Oliver Keatinge Clay

Despite decades of effort, the Collatz conjecture remains neither proved, nor refuted by a counterexample, nor formally shown to be undecidable. This note introduces the Collatz problem and probes its logical depth with a test question: can the search space for counterexamples be iteratively reduced, and when would it help?

Clinical isolates of a fungal pathogen from a single region or country often exhibit structural clonality or phylogenetic clustering at the sequence or MLST level; such population structure can persist also in larger samples. In efforts to improve causal understanding of pathogenesis at the molecular level, genome-wide association screening methods initially designed for other kingdoms have been applied to fungi. The example of a Colombian dataset of 28 clinical Cryptococcus neoformans VNI isolates indicates where the output from standard pipelines may need to be analyzed in new ways in order to efficiently extract hypotheses for experiments from fungal genotype-phenotype data. Collections of clinical isolates of a human fungal pathogen can consist of clusters of genetically similar isolates. Such clustering complicates the screening for genetic associations with clinically relevant traits. We propose new methods, illustrating them for the fungus causing Cryptococcosis.

Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma spp. that results in significant morbidity and mortality in persons with HIV/AIDS and can also affect immunocompetent individuals. Although some PCR and antigen-detection assays have been developed, conventional diagnosis has largely relied on culture, which can take weeks. Our aim was to provide a proof of principle for rationally designing and standardizing PCR assays based on Histoplasma-specific genomic sequences. Via automated comparisons of aligned genome contigs/scaffolds and gene (sub)sequences, we identified protein-coding genes that are present in existing sequences of Histoplasma strains but not in other genera. Two of the genes, PPK and CFP4, were used for designing primer sets for conventional and real-time PCR assays. Both resulted in a 100% analytical specificity in vitro and detected 62/62 H. capsulatum isolates using purified DNA. We also obtained positive detections of 2/2 confirmed H. capsulatum clinical FFPE (formalin-fixed paraffin-embedded) samples using both primer sets. Positive control plasmid 10-fold serial dilutions confirmed the analytical sensitivity of the assays. The findings suggest that these novel primer sets should allow for detection sensitivity and reduce false positive results/cross-reactions. New assays for detecting pathogenic fungi, constructed along these lines, could be simple and affordable to implement.

Candida auris is an emerging fungal pathogen capable of causing invasive infections in humans. Since its first appearance around 1996, it has been isolated in countries spanning five continents. C. auris is a yeast that has the potential to cause outbreaks in hospitals, can survive in adverse conditions, including dry surfaces and high temperatures, and has been frequently misidentified by traditional methods. Furthermore, strains have been identified that are resistant to two and even all three of the main classes of antifungals currently in use. Several nuclear genome assemblies of C. auris have been published representing different clades and continents, yet until recently, the mitochondrial genomes (mtDNA chromosomes) of this species and the closely related species of C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii had not been analyzed in depth. We used reads from PacBio and Illumina sequencing to obtain a de novo reference assembly of the mitochondrial genome of the C. auris clade I isolate B8441 from Pakistan. This assembly has a total size of 28.2 kb and contains 13 core protein-coding genes, 25 tRNAs and the 12S and 16S ribosomal subunits. We then performed a comparative analysis by aligning Illumina reads of 129 other isolates from South Asia, Japan, South Africa, and South America with the B8441 reference. The clades of the phylogenetic tree we obtained from the aligned mtDNA sequences were consistent with those derived from the nuclear genome. The mitochondrial genome revealed a generally low genetic variation within clades, although the South Asian clade displayed two sub-branches including strains from both Pakistan and India. In particular, the 86 isolates from Colombia and Venezuela had mtDNA sequences that were all identical at the base level, i.e., a single conserved haplotype or mitochondrial background that exhibited characteristic differences from the Pakistan reference isolate B8441, such as a unique 25-nt insert that may affect function.

The LUFS domain (LUG/LUH, Flo8, single‐strand DNA‐binding protein [SSBP]) is a well‐conserved and apparently ancient region found in diverse proteins and taxa. This domain, which has as its most obvious structural feature a series of three helices, has been identified in transcriptional regulator proteins of animals, plants, and fungi. Recently, in these pages (Wang et al., Protein Sci., 2019, 28:788–793), the first crystal structure of a LUFS domain was reported, for the human SSBP2, a transcriptional repressor. We briefly address how the new insights into LUFS structures might contribute to a better understanding of an important transcriptional activator of yeasts that contains the LUFS domain, Flo8, and consider how a focus on the LUFS domain and its variation could help us to understand etiologies of drug resistance in a recently emerged pathogenic fungus, Candida auris.

Dimorphic fungal pathogens cause a significant human disease burden and unlike most fungal pathogens affect immunocompetent hosts. To examine the origin of virulence of these fungal pathogens, we compared genomes of classic systemic, opportunistic, and non-pathogenic species, including Emmonsia and two basal branching, non-pathogenic species in the Ajellomycetaceae, Helicocarpus griseus and Polytolypa hystricis . We found that gene families related to plant degradation, secondary metabolites synthesis, and amino acid and lipid metabolism are retained in H. griseus and P. hystricis . While genes involved in the virulence of dimorphic pathogenic fungi are conserved in saprophytes, changes in the copy number of proteases, kinases and transcription factors in systemic dimorphic relative to non-dimorphic species may have aided the evolution of specialized gene regulatory programs to rapidly adapt to higher temperatures and new nutritional environments. Notably, both of the basal branching, non-pathogenic species appear homothallic, with both mating type locus idiomorphs fused at a single locus, whereas all related pathogenic species are heterothallic. These differences revealed that independent changes in nutrient acquisition capacity have occurred in the Onygenaceae and Ajellomycetaceae, and underlie how the dimorphic pathogens have adapted to the human host and decreased their capacity for growth in environmental niches.