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 zrt2Δ 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 Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

Glutamine synthetase (EC 6.3.1.2, also known as -glutamyl:ammonia ligase) catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine. The enzyme has essential roles in different tissues and species, which has led to its consideration as a drug or herbicide target. In the present paper, we describe studies aimed at the discovery of new antimicrobial agents targeting Mycobacterium tuberculosis, the causative pathogen of tuberculosis. A number of distinct classes of glutamine synthetase inhibitors with IC 50 s µM or better were identified via high-throughput screening. A commercially available purine analogue similar to one of the clusters identified (the diketopurines), 1-[(3,4-dichlorophenyl)-methyl]-3,7-dimethyl-8-morpholin-4-yl-purine-2,6-dione, was also shown to inhibit the enzyme, with a measured IC 50 of 2.8 ± 0.4 µM. Two X-ray structures are presented. One is a complex of the enzyme with the purine analogue alone (2.55 Å resolution), and the other includes the compound together with methionine sulfoximine phosphate, magnesium and phosphate (2.2 Å resolution).The former represents a relaxed, inactive conformation of the enzyme, while the latter is a taut, active one. These structures show that the compound binds at the same position in the nucleotide site, regardless of the conformational state. The ATP-binding site of the human enzyme differs substantially, explaining why it has ~60-fold lower affinity for this compound than the bacterial glutamine synthetase. As part of this work, we devised a new synthetic procedure for generating L-(SR)-methionine sulfoximine phosphate from L-(SR)-methionine sulfoximine, which will facilitate future investigations of novel glutamine synthetase inhibitors.

The cytolytic peptide toxin candidalysin is secreted by the invasive, hyphal form of the human fungal pathogen,Candida albicans. Candidalysin is essential for inducing host cell damage during mucosal and systemicC. albicansinfections, resulting in neutrophil recruitment. Neutrophil influx toC. albicans-infected tissue is critical for limiting fungal growth and preventing the fungal dissemination. Here, we demonstrate that candidalysin secreted by hyphae promotes the stimulation of neutrophil extracellular traps (NETs), while synthetic candidalysin triggers a distinct mechanism for NET-like structures (NLS), which are more compact and less fibrous than canonical NETs. Candidalysin activates NADPH oxidase and calcium influx, with both processes contributing to morphological changes in neutrophils resulting in NLS formation. NLS are induced by leukotoxic hypercitrullination, which is governed by protein arginine deaminase 4 activation via calcium influx and initiation of intracellular signalling events. However, activation of signalling by candidalysin does not suffice to trigger downstream events essential for NET formation, as demonstrated by lack of lamin A/C phosphorylation, an event required for activation of cyclin-dependent kinases that are crucial for NET release. Interestingly, exposure to candidalysin does not immediately restrict the capability of neutrophils to produce reactive oxygen species (ROS), nor to phagocytose particles. Instead, candidalysin triggers ROS production, calcium influx and subsequent activation of downstream signalling that drive morphological alteration and the formation of NLS in a dose- and time-dependent manner. Notably, candidalysin-triggered NLS demonstrate anti-Candidaactivity, which is resistant to nuclease treatment and dependent on the deprivation of Zn2+. This study reveals thatC. albicanshyphae releasing candidalysin concurrently trigger canonical NETs and NLS, which together form a fibrous sticky network that entanglesC. albicanshyphae and inhibits their growth. Importantly, this explains discrepancies of previous studies demonstrating that neutrophil-derived extracellular chromatin structures triggered byC. albicanscan be both dependent and independent of ROS. Our data also demonstrate that while candidalysin hampers neutrophil function, the toxin also increases the capability of neutrophils to entangle hyphae and to restrict their growth, reflecting the importance of human neutrophils in controlling the dissemination ofC. albicans.