Role of micronutrients during fungal infections

Trace metals serve as structural and catalytic cofactors for numerous proteins and thus are crucial for all living organisms. Iron is the fourth most abundant element in the Earth’s crust and the most abundant metal in humans. The presence of redox active iron in living systems is critical for oxygen transport, energy production, DNA repair and replication, gene expression, and transcription. Zinc, the twenty fifth most abundant element in the Earth’s crust, is the second most abundant transition metal in vertebrates. Zinc is a redox inactive trace metal and its availability in organisms is indispensable for the function of more than 300 enzymes, DNA stabilization and synthesis, gene expression, protein synthesis, and immunity. Essential trace metals are required at various concentrations depending on the organism and the cellular function. The disturbance of optimal metals level is extremely harmful for cells, thus metal concentrations are tightly controlled within organisms. Correspondingly, the availability of metals is recognized as the central factor in infections. The host is able to both limit and overload iron and zinc levels in order to inhibit microbial growth in a process known as nutritional immunity. Depending on the pathogen species and their location within the organism, the host regulates metal levels from extreme deficiency in the blood to overload in phagosomes. In return, pathogens have evolved to counteract host mediated metal fluctuations. Under metal starvation pathogens rely on plasma membrane metal importers, chelators production, and host metalloproteins utilization. In response to metal toxicity pathogenic microorganisms make the use of plasma membrane metals exporters and intracellular detoxification systems. Candida albicans is a fungus that co-exists as a harmless commensal in humans, however, when the immune system is compromised or host’s barriers are damaged, C. albicans is able to cause superficial to severe systemic infections. During commensalism and infection, the fungus successfully inhabits host niches that dramatically vary in metals availability. Thus C. albicans is able to precisely regulate metals homeostasis via the comprehensive transcriptional network that controls metals assimilation, storage, detoxification, and mobilization processes.Spurenlemente dienen als strukturelle und katalytische Co-faktoren für viele Proteine und sind somit unabdingbar für alle Lebewesen. Eisen ist das vierthäufigste Element der Erdkruste und das am häufigsten vorkommende Metall im Menschen. Das Vorhandensein von redox-aktivem Eisen in lebenden Organismen ist entscheidend für Sauerstofftransport, Energieerzeugung, DNA-Reparatur und -replikation, Genexpression und Transkription. Zink, das 25häufigste Element der Erdkruste, ist das zweithäufigste Metall in Wirbeltieren. Zink ist ein redox-inaktives Spurenelement und dessen Verfügbarkeit im Organismus ist unerlässlich für die Funktion von mehr als 300 Enzymen, für DNA Stabilisierung und -synthese, Genexpression, Proteinsynthese und das Immunsystem. Essentielle Spurenelemente werden entsprechend des Organismus und der zellulären Funktion in verschiedenen Konzentrationen benötigt. Eine Störung des Metallhaushaltes ist sehr schädlich für Zellen, weshalb die Konzentration an Metallen im Organismus streng reguliert wird. Die Verfügbarkeit an Metallen wird daher auch als ausschlaggebender Faktor während Infektionen angesehen. Der Wirt ist in der Lage Eisen- und Zinklevel sowohl zu beschränken als auch überzudosieren um das Wachstum von Mikroorganismen zu hemmen; ein Prozess der als nährstoffbasierte Immunität („nutritional immunity“) bekannt ist. In Abhängigkeit des Krankheitserregers und dessen Lokalisation im Organismus können Metalle extrem limitiert sein (z.B. im Blut) oder andererseits äußerst hohe Konzentrationen erreichen (z.B. im Phagosom). Im Gegenzug haben Krankheitserreger gelernt, den wirtsvermittelten Metallschwankungen entgegenzuwirken. Bei einem Metallmangel verwenden Krankheitserreger in erhöhtem Maße membrangängige Metallimporter, Chelatoren oder Metalloproteine des Wirts. Bei einem Metallüberschuss benutzen Krankheitserreger verstärkt Metallexporter in der Plasmamembran oder intrazelluläre Entgiftungssysteme. Candida albicans ist ein Hefepilz, der als harmloser Kommensale im Menschen existiert, jedoch unter Immunsuppression oder bei Schädigung der Wirtsbarrieren auch oberflächliche bis hin zu schweren systemischen Infektionen auslösen kann. Während Kommensalismus und Infektion kann der Pilz Wirtsnischen besiedeln, die sich in ihrer Verfügbarkeit an Metallen drastisch unterscheiden. Eine präzise Regulation des Metallgleichgewichts in C. albicans wird durch ein umfassendes Transkriptionsnetzwerk ermöglicht, welches die Metallaufnahme, -lagerung, detoxifizierung und -mobilisierung kontrolliert

Candida albicans Hap43 domains are required under iron starvation but not excess

Iron availability is a central factor in infections, since iron is a critical micronutrient for all living organisms. The host employs both iron limitation and toxicity strategies to control microbial growth, and successful pathogens are able to tightly coordinate iron homeostasis in response to changing iron levels. As a commensal and opportunistic pathogen, Candida albicans copes with both iron deficiency and excess via the precise regulation of iron acquisition, consumption and storage. The C. albicans transcription factor Hap43 is known to be required for the iron starvation response, while specific domains of its ortholog, HapX, in Aspergillus fumigatus, were recently shown to regulate iron uptake and consumptions genes under both low and high iron levels. Therefore, we investigated the contribution of C. albicans Hap43 domains in response to changing iron levels. We found the C-terminus of Hap43 to be essential for the activation of iron uptake genes during iron starvation, whereas, in contrast to A. fumigatus, Hap43 was not required in mediating adaptation to iron resistance. These data indicate that the generally conserved metal acquisition systems in fungal pathogens can show individual adaptations to the host environment

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Azerbaijan Republic

Genetic screening of school children in Masalli region in Azerbaijan Republic identified 23 school children with G6PD enzyme activity different deficiency (from 0 up to 60% activity). Biochemical studies were done/performed for school children with activity efficiency on enzyme preparations from erythrocytes. As to WHO Guidelines, enzymatic preparations were related to the following classes: 2nd class – 13 boys, 3rd class – 6 school boys, 4th class – 4 of them. DNA molecular analysis, isolated from blood of the index patient, was classified as the 2nd class of G6PD enzyme deficiency and has shown the substitution of Guanine nucleotide with Adenine in position 1178. As a result of the mutation in protein in the position 393, substitution of amino acids Arginine with Histidine [G6PD,1178 (G-A) Arg393His] takes place

LIPAD (LRRK2/Luebeck International Parkinson's Disease) Study Protocol:Deep Phenotyping of an International Genetic Cohort

Background: Pathogenic variants in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most common known monogenic cause of Parkinson's disease (PD). LRRK2-linked PD is clinically indistinguishable from idiopathic PD and inherited in an autosomal dominant fashion with reduced penetrance and variable expressivity that differ across ethnicities and geographic regions.Objective: To systematically assess clinical signs and symptoms including non-motor features, comorbidities, medication and environmental factors in PD patients, unaffected LRRK2 pathogenic variant carriers, and controls. A further focus is to enable the investigation of modifiers of penetrance and expressivity of LRRK2 pathogenic variants using genetic and environmental data.Methods: Eligible participants are invited for a personal or online examination which comprises completion of a detailed eCRF and collection of blood samples (to obtain DNA, RNA, serum/plasma, immune cells), urine as well as household dust. We plan to enroll 1,000 participants internationally: 300 with LRRK2-linked PD, 200 with LRRK2 pathogenic variants but without PD, 100 PD patients with pathogenic variants in the GBA or PRKN genes, 200 patients with idiopathic PD, and 200 healthy persons without pathogenic variants.Results: The eCRF consists of an investigator-rated (1 h) and a self-rated (1.5 h) part. The first part includes the Movement Disorder Society Unified Parkinson's Disease Rating, Hoehn &Yahr, and Schwab & England Scales, the Brief Smell Identification Test, and Montreal Cognitive Assessment. The self-rating part consists of a PD risk factor, food frequency, autonomic dysfunction, and quality of life questionnaires, the Pittsburgh Sleep Quality Inventory, and the Epworth Sleepiness as well as the Hospital Anxiety and Depression Scales. The first 15 centers have been initiated and the first 150 participants enrolled (as of March 25th, 2021).Conclusions: LIPAD is a large-scale international scientific effort focusing on deep phenotyping of LRRK2-linked PD and healthy pathogenic variant carriers, including the comparison with additional relatively frequent genetic forms of PD, with a future perspective to identify genetic and environmental modifiers of penetrance and expressivityClinical Trial Registration:ClinicalTrials.gov, NCT04214509

Genotype-phenotype correlations of cystic fibrosis in siblings compound heterozygotes for rare variant combinations: Review of literature and case report

Here, we describe a cystic fibrosis (CF) family with affected siblings, two of whom have a combination of I1234V and 1677delTA variants with classic CF features, the third child with a combination of I1234V and L997F variants with atypical CF, and the apparently healthy mother with a combination of 1677delTA and L997F alleles. Interestingly, the sibling with I1234V and L997F variants had normal sweat test results and had a much milder phenotype than the other two siblings with I1234V and 1677delTA variants, suggesting that this combination is causative for atypical CF. The fact that their mother with the combination of 1677delTA and L997F appears to be healthy suggests that the L997F variant causes different phenotypes in different allele combinations. The current cases show that there is a genotype-phenotype correlation in this disease and underline the importance of genotyping individuals with suspected CF to allow prediction of disease severity and effective treatment

Rapid Large-Scale COVID-19 Testing during Shortages

The Coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in economic and social lockdowns in most countries all over the globe. Early identification of infected individuals is regarded as one of the most important prerequisites for fighting the pandemic and for returning to a ‘New Normal’. Large-scale testing is therefore crucial, but is facing several challenges including shortage of sample collection tools and of molecular biological reagents, and the need for safe electronic communication of medical reports. We present the successful establishment of a holistic SARS-CoV-2 testing platform that covers proband registration, sample collection and shipment, sample testing, and report issuing. The RT-PCR-based virus detection, being central to the platform, was extensively validated: sensitivity and specificity were defined as 96.8% and 100%, respectively; intra-run and inter-run precision were <3%. A novel type of sample swab and an in-house-developed RNA extraction system were shown to perform as good as commercially available products. The resulting flexibility guarantees independence from the current bottlenecks in SARS-CoV-2 testing. Based on our technology, we offered testing at local, national, and global levels. In the present study, we report the results from approx. 18,000 SARS-CoV-2 tests in almost 10,000 individuals from a low-frequency SARS-CoV-2 pandemic area in a homogenous geographical region in north-eastern Germany for a period of 10 weeks (21 March to 31 May 2020). Among the probands, five SARS-CoV-2 positive cases were identified. Comparative analysis of corresponding virus genomes revealed a diverse origin from three of the five currently recognized SARS-CoV-2 phylogenetic clades. Our study exemplifies how preventive SARS-CoV-2 testing can be set up in a rapid and flexible manner. The application of our test has enabled a safe maintenance/resume of critical local infrastructure, e.g., nursing homes where more than 5000 elderlies and caretakers got tested. The strategy outlined by the present study may serve as a blueprint for the implementation of large-scale preventive SARS-CoV-2 testing elsewhere

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