Cell walls of the dimorphic fungal pathogens Sporothrix schenckii and Sporothrix brasiliensis exhibit bilaminate structures and sloughing of extensive and intact layers

This work was supported by the Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), grants E-26/202.974/2015 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grants 229755/2013-5, Brazil. LMLB is a senior research fellow of CNPq and Faperj. NG acknowledged support from the Wellcome Trust (Trust (097377, 101873, 200208) and MRC Centre for Medical Mycology (MR/N006364/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Cell wall polysaccharide and amino acid content of <i>S</i>. <i>schenckii</i> and <i>S</i>. <i>brasiliensis</i> yeast cells at different days of growth.

<p>Cell wall polysaccharide and amino acid content of <i>S</i>. <i>schenckii</i> and <i>S</i>. <i>brasiliensis</i> yeast cells at different days of growth.</p

Uptake by human monocyte-derived macrophages (hMac) of <i>S</i>. <i>schenckii</i> (MYA 4820) and <i>S</i>. <i>brasiliensis</i> (MYA4823) yeast cells cultivated for 4 and 10 days in YPD broth.

<p>The uptake of <i>S</i>. <i>schenckii</i> and <i>S</i>. <i>brasiliensis</i> yeast cells (4 and 10 days) are reported as the mean values of three experiments performed in duplicate with their standard deviation (SD). Unpaired t-test: (a) <i>p</i> ≥ 0.0001 <i>S</i>. <i>brasiliensis</i> compared with <i>S</i>. <i>schenckii</i>– 4 days yeast cells; (B) <i>p</i> ≥ 0.0001 <i>S</i>. <i>brasiliensis</i> compared with <i>S</i>. <i>schenckii</i>– 10 days yeast cells.</p

HFP-TEM of the cell wall of the yeast parasitic phase of <i>S</i>. <i>schenckii</i> MYA4822.

<p><i>S</i>. <i>schenckii</i> yeast cells of the clinical isolate MYA 4822 were grown for 7 days in YPD broth. The TEM images show a cell wall double layer (A) and the complete shedding of a cell wall layer (B), similarly to the process observed in other <i>S</i>. <i>schenckii</i> clinical isolates (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006169#pntd.0006169.g001" target="_blank">Fig 1A</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006169#pntd.0006169.s001" target="_blank">S1 Fig</a>).</p

HFP-TEM of the cell wall of the yeast parasitic phase of <i>S</i>. <i>schenckii</i> (MYA4820) and <i>S</i>. <i>brasiliensis</i> (MYA4823) cultivated for 3, 7 or 10 days in YPD broth, pH 7.8.

<p>The TEM images show a cell wall double layer of <i>S</i>. <i>schenckii</i> and its shedding over time in culture (A) and a thicker and extensive microfibrillar outer layer of <i>S</i>. <i>brasiliensis</i> interconnecting yeast cells (B).</p

HFP-TEM of the cell wall of <i>S</i>. <i>brasiliensis</i> yeast parasitic phase.

<p>(A, B) HFP-TEM images at two magnifications show details of long cell wall fibrils (~400 nm) in <i>S</i>. <i>brasiliensis</i> (10 days in culture) interconnecting the yeast cells. In (B) the fibrils form a physical bridge between two <i>S</i>. <i>brasiliensis</i> yeast cells. Scale bars are indicated in each panel.</p

Infrared spectroscopy of the alkali-insoluble cell wall fraction of <i>S</i>. <i>schenckii</i> (MYA 4820) and <i>S</i>. <i>brasiliensis</i> (MYA 4823).

<p>Infrared spectra of <i>S</i>. <i>schenckii</i> (A) and <i>S</i>. <i>brasiliensis</i> (B) cell wall showing the peaks at energies corresponding to the cell wall polysaccharides chitin (1560 and 1640 cm^-1; blue arrows) and β-glucan (890 cm^-1; purple arrow). The presence of β-glucans (1→3 and 1→6) are evidenced by peaks at 1156, 1076, and 1041 cm^-1 (green arrows).</p

Inner layer cell wall thickness <i>of Sporothrix</i> spp. yeast cells.

<p>Inner layer cell wall thickness <i>of Sporothrix</i> spp. yeast cells.</p

¹H-NMR of the rhamnomannan fraction isolated from <i>S</i>. <i>schenckii</i> and <i>S</i>. <i>brasiliensis</i> yeast cells.

<p>¹H-MNR spectra showing the signals corresponding to the α-L-Rha 1→3 α-D-Man<i>p</i> structure (A and B). The H1 region of the rhamnomannans of <i>S</i>. <i>schenckii</i> (C) and <i>S</i>. <i>brasiliensis</i> (D) is enlarged.</p

<i>S</i>. <i>schenckii</i> and <i>S</i>. <i>brasiliensis</i> rhamnomannan ¹³C-NMR signals, yeast phase.

<p><i>S</i>. <i>schenckii</i> and <i>S</i>. <i>brasiliensis</i> rhamnomannan ¹³C-NMR signals, yeast phase.</p