A Polymorphism in the Chitotriosidase Gene Associated with Risk of Mycetoma Due to <i>Madurella mycetomatis</i> Mycetoma–A Retrospective Study

<div><p>Background</p><p><i>Madurella mycetomatis</i> is the most prevalent causative agent of eumycetoma in Sudan, an infection characterized by the formation of grains. Many patients are exposed to the causative agent, however only a small number develop infection. <i>M</i>. <i>mycetomatis</i> contains chitin in its cell wall, which can trigger the human immune system. Polymorphisms in the genes encoding for the chitin-degrading enzymes chitotriosidase and AMCase were described, resulting in altered chitinase activity. We investigated the association between 4 of these polymorphisms and the incidence of <i>M</i>. <i>mycetomatis</i> mycetoma in a Sudanese population.</p><p>Methodology</p><p>Polymorphisms studied in 112 eumycetoma patients and 103 matched controls included a 24-bp insertion in the chitotriosidase gene (rs3831317), resulting in impaired chitinase activity and single nucleotide polymorphism (SNP) in the AMCase gene (rs61756687), resulting in decreased AMCase activity. Also, a SNP (rs41282492) and a 10-bp insertion in the 5’UTR region of the AMCase gene (rs143789088) were studied, both resulting in increased AMCase activity. DNA was isolated from blood and genotypes were determined using PCR-RFLP.</p><p>Principal Findings</p><p>Histological staining proved the presence of chitin in the fungal grain. The polymorphism resulting in decreased chitotriosidase activity was associated with increased odds of eumycetoma (odds ratio 2.9; p = 0.004). No association was found for the polymorphisms in the genes for AMCase (all p>0.05).</p><p>Conclusion</p><p>Decreased chitotriosidase activity was associated with increased risk of <i>M</i>. <i>mycetomatis</i> mycetoma.</p></div

PCR conditions for the different polymorphisms.

<p>PCR conditions for the different polymorphisms.</p

Distribution of polymorphisms in the genes for chitotriosidase and for AMCase.

<p>*Genotype associated with an impaired, normal or decreased enzyme activity according to previous publications [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004061#pntd.0004061.ref014" target="_blank">14</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004061#pntd.0004061.ref016" target="_blank">16</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004061#pntd.0004061.ref017" target="_blank">17</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004061#pntd.0004061.ref019" target="_blank">19</a>–<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004061#pntd.0004061.ref021" target="_blank">21</a>]</p><p>**Hardy Weinberg Equilibrium (HWE) as assessed by Pearson’s χ² test. A p-value of >0.05 was associated with equilibrium.</p><p>Distribution of polymorphisms in the genes for chitotriosidase and for AMCase.</p

Tissue sections of Mycetoma foot, showing the fungal grain.

<p>Magnification 400x. (A) Haematoxylin and eosin staining. The grain, consisting of cement and fungal hyphae, is colored red. Around the grain, a zone with neutrophils is visible. (B) Grocott’s methenamine silver staining. The hyphae inside the grain are stained black. (C) Calcofluor white staining. Chitin is stained by calcofluor white staining [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004061#pntd.0004061.ref018" target="_blank">18</a>]. This photo illustrates that hyphae inside the grain are stained, and not the cement component of the grain.</p

Study population demographic features.

<p>* One patient had two lesions, one of the foot and one of the hand</p><p>Study population demographic features.</p

Calcofluor White staining of <i>in vitro</i> cultured <i>A. fumigatus</i> hyphae after incubation with recombinant chitotriosidase and recombinant AMCase.

<p>When hyphae were cultured on Sabauroud's agar (A), the cell wall remained regular and intact after incubation with the two recombinant chitinases. When hyphae were cultured on Sabauroud's agar with 1 mg/L caspofungin (B), the cell wall was irregular and disrupted after incubation with the two recombinant chitinases.</p

Grocott staining (A, D, G) and presence of AMCase (B, E, H) and chitotriosidase (C, F, I) in several rats.

<p>Panels A, B, and C show the lung of an uninfected rat. Panels D, E and F show the fungal focus in an infected, untreated rat. Panels G, H and I show the fungal focus in an infected, caspofungin treated rat. Original magnification ×400. All panels represent lungs on day 6 after inoculation. Slides were stained according to the described protocols. In Grocott staining (A, D, G), fungal hyphae are coloured black. Chitotriosidase- or AMCase-presenting cells are coloured red (B, C, E, F, H, I). In uninfected rats, normal morphology can be found in the lungs (A, B, C). In infected rats, normal morphology of alveoli is lost (D, E, F). Grocott staining shows many hyphae (D). An inflammatory response is found around the fungal focus, where chitotriosidase and AMCase are increasingly present (red zones) compared to an uninfected rat (E, F). After treatment with caspofungin, Grocott staining shows fungal material in all infected rats (G). AMCase bound fungal hyphae after treatment with caspofungin (H) and thus hyphae became visible. After treatment with caspofungin, chitotriosidase seemed to also bind the fungal cell wall and locate inside hyphal cells (I).</p

<i>In vitro</i> binding of recombinant chitinases to <i>A. fumigatus</i> hyphae.

<p>Binding of recombinant chitotriosidase (A, B). Binding of recombinant chitotriosidase when incubated in combination with recombinant AMCase (C, D) and binding of recombinant AMCase (E, F). Panels A, C and E show unexposed hyphae. Panels B, D and F show caspofungin-exposed hyphae. A, B: Contrast and brightening were slightly modified in Photoshop due to the lack of colour. C, D, E, F: Photos were not modified in Photoshop. A–B Original magnification ×100. C–F Original magnification ×400. Slides were stained according to the described protocols. Binding of either recombinant enzyme is characterized by a red colour. Recombinant chitotriosidase did not bind to unexposed hyphae (A) or to caspofungin-exposed hyphae (B). When incubated with a combination of recombinant chitotriosidase and recombinant AMCase, recombinant chitotriosidase did bind to unexposed hyphae (arrow) and to conidial heads (C) and seemed to be taken up by the fungal cells after caspofungin exposure (D). Also the cell wall seemed to be lysed at several locations (arrows). Recombinant AMCase did bind to unexposed (E) and to caspofungin-exposed hyphae (F).</p

Chitinase activity and fungal load in immunocompromised rats inoculated with <i>A. fumigatus</i> conidia.

<p>Open squares: uninfected untreated rats; filled circles: infected untreated rats; grey triangles: infected rats, treated with caspofungin at 24 h post infection. Data are means of duplicates. Bars represent medians. For each group n≥4. A. Galactomannan (GM)-index, measured by Platelia assay. According to the manufacturer's manual, GM-index of <0,5 is considered negative; B, chitotriosidase activity, expressed in arbitrary units (a.u.); C, AMCase activity, expressed in arbitrary units (a.u.). * p<0.05; significant difference between the indicated groups.</p