Phylogenetic relationships between <i>C</i>. <i>parapsilosis</i> complex lineages.

<p>Distance between the distinct lineages in <i>C</i>. <i>orthopsilosis</i> and <i>C</i>. <i>metapsilosis</i> have been inferred from heterozygous genomic regions in the hybrid strains. The origin of hybrid lineages have been schematically indicated by super-imposing dashed lines connecting different branches in the phylogeny. Strains representing the different lineages are indicated. Lineages whose existence is inferred from the hybrid strains but for which a representative strain is not available are indicated as unknown.</p

Chromosome blocks for the reference genome, comprising seven chromosomes and two unplaced scaffolds.

<p>Heterozygous regions are in white, while homozygous blocks of at least 5 kb are depicted in grey. Loss of heterozygosity (LOH) detection is described in Materials and Methods.</p

Genomic organisation of mating type locus (MTL) idiomorphs, MTLa (yellow) and MTLα (green), in three <i>Candida</i> species.

<p><i>C</i>. <i>albicans</i> and <i>C</i>. <i>orthopsilosis</i> encode both MTL idiomorphs (A). <i>C</i>. <i>metapsilosis</i> encodes complete MTLα and partial MTLa (B). MTLa introgression in <i>C</i>. <i>metapsilosis</i> was confirmed with PCR and Sanger sequencing. Alignments of Sanger products are denoted with dashed lines. P1f-P1rα and P2fα-P2r align to MTLα. In contrast, P1f-P1ra and P2fa-P2r align to parts of both MTLα and MTLa. The long, horizontal line in P2fa-P2r represents a partial alignment of this Sanger sequence to MTLα and partial MTLa loci.</p

Linear and circular mitochondrial genomes in <i>C</i>. <i>metapsilosis</i> isolates.

<p>(A) PFGE analysis. DNA was isolated in agarose blocks and separated in PFGE as described in Materials and Methods. Only strains containing linear mitochondrial genome show a sharp band about 25–30 kb long. (B) Southern blot analysis. PFGE separated DNA samples were blotted onto a nylon membrane and hybridized with radioactively labeled mtDNA probe. The probe hybridizes with a sharp band (L) in the strain MCO448 with linear mtDNA (lane 1). In the strain PL448 with circular mtDNA (lane 2) the probe reveals a smeary band (23–100 kb) plus two fractions (labeled as CI and CII) corresponding to presumed circular replication intermediates of mtDNA. In both strains, the probe also detected mtDNA molecules trapped in wells that contain branched mtDNA structures resulting from recombinantion processes. (C) PCR analysis. The reactions were performed with primers derived from the subtelomeric genes nad3 and atp6 (localized at opposite ends and oriented toward the termini of linear molecules) on total DNA templates and the PCR products were electrophoretically separated. Only strains with circular mitochondrial genome (PL448 and SZMC21154) exhibit a PCR product derived from the end-to-end junction of the mitochondrial genome. MCO448 (lane 1), PL448 (lane 2), SZMC8095 (lane 3), SZMC8094 (lane 4), SZMC8093 (lane 5), SZMC8092 (lane 6), SZMC8022 (lane 7), SZMC2548 (lane 8), SZMC8029 (lane 9), SZMC21154 (lane 10), SZMC8098 (lane 11). M-molecular marker (Lambda Ladder PFG Marker (New England Biolabs) (in A), Low Range PFG Marker (New England Biolabs) (in B), lambda DNA/PstI (in C)).</p

Phylogeny and genome composition in <i>Candida</i> and related species.

<p>Species translating CUG codon as serine (CTG clade) are denoted with color background: haploid species in light blue and diploid species in dark blue. Common human pathogens (red), moderate pathogens (orange), and rare pathogens (grey) are marked with color circles. Species with heterozygous genome are <i>in italic</i>, while extremely homozygous genomes are in bold. Barcharts with number of virulence-related genes for: i) cell wall (HYR/IFF) and adhesion (ALS/EPA) (pink), ii) oxidative-stress response (SOD, CAT, GPX, YBH) (blue), iii) lipases (LIP, PLB, SRR, FOX) (green), iv) secreted aspartic proteinases (SAP) (red) and v) other virulence-related genes (grey) including drug resistance genes (TPS, ERG), regulators (STE20, WH11, PHO100), iron acquisition (FTR1). Barcharts are scaled from 0 to 34 for all species.</p

Multiple correspondence analysis (MCA) of genome diversity, based on 618,120 SNPs.

<p>The countries of isolation are indicated for all strains. SNPs from four genomic libraries of PL429 (pe300, pe400ov, pe600 and mp5000) were analysed separately, but all four libraries clustered together. For simplicity, the plot shows only PL429 results for one library (pe600).</p

Diagrammatic presentation of the occurrence of lipase-, protease- and pseudohyphae-producer strains in the <i>C. parapsilosis sensu lato</i> complex.

<p>n: number of isolates, upper number in fraction: number of pseudohypha-producer strains, lower number in fraction: number of pseudohypha negative strains.</p

Interactions of different <i>C. parapsilosis sensu lato</i> isolates (see Table S1.) with primary human monocyte-derived macrophages.

<p>(A) Killing efficiency of <i>C. parapsilosis sensu lato</i> species based on CFU-determinations (Cp, <i>C. parapsilosis sensu stricto</i>; Co, <i>C</i><i>. orthopsilosis</i>; Cm, <i>C</i><i>. metapsilosis</i>), (B) killing efficiency of lipase producer vs. non-producer and pseudohypha positive vs. negative strains in the <i>C. parapsilosis sensu lato</i> group [lip+, lipase positive (regardless of pseudohypha production); lip-, lipase negative; psh+, pseudohyphae positive (regardless of lipase production); psh-, pseudohyphae negative], (C) killing efficiency of lipase or pseudohyphae positive vs. negative isolates of <i>C. parapsilosis sensu stricto</i>, (D) killing efficiency of lipase or pseudohyphae producer vs. non-producer strains of <i>C</i><i>. orthopsilosis</i>, (E) host-cell damaging capacity of <i>C. parapsilosis sensu lato</i> species based on the release of LDH (lactate dehydrogenase), (F) host-cell damaging capacity of lipase or pseudohyphae producer vs. non-producer strains in the <i>C. parapsilosis sensu lato</i> group, (G) host-cell damaging capacity of lipase or pseudohyphae positive vs. negative isolates of <i>C. parapsilosis sensu stricto</i>, (H) host-cell damaging capacity of lipase or pseudohyphae producer vs. non-producer strains of <i>C</i><i>. orthopsilosis</i>. Cp, <i>C. parapsilosis sensu stricto</i>; Co, <i>C</i><i>. orthopsilosis</i>; Cm, <i>C</i><i>. metapsilosis</i>. Data points on graphs represent individual strains. Experiments were performed in triplicates. Data were analyzed by the Kruskal-Wallis test (A, E), the Mann-Whitney test (B, D, F, G, H) or the Wilcoxon rank sum test (C). * p<0.05, ** p<0.01, *** p<0.001.</p

Phagocytosis of one representative isolate each of <i>C. parapsilosis sensu stricto</i>, <i>C</i><i>. orthopsilosis</i> and <i>C</i><i>. metapsilosis</i> by J774.2 macrophages.

<p>(A) Light microscopic picures of J774.2 macrophages phagocytosing <i>C. parapsilosis sensu lato</i> species, (B) phagocytosis of <i>C. parapsilosis sensu stricto</i>, <i>C</i><i>. orthopsilosis</i> and <i>C</i><i>. metapsilosis</i> by J774.2 macrophages assessed by quantitative imaging, R2: phagocytosing macrophage population discriminated by the presence of red fluorescence due to ingestion of Alexa Fluor 647-labeled yeast cells, (C) numbers of ingested yeast cell/macrophage in the R2 population in case of <i>C. parapsilosis sensu stricto</i>, <i>C</i><i>. orthopsilosis</i> and <i>C</i><i>. metapsilosis</i> determined by the spot-counting feature of IDEAS software.</p