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Advances in transcriptome sequencing allow for simultaneous interrogation of differentially expressed genes from multiple species originating from a single RNA sample, termed dual or multi-species transcriptomics. Compared to single-species differential expression analysis, the design of multi-species differential expression experiments must account for the relative abundances of each organism of interest within the sample, often requiring enrichment methods and yielding differences in total read counts across samples. The analysis of multi-species transcriptomics datasets requires modifications to the alignment, quantification, and downstream analysis steps compared to the single-species analysis pipelines. We describe best practices for multi-species transcriptomics and differential gene expression.

Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.

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 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.

Candida auris is a newly emerging fungal pathogen of humans and has attracted considerable attention from both the clinical and basic research communities. Clinical isolates of C. auris are often resistant to one or more antifungal agents. To explore how antifungal resistance develops, we performed experimental evolution assays using a fluconazole-susceptible isolate of C. auris (BJCA001). After a series of passages through medium containing increasing concentrations of fluconazole, fungal cells acquired resistance. By sequencing and comparing the genomes of the parental fluconazole-susceptible strain and 26 experimentally evolved strains of C. auris, we found that a portion of fluconazole-resistant strains carried one extra copy of chromosome V. In the absence of fluconazole, C. auris cells rapidly became susceptible and lost the extra copy of chromosome V. Genomic and transcriptome sequencing (RNA-Seq) analyses indicate that this chromosome carries a number of drug resistance-related genes, which were transcriptionally upregulated in the resistant, aneuploid strains. Moreover, missense mutations were identified in the genes TAC1B, RRP6, and SFT2 in all experimentally evolved strains. Our findings suggest that the gain of an extra copy of chromosome V is associated with the rapid acquisition of fluconazole resistance and may represent an important evolutionary mechanism of antifungal resistance in C. auris.

Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis widely reported in the Gran Chaco ecosystem. The disease is caused by different species from the genus Paracoccidioides, which are all endemic to South and Central America. Here, we sequenced and analyzed 31 isolates of Paracoccidioides across South America, with particular focus on isolates from Argentina and Paraguay. The de novo sequenced isolates were compared with publicly available genomes. Phylogenetics and population genomics revealed that PCM in Argentina and Paraguay is caused by three distinct Paracoccidioides genotypes, P. brasiliensis (S1a and S1b) and P. restrepiensis (PS3). P. brasiliensis S1a isolates from Argentina are frequently associated with chronic forms of the disease. Our results suggest the existence of extensive molecular polymorphism among Paracoccidioides species, and provide a framework to begin to dissect the connection between genotypic differences in the pathogen and the clinical outcomes of the disease. Fil: Teixeira, Marcus de Melo. Universidade do Brasília; Brasil Fil: Cattana, Maria Emilia. Universidad Nacional del Nordeste. Instituto de Medicina Regional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Matute, Daniel R.. University of North Carolina; Estados Unidos Fil: Muñoz, José F.. Broad Institute Of Mit And Harvard; Estados Unidos Fil: Arechavala, Alicia. Hospital Francisco J Muñiz; Argentina Fil: Isbell, Kristin. University of North Carolina; Estados Unidos Fil: Schipper, Rafael. Universidade do Brasília; Brasil Fil: Santiso, Gabriela Maria. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; Argentina Fil: Tracogna, Fernanda. Gobierno de la Provincia de Chaco. Hospital Julio Cecilio Perrando.; Argentina Fil: Sosa, María de los Ángeles. Universidad Nacional del Nordeste. Instituto de Medicina Regional; Argentina Fil: Cech, Norma. Hospital 4 de Junio; Argentina Fil: Alvarado, Primavera. Instituto de Biomedicina Dr. Jacinto Convit; Venezuela Fil: Barreto, Laura. Instituto Superior de Formación Docente Salome Ureña; República Dominicana Fil: Chacón, Yone. Provincia de Salta. Ministerio de Salud Pública. Hospital del Milagro; Argentina Fil: Ortellado, Juana. Universidad Nacional de Asunción; Paraguay Fil: Lima, Cleoni Mendes de. Universidade Federal de Rondonia; Brasil Fil: Chang, Marilene Rodrigues. Universidade Federal do Mato Grosso do Sul; Brasil Fil: Niño Vega, Gustavo. Universidad de Guanajuato; México Fil: Yasuda, Maria Aparecida Shikanai. Universidade de Sao Paulo; Brasil Fil: Felipe, Maria Sueli Soares. Universidade Catolica de Brasilia; Brasil Fil: Negroni, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; Argentina Fil: Cuomo, Christina A.. Broad Institute of MIT And Harvard; Estados Unidos Fil: Barker, Bridget. Tgen Northern Arizona University; Estados Unidos Fil: Giusiano, Gustavo Emilio. Universidad Nacional del Nordeste. Instituto de Medicina Regional; Argentina. Universidad Nacional del Nordeste. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina

In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology., Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.

Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, the fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro. Comparative analysis of comprehensive sterol profiles, [3H]-fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and the relative expression of C. auris ERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene, TAC1B, and a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into the fluconazole-susceptible isolate AR0387, was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in-vitro, and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCECandida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrate that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole, and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

Dimorphic fungal pathogens cause a significant human disease burden and unlike most fungal pathogens affect immunocompetent hosts. Most dimorphic fungi are found in the family Ajellomycetaceae, including the genera Histoplasma, Blastomyces, Paracoccidioides, and the recently described Emergomyces. To examine the origin of virulence and host adaptation in these fungal pathogens, we compared the gene content of classic systemic, opportunistic, and non-pathogenic species, including new genomes for Emmonsia species and two closely non-pathogenic species, Helicocarpus griseus and Polytolypa hystricis. We examined differences in gene content between pathogens and environmental fungi, and found that gene families related to plant degradation, synthesis of secondary metabolites, and amino acid and lipid metabolism are retained in H. griseus and P. hystricis. Genes of central importance in dimorphic pathogenic fungi such as heat shock response proteins, morphological switch related proteins are conserved in non-pathogenic species. However, we found that changes in the copy number of proteases, phosphotransferases 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, we found that both more basal 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 but similar host/substrate shift from plants to animals have occurred in the Onygenaceae and Ajellomycetaceae, and highlighted how the dimorphic pathogens from Ajellomycetaceae have adapted to the host immune response and decreased their capacity for life stages in environmental niches.

Candida auris is an emerging fungal pathogen of rising concern due to its increasing incidence, its ability to cause healthcare-associated outbreaks and antifungal resistance. Genomic analysis revealed that early cases of C. auris that were detected contemporaneously were geographically stratified into four major clades. Clade II, also termed East Asian clade, consists of the initial isolates described from cases of ear infection, is less frequently resistant to antifungal drugs and to date, the isolates from this group have not been associated with outbreaks. Here, we generate nearly complete genomes (“telomere-to-telomere”) of an isolate of this clade and of the more widespread Clade IV. By comparing these to genome assemblies of the other two clades, we find that the Clade II genome appears highly rearranged, with 2 inversions and 9 translocations resulting in a substantially different karyotype. In addition, large subtelomeric regions have been lost from 10 of 14 chromosome ends in the Clade II genomes. We find that shorter telomeres and genome instability might be a consequence of a naturally occurring loss-of-function mutation in DCC1 exclusively found in Clade II isolates, resulting in a hypermutator phenotype. We also determine that deleted subtelomeric regions might be linked to clade-specific adaptation as these regions are enriched in Hyr/Iff-like cell surface proteins, novel candidate cell surface proteins, and an ALS-like adhesin. The presence of these cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggests an explanation for the different phenotypes observed between clades.IMPORTANCECandida auris was unknown prior to 2009 and since then it has quickly spread around the world, causing outbreaks in healthcare facilities and representing a high fraction of candidemia cases in some regions. The emergence of C. auris is a major concern, since it is often multidrug-resistant, easily spread between patients, and causes invasive infections. While isolates from three global clades cause invasive infections, isolates from Clade II primarily cause ear infections and have not been implicated in outbreaks, though cases of Clade II infections have been reported on different continents. Here, we describe genetic differences between Clade II and Clades I, III and IV, including a loss-of-function mutation in a gene associated with telomere length maintenance and genome stability, and the loss of cell wall proteins involved in adhesion and biofilm formation, that may suggest an explanation for the lower virulence and potential for transmission of Clade II isolates.

Volume 10, no. 1, e02529-18, 2019, [https://doi.org/10.1128/mBio.02529-18][1]. [Figure 1A][2] spotting images were incorrectly duplicated for the Candida albicans and Candida auris plates. We confirmed the essentiality of HSP90 expression in C. albicans and C. auris with multiple biological

Fungal pathogens pose a serious threat to public health. Candida auris is an emerging fungal pathogen that is often resistant to commonly used antifungal drugs. However, the mechanisms governing drug resistance and virulence in this organism remain largely unexplored. In this study, we adapted a conditional expression system to modulate the transcription of an essential gene, HSP90, which regulates antifungal resistance and virulence in diverse fungal pathogens. We showed that Hsp90 is essential for growth in C. auris and is important for tolerance of the clinically important azole antifungals, which block ergosterol biosynthesis. Further, we established that the Cdr1 efflux transporter regulates azole resistance. Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling. Overall, this work provides a novel insight into mechanisms regulating azole resistance in C. auris and uncovers a distinct developmental program regulated by Hsp90., Candida auris is an emerging fungal pathogen and a serious global health threat as the majority of clinical isolates display elevated resistance to currently available antifungal drugs. Despite the increased prevalence of C. auris infections, the mechanisms governing drug resistance remain largely elusive. In diverse fungi, the evolution of drug resistance is enabled by the essential molecular chaperone Hsp90, which stabilizes key regulators of cellular responses to drug-induced stress. Hsp90 also orchestrates temperature-dependent morphogenesis in Candida albicans, a key virulence trait. However, the role of Hsp90 in the pathobiology of C. auris remains unknown. In order to study regulatory functions of Hsp90 in C. auris, we placed HSP90 under the control of a doxycycline-repressible promoter to enable transcriptional repression. We found that Hsp90 is essential for growth in C. auris and that it enables tolerance of clinical isolates with respect to the azoles, which inhibit biosynthesis of the membrane sterol ergosterol. High-level azole resistance was independent of Hsp90 but dependent on the ABC transporter CDR1, deletion of which resulted in abrogated resistance. Strikingly, we discovered that C. auris undergoes a morphogenetic transition from yeast to filamentous growth in response to HSP90 depletion or cell cycle arrest but not in response to other cues that induce C. albicans filamentation. Finally, we observed that this developmental transition is associated with global transcriptional changes, including the induction of cell wall-related genes. Overall, this report provides a novel insight into mechanisms of drug tolerance and resistance in C. auris and describes a developmental transition in response to perturbation of a core regulator of protein homeostasis.

Candida aurisis an emergent fungal pathogen of rising public health concern due to increasing reports of outbreaks in healthcare settings and resistance to multiple classes of antifungal drugs. While distantly related to the more common pathogensC. albicansandC. glabrata,C. aurisis closely related to three rarely observed and often multidrug-resistant species,C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Here, we generated and analyzed near complete genome assemblies and RNA-Seq-guided gene predictions for isolates from each of the four majorC. aurisclades and forC. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Our analyses mapped seven chromosomes and revealed chromosomal rearrangements betweenC. aurisclades and related species. We found conservation of genes involved in mating and meiosis and identified bothMTLaandMTLαC. aurisisolates, suggesting the potential for mating between clades. Gene conservation analysis highlighted that many genes linked to drug resistance and virulence in other pathogenicCandidaspecies are conserved inC. aurisand related species including expanded families of transporters and lipases, as well as mutations and copy number variants inERG11that confer drug resistance. In addition, we found genetic features of the emerging species that likely underlie differences in virulence and drug response between these and otherCandidaspecies, including genes involved in cell wall structure. To begin to characterize the species-specific genes important for antifungal response, we profiled the gene expression ofC. aurisin response to voriconazole and amphotericin B and found induction of several transporters and metabolic regulators that may play a role in drug resistance. This study provides a comprehensive view of the genomic basis of drug resistance, potential for mating, and virulence in this emerging fungal clade.

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., Author Summary Dimorphic fungal pathogens including Blastomyces are the cause of major fungal diseases in North and South America. The genus Emmonsia includes species infecting small mammals as well as a newly emerging pathogenic species recently reported in HIV-positive patients in South Africa. Here, we synthesize both genome sequencing of four isolates of Blastomyces and two species of Emmonsia as well as deep sequencing of Blastomyces RNA to draw major new insights into the evolution of this group and the pathogen response to infection. We investigate the trajectory of genome evolution of this group, characterizing the phylogenetic relationships of these species, a remarkable genome expansion that formed large isochore-like regions of low GC content in Blastomyces, and variation of gene content, related to host interaction, among the dimorphic fungal pathogens. Using RNA-Seq, we profile the response of Blastomyces to macrophage and mouse pulmonary infection, identifying key pathways and novel virulence factors. The identification of key fungal genes involved in adaptation to the host suggests targets for further study and therapeutic intervention in Blastomyces and related dimorphic fungal pathogens.