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.

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. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the well-characterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field.

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. Whole-genome sequencing uncovered a clonal relationship, with only a few unique differences. Of these, two key changes are expected to explain the phenotypic differences observed in the relapse isolate: loss of a predicted AT-rich interaction domain protein and changes in copy number of the left and right arms of chromosome 12. Gene deletion of the predicted transcriptional regulator produced changes in melanin, capsule, carbon source use, and dissemination in the host, consistent with the phenotype of the relapse isolate. In addition, the deletion mutant displayed altered virulence in the murine model. The observed differences suggest the relapse isolate evolved subsequent to penetration of the central nervous system and may have gained dominance following the administration of antifungal therapy. These data reveal the first molecular insights into how the Cryptococcus neoformans genome changes during infection of humans and the manner in which microevolution progresses in this deadly fungal pathogen.

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. The pleiotropic mutant phenotypes in this H99L (for “Laboratory”) lineage are the result of a deletion in the gene encoding the SAGA Associated Factor Sgf29, a mutation that is also present in the widely-used H99L-derived KN99a/α congenic pair. At a molecular level, loss of this gene results in a reduction in histone H3K9 acetylation. Remarkably, analysis of clinical isolates identified loss of function SGF29 mutations in C. neoformans strains infecting two of fourteen patients, demonstrating not only the first example of hypervirulence in clinical C. neoformans samples, but also parallels between in vitro and in vivo microevolution for hypervirulence in this important pathogen.

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 D 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). This proline is oxidized by the proline oxidase-FAD complex that delivers electrons to the electron transport chain and to O 2 , leading to mitochondrial reactive oxygen species (ROS) overproduction. Coupled activity of proline oxidase and P5C dehydrogenase is therefore important for maintaining ROS homeostasis. In the genome of the fungal pathogen Cryptococcus neoformans, there are two paralogs (PUT1 and PUT5) that encode proline oxidases and a single ortholog (PUT2) that encodes P5C dehydrogenase. Transcription of all three catabolic genes is inducible by the presence of proline. However, through the creation of deletion mutants, only Put5 and Put2 were found to be required for proline utilization. The put2D mutant also generates excessive mitochondrial superoxide when exposed to proline. Intracellular accumulation of ROS is a critical feature of cell death; consistent with this fact, the put2D mutant exhibits a slight, general growth defect. Furthermore, Put2 is required for optimal production of the major cryptococcal virulence factors. During murine infection, the put2D mutant was discovered to be avirulent; this is the first report highlighting the importance of P5C dehydrogenase in enabling pathogenesis of a microorganism.P ROLINE is an important biological proteinogenic amino acid that is necessary for primary metabolism in organisms including animals, plants, fungi, and bacteria. This imino acid, as it is more precisely known, has multiple physiological functions. Apart from being an osmoprotectant, proline can serve as a transient storage of organic nitrogen, stabilize proteins and membranes during stressful conditions, prevent protein aggregation during its folding or refolding, and scavenge reactive oxygen species (ROS) such as superoxide anions (Rudolph and Crowe 1985;Carpenter and Crowe 1988;Ignatova and Gierasch 2006;Kaul et al. 2008). However, the exact mechanism by which proline protects against stresses remains poorly understood. Intracellular proline must be present at appropriate levels to confer stress-protective effects, and an excess of free proline has been shown to be detrimental to cell growth or protein functions in numerous organisms (Davis 2000;Morita et al. 2002;Deuschle et al. 2004; Maxwell and Nomura and Takagi 2004). The transport, anabolism, and catabolism of proline therefore need to be tightly regulated for proper cellular defense, adaptation, and growth.Degradation of proline that takes place in eukaryotic mitochondria or prokaryotic plasma membrane results in highener...