Additional file 1: of Paracoccidioides brasiliensis infection promotes thymic disarrangement and premature egress of mature lymphocytes expressing prohibitive TCRs

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Exploring virulence and immunogenicity in the emerging pathogen <i>Sporothrix brasiliensis</i>

<div><p>Sporotrichosis is a polymorphic chronic infection of humans and animals classically acquired after traumatic inoculation with soil and plant material contaminated with <i>Sporothrix</i> spp. propagules. An alternative and successful route of transmission is bites and scratches from diseased cats, through which <i>Sporothrix</i> yeasts are inoculated into mammalian tissue. The development of a murine model of subcutaneous sporotrichosis mimicking the alternative route of transmission is essential to understanding disease pathogenesis and the development of novel therapeutic strategies. To explore the impact of horizontal transmission in animals (e.g., cat-cat) and zoonotic transmission on <i>Sporothrix</i> fitness, the left hind footpads of BALB/c mice were inoculated with 5×10⁶ yeasts (n = 11 <i>S</i>. <i>brasiliensis</i>, n = 2 <i>S</i>. <i>schenckii</i>, or n = 1 <i>S</i>. <i>globosa</i>). Twenty days post-infection, our model reproduced both the pathophysiology and symptomology of sporotrichosis with suppurating subcutaneous nodules that progressed proximally along lymphatic channels. Across the main pathogenic members of the <i>S</i>. <i>schenckii</i> clade, <i>S</i>. <i>brasiliensis</i> was usually more virulent than <i>S</i>. <i>schenckii</i> and <i>S</i>. <i>globosa</i>. However, the virulence in <i>S</i>. <i>brasiliensis</i> was strain-dependent, and we demonstrated that highly virulent isolates disseminate from the left hind footpad to the liver, spleen, kidneys, lungs, heart, and brain of infected animals, inducing significant and chronic weight loss (losing up to 15% of their body weight). The weight loss correlated with host death between 2 and 16 weeks post-infection. Histopathological features included necrosis, suppurative inflammation, and polymorphonuclear and mononuclear inflammatory infiltrates. Immunoblot using specific antisera and homologous exoantigen investigated the humoral response. Antigenic profiles were isolate-specific, supporting the hypothesis that different <i>Sporothrix</i> species can elicit a heterogeneous humoral response over time, but cross reaction was observed between <i>S</i>. <i>brasiliensis</i> and <i>S</i>. <i>schenckii</i> proteomes. Despite great diversity in the immunoblot profiles, antibodies were mainly derived against 3-carboxymuconate cyclase, a glycoprotein oscillating between 60 and 70 kDa (gp60-gp70) and a 100-kDa molecule in nearly 100% of the assays. Thus, our data broaden the current view of virulence and immunogenicity in the <i>Sporothrix</i>-sporotrichosis system, substantially expanding the possibilities for comparative genomic with isolates bearing divergent virulence traits and helping uncover the molecular mechanisms and evolutionary pressures underpinning the emergence of <i>Sporothrix</i> virulence.</p></div

Immunoreactivity of <i>S</i>. <i>brasiliensis</i> Ss54 (= CBS 132990) proteins resolved by 2DGE with mice sera reveals high cross-reactivity and isoforms of gp60 as major component recognized.

<p><i>S</i>. <i>brasiliensis</i> whole cellular extract (yeast phase) were separated by IEF at pH 4 to 7 in the first dimension, followed by 10% SDS-PAGE in the second dimension. Gels were subsequently silver stained (A) or immunoblotted (B-I) with a 1:200 dilution of infected mice sera indicated at the top. Red dot areas indicate all seroreactive protein spots identified as 3-carboxymuconate cyclase. Immunoblot assay using Ss104 antisera did not detect immunoreactivity (I), similar to the 1D assay. Acidic and basic ends are denoted at the bottom, and the positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels series.</p

Characteristics of <i>Sporothrix</i> spp. isolates.

<p>Characteristics of <i>Sporothrix</i> spp. isolates.</p

Virulence characteristics of <i>Sporothrix</i> isolates based on the murine model of subcutaneous sporotrichosis.

<p>Virulence characteristics of <i>Sporothrix</i> isolates based on the murine model of subcutaneous sporotrichosis.</p

Humoral diversity in experimental sporotrichosis.

<p>(A) Immunoblot patterns of clinical isolates of <i>Sporothrix</i> using exoantigen (green bar) and reference whole cell extracts derived from <i>S</i>. <i>brasiliensis</i> (Ss54 = CBS 132990; yellow bar) and <i>S</i>. <i>schenckii</i> (Ss118 = CBS 132974; red bar). Fungal proteins were resolved on 10% SDS-PAGE, electrotransferred to PVDF membranes, and probed against a pooled mouse serum (homologous antisera n = 5, diluted at 1:200). (B) Frequency and diversity of immunoreactive proteins in experimental sporotrichosis revealed immunodominant molecules in the 100-kDa fractions and oscillating between 60 and 70-kDa (gp60-70).</p

Survival curves for BALB/c mice inoculated subcutaneously with <i>Sporothrix</i> species or PBS (control group).

<p>All mice inoculated with highly invasive <i>S</i>. <i>brasiliensis</i> isolates (Ss174, Ss226, and Ss252) died between week 2 and 16 post-infection. ***<i>P</i> < 0.001 for survival in <i>S</i>. <i>brasiliensis</i> Ss174 (red line), Ss226 (green line), and Ss252 (blue line) compared to the control group (black line). Remaining <i>S</i>. <i>brasiliensis</i> (Ss34, Ss54, Ss66, Ss67, Ss99, Ss104, Ss261, and Ss265), <i>S</i>. <i>schenckii</i> (Ss39 and Ss126) and <i>S</i>. <i>globosa</i> (Ss06) infected groups (black line) behaved similarly to the PBS group and no animal died until the end of the assay.</p

Highly virulent <i>S</i>. <i>brasiliensis</i> isolates induce critical weight loss leading to impaired development and death of infected animals.

<p>Data are expressed as percent weight loss (%) determined by measuring the animal’s weight every week post-inoculation and comparing them to the weight of the animal on the day of inoculation. (A) Significant weight loss in <i>S</i>. <i>brasiliensis</i> Ss66, Ss99, Ss174, and Ss226-infected groups, leading to the death of animals (Ss174 and Ss226) and impaired development compared to the PBS group (<i>P</i> < 0.0001). (B) Moderate development for <i>S</i>. <i>brasiliensis</i> Ss34, Ss54, Ss67, Ss104, Ss252, Ss261, and Ss265 and the <i>S</i>. <i>globosa</i> Ss06-infected group. Despite initial weight loss in early infection, mice began to regain weight between the 2^nd and 5^th week post-inoculation, but this evolution was not similar to the PBS group (<i>P</i> < 0.0001). (C) Normal development for <i>S</i>. <i>schenckii</i> Ss39 and Ss126-infected groups, similar to the PBS group, demonstrating the appearance and spontaneous resolution of <i>Sporothrix</i> infection. Statistical analysis is presented in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005903#pntd.0005903.s002" target="_blank">S2 Table</a>.</p

Murine model of subcutaneous infection by <i>Sporothrix</i> species shows isolate-specific lesion patterns.

<p>BALB/c male mice were inoculated in the left hind footpad with a high load of <i>Sporothrix</i> yeast cells (5×10⁶ cells/animal). (A–F) Left hind footpads of mice infected with <i>S</i>. <i>globosa</i> (Ss06), <i>S</i>. <i>schenckii</i> (Ss126), <i>S</i>. <i>brasiliensis</i> (Ss104), <i>S</i>. <i>brasiliensis</i> (Ss54), <i>S</i>. <i>brasiliensis</i> (Ss174), or <i>S</i>. <i>brasiliensis</i> (Ss226). Mice were imaged 27 days post-infection using the L-Pix Touch (Loccus Biotecnologia, São Paulo, Brazil) imaging system.</p

Detection of anti-gp43 antibody and gp43 antigen in bronchoalveolar lavage (BAL) fluid from patients with or without paracoccidioidomycosis (PCM).

<p>Data are n(%).</p><p>Detection of anti-gp43 antibody and gp43 antigen in bronchoalveolar lavage (BAL) fluid from patients with or without paracoccidioidomycosis (PCM).</p