AIMS The aim of the present work is the study of the mechanisms responsible for the dimorphic transition from yeast-like antagonistic to pseudohyphal pathogenic behaviour of the yeast Pichia fermentans. METHODS The strain DiSAABA 726 P. fermentans was grown statically on liquid cultural media differing for nitrogen and carbon sources for the individuation of the nutritional conditions able to induce dimorphic transition. Yeast crude extracts for proteomic analysis were subjected to 2D Gel electrophoresis. Immunofluorescence analyses were performed with primary anti-tubulin monoclonal antibodies YOL1/34 and secondary antibody FITC conjugated with some modification. Hydrolytic enzymes activities were measured in the culture filtrates concentrated by ultrafiltration. ?-1,3-glucosidase and ?-1,3-glucanase activities were assayed by using ?-glucoside analogues (p-nitrophenyl-?-D-glucopyranoside and salicin) and laminarin, respectively. RESULTS The nutritional condition that induce biofilm formation and separate yeast-like and pseudohyphal growth have been individuated and are currently utilized for the realization of enzymatic and proteomic analyses of P. fermentans. Preliminary results illustrated microtubules organization of the two different morphological phases. The screening of the extracellular lytic enzymes produced by the yeast in the selected growth conditions showed a significant increase in the secretion of ?-1,3-glucosidase, ?-1,3-glucanase and polygalacturonase by the yeast grown in the pseudohyphal form. Preliminary results of proteomic analyses showed changes in the protein profiles from yeast cell and pseudohyphae. CONCLUSIONS Starting from the individuation of the nutritional factors able to induce the pathogenic activity it is possible to develop tools and strategies useful for the study of the biological model P. fermentans. This yeast is poorly characterized from a genetic and physiological point of view, and due to its Dr. Jekyll/Mr. Hyde behaviour requires the elaboration of alternative protocols for the evaluation of the hazard associated to the utilization of yeast antagonists and the suggestion of a new paradigm of bio-safety
Development of tools and strategies for the study of the dimorphic transition of P. fermentans, emergent pathogen in post-harvest
Lucia Giorgetti;Biancaelena Maserti;
2009
Abstract
AIMS The aim of the present work is the study of the mechanisms responsible for the dimorphic transition from yeast-like antagonistic to pseudohyphal pathogenic behaviour of the yeast Pichia fermentans. METHODS The strain DiSAABA 726 P. fermentans was grown statically on liquid cultural media differing for nitrogen and carbon sources for the individuation of the nutritional conditions able to induce dimorphic transition. Yeast crude extracts for proteomic analysis were subjected to 2D Gel electrophoresis. Immunofluorescence analyses were performed with primary anti-tubulin monoclonal antibodies YOL1/34 and secondary antibody FITC conjugated with some modification. Hydrolytic enzymes activities were measured in the culture filtrates concentrated by ultrafiltration. ?-1,3-glucosidase and ?-1,3-glucanase activities were assayed by using ?-glucoside analogues (p-nitrophenyl-?-D-glucopyranoside and salicin) and laminarin, respectively. RESULTS The nutritional condition that induce biofilm formation and separate yeast-like and pseudohyphal growth have been individuated and are currently utilized for the realization of enzymatic and proteomic analyses of P. fermentans. Preliminary results illustrated microtubules organization of the two different morphological phases. The screening of the extracellular lytic enzymes produced by the yeast in the selected growth conditions showed a significant increase in the secretion of ?-1,3-glucosidase, ?-1,3-glucanase and polygalacturonase by the yeast grown in the pseudohyphal form. Preliminary results of proteomic analyses showed changes in the protein profiles from yeast cell and pseudohyphae. CONCLUSIONS Starting from the individuation of the nutritional factors able to induce the pathogenic activity it is possible to develop tools and strategies useful for the study of the biological model P. fermentans. This yeast is poorly characterized from a genetic and physiological point of view, and due to its Dr. Jekyll/Mr. Hyde behaviour requires the elaboration of alternative protocols for the evaluation of the hazard associated to the utilization of yeast antagonists and the suggestion of a new paradigm of bio-safetyI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
