Evaluating the quality of a de novo annotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary-based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeasts Cryptococcus neoformans and Cryptococcus deneoformans as test cases. Our results show that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5–10 million reads per RNA-seq replicate. We also showed that the number of introns predicted is an excellent a priori indicator of the quality of the final de novo annotation. We then used this pipeline to annotate the genome of the RNAi-deficient species Cryptococcus deuterogattii strain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent in C. deuterogattii than in the other RNAi-proficient species C. neoformans and C. deneoformans. In contrast, we observed that antisense transcription was not higher in C. deuterogattii than in the two other Cryptococcus species. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.

Cryptococcus gattii is a human and animal pathogen that infects healthy hosts and caused the Pacific Northwest outbreak of cryptococcosis. The inhalation of infectious propagules can lead to internalization of cryptococcal cells by alveolar macrophages, a niche in which C. gattii cells can survive and proliferate. Although the nutrient composition of macrophages is relatively unknown, the high induction of amino acid transporter genes inside the phagosome indicates a preference for amino acid uptake instead of synthesis. However, the presence of countable errors in the R265 genome annotation indicates significant inhibition of transcriptomic analysis in this hypervirulent strain. Thus, we analyzed RNA-Seq data from in vivo and in vitro cultures of C. gattii R265 to perform the reannotation of the genome. In addition, based on in vivo transcriptomic data, we identified highly expressed genes and pathways of amino acid metabolism that would enable C. gattii to survive and proliferate in vivo. Importantly, we identified high expression in three APC amino acid transporters as well as the GABA permease. The use of amino acids as carbon and nitrogen sources, releasing ammonium and generating carbohydrate metabolism intermediaries, also explains the high expression of components of several degradative pathways, since glucose starvation is an important host defense mechanism.

Antifungal resistance has become more frequent, either due to the emergence of naturally resistant species or the development of mechanisms that lead to resistance in previously susceptible species. Among these fungal species of global threat, Candida auris stands out for commonly being highly resistant to antifungal drugs, and some isolates are pan-resistant. The rate of mortality linked to C. auris infections varies from 28% to 78%. In this study, we characterized C. auris extracellular vesicles (EVs) in the presence of caspofungin, an echinocandin, which is the recommended first line antifungal for the treatment of infections due to this emerging pathogen. Furthermore, we also analyzed the protein and RNA content of EVs generated by C. auris cultivated with or without treatment with caspofungin. We observed that caspofungin led to the increased production of EVs, and treatment also altered the type and quantity of RNA molecules and proteins enclosed in the EVs. There were distinct classes of RNAs in the EVs with ncRNAs being the most identified molecules, and tRNA-fragments (tRFs) were abundant in each of the strains studied. We also identified anti-sense RNAs, varying from 21 to 55 nt in length. The differentially abundant mRNAs detected in EVs isolated from yeast subjected to caspofungin treatment were related to translation, nucleosome core and cell wall. The differentially regulated proteins identified in the EVs produced during caspofungin treatment were consistent with the results observed with the RNAs, with the enriched terms being related to translation and cell wall. Our study adds new information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade treatment and persist in the host. The ability of C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal agents.

Evaluating the quality of a de novo annotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeasts Cryptococcus neoformans and Cryptococcus deneoformans as test cases. Our results demonstrate that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5 to 10 million reads per RNA-seq replicate. We also demonstrated that the number of introns predicted is an excellent a priori indicator of the quality of the final de novo annotation. We then used this pipeline to annotate the genome of the RNAi-deficient species Cryptococcus deuterogattii strain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent in C. deuterogattii than in the other RNAi-proficient species C. neoformans and C. deneoformans. In contrast, the average level of antisense transcription was not higher in C. deuterogattii. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.

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