Origin of fungal hybrids with pathogenic potential from warm seawater environments

Abstract

Hybridisation is a common event in yeasts often leading to genomic variability and adaptation. The yeast Candida orthopsilosis is a human-associated opportunistic pathogen belonging to the Candida parapsilosis species complex. Most C. orthopsilosis clinical isolates are hybrids resulting from at least four independent crosses between two parental lineages, of which only one has been identified. The rare presence or total absence of parentals amongst clinical isolates is hypothesised to be a consequence of a reduced pathogenicity with respect to their hybrids. Here, we sequence and analyse the genomes of environmental C. orthopsilosis strains isolated from warm marine ecosystems. We find that a majority of environmental isolates are hybrids, phylogenetically closely related to hybrid clinical isolates. Furthermore, we identify the missing parental lineage, thus providing a more complete overview of the genomic evolution of this species. Additionally, we discover phenotypic differences between the two parental lineages, as well as between parents and hybrids, under conditions relevant for pathogenesis. Our results suggest a marine origin of C. orthopsilosis hybrids, with intrinsic pathogenic potential, and pave the way to identify pre-existing environmental adaptations that rendered hybrids more prone than parental lineages to colonise and infect the mammalian host.

Fig. 1. Hybrids and the parental B lineage amongst C. orthopsilosis marine isolates.

a Frequency of 27-mers of C. orthopsilosis known parental A strain Co90-125, marine non-hybrid SY36 and SY78 strains as well as hybrid marine strains 2Y721, 2Y220, 4Y98, 4Y106, 4Y108, 4Y222 and 4Y225. Previously known clinical hybrid strain MCO456 and clinical non-hybrid strain s428 (A-lineage) have been added to this figure for comparison purposes. The presence of a single peak indicates non-hybrid strains. Isolates showing a two-peaked profile are hybrids. The first peak (with half of the coverage) corresponds to heterozygous positions, whereas the second one represents homozygous regions. Colours in the plot represent the presence (red) or absence (black) of the k-mers in the reference genome of parent A Co90-125 (ASM31587v1). b Plot of the density of sequence divergence in heterozygous blocks (larger than 100 base pairs) of marine C. orthopsilosis strains. Source data are provided as a Source Data file.

Fig. 2. Reference genome assembly of novel C. orthopsilosis parent B.

a Dot plot assessing the similarity between assemblies of Co90-125 parent A ASM31587v1 and newly identified and assembled parent B strain SY36 (see Methods: Genome assembly C. orthopsilosis parent B). b Species tree showing the phylogenetic relationships between members of the C. parapsilosis species complex. C. orthopsilosis strain MCO456 was chosen as a representative of hybrids while Co90-125 and SY36 represent parents A and B, respectively. c GO term enrichment of Biological Process terms of genes harboured in genomic regions that are parent B specific. GO term enrichment analysis was done using clusterProfiler v. 3.14.3, which performs the hypergeometric test. Source data are provided as a Source Data file.

Fig. 3. Phylogenetic relationships between C. orthopsilosis parental and hybrid lineages.

a Neighbour net splits tree network based on alignments harbouring homozygous variants of all C. orthopsilosis strains. Different hybrid clades are circled in colours. Parent A Co90-125 is highlighted in blue, parent B SY36 in red and all other marine isolates in green. b Maximum-likelihood tree showing phased haplotypes A and B for each hybrid strain at heterozygous positions. A haplotypes are highlighted in blue and B haplotypes in red. c Phylogenetic tree based on heterozygous sites. Sequence alignments of phased haplotype A and B were concatenated for each strain in order to build the tree. Different hybrid clades as well as parental reference strains are highlighted in colours. Source data are provided as a Source Data file.

Fig. 4. Phenotypic differences between a selected subset of C. orthopsilosis strains under different conditions.

Area under the curve (AUC) inferred from measurements taken every 15 min during a period of 24 h in solid medium. a Area under the growth curve of C. orthopsilosis hybrid and parental strains (n = 8 biological replicates over 2 independent experiments) under temperatures ranging from 30 °C to 41.5 °C. Strains from the same clade or parental lineage are grouped together. Clades and parental lineages are represented in different colours. Data are presented as mean values +/− SD. b Fitness area under the curve (fAUC) of C. orthopsilosis hybrid and parental strains (n = 4 biological replicates) in the presence of anidulafungin (ANI) and fluconazole (FLZ). Green circles in the X-axis indicate marine isolates. The centre line in the boxplot indicates the median value, the boxes contain the Q1 and Q3 quartiles (IQR). The whiskers extend up to 1.5 × IQR. c Survival curves of G. mellonella injected with 10⁶C. orthopsilosis cells per larva (n = 15) during a period of 7 days. Difference between strains was significant with a P < 0.001 by the log rank (Mantel-Cox) test. Source data are provided as a Source Data file.

Fig. 5. C. orthopsilosis hybrids inherit different traits from parental lineages.

Overview of phenotypic differences between hybrid and parental lineages. The term “Phenotype” indicates growth of the strain under that specific condition in comparison to “No phenotype” which refers to absence of growth in that condition. The term “Intermediate phenotype” refers to a lower degree of growth than that of “Phenotype”. Clades and parental lineages are indicated in different colours. Green circles indicate marine isolates. Source data are provided as a Source Data file.