Cryptococcus neoformans ADS lyase is an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design.

There is significant clinical need for new antifungal agents to manage infections with pathogenic species such as Cryptococcus neoformans Because the purine biosynthesis pathway is essential for many metabolic processes, such as synthesis of DNA and RNA and energy generation, it may represent a potential target for developing new antifungals. Within this pathway, the bifunctional enzyme adenylosuccinate (ADS) lyase plays a role in the formation of the key intermediates inosine monophosphate and AMP involved in the synthesis of ATP and GTP, prompting us to investigate ADS lyase in C. neoformans. Here, we report that ADE13 encodes ADS lyase in C. neoformans. We found that an ade13Δ mutant is an adenine auxotroph and is unable to successfully cause infections in a murine model of virulence. Plate assays revealed that production of a number of virulence factors essential for dissemination and survival of C. neoformans in a host environment was compromised even with the addition of exogenous adenine. Purified recombinant C. neoformans ADS lyase shows catalytic activity similar to its human counterpart, and its crystal structure, the first fungal ADS lyase structure determined, shows a high degree of structural similarity to that of human ADS lyase. Two potentially important amino acid differences are identified in the C. neoformans crystal structure, in particular a threonine residue that may serve as an additional point of binding for a fungal enzyme-specific inhibitor. Besides serving as an antimicrobial target, C. neoformans ADS lyase inhibitors may also serve as potential therapeutics for metabolic disease; rather than disrupt ADS lyase, compounds that improve the stability the enzyme may be used to treat ADS lyase deficiency disease.

Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans.

Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first example of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.

A genomic safe haven for mutant complementation in Cryptococcus neoformans.

Just as Koch's postulates formed the foundation of early infectious disease study, Stanley Falkow's molecular Koch's postulates define best practice in determining whether a specific gene contributes to virulence of a pathogen. Fundamentally, these molecular postulates state that if a gene is involved in virulence, its removal will compromise virulence. Likewise, its reintroduction should restore virulence to the mutant. These approaches are widely employed in Cryptococcus neoformans, where gene deletion via biolistic transformation is a well-established technique. However, the complementation of these mutants is less straightforward. Currently, one of three approaches will be taken: the gene is reintroduced at the original locus, the gene is reintroduced into a random site in the genome, or the mutant is not complemented at all. Depending on which approach is utilized, the mutant may be complemented but other genes are potentially disrupted in the process. To counter the drawbacks of the current approaches to complementation we have created a new tool to assist in this key step in the study of a gene's role in virulence. We have identified and characterized a small gene-free region in the C. neoformans genome dubbed the "safe haven", and constructed a plasmid vector that targets DNA constructs to this preselected site. The plasmid vector integrates with high frequency, effectively complementing a mutant strain without disrupting adjacent genes. qRT-PCR of the flanking genes on either side of the safe haven site following integration of the targeting vector revealed no changes in their expression, and no secondary phenotypes were observed in a range of phenotypic assays including an intranasal murine infection model. Combined, these data confirm that we have successfully created a much-needed molecular resource for the Cryptococcus community, enabling the reliable fulfillment of the molecular Koch's postulates.