Other Genes Affecting Virulence

Additional genes affecting virulence that do not fall neatly into the categories described above are described in this section. They can be loosely grouped as genes that enable the pathogen to survive in and adapt to the host environment. For example, the host environment is surprisingly poor in several essential elements such as iron and phosphate, and the fungus must activate pathways and genes to overcome these limitations.

Iron uptake: The ability to acquire iron in vivo is essential for most microbial pathogens (reviewed in Haas, 2003). The host contains very little free iron as most of it is bound to carrier proteins such as transferrin. A. fumigatus employs three iron-uptake mechanisms: ferrous iron uptake, reductive iron assimilation, and siderophore-mediated iron uptake, with only the last showing involvement in virulence (Schrettl et al., 2004). Siderophores are small cyclic peptides that function as ferric iron-specific chelators. They are excreted during iron starvation and recovered together with the bound iron by specific transporters. A. fumigatus synthesizes at least five different siderophores, the most common being intracellular ferricrocin and secreted triacetylfusarinine C (Hissen et al., 2004). The pathway involved in their biosynthesis contains at least five genes, including sidA (L-ornithine-N 5-monooxygenase) which catalyzes the first committed step of hydroxamate-type siderophore biosynthesis and sidC a nonribosomal peptide synthase (NRPS) (Eisendle, 2004) (Table 8.1). Siderophore-mediated iron uptake was found to be essential for A. fumigatus virulence in a murine model of invasive aspergillosis. Deletion of sidA results in severely attenuated virulence in immunosuppressed mice (Schrettl et al., 2004; Hissen et al., 2005). Mutant conidia fail to germinate in the infected lungs suggesting that this environment contains very little free iron. Mammals do not contain a sidA ortholog or a siderophore system in general. Therefore, it represents a promising new target for the development of antifungal therapies.

Magnesium uptake: The ability to acquire magnesium in vivo is essential for bacterial pathogens such as Mycobacterium tuberculosis and Salmonella typhimurium because it enables them to survive digestion in the phagolysosome (Maguire, 2006). Magnesium is essential for the growth of A. fumigatus. It contains four genes with putative magnesium acquisition function including Afu4g00930, an ortholog of the prokaryotic CorA and yeast ALR1 magnesium plasma membrane transporters and Afu7g05060, an ortholog of the bacterial MgtC transporters (Table 8.3). Interestingly, A. fumigatus is the only sequenced fungus that contains an MgtC prokaryotic-related transporter. Afu7g05060/MgtC is currently being studied in A.fumigatus (Gastebois and Mouyna, personal communication, 2005).

Phosphate acquisition: A. fumigatus needs about 10 mM free phosphate to grow in culture, whereas serum contains only about 1 mM. Its genome contains four homologs of the S. cerevisiae low-affinity and high-affinity inorganic phosphate

Table 8.3 A. fumigatus putative essential element transporters

Classification*

Genes

Iron uptake

Siderophore biosynthesis sidA (Afu2g07680). sidC (Afu1g17200). Afu3g03400. Afu1g04450

Magnesium uptake Magnesium transporters

Afu4g00930 (CorA-like). Afu7g05060 (MgtC-like) Afu5g05830. Afu6g02550

Phosphate uptake Phosphate transporters Acid phosphatases

Afu7g06350. Afu1g04290. Afu4g09210. Afu2g10690 PhoA (Afu1g03570). Afu4g01070. Afu4g03660. Afu5g01330. Afu6g1330. and Afu3g14570

Zinc uptake Zinc transporters zrfA (Afu1g01550). zrfB (Afu2g03860). Afu6g00470. Afu8g04010. Afu2g01460. Afu6g14170. Afu4g09560

* Classification based on A. fumigatus genome annotation (Nierman et al., 2005) and available at http://www.genome.jp/kegg-bin/show_organism?menu_type=gene_catalogs&org=afm.

* Underlined: genes that have been studied to date.

transporters, and at least six secreted acid phosphatases including PhoA (Table 8.3) and six phospholipases (Table 8.1) that release phosphate from organic sources. Only one of these 14 genes, PhoA, has been studied. It is a GPI-anchored cell wall protein with phosphate-repressible acid phosphatase activity (Bernard et al., 2002). The deletion of phoA has not been described.

Zinc uptake: Zinc is an essential micronutrient that A. fumigatus must obtain from the environment in order to grow. The A. fumigatus genome contains seven putative zinc transporters including ZrfA and ZrfB (Table 8.3). Deletion of both ZrfA and ZrfB dramatically reduces growth under acid, zinc-limiting conditions (Vicentefranqueira et al., 2005). Their role in virulence has not been described. The other five transporters have not been studied to date.

Signaling pathways: Signaling pathways are key elements in sensing and transmitting the response of cells to environmental conditions. The major known fungal-signaling pathways are described below, with an emphasis on their role in the growth and virulence of A. fumigatus.

Histidine kinases: Histidine kinases have an important role in virulence of both plant and human pathogens. They act primarily as sensors that respond to changes in osmolarity, resistance to fungicides and cell wall assembly (Nemecek et al., 2006). The A. fumigatus genome contains approximately 15 putative histidine kinase genes of which only two, fos-1 and tcsB were studied (Table 8.4). Deletion of fos-1 results in no obvious defect during growth on agar, but the mutant is attenuated in virulence in infected mice (Pott et al., 2000; Clemons et al., 2002). Deletion of tcsB results in no obvious phenotype suggesting there is some redundancy in this family (Du et al., 2006). Mammalian cells do not have any two-component or phosphorelay systems, suggesting they might be useful antifungal drug targets for treatment of human mycoses.

G-proteins The fungal G-protein superfamily is divided into the small G-protein and the heterotrimeric receptor coupled G-protein families.

200

N. Osherov

Table 8.4 A. fumigatus putative signaling pathway genes

Classification*

Genes

Histidine kinases

fos-1 (Afu6g10240), tcsB (Afu2g0066), Afu4g01020,

Afu4g00660,

Afu4g00320, Afu4g02900, Afu8g06140, Afu7g08550,

Afu2g03560, Afu6g09260,

Afu4g07400, Afu5g10020, Afu3g12550, Afu3g12530,

Afu3g07130

Small G-proteins

Ras-like

rasA (Afu5g11230), rasB (Afu2g07770), rsr1 (Afu5g08950),

rhbA (Afu5g05480), Afu3g05770, Afu7g02540,

Afu1g02190, Afu1g07680, Afu2g15570

Rho-like

Afu4g04810

Rac-like

Rho1 (Afu6g06900), rho2 (Afu3g10340), rho3 (Afu3g06690),

rho4 (Afu5g14060), Afu2g05740 Afu3g06300

MAP-kinase pathway

MAPKKK

Afu1g10940, Afu5g06420, Afu3g11080

MAPKK

Afu1g05800, Afu1g15950, Afu3g05900

MAPK

sakA (Afu1g12940), mpkA (Afu4g13720) mpkB (Afu6g12820),

mpkC (Afu5g09100)

cAMP signaling

PKA catalytic subunits

pkaC (Afu2g12200), Afu1g06400, Afu5g08570

PKA regulatory subunits

pkaR (Afu3g10000)

Adenylate cyclase

acyA (Afu6g08520)

* Classification based on A. fumigatus genome annotation. (Nierman et al., 2005.) Available at http://www.genome.jp/kegg-bin/show_organism?menu_type=gene_catalogs &org=afm.

* Underlined: genes that have been studied to date.

* Classification based on A. fumigatus genome annotation. (Nierman et al., 2005.) Available at http://www.genome.jp/kegg-bin/show_organism?menu_type=gene_catalogs &org=afm.

* Underlined: genes that have been studied to date.

Small G-proteins such as ras, rho, rac, and cdc42 have a major role in fungal nutrient sensing, polarity and growth (Wendland, 2001). The A. fumigatus genome contains at least 16 genes encoding small G-proteins, of which only three rasA, rasB, rhbA were studied (Table 8.4). The genes rasA and rasB appear to have different, but overlapping roles in the germination, vegetative growth and asexual development of A. fumigatus (Fortwendel et al., 2004). The virulence of these mutants was not reported. RhbA is a small G-protein that is upregulated when A. fumigatus is grown under nitrogen starvation and in culture with human endothe-lial cells. Deletion of rhbA results in slow growth on medium containing a poor nitrogen source and reduced virulence in mice (Panepinto et al., 2003). This might be because of their reduced ability to utilize nitrogen sources in infected tissue.

Heterotrimeric receptor-coupled G-proteins which interact with G-protein coupled transmembrane receptors (GPCRs) are involved in growth, development, mating, biosynthesis of secondary metabolites and virulence (Yu & Keller, 2005; Lafon et al., 2006). The A. fumigatus genome contains 15 putative GPCRs, three G-alpha subunit, one G-beta, and one G-gamma encoding genes. They also contain five homologs of RGS (regulator of G-protein signaling) predicted to participate in attenuation of G-protein activity (Lafon et al., 2006). Of these, only gpaA (Afu1g13140) and gpaB (Afu1g12930) encoding G-alpha subunits were studied in A. fumigatus. Deletion of gpaB resulted in decreased conidiation (but normal growth rates), increased sensitivity to macrophage killing. Most importantly, the gpaB null mutant showed greatly reduced virulence in a low-dose inhalation mouse-infection model, despite the fact that the mutant conidia germinated and colonized the infected lungs normally (Leibman et al., 2003, 2005).

MAPK pathways: MAP-kinase (MAPK) cascades have a pivotal role in fungal nutrient sensing, osmolarity response, cell wall integrity and pheromone response pathways. These kinase cascades contain three protein kinases (MAPKKK, MAPKK, and MAPK) that act in series by phosphorylation (May et al., 2005). The A. fumigatus genome contains three putative MAPKKK, three MAPKK, and four MAPK genes (Table 8.4). Of these 10 genes, only two, the Hog-family sakA/ MAPK and mpkC have been studied. Deletion of sakA results in an inability to grow under hypotonic stress, sensitivity to oxidative stress, and impaired nitrogen sensing (Xue et al., 2004, Du et al., 2006), whereas deletion of mpkC resulted in impaired conidial germination and growth in the presence of sorbitol and mannitol as sole carbon sources (Reyes et al., 2006, in press). The virulence of these strains was not reported.

cAMP signaling: The cyclic AMP-dependent protein kinase (PKA) regulates morphology, stress response and virulence in a number of fungal pathogens of humans and plants. The A. fumigatus genome contains three PKA catalytic subunits, one regulatory subunit and one gene encoding adenylate cyclase (Table 8.4). Of these five genes, three, acyA adenylate cyclase, pkaC catalytic subunit and pkaR regulatory subunit, were studied. Deletion of acyA results in decreased growth and conidiation and increased sensitivity to macrophages (Liebmann et al., 2004).

Deletion of A. fumigatus pkaC and pkaR results in decreased growth and reduced tolerance to oxidative stress. Reduced virulence in mice was also seen, although this is most likely a general outcome of impaired growth (Liebmann et al., 2004; Zhao et al., 2006).

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