AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replication, transcription, and energy metabolism, and the same rationale applies when considering the pathway as an antifungal target. Here, we describe the identification and characterization of C. neoformans 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/5'-inosine monophosphate cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final two enzymatic steps in the formation of the first purine base inosine monophosphate. We demonstrate that mutants lacking the ATIC-encoding ADE16 gene are adenine and histidine auxotrophs that are unable to establish an infection in a murine model of virulence. In addition, our assays employing recombinantly expressed and purified C. neoformans ATIC enzyme revealed Km values for its substrates AICAR and 5-formyl-AICAR are 8-fold and 20-fold higher, respectively, than in the human ortholog. Subsequently, we performed crystallographic studies that enabled the determination of the first fungal ATIC protein structure, revealing a key serine-to-tyrosine substitution in the active site, which has the potential to assist the design of fungus-specific inhibitors. Overall, our results validate ATIC as a promising antifungal drug target.

Best practices in DNA methylation: lessons from inflammatory bowel disease, psoriasis and ankylosing spondylitis.

Advances in genomic technology have enabled a greater understanding of the genetics of common immune-mediated diseases such as ankylosing spondylitis (AS), inflammatory bowel disease (IBD) and psoriasis. The substantial overlap in genetically identified pathogenic pathways has been demonstrated between these diseases. However, to date, gene discovery approaches have only mapped a minority of the heritability of these common diseases, and most disease-associated variants have been found to be non-coding, suggesting mechanisms of disease-association through transcriptional regulatory effects.Epigenetics is a major interface between genetic and environmental modifiers of disease and strongly influence transcription. DNA methylation is a well-characterised epigenetic mechanism, and a highly stable epigenetic marker, that is implicated in disease pathogenesis. DNA methylation is an under-investigated area in immune-mediated diseases, and many studies in the field are affected by experimental design limitations, related to study design, technical limitations of the methylation typing methods employed, and statistical issues. This has resulted in both sparsity of investigations into disease-related changes in DNA methylation, a paucity of robust findings, and difficulties comparing studies in the same disease.In this review, we cover the basics of DNA methylation establishment and control, and the methods used to examine it. We examine the current state of DNA methylation studies in AS, IBD and psoriasis; the limitations of previous studies; and the best practices for DNA methylation studies. The purpose of this review is to assist with proper experimental design and consistency of approach in future studies to enable a better understanding of the functional role of DNA methylation in immune-mediated disease.