Crucial Role of Antioxidant Proteins and Hydrolytic Enzymes in Pathogenicity of Penicillium expansum (2006)

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase. Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase. Penicillium expansum is a widely spread fungal pathogen that causes blue mold rot in a variety of fruits, including apples, pears, peaches, and cherries (1Marek P. Annamalai T. Venkitanarayanan K. Detection of Penicillium expansum by polymerase chain reaction..Int. J. Food Microbiol. 2003; 89: 139-144Crossref PubMed Scopus (63) Google Scholar). It causes significant economic losses to the fruit industry and is also of potential public health concern because it produces toxic secondary metabolites, including patulin, citrinin, and chaetoglobosins (2Andersen B. Smedsgaard J. Frisvad J.C. Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products..J. Agric. Food Chem. 2004; 52: 2421-2428Crossref PubMed Scopus (233) Google Scholar). Patulin is produced by various filamentous fungi with P. expansum being generally regarded as one of the main producers (3Lennox J.E. McElroy L.J. Inhibition of growth and patulin synthesis in Penicillium expansum by potassium sorbate and sodium propionate in culture..Appl. Environ. Microbiol. 1984; 48: 1031-1033Crossref PubMed Google Scholar). Because of the potential carcinogenic effects of this mycotoxin, Europe and the United States have established a maximum limit for patulin contamination in apple-based products (3Lennox J.E. McElroy L.J. Inhibition of growth and patulin synthesis in Penicillium expansum by potassium sorbate and sodium propionate in culture..Appl. Environ. Microbiol. 1984; 48: 1031-1033Crossref PubMed Google Scholar). Control of decay caused by P. expansum has become important for ensuring the quality and safety of various fruits. Currently the disease caused by P. expansum is mainly controlled by the intensive use of synthetic fungicides. However, concerns about public health and the development of fungicide resistance by pathogens have prompted the search for alternative methods (4Janisiewicz W.J. Enhancement of biocontrol of blue mold with the nutrient analog 2-deoxy-d-glucose on apples and pears..Appl. Environ. Microbiol. 1994; 60: 2671-2676Crossref PubMed Google Scholar). This requires more knowledge about the pathogenesis at the biochemical and molecular level. During the course of invasion, a plant pathogen encounters the defense strategies of the host, including the accumulation of barrier-forming substances and the production of antimicrobial compounds that act directly to prevent pathogen invasion (5Campo S. Carrascal M. Coca M. Abián J. Segundo B.S. The defense response of germinating maize embryos against fungal infection: a proteomics approach..Proteomics. 2004; 4: 383-396Crossref PubMed Scopus (131) Google Scholar). In addition, the pathogen will also face the attack of reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; PDB, potato dextrose broth; YNB, 0.67% yeast nitrogen base without amino acids plus 2% dextrose; 2D, two-dimensional; CBB, Coomassie Brilliant Blue; DCHF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DNP, 2,4-dinitrophenylhydrazone; TEMED, N,N,N′,N′-tetramethylethylenediamine; EST, expressed sequence tag. primarily superoxide (O2) and hydrogen peroxide (H2O2), generated by the host at the site of infection (5Campo S. Carrascal M. Coca M. Abián J. Segundo B.S. The defense response of germinating maize embryos against fungal infection: a proteomics approach..Proteomics. 2004; 4: 383-396Crossref PubMed Scopus (131) Google Scholar). These ROS may cause oxidative damage to cell components including proteins, lipids, and nucleic acids (6Yan S. Tang Z. Su W. Sun W. Proteomic analysis of salt stress-responsive proteins in rice root..Proteomics. 2005; 5: 235-244Crossref PubMed Scopus (409) Google Scholar, 7Reverter-Branchat G. Cabiscol E. Tamarit J. Ros J. Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae..J. Biol. Chem. 2004; 279: 31983-31989Abstract Full Full PubMed Scopus Google Scholar). against ROS, pathogens have defense including catalase and superoxide as as the as glutathione and and the of S. PubMed Scopus Google Scholar, P. P. W. The molecular against reactive oxygen species in Microbiol. PubMed Scopus Google Scholar). B. Oxidative stress is in cell in Saccharomyces S. 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In study, of P. expansum were in the absence and presence of borate for at The were with DCHF-DA, and observed under a of the with DCHF-DA, that were (ROS) produced at that However, with the of ROS were in of P. expansum. with more under borate stress were with the DCHF-DA, indicating that of produced in to borate. The levels of ROS in are generated by metabolism G. Cabiscol E. Tamarit J. Ros J. Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae..J. Biol. 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M. of major proteins of PubMed Scopus Google that of the major proteins as dehydrogenase, and in the pathogen proteins, is of In the of a protein on the of Biol. Chem. Full Full PubMed Scopus Google it to a virulence that to the infection of the Additionally two proteins and which significant to proteins with in the protein to may potential virulence and their to In we have a proteome analysis to find two antioxidant enzymes, catalase and glutathione S-transferase, in P. expansum the identified These two proteins may for the virulence of this fungal pathogen because the of expression of proteins in a of ROS and of carbonylated proteins in Additionally identified in the extracellular and expression repressed in the presence of borate. These may for the of and potential for for in the proteomics and for in the analysis of also for and of the