title: Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans.
abstract: 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.
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Ross D
authors.initials: RD
authors.lastname: Blundell
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.|Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Williams
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Samantha D M
authors.initials: SD
authors.lastname: Arras
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Chitty
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Kirsten L
authors.initials: KL
authors.lastname: Blake
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.|Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.|MX Beamlines, Australian Synchrotron , 800 Blackburn Road, Clayton, Victoria 3168, Australia.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Ericsson
authors.affiliation: College of Pharmacy, University of Kentucky , Lexington, Kentucky 40536-0596, United States.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Tibrewal
authors.affiliation: College of Pharmacy, University of Kentucky , Lexington, Kentucky 40536-0596, United States.
authors.forename: Jurgen
authors.initials: J
authors.lastname: Rohr
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Y Q Andre E
authors.initials: YQ
authors.lastname: Koh
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.|Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kappler
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Avril A B
authors.initials: AA
authors.lastname: Robertson
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Butler
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Cooper
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.|Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: James A
authors.initials: JA
authors.lastname: Fraser
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000265:Adenylosuccinate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003453:Cryptococcosis / Q000382:microbiology*; D003455:Cryptococcus neoformans / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D005260:Female; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D014774:Virulence
chemical_list: D005656:Fungal Proteins; D000255:Adenosine Triphosphate; D000265:Adenylosuccinate Synthase
keywords: Cryptococcus neoformans; X-ray crystallography; antifungal; molecular genetics; target verification