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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.

Glutamine synthetase (GS, EC 6.3.1.2; also known as gamma-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 have led to its consideration as a drug or an herbicide target. In this article, 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 GS inhibitors with an IC(50) of micromolar value 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 2.5+/-0.4 microM. Two X-ray structures are presented: one is a complex of the enzyme with the purine analogue alone (2.55-A resolution), and the other includes the compound together with methionine sulfoximine phosphate, magnesium and phosphate (2.2-A 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 an approximately 60-fold lower affinity for this compound than the bacterial GS. 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 GS inhibitors.