Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus

Abstract

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.

Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis

“…Previous investigations have analysed the role of different fungal proteases in virulence of A. fumigatus. Main proteases, such as the alkaline serine protease Alp, the metalloprotease Mep and an aspartic protease, have been found to be produced during infection, but deletion of the coding sequences did not lead to any visible phenotype and the corresponding mutants retained their virulence in murine infection models (Reichard et al, 1990;1997;Monod et al, 1993a,b;Tang et al, 1993;Jaton-Ogay et al, 1994;Smith et al, 1994). Recently, a new class of secreted proteases, the so-called sedolisins, have been identified, but the impact of these proteases in virulence has not been clarified yet (Reichard et al, 2006).…”

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

“…Aspergilli that cause invasive aspergillosis are known to express genes that encode serine proteinases (34)(35)(36)(37), metalloproteinases (38)(39)(40), and aspartic proteinases (41). Disruption of one or two of these does not show a significant decrease in virulence (15)(16)(17). When one aspartic proteinase gene was disrupted, immunologically crossreactive proteinase(s) was found to be associated with the fungal wall (42), suggesting the presence of multiple aspartic proteinases.…”

“…A variety of lines of evidence for their role in pathogenesis, including the correlation between the level of such enzymes and virulence, immunocytochemical evidence for the secretion of the enzyme during infection, protection of the host by inhibition of the enzymes with selective inhibitors or specific antibodies, and enhancement of virulence by gene transfer, have been challenged on the basis of the results of disruption experiments on genes that encode cutinase and cell wall-degrading enzymes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In the case of animal pathogens, single and double proteinase gene disruption failed to show a clear decrease in virulence (15)(16)(17). The inability to demonstrate a drastic decrease in virulence by such gene disruption has been interpreted as casting doubt on the role of extracellular enzymes that degrade physical barriers of the host in pathogenesis (18).…”

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium

“…In addition, A. fumigatus secretes leucine aminopeptidases (Laps), which are non-specific monoaminopeptidases, dipeptidyl-peptidases (DppIV and DppV) and a prolylpeptidase (AfuS28) as exopeptidases (Beauvais et al, 1997a, b;Monod et al, 2005;Sriranganadane et al, 2010). alpD mepD knockout mutants are unable to grow in a medium containing protein as the sole nitrogen source at neutral and high pH while single alpD and mepD mutants produced 30 and 70 % of the proteolytic activity of the wild-type strain, respectively (Monod et al, 1993a, b;Jaton-Ogay et al, 1994).…”

“…Like many other ascomycetes from the soil, A. fumigatus grows well in media containing protein as the sole nitrogen and carbon source and shows secreted proteolytic activity (Reichard et al, 1990;Monod et al, 1991;Sriranganadane et al, 2010). At neutral pH, the fungus secretes two endoproteases, an alkaline protease (Alp) of the subtilisin family (Reichard et al, 1990;Monod et al, 1991) and a metalloprotease (Mep) of the fungalisin family (Monod et al, 1993a, b;Jaton-Ogay et al, 1994). In addition, A. fumigatus secretes leucine aminopeptidases (Laps), which are non-specific monoaminopeptidases, dipeptidyl-peptidases (DppIV and DppV) and a prolylpeptidase (AfuS28) as exopeptidases (Beauvais et al, 1997a, b;Monod et al, 2005;Sriranganadane et al, 2010).…”