Environmental stress response of the hyphomycetous entomopathogenic fungi
Xavier, Ilungo Johnny
This study was aimed at characterizing the response of entomopathogenic fungi (EPF) to environmental stress conditions. The growth temperature requirements of six Beauveria brongniartii strains were determined. The various developmental classes of, and the production of extracellular proteases by the six strains were strain- and growth temperature-specific. One dimensional SDS-PAGE combined with zymograms revealed that the molecular masses of the extracellular proteases of these fungi ranged from 27 to 54 kDa. The heat shock response of 'B. brongniartii, B. bassiana, Metarhizium anisopliae, Paecilomyces farinosus, Verticillium lecanii', and ' Tolypocladium nivea' strains was studied using two-dimensional (2D) gel electrophoresis. Analysis of the polypeptide map obtained by 2-D gels of heat-shocked cells indicated three patterns: unique heat shock proteins (HSPs) were (1) induced, (2) enhanced, or (3) repressed. Some HSPs were unique for each strain tested while some were shared. Exposure of 'B. bassiana, Metarhizium anisopliae, Paecilomyces farinosus, Verticillium lecanii', and 'Tolypocladium nivea' strains to a heat shock of 45°C for 1 h conferred thermotolerance i.e., the increased ability to survive exposure to a lethal temperature of 55°C. Heat shock also induced the synthesis of proteases in 'Beauveria' spp. The heat shock-induced protease of 'B. vermiconia' (molecular mass of 45 kDa and pI of 9.0) was characterized as a serine protease. A strong immunological cross reactivity between the HSP 70 proteins of all EPF tested and the HSP 70 and SSA4 HSP of 'Saccharomyces cerevisiae' was established using Western blotting. At least four isoforms of the HSP 70 were identified in ' B. bassiana' using 2-D Western blotting. However, EPF homologues of the DnaK, DnaJ and GrpE HSPs of 'Escherichia coli' could not be detected. In 'B. bassiana' and 'M. anisopliae', several UV- and starvation-induced proteins were identified, some of which overlapped with HSPs. Starvation of 'B. brongniartii' 979 also resulted in the production of exudate containing an alkaline, serine protease (molecular mass of 34.5 kDa and pI of 9.5). Injecting the migratory grasshopper ' Melanoplus sanguinipes' with the exudate resulted in rapid insect mortality, thereby demonstrating the toxic nature of the protease. Characterization of the EPF heat shock factor (HSF) and heat shock element (HSE) binding activities using gel mobility shift assays showed the presence of both constitutive and induced HSF-HSE binding complexes. Heat shock resulted in increased amounts and decreased mobilities of these complexes. The monomeric molecular mass of both heat shocked and control HSFs was determined to be 85.8 kDa by UV-crosslinking, and the apparent molecular masses of the native HSE-HSF complexes as determined by pore exclusion gradient gel electrophoresis was 260 and 300 kDa, respectively. This suggested that both complexes consisted of HSF trimers. Proteolytic band clipping assays revealed an identical partial cleavage profile for both complexes suggesting that both are formed by the same protein. The difference in mobility between the complexes was not abolished by enzymatic dephosphorylation and deglycosylation indicating a conformational change of HSF rather than phosphorylation and glycosylation. This study constitutes the first comprehensive biochemical and molecular characterization of the environmental stress response of EPF.