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An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity
Author(s) -
Tang C. M.,
Cohen J.,
Holden D. W.
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb00891.x
Subject(s) - biology , aspergillus fumigatus , mutant , microbiology and biotechnology , hygromycin b , elastase , protease , extracellular , gene , biochemistry , enzyme
Summary Invasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus , is a life‐threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase‐chain‐reaction‐based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS‐polyacrylamide gel etectrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild‐type cells, the mutant had undetectable activity on azocollagen and elastin‐Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus , and that the mutant strain will be useful in assessing the role of AFAlp in pathogenicity.