Active Matrix Metalloprotease-9 Is Associated with the Collagen Capsule Surrounding the Madurella mycetomatis Grain in Mycetoma

Kirsten Geneugelijk,Ahmed H Fahal,Wendy Kloezen,Wendy W J Van De Sande

doi:10.1371/journal.pntd.0002754

Kirsten Geneugelijk, Ahmed H Fahal + Show 2 more

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https://doi.org/10.1371/journal.pntd.0002754

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Abstract

Madurella mycetomatis is the main causative organism of eumycetoma, a persistent, progressive granulomatous infection. After subcutaneous inoculation M. mycetomatis organizes itself in grains inside a granuloma with excessive collagen accumulation surrounding it. This could be contributing to treatment failure towards currently used antifungal agents. Due to their pivotal role in tissue remodelling, matrix metalloproteinases-2 (MMP-2) and 9 (MMP-9) or tissue inhibitor of metalloproteinases (TIMP) might be involved in this process. Local MMP-2 and MMP-9 expression was assessed by immunohistochemistry while absolute serum levels of these enzymes were determined in mycetoma patients and healthy controls by performing ELISAs. The presence of active MMP was determined by gelatin zymography. We found that both MMP-2 and MMP-9 are expressed in the mycetoma lesion, but the absolute MMP-2, -9, and TIMP-1 serum levels did not significantly differ between patients and controls. However, active MMP-9 was found in sera of 36% of M. mycetomatis infected subjects, whereas this active form was absent in sera of controls (P<0.0001). MMP-2, MMP-9, and TIMP-1 polymorphisms in mycetoma patients and healthy controls were determined through PCR-RFLP or sequencing. A higher T allele frequency in TIMP-1 (+372) SNP was observed in male M. mycetomatis mycetoma patients compared to controls. The presence of active MMP-9 in mycetoma patients suggest that MMP-9 is activated or synthesized by inflammatory cells upon M. mycetomatis infection. Inhibiting MMP-9 activity with doxycycline could prevent collagen accumulation in mycetoma, which in its turn might make the fungus more accessible to antifungal agents.

Highlights

Madurella mycetomatis is the most prevalent causative organism of eumycetoma, a persistent, progressive granulomatous infection involving subcutaneous tissues and bones [1]
matrix metalloproteinases-2 (MMP-2) and Matrix Metalloproteinases (MMPs)-9 are expressed by immune-cells surrounding the fungal grain
In order to determine if the gelatinases MMP-2 and MMP-9 play a role in the encapsulation of the mycetoma grain, the presence of these two MMPs was demonstrated by immunohistochemical staining of tissue sections of patients infected with M. mycetomatis (Figures 1c and 1d)

Summary

Introduction

Madurella mycetomatis is the most prevalent causative organism of eumycetoma, a persistent, progressive granulomatous infection involving subcutaneous tissues and bones [1]. Treatment with the currently used antifungal agents, ketoconazole and itraconazole, only facilitates surgical removal of mycetoma lesions as they induce encapsulation of the fungal grain with fibrous tissue [3,4]. MMP-2 and MMP-9 have the ability to degrade a variety of ECM constituents (e.g. gelatin, elastin, and various types of collagen) [6,7]. Since both MMP-2 and MMP-9 are zymogens, proteolytic activation is prerequisite to become completely active [7]. The exact mechanism(s) explaining these observations have to be clarified, it is hypothesized that MMPs induce de novo ECM accumulation through its digestion of ECM constituents. Another explanation might be that MMPs provoke collagen accumulation via another pathway than ECM digestion

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