Identification and characterisation of sPEPs in Cryptococcus neoformans.

Short open reading frame (sORF)-encoded peptides (sPEPs) have been found across a wide range of genomic locations in a variety of species. To date, their identification, validation, and characterisation in the human fungal pathogen Cryptococcus neoformans has been limited due to a lack of standardised protocols. We have developed an enrichment process that enables sPEP detection within a protein sample from this polysaccharide-encapsulated yeast, and implemented proteogenomics to provide insights into the validity of predicted and hypothetical sORFs annotated in the C. neoformans genome. Novel sORFs were discovered within the 5' and 3' UTRs of known transcripts as well as in "non-coding" RNAs. One novel candidate, dubbed NPB1, that resided in an RNA annotated as "non-coding", was chosen for characterisation. Through the creation of both specific point mutations and a full deletion allele, the function of the new sPEP, Npb1, was shown to resemble that of the bacterial trans-translation protein SmpB.

Broadening the spectrum of fluorescent protein tools for use in the encapsulated human fungal pathogen Cryptococcus neoformans.

Green fluorescent protein (GFP) and its counterparts are modern molecular biology research tools indispensable in many experimental systems. Within fungi, researchers studying Saccharomyces cerevisiae and other model ascomycetes have access to a wide variety of fluorescent proteins. Unfortunately, many of these tools have not crossed the phylum divide into the Basidiomycota, where only GFP S65T, Venus, Ds-Red, and mCherry are currently available. To address this, we searched the literature for potential candidates to be expressed in the human fungal pathogen Cryptococcus neoformans and identified a suite of eight more modern fluorescent proteins that span the visible spectrum. A single copy of each fluorophore was heterologously expressed in Safe Haven 1 and their fluorescence intensities compared in this encapsulated yeast. mTurquoise2, mTFP1, Clover, mNeonGreen, mRuby3, and Citrine were highly visible under the microscope, whereas Superfolder GFP and mMaroon1 were not. Expressed fluorophores did not impact growth or virulence as demonstrated by an in vitro spotting assay and murine inhalation model, respectively.

amdS as a dominant recyclable marker in Cryptococcus neoformans.

While the fungal pathogen Cryptoccocus neoformans is a leading cause of death in immunocompromised individuals, the molecular toolkit currently available to study this important pathogen is extremely limited. To enable an unprecedented level of control over manipulation of the genome, we have developed a dominant recyclable marker by expanding on the classic studies of the amdS gene by Michael J. Hynes and John Pateman. The ascomycete Aspergillus nidulans employs the acetamidase AmdS to hydrolyse acetamide to ammonium and acetate, which serve as a nitrogen and carbon source, respectively. Acetamidase activity has never been reported in the Basidiomycota. Here we have successfully demonstrated that acetamide can be utilized as a good nitrogen source in C. neoformans heterologously expressing amdS and that this activity does not influence virulence, enabling it to be used as a basic dominant selectable marker. The expression of this gene in C. neoformans also causes sensitivity to fluoroacetamide, permitting counterselection. Taking advantage of this toxicity we have modified our basic marker to create a comprehensive series of powerful and reliable tools to successfully delete multiple genes in the one strain, generate markerless strains with modifications such as fluorescent protein fusions at native genomic loci, and establish whether a gene is essential in C. neoformans.

The Long History of the Diverse Roles of Short ORFs: sPEPs in Fungi.

Since the completion of the genome sequence of the model eukaryote Saccharomyces cerevisiae, there have been significant advancements in the field of genome annotation, in no small part due to the availability of datasets that make large-scale comparative analyses possible. As a result, since its completion there has been a significant change in annotated ORF size distribution in this first eukaryotic genome, especially in short ORFs (sORFs) predicted to encode polypeptides less than 150 amino acids in length. Due to their small size and the difficulties associated with their study, it is only relatively recently that these genomic features and the sORF-encoded peptides (sPEPs) they encode have become a focus of many researchers. Yet while this class of peptides may seem new and exciting, the study of this part of the proteome is nothing new in S. cerevisiae, a species where the biological importance of sPEPs has been elegantly illustrated over the past 30 years. Here the authors showcase a range of different sORFs found in S. cerevisiae and the diverse biological roles of their encoded sPEPs, and provide an insight into the sORFs found in other fungal species, particularly those pathogenic to humans.

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. 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.

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. neoformans genome. Each sirtuin gene was deleted and a wide range of phenotypic tests performed to gain insight into the potential roles they play. Given the pleiotropic nature of sirtuins in other species, it was surprising that only two of the five deletion strains revealed mutant phenotypes in vitro. However, cryptic consequences of the loss of each sirtuin were identified through whole cell proteomics, and mouse infections revealed a role in virulence for SIR2, HST3 and HST4. The most intriguing phenotype was the repeated inability to complement mutant phenotypes through the reintroduction of the wild-type gene. These data support the model that regulation of sirtuin activity may be employed to enable a drastic alteration of the epigenetic landscape and virulence of C. neoformans.