The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution. The capacity of disease-causing microbes to acquire nutrients from their host is one of the most fundamental aspects of infection. Host organisms therefore restrict microbial access to certain key nutrients in a process known as nutritional immunity. Recently, it was found that infected vertebrates sequester zinc from invading microorganisms to control infection. Therefore, the mechanisms of microbial zinc acquisition represent potential virulence attributes. Here we report the molecular mechanism of host-derived zinc acquisition by the major human fungal pathogen, Candida albicans. We show that C. albicans utilises a secreted protein, the pH-regulated antigen 1 (Pra1), to bind zinc from its environment. Pra1 then reassociates with the fungal cell via a syntenically encoded (genetically-linked) membrane transporter (Zrt1) to acquire this essential metal. Deletion of PRA1 prevented utilisation of host zinc and damage of host cells in the absence of exogenous zinc. Finally, we demonstrate that this zinc-scavenging locus arose in an ancient fungal lineage and remains conserved in many contemporary species. Syntenically arranged zinc acquisition systems have evolved independently in the fungal and bacterial kingdoms, suggesting that such an arrangement is evolutionary beneficial for microorganisms.

Propionate is used to protect bread and animal feed from moulds. The mode of action of this short‐chain fatty acid was studied using Aspergillus nidulans as a model organism. The filamentous fungus is able to grow slowly on propionate, which is oxidized to acetyl‐CoA via propionyl‐CoA, methylcitrate and pyruvate. Propionate inhibits growth of A. nidulans on glucose but not on acetate; the latter was shown to inhibit propionate oxidation. When grown on glucose a methylcitrate synthase deletion mutant is much more sensitive towards the presence of propionate in the medium as compared to the wild‐type and accumulates 10‐fold higher levels of propionyl‐CoA, which inhibits CoA‐dependent enzymes such as pyruvate dehydrogenase, succinyl‐CoA synthetase and ATP citrate lyase. The most important inhibition is that of pyruvate dehydrogenase, as this affects glucose and propionate metabolism directly. In contrast, the blocked succinyl‐CoA synthetase can be circumvented by a succinyl‐CoA:acetate/propionate CoA‐transferase, whereas ATP citrate lyase is required only for biosynthetic purposes. In addition, data are presented that correlate inhibition of fungal polyketide synthesis by propionyl‐CoA with the accumulation of this CoA‐derivative. A possible toxicity of propionyl‐CoA for humans in diseases such as propionic acidaemia and methylmalonic aciduria is also discussed.

The pathway of the oxidation of propionate to pyruvate in Escherichia coli involves five enzymes, only two of which, methylcitrate synthase and 2‐methylisocitrate lyase, have been thoroughly characterized. Here we report that the isomerization of (2S,3S)‐methylcitrate to (2R,3S)‐2‐methylisocitrate requires a novel enzyme, methylcitrate dehydratase (PrpD), and the well‐known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle. AcnB was purified as 2‐methylaconitate hydratase from E. coli cells grown on propionate and identified by its N‐terminus. The enzyme has an apparent K_m of 210 µm for (2R,3S)‐2‐methylisocitrate but shows no activity with (2S,3S)‐methylcitrate. On the other hand, PrpD is specific for (2S,3S)‐methylcitrate (K_m = 440 µm) and catalyses in addition only the hydration of cis‐aconitate at a rate that is five times lower. The product of the dehydration of enzymatically synthesized (2S,3S)‐methylcitrate was designated cis‐2‐methylaconitate because of its ability to form a cyclic anhydride at low pH. Hence, PrpD catalyses an unusual syn elimination, whereas the addition of water to cis‐2‐methylaconitate occurs in the usual anti manner. The different stereochemistries of the elimination and addition of water may be the reason for the requirement for the novel methylcitrate dehydratase (PrpD), the sequence of which seems not to be related to any other enzyme of known function. Northern‐blot experiments showed expression of acnB under all conditions tested, whereas the RNA of enzymes of the prp operon (PrpE, a propionyl‐CoA synthetase, and PrpD) was exclusively present during growth on propionate. 2D gel electrophoresis showed the production of all proteins encoded by the prp operon during growth on propionate as sole carbon and energy source, except PrpE, which seems to be replaced by acetyl‐CoA synthetase. This is in good agreement with investigations on Salmonella enterica LT2, in which disruption of the prpE gene showed no visible phenotype.

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.