Characterisation of an line that completely lacks ribonucleotide reduction yields insights into the evolution of parasitism and endosymbiosis.

Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides.

Creeping yeast: a simple, cheap and robust protocol for the identification of mating type in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an exceptional genetic system, with genetic crosses facilitated by its ability to be maintained in haploid and diploid forms. Such crosses are straightforward if the mating type/ploidy of the strains is known. Several techniques can determine mating type (or ploidy), but all have limitations.

Convergent microevolution of Cryptococcus neoformans hypervirulence in the laboratory and the clinic.

Reference strains are a key component of laboratory research, providing a common background allowing for comparisons across a community of researchers. However, laboratory passage of these strains has been shown to lead to reduced fitness and the attenuation of virulence in some species. In this study we show the opposite in the fungal pathogen Cryptococcus neoformans, with analysis of a collection of type strain H99 subcultures revealing that the most commonly used laboratory subcultures belong to a mutant lineage of the type strain that is hypervirulent.

Sirtuins in the phylum Basidiomycota: A role in virulence in Cryptococcus neoformans.

Virulence of Cryptococcus neoformans is regulated by a range of transcription factors, and is also influenced by the acquisition of adaptive mutations during infection. Beyond the temporal regulation of virulence factor production by transcription factors and these permanent microevolutionary changes, heritable epigenetic modifications such as histone deacetylation may also play a role during infection. Here we describe the first comprehensive analysis of the sirtuin class of NAD+ dependent histone deacetylases in the phylum Basidiomycota, identifying five sirtuins encoded in the C.

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.

Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus neoformans.

Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C.

Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ).

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.

Reactive oxygen species homeostasis and virulence of the fungal pathogen Cryptococcus neoformans requires an intact proline catabolism pathway.

Degradation of the multifunctional amino acid proline is associated with mitochondrial oxidative respiration. The two-step oxidation of proline is catalyzed by proline oxidase and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase, which produce P5C and glutamate, respectively. In animal and plant cells, impairment of P5C dehydrogenase activity results in P5C-proline cycling when exogenous proline is supplied via the actions of proline oxidase and P5C reductase (the enzyme that converts P5C to proline).

Comparative Genomics of Serial Isolates of Cryptococcus neoformans Reveals Gene Associated With Carbon Utilization and Virulence.

The opportunistic fungal pathogen Cryptococcus neoformans is a leading cause of mortality among the human immunodeficiency virus/acquired immunodeficiency syndrome population and is known for frequently causing life-threatening relapses. To investigate the potential contribution of in-host microevolution to persistence and relapse, we have analyzed two serial isolates obtained from a patient with acquired immunodeficiency syndrome who suffered an initial and relapse episode of cryptococcal meningoencephalitis. Despite being identical by multilocus sequence typing, the isolates differ phenotypically, exhibiting changes in key virulence factors, nutrient acquisition, metabolic profiles, and the ability to disseminate in an animal model.