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. 2015 Jul 14;9(7):e0003926.
doi: 10.1371/journal.pntd.0003926. eCollection 2015.

A Madurella mycetomatis Grain Model in Galleria mellonella Larvae

Affiliations

A Madurella mycetomatis Grain Model in Galleria mellonella Larvae

Wendy Kloezen et al. PLoS Negl Trop Dis. .

Abstract

Eumycetoma is a chronic granulomatous subcutaneous infectious disease, endemic in tropical and subtropical regions and most commonly caused by the fungus Madurella mycetomatis. Interestingly, although grain formation is key in mycetoma, its formation process and its susceptibility towards antifungal agents are not well understood. This is because grain formation cannot be induced in vitro; a mammalian host is necessary to induce its formation. Until now, invertebrate hosts were never used to study grain formation in M. mycetomatis. In this study we determined if larvae of the greater wax moth Galleria mellonella could be used to induce grain formation when infected with M. mycetomatis. Three different M. mycetomatis strains were selected and three different inocula for each strain were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 4 mg/larvae. Larvae were monitored for 10 days. It appeared that most larvae survived the lowest inoculum, but at the highest inoculum all larvae died within the 10 day observation period. At all inocula tested, grains were formed within 4 hours after infection. The grains produced in the larvae resembled those formed in human and in mammalian hosts. In conclusion, the M. mycetomatis grain model in G. mellonella larvae described here could serve as a useful model to study the grain formation and therapeutic responses towards antifungal agents in the future.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Different morphologies of Madurella mycetomatis strains used to infect Galleria mellonella larvae.
Strains mm55 (panel A), mm68 (panel B) and cn796 (panel C) grown on Sabouraud agar for 4 weeks at 37°C. Strain mm68 clearly has a different morphology than strains mm55 and cn796.
Fig 2
Fig 2. Survival of Galleria mellonella larvae infected with Madurella mycetomatis.
In panel A, the survival curves of the control larvae are shown. For each experiment three different controls were used: untouched larvae, larvae only pricked by the needle and larvae injected with PBS. As is seen in this panel, high survival was obtained for all control groups. In panel B, the survival of larvae infected with 0.04, 0.4 and 4 mg mm55 are shown. More than 90% of the larvae survived when they were infected with only 0.04 mg mm55, no larvae survived when they were infected with 4 mg mm55. In panel C, the results for strain mm68 are shown. As is seen in this graph, when larvae were infected with 4 mg mm68, still some survival is noted. In panel D, the survival of larvae infected with 0.04, 0.4 and 4 mg cn796 are shown. Again at the highest inoculum all larvae died.
Fig 3
Fig 3. Melanization of Galleria mellonella larvae during M. mycetomatis infection.
In panel A, three larvae infected with 0.04, 0.4 and 4 mg mm55 are shown. As is seen, the larvae infected with 0.04 mg mm55 is not melanised (left larvae), the larvae infected with 0.4 mg mm55 is slightly melanised (middle larvae) and the larvae infected with 4 mg mm55 is heavily melanised (right larvae). In panels B, C and D, the melanization of the haemolymph is demonstrated by measuring the OD405nm of the haemolymph at time points; 4h, 24h, 72h, 168h and 700h after infection. In panel B, the melanization of larvae infected with mm55 is shown, in panel C those infected with mm68 and in panel D those infected with cn796. At all time-points it appeared that the larvae infected with the highest inoculum of M. mycetomatis were the most melanised, those with the lowest inoculum, were the least melanised.
Fig 4
Fig 4. Grain formation in human, mouse and larvae of Galleria mellonella.
In panels A, B and C the presence of grains in tissue is shown. In panel A, black grains are clearly visible inside an excised part of the mycetoma lesion of a human patient. In panel B, grain formation inside a Balb/c mouse is shown. In panel C, grains inside a dissected G. mellonella larvae are demonstrated. In panel D, E and F, 100 times magnified HE slides of mycetoma grains show that the grains are surrounded with immune cells, and a large zone of neutrophils is present in both human and mice. In G. mellonella larvae these immune cells are not present yet (panel F) at a time point of 3 days after infection. In grains obtained from human, mouse and G. mellonella larvae fungal hyphae can be seen as demonstrated with Grocott staining (panels G, H, I). With Sirius Red staining a clear encapsulation of grains with collagen is seen in human, mouse and G. mellonella larvae (panels J, K, L).
Fig 5
Fig 5. Grain formation over time.
Grain formation of M. mycetomatis strain mm55 within Galleria mellonella larvae (100 times magnified). At 4h, 24h, 3 days and 7 days post infection grain formation is shown. Histological slides are stained with HE (panels A,B,C,D), Grocott (E,F,G,H) to demonstrate the presence of fungal hyphae and Sirius red to demonstrate the presence of collagen(I, J, K, L). As is seen in panels A, E and I small grains are already visible at 4h after infection. Hardly any immune cells are present (panel A), and no collagen is seen surrounding the grain (panel I). After 24h the grains are larger and cement material starts to form (panel B). At 3 days post infection, the grain has become encapsulated with collagen (panel K). This collagen capsule disappears at day 7 (panel L), a time point at which many immune cells are shown to be present surrounding the grain (panel D).
Fig 6
Fig 6. Assessment of fungal burden over time.
In panels A, B and C, the number of colony forming units per larvae are shown. At time points 4h, 24h, 72h, 168h and 700h after infection larvae were sacrificed and homogenized. For each larvae, 250 μl undiluted, 50 μl undiluted and 50 μl 1:10 diluted suspension was plated out on Sabouraud-gentamicin agar. In panel A, the fungal burden of larvae infected with 4 mg/larvae (red), 0.4 mg/larvae (blue) or 0.04 mg/larvae (green) mm55 is shown. In panels B and C, the same is demonstrated for strains mm68 and cn796. At all time-points it appeared that the larvae infected with the highest inoculum of M. mycetomatis had the highest concentration of M. mycetomatis in their haemolymph. For the lowest concentration, the CFU dropped below the detection limit as early as 3 days after infection.

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