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Protoplast-mediated transformation of Madurella mycetomatis using hygromycin resistance as a selection marker

Summary

Scientists have successfully developed a genetic engineering method for Madurella mycetomatis, the fungus that causes mycetoma, a serious tropical disease. They used a technique to remove the fungal cell wall and insert genes into the cells, creating strains that produce green fluorescent protein (GFP). This breakthrough enables researchers to better understand how this fungus causes disease and to develop new treatments.

Background

Madurella mycetomatis is the primary causative agent of mycetoma, a chronic granulomatous infection affecting subcutaneous tissue with limited treatment options. Currently, no genetic toolbox exists for M. mycetomatis to study its pathogenic mechanisms at the molecular level.

Objective

To develop a protoplast-mediated transformation protocol for M. mycetomatis using hygromycin as a selection marker and to create a GFP-expressing reporter strain to facilitate pathogenicity studies and drug discovery research.

Results

M. mycetomatis showed high sensitivity to hygromycin B at concentrations as low as 10 μg/ml. Successfully generated Mm55-hygR transformants with an average transformation rate of 3 transformants per 1×10⁵ protoplasts. Created a cytoplasmic GFP-expressing strain (Mm55-GFP) that retained pathogenicity in Galleria mellonella larvae with no significant differences in survival, grain formation, or infection burden compared to wildtype.

Conclusion

A protoplast-mediated transformation protocol was successfully established for M. mycetomatis with hygromycin as an effective selection marker. GFP can serve as a functional reporter for this species, enabling future studies on virulence factors, protein localization, and drug screening applications.
  • Published in:PLoS Neglected Tropical Diseases,
  • Study Type:Experimental Research,
  • Source: 10.1371/journal.pntd.0012092, PMID: 38578808
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