacterial-host interactions. However, the dynamics of nutrient availability within local host niches during fungal infection are poorly defined. We have combined laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS), MALDI imaging and immunohistochemistry with microtranscriptomics to examine iron homeostasis in the host and pathogen in the murine model of systemic candidiasis. Dramatic changes in the renal iron landscape occur during disease progression. The infection perturbs global iron homeostasis in the host leading to iron accumulation in the renal medulla. Paradoxically, this is accompanied by nutritional immunity in the renal cortex as iron exclusion zones emerge locally around fungal lesions. These exclusion zones correlate with immune infiltrates and haem oxygenase 1-expressing host cells. This local nutritional immunity decreases iron availability, leading to a switch in iron acquisition mechanisms within mature fungal lesions, as revealed by laser capture microdissection and qRT-PCR analyses. Therefore, a complex interplay of systemic and local events influences iron homeostasis and pathogen-host dynamics during disease progression.

Isotope ratio measurements in biological tissues using LA-ICP-MS – possibilities, limitations, and perspectives

This article describes the advantages and possibilities of using LA-ICP-MS for isotope ratio measurements, briefly comparing quadrupole, sector-field and multi-collector systems. It also discusses problems and limitations of the respective set-ups. A special focus lies on biological samples from stable isotope tracer experiments and on spatially resolved isotope ratio measurements including imaging of isotope ratios. The article explores if there are valid reasons why until now almost no one used LA-ICP-MS to image isotope ratios. It wants to encourage the ICP-MS community to realise the potential of using LA-ICP-MS for isotope ratio measurements in biological samples and to hopefully spur further developments and applications.

Multi-elemental bio-imaging of rat tissue from a study investigating the bioavailability of bismuth from shotgun pellets

AbstractIn recent years, bismuth has been promoted as a “green element” and is used as a substitute for the toxic lead in ammunition and other applications. However, the bioavailability and toxicity of bismuth is still not very well described. Following a hunting accident with bismuth-containing shots, a bioavailability study of bismuth from metal pellets inoculated into rat limb muscles was carried out. Bismuth could be found in urine and blood of the animals. Bio-imaging using laser ablation ICP-MS of thin sections of the tissue around the metal implant was carried out to find out more about the distribution of the metal diffusing into the tissue. Two laser ablation systems with different ablation cell designs were compared regarding their analytical performance. Low concentrations of bismuth showing a non-symmetrical pattern were detected in the tissue surrounding the metal implant. This was partly an artefact from cutting the thin sections but also bio-mobilisation of the metals of the implant could be seen. An accumulation of zinc around the implant was interpreted as a marker of inflammation. Challenges regarding sample preparation for laser ablation and bio-imaging of samples of diverse composition became apparent during the analysis. Figure 

Host-Imposed Copper Poisoning Impacts Fungal Micronutrient Acquisition during Systemic Candida albicans Infections

Nutritional immunity is a process whereby an infected host manipulates essential micronutrients to defend against an invading pathogen. We reveal a dynamic aspect of nutritional immunity during infection that involves copper assimilation. Using a combination of laser ablation inductively coupled mass spectrometry (LA-ICP MS) and metal mapping, immunohistochemistry, and gene expression profiling from infected tissues, we show that readjustments in hepatic, splenic and renal copper homeostasis accompany disseminated Candida albicans infections in the mouse model. Localized host-imposed copper poisoning manifests itself as a transient increase in copper early in the kidney infection. Changes in renal copper are detected by the fungus, as revealed by gene expression profiling and fungal virulence studies. The fungus responds by differentially regulating the Crp1 copper efflux pump (higher expression during early infection and down-regulation late in infection) and the Ctr1 copper importer (lower expression during early infection, and subsequent up-regulation late in infection) to maintain copper homeostasis during disease progression. Both Crp1 and Ctr1 are required for full fungal virulence. Importantly, copper homeostasis influences other virulence traits—metabolic flexibility and oxidative stress resistance. Our study highlights the importance of copper homeostasis for host defence and fungal virulence during systemic disease.

Biosynthesis of the fluorinated natural product nucleocidin in Streptomyces calvus is dependent on the bldA specified Leu‐tRNAUUA molecule

Nucleocidin is one of the very few natural products known to contain fluorine. Mysteriously, the nucleocidin producer Streptomyces calvus ATCC 13382 has not been observed to synthesize the compound since its discovery in 1956. Here, we report that complementation of S. calvus ATCC 13382 with a functional bldA‐encoded Leu‐tRNAUUA molecule restores the production of nucleocidin. Nucleocidin was detected in culture extracts by 19F NMR spectroscopy, HPLC‐ESI‐MS, and HPLC‐continuum source molecular absorption spectroscopy for fluorine‐specific detection. The molecule was purified from a large‐scale culture and definitively characterized by NMR spectroscopy and high‐resolution MS. The nucleocidin biosynthetic gene cluster was identified by the presence of genes encoding the 5′‐O‐sulfamate moiety and confirmed by gene disruption. Two of the genes within the nucleocidin biosynthetic gene cluster contain TTA codons, thus explaining the dependence on bldA and resolving a 60‐year‐old mystery.

In vivo formation of natural HgSe nanoparticles in the liver and brain of pilot whales

To understand the biochemistry of methylmercury (MeHg) that leads to the formation of mercury-selenium (Hg-Se) clusters is a long outstanding challenge that promises to deepen our knowledge of MeHg detoxification and the role Se plays in this process. Here, we show that mercury selenide (HgSe) nanoparticles in the liver and brain of long-finned pilot whales are attached to Se-rich structures and possibly act as a nucleation point for the formation of large Se-Hg clusters, which can grow with age to over 5 μm in size. The detoxification mechanism is fully developed from the early age of the animals, with particulate Hg found already in juvenile tissues. As a consequence of MeHg detoxification, Se-methionine, the selenium pool in the system is depleted in the efforts to maintain essential levels of Se-cysteine. This study provides evidence of so far unreported depletion of the bioavailable Se pool, a plausible driving mechanism of demonstrated neurotoxic effects of MeHg in the organism affected by its high dietary intake.

Possible link between Hg and Cd accumulation in the brain of long-finned pilot whales (Globicephala melas)

The bioaccumulation of metals was investigated by analysis of liver, kidney, muscle and brain tissue of a pod of 21 long-finned pilot whales (Globicephala melas) of all ages stranded in Scotland, UK. The results are the first to report cadmium (Cd) passage through the blood-brain barrier of pilot whales and provide a comprehensive study of the long-term (up to 35 years) mammalian exposure to the environmental pollutants. Additionally, linear accumulation of mercury (Hg) was observed in all studied tissues, whereas for Cd this was only observed in the liver. Total Hg concentration above the upper neurochemical threshold was found in the sub-adult and adult brains and methylmercury (MeHg) of 2.2mg/kg was found in the brain of one individual. Inter-elemental analysis showed significant positive correlations of Hg with selenium (Se) and Cd with Se in all studied tissues. Furthermore, differences in the elemental concentrations in the liver and brain tissues were found between juvenile, sub-adult and adult groups. The highest concentrations of manganese, iron, zinc, Se, Hg and MeHg were noted in the livers, whereas Cd predominantly accumulated in the kidneys. High concentrations of Hg and Cd in the tissues of pilot whales presented in this study reflect ever increasing toxic stress on marine mammals.

Imaging of trace elements in tissues: with a focus on laser ablation inductively coupled plasma mass spectrometry.

Purpose of reviewElemental imaging techniques are capable of showing the spatial distribution of elements in a sample. Their application in biomedical sciences is promising, but they are not yet widely employed. The review gives a short overview about techniques available and then focuses on the advantages of using laser ablation inductively coupled plasma mass spectrometry for elemental bioimaging. Current examples for the use of elemental imaging with medical context are given to illustrate the potential of this type of analysis for clinical applications. Recent findingsRecently, synchrotron-based techniques and laser ablation inductively coupled plasma mass spectrometry have been successfully applied to analyse the spatial distribution of elements in biological samples of medical relevance. SummaryElemental bioimaging methods have a great potential for medical applications. They are complementary to molecular imaging and histological staining and are especially attractive when used in combination with stable isotope tracer experiments.

Plasma zinc's alter ego is a low-molecular-weight humoral factor

Mild dietary zinc deprivation in humans and rodents has little effect on blood plasma zinc levels, and yet cellular consequences of zinc depletion can be detected in vascular and other tissues. We proposed that a zinc‐regulated humoral factor might mediate the effects of zinc deprivation. Using a novel approach, primary rat vascular smooth muscle cells (VSMCs) were treated with plasma from zinc‐deficient (<1 mg Zn/kg) or zinc‐adequate (35 mg Zn/kg, pair‐fed) adult male rats, and zinc levels were manipulated to distinguish direct and indirect effects of plasma zinc. Gene expression changes were analyzed by microarray and qPCR, and incubation of VSMCs with blood plasma from zinc‐deficient rats strongly changed the expression of >2500 genes, compared to incubation of cells with zinc‐adequate rat plasma. We demonstrated that this effect was caused by a low‐molecular‐weight (~2‐kDa) zinc‐regulated humoral factor but that changes in gene expression were mostly reversed by adding zinc back to zinc‐deficient plasma. Strongly regulated genes were overrepresented in pathways associated with immune function and development. We conclude that zinc deficiency induces the production of a low‐molecular‐weight humoral factor whose influence on VSMC gene expression is blocked by plasma zinc. This factor is therefore under dual control by zinc.— Ou, O., Allen‐Redpath, K., Urgast, D., Gordon, M.‐J., Campbell, G., Feldmann, J., Nixon, G. F., Mayer, C.‐D., Kwun, I.‐S., and Beattie, J. H. Plasma zincs alter ego is a low‐molecular‐weight humoral factor. FASEB J. 27, 3672–3682 (2013). www.fasebj.org

The mechanisms of detoxification of As(III), dimethylarsinic acid (DMA) and As(V) in the microalga Chlorella vulgaris

The response of Chlorella vulgaris when challenged by As(III), As(V) and dimethylarsinic acid (DMA) was assessed through experiments on adsorption, efflux and speciation of arsenic (reduction, oxidation, methylation and chelation with glutathione/phytochelatin [GSH/PC]). Our study indicates that at high concentrations of phosphate (1.62mM of HPO4(2-)), upon exposure to As(V), cells are able to shift towards methylation of As(V) rather than PC formation. Treatment with As(V) caused a moderate decrease in intracellular pH and a strong increase in the concentration of free thiols (GSH). Passive surface adsorption was found to be negligible for living cells exposed to DMA and As(V). However, adsorption of As(III) was observed to be an active process in C. vulgaris, because it did not show saturation at any of the exposure periods. Chelation of As(III) with GS/PC and to a lesser extent hGS/hPC is a major detoxification mechanism employed by C. vulgaris cells when exposed to As(III). The increase of bound As-GS/PC complexes was found to be strongly related to an increase in concentration of As(III) in media. C. vulgaris cells did not produce any As-GS/PC complex when exposed to As(V). This may indicate that a reduction step is needed for As(V) complexation with GSH/PC. C. vulgaris cells formed DMAS(V)-GS upon exposure to DMA independent of the exposure period. As(III) triggers the formation of arsenic complexes with PC and homophytochelatins (hPC) and their compartmentalisation to vacuoles. A conceptual model was devised to explain the mechanisms involving ABCC1/2 transport. The potential of C. vulgaris to bio-remediate arsenic from water appeared to be highly selective and effective without the potential hazard of reducing As(V) to As(III), which is more toxic to humans.