<i>Paracoccidioides - Figure 1 </i> Species Complex: Ecology, Phylogeny, Sexual Reproduction, and Virulence

<p>(A) Median-joining haplotypic network distribution of the <i>Paracoccidioides</i> genus and <i>L. loboi</i> based on the <i>gp43</i> marker. The size of the circumference is proportional to the haplotype frequency, and colors vary according to the sampling location of each haplotype. Red dots (median vectors) are hypothetical missing intermediates, and black dots represent each mutation site. (B) Schematic representation of serum/antigen compatibility among patients and isolates from Mato Grosso and São Paulo used in serological tests. The illustration shows the low immunogenic specificity when serum and antigenic pool from different states are reacted <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004397#ppat.1004397-Batista1" target="_blank">[41]</a>.</p

Population structure of <i>Histoplasma capsulatum</i> deduced by Bayesian Analysis of Population Structure (BAPS).

<p>A) Structure plots of 205 isolates revealing 6 different major populations (Clusters 1–6). Phylogenetic species were assigned to each of the six deduced populations as follows: Cluster 1 representing the phylogenetic species NAm 1, Cluster 2 representing the phylogenetic species RJ, Cluster 3 containing LAm B, Cluster 4 constituted by phylogenetic species NAm 2, Cluster 5 constituted by LAm A1, LAm A2, BR1-4, and the paraphyletic low supported clades Eurasia, Unknown 1 and Unknown 2 and Cluster 6 containing Netherlands, Panama, Africa, Australia and BAC1. B) Bayesian population tree based on substructures of the 6 initial clusters deduced by BAPS. Gene flow is represented by mixture isolates that are annotated with brackets along the tree.</p

Extended Bayesian Skyline Plot (EBSP) of <i>Histoplasma capsulatum sensu lato</i>.

<p>EBSPs represents population size changes over time and divergence dating was inferred using BEAST v1.8.2 based on conservative intervals of nucleotide substitutions rates and dates (0.00043–0.00656 subst/site/lineage/My; 0.0–15 Ma) that encompass values obtained by Kasuga et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004732#pntd.0004732.ref040" target="_blank">40</a>]. Y-axes are effective population size divided by generation time. X-axes are in millions of years. Confidence intervals of each dated phylogenetic species were added to the nodes.</p

Maximum Likelihood (ML) tree of <i>Histoplasma capsulatum</i> generated by IQ-TREE software for 232 taxa through 4 different loci (<i>arf</i>, <i>ole1</i>, <i>tub</i> and <i>anti-H</i> loci) reveals at least monophyletic braches as following: NAm 1, NAm 2, RJ, LAm B, NAm LAm A1, LAm A2, BR1-4, and Cluster 6 containing Netherlands, Panama, Africa, Australia and BAC1.

<p>Dual branch support, inferred by non-parametric bootstrap for ML analysis, combined with posterior probabilities obtained for the BI analysis, was added to the branches. Monophyletic branches that were supported by two methods (Bootstrap≥70/Posterior Probabilities≥0.95) were designated high confidence clades. We also identified possible in-group variation that may be associated with specific niches. Low supported clades such as Eurasia, Unknown 1 and Unknown 2 were detected but do not follow our monophyletic branches supporting criteria.</p

Differences in Cell Morphometry, Cell Wall Topography and Gp70 Expression Correlate with the Virulence of <i>Sporothrix brasiliensis</i> Clinical Isolates

<div><p>Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including <i>Sporothrix schenckii</i> and <i>Sporothrix brasiliensis</i>. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of <i>S. brasiliensis</i> compared with two reference strains of <i>S. schenckii</i>. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as <i>S. brasiliensis</i>. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with <i>S. schenckii</i>. A single <i>S. brasiliensis</i> isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent <i>S. brasiliensis</i> isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the <i>S. schenckii</i> and <i>S. brasiliensis</i> genomes<i>.</i></p> </div

<i>In silico</i> characterization of gp70 in <i>S. schenckii</i> (1099-18) and <i>S. brasiliensis</i> (5110).

<p>Identification of important domains and putative glycosylation sitesin the gp70 protein sequence using InterPro and EnsembleGly. The gene and protein sequences of gp70 were deposited in the GenBank and EMBL-EBI, respectively.</p

Western blot analysis of the cell surface gp70 of <i>S. brasiliensis</i>.

<p>The gp70 antigen was revealed on cell surface extracts of the yeast parasitic phase of two <i>S. schenckii</i> reference strains (lanes 1 and 2) and several clinical isolates of <i>S. brasiliensis</i> (lanes 3 to 8) by a monoclonal antibody anti-gp70, mAb P6E7. A purified gp70 was used as a positive control and in C is shown the negative control of the mAb P6E7. Correspondent strains in lanes (1) 1099-18; (2) IPEC 15383; (3) IPEC 17943; (4) 5110; (5) Ss 54; (6) UFTM 01; (7) HUPE 114500 and (8) HUPE 114158. The amount of protein loaded in lanes 1 to 8 was 5 µg..</p

Morphology and morphometry of the yeast phase of <i>S. schenckii</i> and <i>S. brasiliensis</i> clinical isolates.

<p>(A, B) Scanning electron microscopy of 1099-18 and IPEC 15383 of <i>S. schenckii</i>, respectively; (C, D) IPEC 17943 and (D) 5110, <i>S. brasiliensis</i> Scanning electron microscopy of IPEC 17943 and 5110 strains of <i>S. brasiliensis</i>, respectively. (E) Morphometric analysis of yeast cells of <i>S. schenckii</i> and <i>S. brasiliensis</i> isolates showing the cell mean size (<u>+</u> standard deviation). Fifty eight yeast cells of each strain were analyzed. * <i>p</i>< 0.05 relative to all other isolates.</p

Distribution and subcellular localization of gp70 on yeast cells of <i>Sporothrix</i> clinical isolates.

<p>Transmission electron micrograph of <i>S. schenckii</i> or <i>S. brasiliensis</i> yeast cells incubated with the monoclonal anti-gp 70 antibody followed by a mouse anti-IgG gold-conjugated antibody. Is shown in (A) <i>S. schenckii</i> 1099-18, (B) <i>S. schenckii</i> IPEC 15383, (C) <i>S. brasiliensis</i> IPEC 17943 and (D) <i>S. brasiliensis</i> 5110. Arrows indicate localization of gp70 on the cell wall (black arrows), in the cytoplasm (white arrows) and at the extracellular compartment (arrowheads). Images featured were digitally magnified.</p

Gp70 expression on the cell wall of <i>S. schenckii</i> and <i>S. brasiliensis</i> clinical isolates.

<p>The expression of gp70 in cell wall extracts (see Methods) was verified by western blot analysis of <i>S. schenckii</i> 1099-18 (A) and IPEC 15383 (B); and (C) of <i>S. brasiliensis</i> IPEC 17943 (C) and 5110 (D). The amount of protein loaded was 5 µg (A-C) and 15µg (D). (E-M) Scanning electron microscopy showing the backscattered electron imaging (I-M) of yeast cells of <i>S. schenckii</i> 1099-18 (E, I), IPEC 15383 (F, J) and of <i>S. brasiliensis</i> IPEC 17943 (G, L) and 5110 (H, M) which were incubated with a monoclonal antibody anti-gp70 followed by a mouse anti-IgG gold-conjugate. E-H, Scale bar 1.0 µm.</p