Disruption of the serine proteinase gene (sep) in Aspergillus flavus leads to a compensatory increase in the expression of a metalloproteinase gene (mep20)

Abstract

The serine proteinase gene (sep) in Aspergillus flavus was disrupted by homologous recombination with a hygromycin resistance gene as the marker. The gene-disrupted mutant GR-2 contained a single-copy insertion of the marker gene and did not express the sep gene. Serine proteinase activity, 36-kDa protein labeled by 3 H-diisopropylfluorophosphate, and immunologically detectable proteinase were not detected in the culture fluid of GR-2. Despite the absence of the serine proteinase, the total elastinolytic activity levels in the mutant and the wild-type A. flavus were comparable. Immunoblots revealed that the mutant secreted greater amounts of an elastinolytic metalloproteinase gene (mep20) product than did the wild type. Furthermore, mep20 mRNA levels, measured by RNase protection assay, in the mutant were higher than those in the wild type. Inhibition of the serine proteinase by Streptomyces subtilisin inhibitor (SSI) in the culture medium of wild-type A. flavus also resulted in an elevation of mep20 gene products. Although no serine proteinase activity could be detected, the level of elastinolytic activity of the SSI-treated culture was comparable to that of the control. Immunoblots revealed that the addition of SSI caused an elevation in the levels of metalloproteinase and its mRNA. These results suggest that the expression of the genes encoding serine and metalloproteinases are controlled by a common regulatory system and the fungus has a mechanism to sense the status of extracellular proteolytic activities.Extracellular hydrolases are produced by many microbes to degrade and obtain nutrients from polymers found in their environment. In addition, microbial proteinases have been implicated in the colonization and virulence of a number of bacteria and fungi (9,12,21,30,32). In the invasive lung infections of immunocompromised hosts, aspergilli must penetrate proteinaceous barriers such as elastin, collagen, and laminin (45), and thus proteinases are thought to be virulence factors (5,20,27,35,37,44). Aspergillus fumigatus and A. flavus produce serine proteinase and metalloproteinases that are capable of hydrolyzing such polymers (19,25,28,(33)(34)(35)(36)(37). A serine proteinase is produced as a major extracellular protein when these fungi are grown in a medium containing either elastin or collagen as the sole nitrogen source (19,20,35,36). The serine proteinase genes (sep) in both fungi show about 80% amino acid sequence identity (15, 33). In addition, A. fumigatus produces a 42-kDa metalloproteinase and A. flavus produces a 23-kDa metalloproteinase (25,28,34,36,42). In A. flavus, the levels of the 23-kDa metalloproteinase and serine proteinase are almost comparable (33,34,36), while in A. fumigatus, the 42-kDa metalloproteinase is produced in amounts less than those of the serine proteinase (25, 28). The gene for the 23-kDa metalloproteinase in A. flavus would encode a protein with a theoretical molecular mass of 20 kDa, and, therefore, this gene was designated mep20 and the protein was designated MEP20 (34)...

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit

“…niger are encoded by a family of diverged genes [42]. A similar phenomenon that disruption of serine proteinase caused an increase in metalloproteinase has also been found in Aspergillus flavus [43]. Even so, the lack of pgxB still dramatically reduced lesion diameter on apples.…”

The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance

“…RNase protection assays (RPAs) were conducted essentially as described previously (Ramesh and Kolattukudy, 1996). Briefly, total RNA was isolated from strains AC‐168, DSM‐468 and DSM‐491 using the Qiagen RNA easy kit with one modification to the suggested protocol: the initial resuspension of bacterial cell pellets was performed in 0.05 M Tris‐HCl, pH 8.0, 0.1 M EDTA, 25% sucrose and 300 μg ml −1 lysozyme.…”

Two Novel Genes Induced by Hard-Surface Contact of Colletotrichum gloeosporioides Conidia

“…Both possibilities have been suggested for other genes. For example, disruption of genes in pathogenic fungi encoding various degradative enzymes or toxins, singly or in combination, did not provide unambiguous evidence for their function in pathogenicity, although such enzymes are thought to be important for infection (16,20,25,37). Madhani et al (29) showed that several downstream effectors are under the regulation of the Kss1 mitogen-activated protein kinase (MAPK) signaling pathway which controls dimorphic development.…”