Highly Recombinant VGII Cryptococcus gattii Population Develops Clonal Outbreak Clusters through both Sexual Macroevolution and Asexual Microevolution

ABSTRACTAn outbreak of the fungal pathogen Cryptococcus gattii began in the Pacific Northwest (PNW) in the late 1990s. This outbreak consists of three clonal subpopulations: VGIIa/major, VGIIb/minor, and VGIIc/novel. Both VGIIa and VGIIc are unique to the PNW and exhibit increased virulence. In this study, we sequenced the genomes of isolates from these three groups, as well as global isolates, and analyzed a total of 53 isolates. We found that VGIIa/b/c populations show evidence of clonal expansion in the PNW. Whole-genome sequencing provided evidence that VGIIb originated in Australia, while VGIIa may have originated in South America, and these were likely independently introduced. Additionally, the VGIIa outbreak lineage may have arisen from a less virulent clade that contained a mutation in the MSH2 ortholog, but this appears to have reverted in the VGIIa outbreak strains, suggesting that a transient mutator phenotype may have contributed to adaptation and evolution of virulence in the PNW outbreak. PNW outbreak isolates share genomic islands, both between the clonal lineages and with global isolates, indicative of sexual recombination. This suggests that VGII C.gattii has undergone sexual reproduction, either bisexual or unisexual, in multiple locales contributing to the production of novel, virulent subtypes. We also found that the genomes of two basal VGII isolates from HIV+ patients contain an introgression tract spanning three genes. Introgression substantially contributed to intra-VGII polymorphism and likely occurred through sexual reproduction with VGI. More broadly, these findings illustrate how both microevolution and sexual reproduction play central roles in the development of infectious outbreaks from avirulent or less virulent progenitors.IMPORTANCECryptococcus gattii is the causative agent responsible for ongoing infections in the Pacific Northwest of the United States and western Canada. The incidence of these infections increased dramatically in the 1990s and remains elevated. These infections are attributable to three clonal lineages of C.gattii, VGIIa, VGIIb, and VGIIc, with only VGIIa identified once previously in the Pacific Northwest prior to the start of the outbreak, albeit in a less virulent form. This study addresses the origin and emergence of this outbreak, using whole-genome sequencing and comparison of both outbreak and global isolates. We show that VGIIa arose mitotically from a less virulent clonal group, possibly via the action of a mutator phenotype, while VGIIb was likely introduced from Australia, and VGIIc appears to have emerged in the United States or in an undersampled locale via sexual reproduction. This work shows that multiple processes can contribute to the emergence of an outbreak

Analysis of a Food-Borne Fungal Pathogen Outbreak: Virulence and Genome of a Mucor circinelloides Isolate from Yogurt

ABSTRACTFood-borne pathogens are ongoing problems, and new pathogens are emerging. The impact of fungi, however, is largely underestimated. Recently, commercial yogurts contaminated with Mucor circinelloides were sold, and >200 consumers became ill with nausea, vomiting, and diarrhea. Mucoralean fungi cause the fatal fungal infection mucormycosis, whose incidence has been continuously increasing. In this study, we isolated an M.circinelloides strain from a yogurt container, and multilocus sequence typing identified the strain as Mucor circinelloides f. circinelloides. M. circinelloides f. circinelloides is the most virulent M.circinelloides subspecies and is commonly associated with human infections, whereas M.circinelloides f. lusitanicus and M.circinelloides f. griseocyanus are less common causes of infection. Whole-genome analysis of the yogurt isolate confirmed it as being close to the M. circinelloides f. circinelloides subgroup, with a higher percentage of divergence with the M.circinelloides f. lusitanicus subgroup. In mating assays, the yogurt isolate formed sexual zygospores with the (−) M. circinelloides f. circinelloides tester strain, which is congruent with its sex locus encoding SexP, the (+) mating type sex determinant. The yogurt isolate was virulent in murine and wax moth larva host systems. In a murine gastromucormycosis model, Mucor was recovered from fecal samples of infected mice for up to 10days, indicating that Mucor can survive transit through the GI tract. In interactions with human immune cells, M.circinelloides f. lusitanicus induced proinflammatory cytokines but M. circinelloides f. circinelloides did not, which may explain the different levels of virulence in mammalian hosts. This study demonstrates that M.circinelloides can spoil food products and cause gastrointestinal illness in consumers and may pose a particular risk to immunocompromised patients.IMPORTANCEThe U.S. FDA reported that yogurt products were contaminated with M. circinelloides, a mucoralean fungal pathogen, and >200 consumers complained of symptoms, including vomiting, nausea, and diarrhea. The manufacturer voluntarily withdrew the affected yogurt products from the market. Compared to other food-borne pathogens, including bacteria, viruses, and parasites, less focus has been placed on the risk of fungal pathogens. This study evaluates the potential risk from the food-borne fungal pathogen M. circinelloides that was isolated from the contaminated commercial yogurt. We successfully cultured an M.circinelloides isolate and found that the isolate belongs to the species M.circinelloides f. circinelloides, which is often associated with human infections. In murine and insect host models, the isolate was virulent. While information disseminated in the popular press would suggest this fungal contaminant poses little or no risk to consumers, our results show instead that it is capable of causing significant infections in animals

Cryptococcus: from environmental saprophyte to global pathogen.

Cryptococcosis is a globally distributed invasive fungal infection that is caused by species within the genus Cryptococcus which presents substantial therapeutic challenges. Although natural human-to-human transmission has never been observed, recent work has identified multiple virulence mechanisms that enable cryptococci to infect, disseminate within and ultimately kill their human host. In this Review, we describe these recent discoveries that illustrate the intricacy of host-pathogen interactions and reveal new details about the host immune responses that either help to protect against disease or increase host susceptibility. In addition, we discuss how this improved understanding of both the host and the pathogen informs potential new avenues for therapeutic development

CryptoCEN: A Co-Expression Network for Cryptococcus neoformans reveals novel proteins involved in DNA damage repair.

Elucidating gene function is a major goal in biology, especially among non-model organisms. However, doing so is complicated by the fact that molecular conservation does not always mirror functional conservation, and that complex relationships among genes are responsible for encoding pathways and higher-order biological processes. Co-expression, a promising approach for predicting gene function, relies on the general principal that genes with similar expression patterns across multiple conditions will likely be involved in the same biological process. For Cryptococcus neoformans, a prevalent human fungal pathogen greatly diverged from model yeasts, approximately 60% of the predicted genes in the genome lack functional annotations. Here, we leveraged a large amount of publicly available transcriptomic data to generate a C. neoformans Co-Expression Network (CryptoCEN), successfully recapitulating known protein networks, predicting gene function, and enabling insights into the principles influencing co-expression. With 100% predictive accuracy, we used CryptoCEN to identify 13 new DNA damage response genes, underscoring the utility of guilt-by-association for determining gene function. Overall, co-expression is a powerful tool for uncovering gene function, and decreases the experimental tests needed to identify functions for currently under-annotated genes

Mapping <i>C</i>. <i>neoformans</i> RNAseq runs.

A. A total of 1,523 RNAseq runs from 34 identified studies are scatter-plotted as the number of genes with nonzero expression versus the fraction transcripts that map exactly once. B. The RNAseq runs that have nonzero expression for at least half of the genes which are used to construct the CryptoCEN network. (TIF)</p

DNA damage.

Tab 1) co-expression. Tab 2) phenotype summary. Tab 3) reciprocal co-expression. (XLSX)</p

Retrospective predictive accuracy of CryptoCEN.

A. A re-embedded gene by expression matrix, scanning the key embedding parameters umap_a and umap_b over the ranges [20, 30, 40, 50, 60] and [0.45, 0.5, 0.55], respectively, while keeping the remaining parameters fixed (prereduction of dimension to 500 using PCA, n_neighbors = 30, negative_sample_rate = 50, umap_repulsion_strength = 3, n_epochs = 2000). Rows are umap_a and the columns are umap_b. The points are clustered using leiden clustering using with a resolution parameter of 1e-3 and points are colored by the cluster index. B. UpSet plot of the retrospective prediction accuracy, as determined by the neighbor voting guilt-by-association (GBA) area under the ROC curve (AUROC). AUROCs values range between 0.5 for random predictor and 1 for a perfect predictor. As data sources are combined, the prediction accuracy increases. Each annotated GO term is colored by ontology biological process (BP), cellular component (CC), or molecular function (MF). C. Enrichment of co-expression in the gene by expression matrix UMAP. For each cluster, we selected inter and intra-cluster associations. We then computed the area under the receiver operator characteristic (AUROC) for the enrichment of the intra-cluster associations over the inter-cluster associations based on the co-expression score. Enrichment within each cluster is indicated by color. (TIF)</p

Evolutionary constraints on co-expression.

A) Phylogeny used for co-evolution. Tips indicate each species, scale bar indicates 0.05 substitutions/site. B) Co-expression vs. co-evolution scores over the 5,264 overlapping gene sets does not show a positive correlation (correlation coefficient is -0.001). C) There is no difference in histone localization between the strains. Histones were marked by fusion of H4 with GFP. Cells were incubated in liquid YPD at 30°C before imaging. Images taken at 40X magnification, scale = 5 microns. D) There is no difference in nocodazole sensitivity between the cdc42Δ or cdc420Δ mutant strains. The indicated strains were grown overnight at 30°C in liquid YPD medium and then serially diluted onto YPD or plates containing 0.3 μM nocodazole and incubated for 2 days before imaging. (TIF)</p

Co-Expression scores.

Elucidating gene function is a major goal in biology, especially among non-model organisms. However, doing so is complicated by the fact that molecular conservation does not always mirror functional conservation, and that complex relationships among genes are responsible for encoding pathways and higher-order biological processes. Co-expression, a promising approach for predicting gene function, relies on the general principal that genes with similar expression patterns across multiple conditions will likely be involved in the same biological process. For Cryptococcus neoformans, a prevalent human fungal pathogen greatly diverged from model yeasts, approximately 60% of the predicted genes in the genome lack functional annotations. Here, we leveraged a large amount of publicly available transcriptomic data to generate a C. neoformans Co-Expression Network (CryptoCEN), successfully recapitulating known protein networks, predicting gene function, and enabling insights into the principles influencing co-expression. With 100% predictive accuracy, we used CryptoCEN to identify 13 new DNA damage response genes, underscoring the utility of guilt-by-association for determining gene function. Overall, co-expression is a powerful tool for uncovering gene function, and decreases the experimental tests needed to identify functions for currently under-annotated genes.</div

<i>Cryptococcus</i> Co-Expression analysis identifies both known and unknown gene clusters.

A) A gene-by-environment heatmap generated from collected C. neoformans RNAseq experiments from the SRA. The genes are on the y axis, and conditions are on the x axis. B) A gene-by-gene heatmap generated from Spearman rank correlation. C) Representative gene-by-gene expression patterns for pairs of genes at four different co-expression scores. Each dot represents the expression in a single RNAseq run, and dots are colored by study. D) Embedding of the C. neoformans Co-Expression Network (CryptoCEN) using UMAP for dimensionality reduction. Twenty-two clusters were identified using Louvain modularity clustering. The genes in each cluster were analyzed for biological process GO term enrichment using Fisher’s exact test through FungiDB, and the most significantly enriched specific term after Bonferroni correction for multiple testing was used for labelling. Clusters without significant GO term enrichment after multiple hypothesis testing correction were labeled only by cluster number. E) Genes in cluster 6 sub-clusters were analyzed for both biological process and cellular component GO term enrichment using Fisher’s exact test through FungiDB. F) Genes in cluster 16 sub-clusters were analyzed for both biological process and cellular component GO term enrichment using Fisher’s exact test through FungiDB.</p