Natamycin and the germinating spore

Abstract

Fungi cause enormous food losses worldwide due to crop infection and food spoilage. Contamination by fungi often starts with dispersal vehicles (spores or conidia) that are dispersed either by air and water. A crucial step in fungal contamination is the process of germination, which is followed by mycelial growth throughout the food product. Natamycin is widely used as an anti-fungal to prevent outgrowth of fungi on surfaces of food products. It is a polyene antibiotic that binds specifically to ergosterol in model membranes and fungal cells. Surprisingly, despite the long use of natamycin, its mode of action is an enigma. Early observations have shown that conidia are less sensitive to polyene antibiotics than growing fungal hyphae. To study sterol localization during germination, the polyene antibiotic filipin was used as a fluorescent sterol marker. Ergosterol could not be detected by filipin staining in dormant and swollen conidia of Penicillium discolor. However, after 6 h of incubation in medium a fluorescent cap was observed at the presumptive site of germ tube emergence. The fluorescence intensity of the cap increased as the formation of the germ tube proceeded. This indicates an important role of ergosterol in polarized growth. The effect of natamycin on germinating conidia of P. discolor was tested and compared with other polyene antibiotics namely, filipin and nystatin. The fluorescent vital dye TOTO-1 showed that the integrity of the plasma membrane is maintained in the presence of natamycin. In contrast, the polyenes filipin and nystatin caused dose- and time dependent influx of these fluorescent compounds. Electron spin resonance (ESR) studies confirmed the integrity of the conidial membrane in the presence of natamycin, but showed differences between filipin and nystatin. These results suggests that natamycin unlike other well-known polyenes does not form membrane disrupting complexes, but interferes with ergosterol functioning in the cell. Experiments with the endocytic marker FM4-64 suggest that natamycin can be used as a specific inhibitor for early endocytosis. The transcriptome of Aspergillus niger was analysed during germination of conidia in the absence or presence of the natamycin. A surprising number of different pre-packed transcripts were observed in dormant conidia. Most changes in the transcriptome were observed during the first two hours after inoculation. This was accompanied by a reduction in cellular microviscosity and degradation of compatible solutes as trehalose and mannitol..Differential expression had dropped notable after 2 hours and down-regulation of transcripts was dominant. Between 2 and 8 hours of germination many morphological important changes were observed. Conidia grew initially isotropically and then showed polarized growth. After 6-7 hours, 1 cycle of mitosis before or during the formation of germ tubes. No mitosis and germ tube formation was observed in the presence of 3 ?M natamycin. In 10 ?M natamycin conidia even did not swell and remained small. Interestingly, the transcriptome of natamycin-treated conidia was identical to untreated conidia after 2 h of incubation. However, natamycin-treated conidia showed strongly affected gene expression after 8 h.