New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons.

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.

Hypertension and the roles of the 9p21.3 risk locus: Classic findings and new association data.

BACKGROUND: The band 9p21.3 contains an established genomic risk zone for cardiovascular disease (CVD). Since the initial 2007 Wellcome Trust Case Control Consortium study (WTCCC), the increased CVD risk associated with 9p21.3 has been confirmed by multiple studies in different continents. However, many years later there was still no confirmed report of a corresponding association of 9p21.3 with hypertension, a major CV risk factor, nor with blood pressure (BP). THEORY: In this contribution, we review the bipartite haplotype structure of the 9p21.3 risk locus: one block is devoid of protein-coding genes but contains the lead CVD risk SNPs, while the other block contains the first exon and regulatory DNA of the gene for the cell cycle inhibitor p15. We consider how findings from molecular biology offer possibilities of an involvement of p15 in hypertension etiology, with expression of the p15 gene modulated by genetic variation from within the 9p21.3 risk locus. RESULTS: We present original results from a Colombian study revealing moderate but persistent association signals for BP and hypertension within the classic 9p21.3 CVD risk locus. These SNPs are mostly confined to a 'hypertension island' that spans less than 60 kb and coincides with the p15 haplotype block. We find confirmation in data originating from much larger, recent European BP studies, albeit with opposite effect directions. CONCLUSION: Although more work will be needed to elucidate possible mechanisms, previous findings and new data prompt reconsidering the question of how variation in 9p21.3 might influence hypertension components of cardiovascular risk.

Mitochondrial Genome Sequences of the Emerging Fungal Pathogen Candida auris.

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.

Updates and Comparative Analysis of the Mitochondrial Genomes of Paracoccidioides spp. Using Oxford Nanopore MinION Sequencing.

The mitochondrial genome of the Paracoccidioides brasiliensis reference isolate Pb18 was first sequenced and described by Cardoso et al. (2007), as a circular genome with a size of 71.3 kb and containing 14 protein coding genes, 25 tRNAs, and the large and small subunits of ribosomal RNA. Later in 2011, Desjardins et al. (2011) obtained partial assemblies of mitochondrial genomes of P. lutzii (Pb01), P. americana (Pb03), and P. brasiliensis sensu stricto (Pb18), although with a size of only 43.1 kb for Pb18. Sequencing errors or other limitations resulting from earlier technologies, and the advantages of NGS (short and long reads), prompted us to improve and update the mtDNA sequences and annotations of two Paracoccidioides species. Using Oxford Nanopore and Illumina read sequencing, we generated high-quality complete de novo mitochondrial genome assemblies and annotations for P. brasiliensis (Pb18) and P. americana (Pb03). Both assemblies were characterized by an unusually long spacer or intron region (>50 kb) between exons 2 and 3 of the nad5 gene, which was moderately conserved between Pb03 and Pb18 but not similar to other reported sequences, except for an unassigned contig in the 2011 assembly of Pb03. The reliability of the insert missing from previous mtDNA genome assemblies was confirmed by inspection of the individual Nanopore read sequences containing nad5 coding DNA, and experimentally by PCR for Pb18. We propose that the insert may aid replication initiation and may be excised to produce a smaller structural variant. The updated mtDNA genomes should enable more accurate SNP and other comparative or evolutionary analyses and primer/probe designs. A comparative analysis of the mtDNA from 32 isolates of Paracoccidioides spp., using the SNPs of the aligned mitochondrial genomes, showed groupings within the brasiliensis species complex that were largely consistent with previous findings from only five mitochondrial loci.

The LUFS domain, its transcriptional regulator proteins, and drug resistance in the fungal pathogen Candida auris.

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.

Draft Genome Sequences of Two Sporothrix schenckii Clinical Isolates Associated with Human Sporotrichosis in Colombia.

Sporothrix schenckii is a thermodimorphic fungal pathogen with a high genetic diversity. In this work, we present the assembly and similarity analysis of the whole-genome sequences of two clinical isolates from Colombia of S. schenckiisensu stricto.

Genome analysis reveals evolutionary mechanisms of adaptation in systemic dimorphic fungi.

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.

Novel taxa of thermally dimorphic systemic pathogens in the Ajellomycetaceae (Onygenales).

Recent discoveries of novel systemic fungal pathogens with thermally dimorphic yeast-like phases have challenged the current taxonomy of the Ajellomycetaceae, a family currently comprising the genera Blastomyces, Emmonsia, Emmonsiellopsis, Helicocarpus, Histoplasma, Lacazia and Paracoccidioides. Our morphological, phylogenetic and phylogenomic analyses demonstrated species relationships and their specific phenotypes, clarified generic boundaries and provided the first annotated genome assemblies to support the description of two new species. A new genus, Emergomyces, accommodates Emmonsia pasteuriana as type species, and the new species Emergomyces africanus, the aetiological agent of case series of disseminated infections in South Africa. Both species produce small yeast cells that bud at a narrow base at 37°C and lack adiaspores, classically associated with the genus Emmonsia. Another novel dimorphic pathogen, producing broad-based budding cells at 37°C and occurring outside North America, proved to belong to the genus Blastomyces, and is described as Blastomyces percursus.

Genome Diversity, Recombination, and Virulence across the Major Lineages of Paracoccidioides.

The Paracoccidioides genus includes two species of thermally dimorphic fungi that cause paracoccidioidomycosis, a neglected health-threatening human systemic mycosis endemic to Latin America. To examine the genome evolution and the diversity of Paracoccidioides spp., we conducted whole-genome sequencing of 31 isolates representing the phylogenetic, geographic, and ecological breadth of the genus. These samples included clinical, environmental and laboratory reference strains of the S1, PS2, PS3, and PS4 lineages of P. brasiliensis and also isolates of Paracoccidioides lutzii species. We completed the first annotated genome assemblies for the PS3 and PS4 lineages and found that gene order was highly conserved across the major lineages, with only a few chromosomal rearrangements. Comparing whole-genome assemblies of the major lineages with single-nucleotide polymorphisms (SNPs) predicted from the remaining 26 isolates, we identified a deep split of the S1 lineage into two clades we named S1a and S1b. We found evidence for greater genetic exchange between the S1b lineage and all other lineages; this may reflect the broad geographic range of S1b, which is often sympatric with the remaining, largely geographically isolated lineages. In addition, we found evidence of positive selection for the GP43 and PGA1 antigen genes and genes coding for other secreted proteins and proteases and lineage-specific loss-of-function mutations in cell wall and protease genes; these together may contribute to virulence and host immune response variation among natural isolates of Paracoccidioides spp. These insights into the recent evolutionary events highlight important differences between the lineages that could impact the distribution, pathogenicity, and ecology of Paracoccidioides. IMPORTANCE Characterization of genetic differences between lineages of the dimorphic human-pathogenic fungus Paracoccidioides can identify changes linked to important phenotypes and guide the development of new diagnostics and treatments. In this article, we compared genomes of 31 diverse isolates representing the major lineages of Paracoccidioides spp. and completed the first annotated genome sequences for the PS3 and PS4 lineages. We analyzed the population structure and characterized the genetic diversity among the lineages of Paracoccidioides, including a deep split of S1 into two lineages (S1a and S1b), and differentiated S1b, associated with most clinical cases, as the more highly recombining and diverse lineage. In addition, we found patterns of positive selection in surface proteins and secreted enzymes among the lineages, suggesting diversifying mechanisms of pathogenicity and adaptation across this species complex. These genetic differences suggest associations with the geographic range, pathogenicity, and ecological niches of Paracoccidioides lineages.

The Dynamic Genome and Transcriptome of the Human Fungal Pathogen Blastomyces and Close Relative Emmonsia.

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.

Genome update of the dimorphic human pathogenic fungi causing paracoccidioidomycosis.

Paracoccidiodomycosis (PCM) is a clinically important fungal disease that can acquire serious systemic forms and is caused by the thermodimorphic fungal Paracoccidioides spp. PCM is a tropical disease that is endemic in Latin America, where up to ten million people are infected; 80% of reported cases occur in Brazil, followed by Colombia and Venezuela. To enable genomic studies and to better characterize the pathogenesis of this dimorphic fungus, two reference strains of P. brasiliensis (Pb03, Pb18) and one strain of P. lutzii (Pb01) were sequenced [1]. While the initial draft assemblies were accurate in large scale structure and had high overall base quality, the sequences had frequent small scale defects such as poor quality stretches, unknown bases (N's), and artifactual deletions or nucleotide duplications, all of which caused larger scale errors in predicted gene structures. Since assembly consensus errors can now be addressed using next generation sequencing (NGS) in combination with recent methods allowing systematic assembly improvement, we re-sequenced the three reference strains of Paracoccidioides spp. using Illumina technology. We utilized the high sequencing depth to re-evaluate and improve the original assemblies generated from Sanger sequence reads, and obtained more complete and accurate reference assemblies. The new assemblies led to improved transcript predictions for the vast majority of genes of these reference strains, and often substantially corrected gene structures. These include several genes that are central to virulence or expressed during the pathogenic yeast stage in Paracoccidioides and other fungi, such as HSP90, RYP1-3, BAD1, catalase B, alpha-1,3-glucan synthase and the beta glucan synthase target gene FKS1. The improvement and validation of these reference sequences will now allow more accurate genome-based analyses. To our knowledge, this is one of the first reports of a fully automated and quality-assessed upgrade of a genome assembly and annotation for a non-model fungus.

GC3 of genes can be used as a proxy for isochore base composition: a reply to Elhaik et al.

In an article published in these pages, Elhaik et al. (Elhaik E, Landan G, Graur D. 2009. Can GC content at third-codon positions be used as a proxy for isochore composition? Mol Biol Evol. 26:1829-1833) asked if GC3, the GC level of the third-codon positions in protein-coding genes, can be used as a "proxy" to estimate the GC level of the surrounding isochore. We use available data to directly answer this simple question in the affirmative and show how the use of indirect methods can lead to apparently conflicting conclusions. The answer reasserts that in human and other vertebrates, genes have a strong tendency to reside in compositionally corresponding isochores, which has far-reaching implications for genome structure and evolution.