Offensive Virulence Factors

Offensive virulence factors are molecules that enable the fungus to interact directly with the host, causing it damage. They include secreted enzymes such as proteases, peptidases and phospholipases, secreted toxins, and adhesins.


Proteases (also known as peptidases) play a major role in the virulence of several fungal pathogens by digesting host proteins and thereby enhancing tissue penetration (Monod et al., 2002). During the previous decade, considerable efforts were made to identify A. fumigatus proteases and assess their role in infection and virulence (Tomee & Kauffmann, 2000; Monod et al., 2002). The major findings were that secreted A. fumigatus proteases induce pro-inflammatory cytokine release in infected macrophages and epithelial cells, thereby alerting the immune system (Kauffmann et al., 2000). Infected lung epithelial cells also undergo major changes to the actin cytoskeleton, leading to cell peeling and death (Tomee et al., 1997; Kogan et al., 2004). However, deletion analyses of selected proteases (Alpl, MEP, PEP1), have failed to conclusively demonstrate a significant role in virulence in animal models (Monod et al., 2002). This is probably a result of the large number of proteases secreted by A. fumigatus and the functional redundancy between them (Robson et al., 2005). Its genome encodes for at least 38 proteases (Table 8.1). Of these, 15 are predicted secreted endoproteases (cleaving peptide bonds internally, within a polypeptide), including eight aspartic proteases, four serine/subtilisin proteases and three metalloproteases. Only five were studied to date (Table 8.1, underlined). The A. fumigatus genome also contains at least 23 predicted secreted exopeptidases (cleaving peptide bonds only at the N- or C-terminal of the polypeptide chain) (Table 8.1). Of these only two (DppIV and DppV) were cloned, characterized enzymatically but not deleted (Beauvais et al., 1997a, b). DppV is

Table 8.1 A. fumigatus putative proteases, lysophospholipases, and non-ribosomal peptide synthase (NRPS) genes




Aspartic endopeptidase

PEP1/aspereillopepsin A/I/ F/asp f10*. PEP2,

OpsB,CtsD, AfU6g03260/AP1, Afu4g09400/AP3,

Afu3g01220, Afu2g15950

Serine endopeptidase

Alpl/subtilisin N, Alp2/asp f13, AorO,



MEP/Asp f5, Afu6g14420 Afu4g13750


CpdS/Afu6g00310, Afu8g04120, Afu5g01200,


Afu5g01200, Afu2g04370, Afu1g00420, Afu6g13540,

Afu4g07270, Afu1g08940, Afu5g14610,

Afu5g07330, Afu3g12210, Afu2g0879

Dipeptidyl peptidases

DppIV, DppV, Afu2g0903

Tripeptidyl peptidases

Afu4g03490, Afu4g14000, Afu3g0893

Serine exodopeptidases

Afu4g03790, Afu2g01250, Afu2g17330




Phospholipase A


Phospholipase B


Phospholipase C

Afu7g04910, Afu1g17590

Phospholipase D


Non-ribosomal peptide

gliP, sidC, pes1, Afu6g08560, Afu6g09610,

synthases (NRPS)

Afu6g12050, Afu6g12080, Afu4g14440,

Afu5g12730, Afu5g10120, Afu15270,

Afu3g13730, Afu3g12920

* Classification based on A. fumigatus genome annotation (Nierman 2005) and available at http:// = gene_catalogs & org = afm. All genes except the non-ribosomal peptide synthases contain a putative signal peptide sequence as analyzed by Signal P 3.0 (

* Underlined: Genes that have been studied to date.

* Classification based on A. fumigatus genome annotation (Nierman 2005) and available at http:// = gene_catalogs & org = afm. All genes except the non-ribosomal peptide synthases contain a putative signal peptide sequence as analyzed by Signal P 3.0 (

* Underlined: Genes that have been studied to date.

strongly antigenic and is useful for the diagnosis of aspergilloma and ABPA (Sarfati et al., 2006).


Phospholipases are a heterogeneous group of enzymes which hydrolyze one or more ester bonds in phosphoglycerides. They have an important role in the virulence of bacteria and several pathogenic fungi including Candida species and Cryptococcus neoformans (Ghannoum, 2000). Spectrometric analysis of A. fumigatus filtrates indicated phospholipase A, B, C, and D activities (Birch et al., 1996). The A. fumigatus genome contains six predicted secreted phospholipases (Table 8.1). Of these, two phospholipase B (PLB) genes, AfPLBl and AfPLB3, were cloned and studied. These proteins contain a signal peptide sequence and are secreted enzymes (Shen et al., 2004). None of the A. fumigatus phospholipases were deleted to date and their contribution to virulence remains unknown.


A. fumigatus produces a rich cocktail of toxins and apparently uses them to fight off competition and predators in its natural environment. They include the low molecular weight toxic secondary metabolites (gliotoxin, helvolic acid/fumigacin, fumagillin, fumigaclavine A-C, festuclavine, aurasperone C, aflatoxin B1, and G1 and verruculogen), and small proteins inhibiting ribosome function (Restrictocin/ mitogillin/As f1) or inducing erythrocyte hemolysis (Asp-hemolysin) (Kamei & Watanabe, 2005; Rementeria et al., 2005; Yu & Keller, 2005). Gliotoxin and restric-tocin are the only two toxins produced at toxic levels in infected animals and patients by most clinical strains of A. fumigatus (Kamei & Watanabe, 2005; Lewis et al., 2005). Deletion of the gene encoding restrictocin did not affect fungal virulence in mice (Smith et al., 1993).

Until recently, the involvement of the toxic secondary metabolites in virulence had only been demonstrated in vitro, through their immunosuppressive action on macrophages, and inhibitory effects on epithelial cell function. Identifying the genes responsible for their synthesis has proven difficult. The sequenced A. fumigatus genome indicates the potential for the biosynthesis of up to 40 novel metabolites from 26 clusters of genes encoding predicted prenyltransferases, oxidoreductases, methyltransferases, transporters, 13 nonribosomal peptide synthetases (NRPSs) (Table 8.1), polyketide synthases (PKSs), and transcription factors. Thirteen of these clusters are unique to A. fumigatus. (Nierman et al., 2005). The gene clusters involved in ergot alkaloid biosynthesis and gliotoxin biosynthesis were identified (Gardiner & Howlett, 2005; Coyle & Panaccione, 2005).

dmaW and Ergot Alkaloid Biosynthesis

Deletion of dmaW, encoding dimethylallyltryptophan synthase, a key enzyme in ergot alkaloid biosynthesis, eliminates the biosynthesis of all known ergot alkaloids (fumigaclavine A-C, festuclavine) from A. fumigatus. However, the virulence of this strain has yet to be characterized (Coyle & Panaccione, 2005).

gliP and Gliotoxin Biosynthesis

Deletion of the NRPS gliP, the first enzyme in the pathway for gliotoxin biosynthesis, results in an A. fumigatus strain which fails to produce gliotoxin. In vitro, the culture supernatant of the gliP-deficient strains has a reduced cytotoxic effect on both macrophage-like cells and T cell lines. Surprisingly, the gliP null mutant is normally virulent in immunosuppressed mice, indicating that gliotoxin alone is not a virulence factor in A. fumigatus (Cramer et al., 2006; Kupfahl et al., 2006; May GS submitted) A possible explanation for this result is that in vivo, gliotoxin acts in combination with additional secreted toxins. This is supported by the recent work of Bok et al. (2005): Deletion of laeA, a transcription factor and global regulator of secondary metabolite biosynthesis, blocked production of all major types of secondary metabolites including gliotoxin and resulted in decreased virulence in immunosuppressed mice.


Deletion of the NRPS pes1 results in significantly reduced virulence in the Galleria insect larva model system and increased sensitivity to oxidative stress in culture and during neutrophil-mediated phagocytosis. The mutant also exhibits smoother conidial surface morphology and increased hydrophobicity (Reeves et al., 2006).

In the coming years, deletion of additional gene clusters and transcription factors involved in secondary metabolite biosynthesis will greatly enhance our understanding of their role in virulence.


Fungal adhesion to host tissues is mediated primarily by adhesins (Verstrepen & Klis, 2006). Adhesins have been cloned in several fungi, including S. cerevisiae and C. albicans. All fungal adhesins share a common three-domain structure which includes a glycophosphatidylinositol-anchored (GPI) C-terminal part, a large middle domain characterized by the presence of multiple serine- and threonine-rich repeats and an N-terminal part containing a carbohydrate or peptide-binding domain. The repeats undergo frequent recombination dependent expansion or contraction resulting in isolates containing different length versions of this region. Adhesins with more repeats generally show greater adherence, whereas shorter adhesins with less repeats generally exhibit decreased adhesion, possibly because the N-terminal domain remains buried in the cell wall. Several putative A. fumigatus adhesins have been characterized biochemically but none were cloned (reviewed in Latge, 1999). BLAST homology searches with sequences of known fungal adhesins (e.g. S. cerevisiae FLO genes, C. albicans ALS or Hwp1 genes among others) have failed to identify significant homologs in Aspergillus spp. However, the genome of A. fumigatus contains at least 20 genes with a putative C-terminal GPI anchor and multiple tandem repeats. Of these, four genes (Afu3g08990, Afu2g05150 (MP-2), Afu4g09600, and Afu6g14090) were shown to vary in the number of repeats in different A. fumigatus isolates (Levdansky et al., submitted). Deletion of theses genes will help clarify whether they are involved in adhesion or host interactions.

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