Defensive Virulence Factors

Defensive virulence factors are traits that enable the fungus to proliferate in the hostile environment of the human body. They include the ability to grow at human body temperature (thermotolerance), the protective capacity of the fungal cell wall and its components (polysaccharides, proteins, pigments) and the ability to degrade reactive oxygen species produced by the host immune system.

Thermotolerance

A. fumigatus thermotolerance is a trait critical for survival in decomposing organic matter. However, genetic evidence suggests that whereas the ability of A. fumigatus to grow at 37°C is important for virulence, the ability to grow at the high temperatures in which this fungus specializes (i.e. above 48°C), is not. Seven A. fumigatus chemical mutants that grow normally up to 42°C, but fail to grow at 48°C displayed wild-type virulence in mice (Chang et al., 2004). Also, a mutant disrupted for CgrA, a nucleolar gene involved in ribosome biogenesis, which grew normally at 25°C but poorly at 37°C exhibited reduced virulence in mice (Bhabhra et al., 2004).

Recent microarray analysis following a shift of growth to elevated temperatures (37°C and 48°C), identified many upregulated A. fumigatus heat-shock genes which may have a role in thermotolerance (Nierman et al., 2005). Interestingly, the heat-responsive genes activated in A. fumigatus differ greatly from those in the non-pathogenic yeast S. cerevisiae. Recently, proteomic analysis of A. fumigatus has been developed, and this should yield further insights into the response to heat at the protein level (Kniemeyer et al., 2006).

In the near future, a comparison of the genome-wide heat-shock response of A. fumigatus to related, non thermophylic Aspergilli, such as A. nidulans, and other fungal pathogens, may identify genes or pathways that enable A. fumigatus to withstand high temperatures and affect pathogenicity.

The Fungal Cell Wall

The cell wall of A. fumigatus can be viewed as a multifactorial protective shield. It is composed of a polysaccharide skeleton of glucan, chitin, and galactomannan polymers interlaced and coated with cell wall-associated proteins (CWAPs) and embedded pigments (Latge et al., 2005). Past studies have identified pigments such as melanin and CWAPs such as the hydrophobins as important protective cell wall-virulence factors and these are described below. However, the recent analysis of the A. fumigatus genome has identified over 80 putative plasma membrane or cell wall-associated GPI-anchored proteins of which almost half are of unknown function, providing a rich starting point for the discovery of additional protective virulence factors (Table 8.2) (Nierman et al., 2005).

Cell wall mutants: Recently, two A. fumigatus cell wall mutants (A ecm33 and A ags3) which are hypervirulent in infected immunosuppressed mice have been described (Romano et al., 2006; Maubon et al., 2006). The reason for the hypervir-ulence of these mutants is not entirely clear. Hypervirulence in the A ags3 mutant could be a result of its faster germination (thereby evading macrophage killing) or higher concentrations of conidial melanin (leading to increased resistance to ROS).

Table 8.2 A. fumigatus genes encoding for putative GPI-anchored proteins

Classification*

Genes

Glycosyl hydrolase

Afu2g00680, Afu4g00620, Afu3g00700

Glucanosyl transferase

Afu1g16190 (CrfPf, Afu2g01170 (GEW), Afu2g03120

(Utr2p-liket), Afu2g14360S/T, Afu3g00270 (Bgl2p-like) S/T,

Afu6g08510 (Crf1-like) S/T, Afu6g11390 (GEL2), Afu8g06030

(MutA-like), Afu3g03080, Afu6g12410 (GEL7), Afu8g02130

S/T+C(GEL5), Afu2g12850 (GEL3), Afu3g13200(GEL6),

Afu2g05340(GEL4)

Spore coat hydrophobin

Afu5g09580 (HYP1), Afu8g07060

Aspartic protease

Afu4g07040 (CtsD), Afu6g05350 (OpsB-like),

Chitin-related proteins

Afu5g03760S/T+C (ChiA1), Afu6g10430*(chitin deacetylase),

Afu6g00500 (chitosanase)

CFEMa-domain protein

Afu6g10580 S/T (proline-rich antigen 2-like) f,

Afu6g14090 fS/T Afu6g06690

ecm33-like

Afu4g06820 (SPS-2 like; AfuEcm33;spot10f) S/T

Immunoreactive protein

Afu2g05150S/T+C (MP-2), Afu6g02800 (spot 19 *) S/T,

Afu4g03240 (MP-1) S/T

SUNb domain protein

Afu1g13940(Adg3p-like) S/T+C

Lysophospholipase

Afu3g14680 (Plb3), Afu4g08720,

Alpha-amylase

Afu2g03230 (AmyA), Afu2g13460 (AmyA-like), Afu3g00900

Agglutinin/Ecm

(AmyA) Afu4g09600 (agglutinin-like) f,

Afu8g05410* (ecm-like) f

Amino acid permease

Afu6g00410

Acid phosphatase

Afu1g03570 (phoA)

Sexual development

Afu5g10400 (LsdA-like)

WSC domain protein

Afu3g07050 S/T,

Acetyltransferase

Afu8g04000

Conserved hypotheticalc

Afu1g03630 S/T, Afu1g05790 S/T, Afu1g09510 S/T,

Afu1g09590, Afu1g11220 S/T, Afu2g01140 S/T,

Afu3g00880 S/T, Afu3g08990 S/T, Afu3g13110 S/T+C,

Afu3g14210 S/T, Afu4g12370, Afu5g08800, Afu7g00450 S/T,

Afu7g00580 S/T, Afu7g00970 S/T+C, Afu7g03970 S/T+C,

Afu8g02450 S/T, Afu8g04370 S/T, Afu5g07800,

Afu5g09960* S/T+C, Afu6g13710, Afu7g02440 S/T,

Afu8g04860S/T, Afu1g09650 S/T+C, Afu1g11680,

Afu1g13760, Afu2g02440 S/T, Afu3g01150 S/T,

Afu3g13640 S/T, Afu4g03500, Afu5g01920, Afu8g00830 S/T+,

Hypothetical1

Afu5g10010 S/TAfu4g06370 S/T, Afu8g01770 S/T+C,

Afu6g14010 S/T, Afu2g07800 S/T

* Classification was based on functional, structural, or immunological criteria. Underlined - Genes that have been studied to date.

S/T - serine/threonine-rich protein.

S/T+C - serine/threonine and cysteine-rich protein.

a -An eight-cysteine-containing domain unique to a group of fungal membrane and cell wall proteins (Kulkarni et al., 2003).

b - Similar to S. cerevisiae proteins of the SUN family (Simlp, Uthlp, Nca3p, Sun4p) that may participate in DNA replication, autophagy and cell death.

c - ORFs with significant homology (BlastP value <10-10) to other uncharacterized proteins. d - ORFs with no significant homology to any other proteins.

e - Annotation provided by CADRE unless otherwise specified (http://www.cadre.man.ac.uk/). f - Annotation performed by BlastP search (BlastP value <10-10).

* - Previously identified by Bruneau et al. (2001).

* Classification was based on functional, structural, or immunological criteria. Underlined - Genes that have been studied to date.

S/T - serine/threonine-rich protein.

S/T+C - serine/threonine and cysteine-rich protein.

a -An eight-cysteine-containing domain unique to a group of fungal membrane and cell wall proteins (Kulkarni et al., 2003).

b - Similar to S. cerevisiae proteins of the SUN family (Simlp, Uthlp, Nca3p, Sun4p) that may participate in DNA replication, autophagy and cell death.

c - ORFs with significant homology (BlastP value <10-10) to other uncharacterized proteins. d - ORFs with no significant homology to any other proteins.

e - Annotation provided by CADRE unless otherwise specified (http://www.cadre.man.ac.uk/). f - Annotation performed by BlastP search (BlastP value <10-10).

* - Previously identified by Bruneau et al. (2001).

Hypervirulence in the A ecm33 mutant could also occur because of its faster germination. Other factors may include its tendency towards conidial clumping (leading to occlusion of blood vessels) or increased immunogenicity (because of changes in the cell wall structure and exposure of immunogenic antigens). Increased immuno-genicity and the induction of septic shock in infected animals, was previously shown in hypervirulent S. cerevisiae and C. glabrata mutants (Wheeler et al., 2003; Kamran et al., 2004).

Pigments: Pigments such as melanin and its derivatives are important protective virulence factors in fungal pathogens of both plants and humans. They provide mechanical strength, UV protection and the scavenging of free oxygen radicals (Gomez, 2003). Melanins are dark-brown or black pigments formed by the oxida-tive polymerization of phenolic compounds. In A. fumigatus melanin is produced by a cluster of six genes via the DHN-melanin pathway (reviewed in Brakhage and Leibmann, 2005). Homologs of this cluster are found in most filamentous fungi. Melanin is found as discrete black particles in the cell wall of dormant conidia. In culture, it is rapidly degraded during germination, disappearing by the time of germ-tube emergence (Youngchim et al., 2004). However, melanin or its intermediates might also be produced during invasive growth: PksP, the enzyme involved in the first step of melanin biosynthesis, which is active in vitro only during conid-iogenesis, continues to be expressed during hyphal growth in the lungs of infected mice (Langfelder et al., 2001). This activation may occur via the PKA-dependent cAMP pathway as reduced PksP transcription is seen in mutants of this pathway (Liebmann et al., 2003).

A mutant producing pigmentless white conidia was generated by deletion of PksP (Alb1) (Tsai et al., 1999). This mutant was more sensitive to oxygen radicals and macrophages, showed increased ingestion by neutrophils and reduced virulence in a mouse model for IA (Jahn et al., 1997; Tsai et al., 1998). Deletion of the five additional genes participating in the DHN-melanin pathway Abr1/Abr2, Arp1/ Arp2 and Ayg1 results in brown, pink and yellowish-green conidia, respectively, but the virulence of these strains was not reported (Tsai et al., 1999, 2001).

It is important to note that many non-pathogenic fungi also contain melanin; therefore it is unlikely that melanin alone is responsible for the virulence of A. fumigatus.

Hydrophobins: Fungal hydrophobins are small cysteine-rich proteins involved in generating a thin layer of proteinaceous rod-like structures on the surface of aerial hyphae and conidia (Linder et al., 2005). This hydrophobic layer, by retarding water, improves conidial dispersion in the atmosphere. A. fumigatus contains six hydrophobin genes of which two, rodA and rodB were studied. They are conidial-specific proteins, disappearing during germination and hyphal growth. Deletion of rodA results in a loss of conidial rodlet structure, increased wettability, reduced conidial adhesion to some substrates, increased sensitivity to killing by alveolar macrophages but normal virulence in mice (Thau et al., 1994). This suggests that rodA forms a protective conidial-specific retardant barrier against macrophage oxidants. Deletion of rodB does not result in any observable phenotype despite its similarity to rodA (Paris et al., 2003).

ROS protection (catalases, SODs, antioxidants): During infection, A. fumigatus is attacked by reactive oxygen species (superoxide O2-, hydrogen peroxide H2O2, and hydroxyl radicals OH) produced by neutrophils and macrophages. It is therefore logical to assume that detoxification of H2O2 and O2- by A. fumigatus catalases, superoxide dismutases (SODs), and antioxidants such as glutathione might function as defensive virulence factors. The A. fumigatus genome contains five catalase and two SOD-encoding genes. The catalases include the closely related catA, catB/cat1, catC, and catE genes and the more distantly related cat2 gene encoding a bifunctional catalase/peroxidase. Deletion analysis of catA, catB/cat1, and cat2 indicated that only the last two together are involved in virulence, exhibiting delayed infection in a rat model of aspergillosis (Calera et al., 1997; Paris et al., 2003b). This limited effect might be due to functional redundancy or compensatory mechanisms.

The SODs include the immunogenic-secreted Cu/Zn SOD1 and intracellular Mn-SOD SodA/Asp f 6 (Holdom et al., 2000; Fluckiger et al., 2002). Neither SOD1 nor SodA have yet been deleted, so their role in virulence remains unknown.

Additional protection against oxidants is afforded by the antioxidant glutathione (GSH). GSH is a tripeptide (Glu-Cys-Gly) generated by the glutaredoxin and thioredoxin protein systems (reviewed in Grant, 2001). Mutants in these pathways have not been constructed in A. fumigatus and their role in virulence remains unknown (Chauhan et al., 2006).

Efflux pumps: A. fumigatus efflux pumps are involved in antifungal resistance and might also be used to detoxify components of the immune system. The A. fumigatus genome contains at least 327 genes encoding multidrug resistance (MDR) efflux transporters, at least threefold more than in the yeast S. cerevisiae, but roughly equal to that of Aspergillus nidulans and Aspergillus oryzae. These include 49 genes of the ATP-binding cassette (ABC) and 278 genes of the major facilitator superfamily (MFS) classes (Nierman et al., 2005; Ferreira et al., 2005). None have yet been deleted. Transcriptional analysis indicates that four ABC family members (MDR1, MDR2, atrF, abcA, and MDR4) (Tobin et al., 1997; Slaven et al., 2002; Langfelder et al., 2002; Nascimento et al., 2003) and one MFS member (MDR3)(Nascimento et al., 2003) are upregulated in azole-resistant isolates of A. fumigatus exposed to the drug. Five additional ABC transporters (abcA-E) and three MFS members (mfsA-E) are upregulated in wild-type A. fumigatus in response to voriconazole (da Silva, 2006).

Large-scale inactivation experiments for A. fumigatus transporter-encoding genes and the identification and deletion of their transcriptional repressors and activators could help define which of these genes are involved in resistance to drugs and toxic molecules in this species.

Aspergillus fumigatus biofilms: Fungal biofilms are three-dimensional structures composed of cells embedded in an extracellular matrix. Biofilms of the pathogenic yeast C. albicans are intrinsicaly resistant to almost all antifungals in clinical use (reviewed in d'Enfert, 2006). A. fumigatus in culture produces an amorphous extracellular matrix which can be visualized by cryo-scanning electron microscopy and confocal microscopy (Beauvais et al., Cell Microbiol., accepted for publication).

Further studies to reveal the components of the matrix and its role in defending the fungus against the host defenses and antifungal drugs are in progress.

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