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

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies

The putative role of zinc homeostasis in grain formation by Madurella mycetomatis during mycetoma infection

Madurella mycetomatis is the main cause of mycetoma, a chronic, granulomatous skin infection of the subcutaneous tissue. One of the main virulence factors is the formation of grains, which are difficult to treat with the currently available antifungal drugs. Studies have indicated that zinc homeostasis could be an important factor for grain formation. Therefore, in this review the mechanisms behind zinc homeostasis in other fungal species were summarized and an in silico analysis was performed to identify the components of zinc homeostasis in M. mycetomatis. Orthologues for many of the zinc homeostasis components found in other fungal species could also be identified in M. mycetomatis, including those components that have been identified to play a role in biofilm formation, a process which has some parallels with grain formation. Zinc homeostasis may well play an important role in the process of grain formation and, therefore, more knowledge on this subject in M. mycetomatis is required as it may lead to novel therapies to combat this debilitating disease

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

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies

The effect of the novel antifungal drug F901318 (Olorofim) on the growth and viability of Aspergillus fumigatus

We thank our colleagues at F2G Ltd. for their support in this work, as well as the Manchester Fungal Infection Group at the University of Manchester. We also thank Gillian Milne and the Microscopy and Histology Core Facility at the Medical Research Council Centre for Medical Mycology at the University of Aberdeen for their assistance with TEM. We also thank John Rex for critical reading of the manuscript. The Microscopy and Histology Core Facility at the Medical Research Council Centre for Medical Mycology at the University of Aberdeen is supported by grant number MR/N006364/1. S.D.P. and M.C.A. are supported by the European Marie Curie ITN FungiBrain grant PITN-GA-2013-607963. S.D.P. was the primary author and performed the live-cell imaging microscopy, TEM, viability staining, and the analysis of the data. N.B. performed the postexposure effect assays. M.C.A. assisted with TEM. G.E.M.S. designed F901318. N.B., A.C.B., D.L., M.B., N.D.R., and J.D.O. provided guidance and assistance during this project and in the preparation of the manuscript.Peer reviewedPublisher PD

Plasmids used in this study.

In this table a description and reference for each of the plasmids used in this study are listed. (XLSX)</p

Survival of Mm55-GFP infected larvae and the burden of infection.

A) Survival curve of Mm55-GFP (indicated in green, n = 77) and Mm55 wild type (indicated in black, n = 41) infected larvae. The dotted line indicates the uninfected PBS control (n = 72). The raw data is presented in Tabel S7. Both Mm55-GFP and Mm55 wild type significantly differ compared with the control (p = <0.0001 and p = <0.0001 respectively).</p

Transformation rate.

In this the data from 6 transformation experiments is shown. For each transformation experiment, the number of protoplasts present in the transformation mix, the number of resulting transformants and the number of transformants/1,00E+05 protoplasts is shown. Based on these results the average transformation rate was determined. (XLSX)</p

Grain comparison between Mm55 and Mm55-GFP.

A) H&E stain of a grain in Mm55 wild type infected G. mellonella, 100 × magnification. B) H&E stain of a grain in Mm55-GFP infected G. mellonella, 100 × magnification. C&D) Burden of infection measured in the total number of grains counted (S8C Table) and the respective calculated total grain size (S9D Table). For each time point and strain n = 5, except for time point t = 24h for Mm55-GFP (n = 4) and t = 168h (n = 3) for both strains. p-Values are calculated per condition and time-point with Mann-Whitney. Values are shown above each time-point. No significant differences were noted in the total number of grains (C) or the total grain size (D).</p