Strain and temperature dependent aggregation of Candida auris is attenuated by inhibition of surface amyloid proteins.

Candida auris is a multi-drug resistant human fungal pathogen that has become a global threat to human health due to its drug resistant phenotype, persistence in the hospital environment and propensity for patient to patient spread. Isolates display variable aggregation that may affect the relative virulence of strains. Therefore, dissection of this phenotype has gained substantial interest in recent years. We studied eight clinical isolates from four different clades (I-IV); four of which had a strongly aggregating phenotype and four of which did not. Genome analysis identified polymorphisms associated with loss of cell surface proteins were enriched in weakly-aggregating strains. Additionally, we identified down-regulation of chitin synthase genes involved in the synthesis of the chitinous septum. Characterisation of the cells revealed no ultrastructural defects in cytokinesis or cell separation in aggregating isolates. Strongly and weakly aggregating strains did not differ in net surface charge or in cell surface hydrophobicity. The capacity for aggregation and for adhesion to polystyrene microspheres were also not correlated. However, aggregation and extracellular matrix formation were all increased at higher growth temperatures, and treatment with the amyloid protein inhibitor Thioflavin-T markedly attenuated aggregation. Genome analysis further indicated strain specific differences in the genome content of GPI-anchored proteins including those encoding genes with the potential to form amyloid proteins. Collectively our data suggests that aggregation is a complex strain and temperature dependent phenomenon that may be linked in part to the ability to form extracellular matrix and cell surface amyloids.

Fluconazole resistant Candida auris clinical isolates have increased levels of cell wall chitin and increased susceptibility to a glucosamine-6-phosphate synthase inhibitor.

In 2009 Candida auris was first isolated as fungal pathogen of human disease from ear canal of a patient in Japan. In less than a decade, this pathogen has rapidly spread around the world and has now become a major health challenge that is of particular concern because many strains are resistant to multiple class of antifungal drugs. The lack of available antifungals and rapid increase of this fungal pathogen provides an incentive for the development of new and more potent anticandidal drugs and drug combinatorial treatments. Here we have explored the growth inhibitory activity against C. auris of a synthetic dipeptide glutamine analogue, L-norvalyl-N 3-(4-methoxyfumaroyl)-L-2,3- diaminopropanoic acid (Nva-FMDP), that acts as an inhibitor of glucosamine-6-phosphate (GlcN-6-P) synthase - a key enzyme in the synthesis of cell wall chitin. We observed that in contrast to FLC susceptible isolates of C. auris, FLC resistant isolates had elevated cell wall chitin and were susceptible to inhibition by Nva-FMDP. The growth kinetics of C. auris in RPMI-1640 medium revealed that the growth of FLC resistant isolates were 50-60% more inhibited by Nva-FMDP (8 µ g/ml) compared to a FLC susceptible isolate. Fluconazole resistant strains displayed increased transcription of CHS1, CHS2 and CHS3, and the chitin content of the fluconazole resistant strains was reduced following the Nva-FMDP treatment. Therefore, the higher chitin content in FLC resistant C. auris isolates may make the strain more susceptible to inhibition of the antifungal activity of the Nva-FMDP peptide conjugate.

Crosstalk between the calcineurin and cell wall integrity pathways prevents chitin overexpression in Candida albicans.

Echinocandins such as caspofungin are frontline antifungal drugs that compromise ß-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by upregulation of chitin synthesis, thereby sustaining cell wall integrity (CWI). When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a subpopulation of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable supra-high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway. This article has an associated First Person interview with the first author of the paper.