Objectives: In the present study we demonstrate the potent antifungal properties of human beta defensins (hbD1, hbD2 & hbD3) against representative clinical isolates of filamentous fungi. Methods: Defensins. Recombinant peptides of hbD1, hbD2 & hbD3 were purchased from Peprotech EC, UK. Lyophilised peptides were reconstituted in 0.1% acetic acid and stored in working aliquots ranging from 1 to 0.002 mg/ml. Fungal strains & AFST. The filamentous fungi used in this study were Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans and Aspergillus terreus, deriving from suspected cases of invasive mycotic infections. Presumptive species identification was done microscopically by the lactophenol blue method and further validated by determining their carbon assimilation profiles using the ID32C system (Biomí©rieux, Marcy d’ Etoile). Routine antifungal susceptibility testing (AFST) for itraconazole, fluconazole, voriconazole, posaconazole, caspofungin and amphotericin B were performed by means of E-test (AB Biomí©rieux) according to the manufacturers’ instructions. Antifungal defensin assay. Minimum Fungicidal Concentrations (MFC) of hbD1, hbD2 & hbD3 were determined by the microdilution assay as described elsewhere. Briefly, fungal spores were harvested from mature colonies grown on SDA agar at 30C and resuspended in 10mM sodium phosphate Buffer (PB) to obtain a final concentration of 10^6 cells/ml. Aliquots of diluted cells were exposed to varying concentrations of defensins for 24h at 30C in sterile 96-well microtitre plates and growth inhibition was assessed by measuring culture absorbance at 595nm using a microplate reader. Cell suspensions in buffer alone and/or acetic acid served as negative controls. Results: Inhibition of fungal growth was evident with all three defensins tested and was accompanied by fungicidal effects as demonstrated by re-inoculation of the fungi to SDA plates. Among those tested, hbD3 appears to have the strongest potency as compared to those of hbD1 &2. Conclusions: In recent years, intensive work on defensins has expanded our knowledge on innate defense mechanisms and may provide us novel therapeutic options for combating serious and emerging infections as invasive mycoses. The present study, provides a first indication of specific antifungal activity of those endogenous peptides against clinically important filamentous fungi and highlights the need for further analysis of their mode of action.
Full conference title:
19th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 19th (2009)