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

Widespread inter- and intra-domain horizontal gene transfer of d-amino acid metabolism enzymes in eukaryotes

Analysis of the growing number of available fully-sequenced genomes has shown that Horizontal Gene Transfer (HGT) in eukaryotes is more common than previously thought. It has been proposed that genes with certain functions may be more prone to HGT than others, but we still have a very poor understanding of the selective forces driving eukaryotic HGT. Recent work uncovered that D-amino acid racemases have been commonly transferred from bacteria to fungi, but their role in the receiving organisms is currently unknown. Here, we set out to assess whether D-amino acid racemases are commonly transferred to and between eukaryotic groups. For this we performed a global survey that used a novel automated phylogeny-based HGT-detection algorithm (Abaccus). Our results revealed that at least 7.0% of the total eukaryotic racemase repertoire is the result of inter- or intra-domain HGT. These transfers are significantly enriched in plant-associated fungi. For these, we hypothesize a possible role for the acquired racemases allowing to exploit minoritary nitrogen sources in plant biomass, a nitrogen-poor environment. Finally, we performed experiments on a transferred aspartate-glutamate racemase in the fungal human pathogen Candida glabrata, which however revealed no obvious biological role

The Hyphal-Associated Adhesin and Invasin Als3 of Candida albicans Mediates Iron Acquisition from Host Ferritin

Iron sequestration by host iron-binding proteins is an important mechanism of resistance to microbial infections. Inside oral epithelial cells, iron is stored within ferritin, and is therefore not usually accessible to pathogenic microbes. We observed that the ferritin concentration within oral epithelial cells was directly related to their susceptibility to damage by the human pathogenic fungus, Candida albicans. Thus, we hypothesized that host ferritin is used as an iron source by this organism. We found that C. albicans was able to grow on agar at physiological pH with ferritin as the sole source of iron, while the baker's yeast Saccharomyces cerevisiae could not. A screen of C. albicans mutants lacking components of each of the three known iron acquisition systems revealed that only the reductive pathway is involved in iron utilization from ferritin by this fungus. Additionally, C. albicans hyphae, but not yeast cells, bound ferritin, and this binding was crucial for iron acquisition from ferritin. Transcriptional profiling of wild-type and hyphal-defective C. albicans strains suggested that the C. albicans invasin-like protein Als3 is required for ferritin binding. Hyphae of an Δals3 null mutant had a strongly reduced ability to bind ferritin and these mutant cells grew poorly on agar plates with ferritin as the sole source of iron. Heterologous expression of Als3, but not Als1 or Als5, two closely related members of the Als protein family, allowed S. cerevisiae to bind ferritin. Immunocytochemical localization of ferritin in epithelial cells infected with C. albicans showed ferritin surrounding invading hyphae of the wild-type, but not the Δals3 mutant strain. This mutant was also unable to damage epithelial cells in vitro. Therefore, C. albicans can exploit iron from ferritin via morphology dependent binding through Als3, suggesting that this single protein has multiple virulence attributes

Ncs2* mediates in vivo virulence of pathogenic yeast through sulphur modification of cytoplasmic transfer RNA.

Fungal pathogens threaten ecosystems and human health. Understanding the molecular basis of their virulence is key to develop new treatment strategies. Here, we characterize NCS2*, a point mutation identified in a clinical baker's yeast isolate. Ncs2 is essential for 2-thiolation of tRNA and the NCS2* mutation leads to increased thiolation at body temperature. NCS2* yeast exhibits enhanced fitness when grown at elevated temperatures or when exposed to oxidative stress, inhibition of nutrient signalling, and cell-wall stress. Importantly, Ncs2* alters the interaction and stability of the thiolase complex likely mediated by nucleotide binding. The absence of 2-thiolation abrogates the in vivo virulence of pathogenic baker's yeast in infected mice. Finally, hypomodification triggers changes in colony morphology and hyphae formation in the common commensal pathogen Candida albicans resulting in decreased virulence in a human cell culture model. These findings demonstrate that 2-thiolation of tRNA acts as a key mediator of fungal virulence and reveal new mechanistic insights into the function of the highly conserved tRNA-thiolase complex

The Fungal Pathogen Candida glabrata Does Not Depend on Surface Ferric Reductases for Iron Acquisition

Iron acquisition is a crucial virulence determinant for many bacteria and fungi, including the opportunistic fungal pathogens Candida albicans and C. glabrata. While the diverse strategies used by C. albicans for obtaining iron from the host are well-described, much less is known about the acquisition of this micronutrient from host sources by C. glabrata – a distant relative of C. albicans with closer evolutionary ties to Saccharomyces cerevisiae, which nonetheless causes severe clinical symptoms in humans. Here we show that C. glabrata is much more restricted than C. albicans in using host iron sources, lacking, for example, the ability to grow on transferrin and hemin/hemoglobin. Instead, C. glabrata is able to use ferritin and non-protein-bound iron (FeCl3) as iron sources in a pH-dependent manner. As in other fungal pathogens, iron-dependent growth requires the reductive high affinity (HA) iron uptake system. Typically highly conserved, this uptake mechanism normally relies on initial ferric reduction by cell-surface ferric reductases. The C. glabrata genome contains only three such putative ferric reductases, which were found to be dispensable for iron-dependent growth. In addition and in contrast to C. albicans and S. cerevisiae, we also detected no surface ferric reductase activity in C. glabrata. Instead, extracellular ferric reduction was found in this and the two other fungal species, which was largely dependent on an excreted low-molecular weight, non-protein ferric reductant. We therefore propose an iron acquisition strategy of C. glabrata which differs from other pathogenic fungi, such as C. albicans, in that it depends on a limited set of host iron sources and that it lacks the need for surface ferric reductases. Extracellular ferric reduction by a secreted molecule possibly compensates for the loss of surface ferric reductase activity in the HA iron uptake system

Comparative Study on Alternative Splicing in Human Fungal Pathogens Suggests Its Involvement During Host Invasion

Alternative splicing (AS) is an important regulatory mechanism in eukaryotes but only little is known about its impact in fungi. Human fungal pathogens are of high clinical interest causing recurrent or life-threatening infections. AS can be well-investigated genome-wide and quantitatively with the powerful technology of RNA-Seq. Here, we systematically studied AS in human fungal pathogens based on RNA-Seq data. To do so, we investigated its effect in seven fungi during conditions simulating ex vivo infection processes and during in vitro stress. Genes undergoing AS are species-specific and act independently from differentially expressed genes pointing to an independent mechanism to change abundance and functionality. Candida species stand out with a low number of introns with higher and more varying lengths and more alternative splice sites. Moreover, we identified a functional difference between response to host and other stress conditions: During stress, AS affects more genes and is involved in diverse regulatory functions. In contrast, during response-to-host conditions, genes undergoing AS have membrane functionalities and might be involved in the interaction with the host. We assume that AS plays a crucial regulatory role in pathogenic fungi and is important in both response to host and stress conditions

Synthesis and evaluation of novel furanones as biofilm inhibitors in opportunistic human pathogens

Diseases caused by biofilm-forming pathogens are becoming increasingly prevalent and represent a major threat to human health. This trend has prompted a search for novel inhibitors of microbial biofilms which could, for example, be used to potentiate existing antibiotics. Naturally-occurring, halogenated furanones isolated from marine algae have proven to be effective biofilm inhibitors in several bacterial species. In this work, we report the synthesis of a library of novel furanones and their subsequent evaluation as biofilm inhibitors in several opportunistic human pathogens including S. enterica, S. aureus, E. coli, S. maltophilia, P. aeruginosa and C. albicans. A number of the most potent compounds were subjected to further analysis by confocal laser-scanning microscopy for their effects on P. aeruginosa and C. albicans biofilms individually, in addition to mixed polymicrobial biofilms. Lastly, we investigated the impact of a promising candidate on survival rates in vivo using a Galleria mellonella model

Synthesis and evaluation of novel furanones as biofilm inhibitors in opportunistic human pathogens.

Diseases caused by biofilm-forming pathogens are becoming increasingly prevalent and represent a major threat to human health. This trend has prompted a search for novel inhibitors of microbial biofilms which could, for example, be used to potentiate existing antibiotics. Naturally-occurring, halogenated furanones isolated from marine algae have proven to be effective biofilm inhibitors in several bacterial species. In this work, we report the synthesis of a library of novel furanones and their subsequent evaluation as biofilm inhibitors in several opportunistic human pathogens including S. enterica, S. aureus, E. coli, S. maltophilia, P. aeruginosa and C. albicans. A number of the most potent compounds were subjected to further analysis by confocal laser-scanning microscopy for their effects on P. aeruginosa and C. albicans biofilms individually, in addition to mixed polymicrobial biofilms. Lastly, we investigated the impact of a promising candidate on survival rates in vivo using a Galleria mellonella model

Candida albicans Scavenges Host Zinc via Pra1 during Endothelial Invasion

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