Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C-type lectins represent a family of receptors, which recognize pathogen-specific carbohydrates. One of them is beta1-3 glucan, a major component of the fungal cell wall. Here we provide evidence that beta1-3 glucan plays an important role for the elimination of A. fumigatus conidia. Laminarin, a soluble beta1-3 glucan and antibodies to dectin-1, a well known beta1-3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible beta1-3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of beta1-3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild-type conidia are phagocytosed with high efficiency thereby confirming the relevance of beta1-3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2-mediated recognition of A. fumigatus and the phagocytic response.     

Two alpha(1-3) glucan synthases with different functions in Aspergillus fumigatus

Alpha(1-3) glucan is a main component of the Aspergillus fumigatus cell wall. In spite of its importance, synthesis of this amorphous polymer has not been investigated to date. Two genes in A. fumigatus, AGS1 and AGS2, are highly homologous to the AGS genes of Schizosaccharomyces pombe, which encode putative alpha(1-3) glucan synthases. The predicted Ags proteins of A. fumigatus have an estimated molecular mass of 270 kDa. AGS1 and AGS2 were disrupted in A. fumigatus. Both Deltaags mutants have similar altered hyphal morphologies and reduced conidiation levels. Only Deltaags1 presented a reduction in the alpha(1-3) glucan content of the cell wall. These results showed that Ags1p and Ags2p were functionally different. The cellular localization of the two proteins was in agreement with their different functions: Ags1p was localized at the periphery of the cell in connection with the cell wall, whereas Ags2p was intracellularly located. An original experimental model of invasive aspergillosis based on mixed infection and quantitative PCR was developed to analyze the virulence of A. fumigatus mutant and wild-type strains. Using this model, it was shown that the cell wall and morphogenesis defects of Deltaags1 and Deltaags2 were not associated with a reduction in virulence in either mutant. This result showed that a 50% reduction in the content of the cell wall alpha(1-3) glucan does not play a significant role in A. fumigatus pathogenicity.     

α1,3 glucans are dispensable in Aspergillus fumigatus

A triple α1,3 glucan synthase mutant of Aspergillus fumigatus obtained by successive deletions of the three α1,3 glucan synthase genes (AGS1, AGS2, and AGS3) has a cell wall devoid of α1,3 glucans. The lack of α1,3 glucans affects neither conidial germination nor mycelial vegetative growth and is compensated by an increase in β1,3 glucan and/or chitin content.     

Prenylation of Rho1p is required for activation of yeast 1, 3-beta-glucan synthase

One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeast Saccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1, 3-beta-glucan synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the beta subunit of geranylgeranyl transferase type I called cal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-beta-glucan synthase. The 1,3-beta-glucan synthase activity of the cal1-1 membrane was significantly reduced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1, 3-beta-glucan synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. Glutathione S-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction. The modified form of GST-Rho1p was specifically able to restore the 1,3-beta-glucan synthase of the rho1-3 membrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p. These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-beta-glucan synthase in vitro. Increased cytoplasmic levels of divalent cations such as Ca(2+) restored both Rho1p modification and the 1,3-beta-glucan synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.     

Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p. Using the two-hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP-bound form of Rho1p. This gene was identified as BNI1, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus. A temperature-sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis. Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase. These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p.     

Rho1p mutations specific for regulation of beta(1-->3)glucan synthesis and the order of assembly of the yeast cell wall

In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(1-->3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non-permissive temperature was used to isolate in vitro-generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the beta(1-->3) glucan synthesis function of Rho1p. At the non-permissive temperature (37 degrees C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, beta(1-->6)glucan is linked to both beta(1-->3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [(14)C]-glucose into beta(1-->3)glucan at 37 degrees C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and beta(1-->6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the beta(1-->3)-beta(1-->6)glucan complex was synthesized at almost normal levels. As beta(1-->3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is beta(1-->3)glucan, beta(1-->6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.     

AGS3, an alpha(1-3)glucan synthase gene family member of Aspergillus fumigatus, modulates mycelium growth in the lung of experimentally infected mice

The cell wall of human fungal pathogen Aspergillus fumigatus protects the fungus against threats from environment and interacts with the host immune system. Alpha(1-3)glucan is the major polysaccharide of Aspergillus fumigatus cell wall, and it has been shown to contribute to the virulence of diverse fungal pathogens. In A. fumigatus, three putative alpha(1-3)glucan synthase genes AGS1, AGS2 and AGS3 have been identified. AGS1 is responsible for cell wall alpha(1-3)glucan biosynthesis, but strains with deletions of either AGS1 or AGS2 are not defective in virulence [Beauvais, A., Maubon, D., Park, S., Morelle, W., Tanguy, M., Huerre, M., Perlin, D.S., Latgé, J. P., 2005. Two alpha(1-3) glucan synthases with different functions in Aspergillus fumigatus. Appl. Environ. Microbiol. 71, 1531-1538]. In contrast, we present evidence that AGS3 is also responsible for cell wall alpha(1-3)glucan biosynthesis and can modulate the virulence of A. fumigatus. An AGS3 deletion strain was found to produce faster and more robust disease than the parental strain in an experimental mouse model of aspergillosis. The apparent hyper-virulence in the AGS3-deleted mutant was correlated with an increased melanin content of the conidial cell wall, a better resistance to reactive oxygen species and a quicker germination rate. These results suggest an indirect role for AGS3 in virulence through an adaptive mechanism.     

Neurospora crassa FKS protein binds to the (1,3)beta-glucan synthase substrate,UDP-glucose

The essential fungal cell-wall polymer (1,3)beta-glucan is synthesized by the enzyme (1,3)beta-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-glucan synthase. We have purified (1,3)beta-glucan synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[beta-(32)P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H(+)-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-glucan synthase.     

Receptor internalization in yeast requires the Tor2-Rho1 signaling pathway

Efficient internalization of proteins from the cell surface is essential for regulating cell growth and differentiation. In a screen for yeast mutants defective in ligand-stimulated internalization of the alpha-factor receptor, we identified a mutant allele of TOR2, tor2G2128R. Tor proteins are known to function in translation initiation and nutrient sensing and are required for cell cycle progression through G1. Yeast Tor2 has an additional role in regulating the integrity of the cell wall by activating the Rho1 guanine nucleotide exchange factor Rom2. The endocytic defect in tor2G2128R cells is due to disruption of this Tor2 unique function. Other proteins important for cell integrity, Rom2 and the cell integrity sensor Wsc1, are also required for efficient endocytosis. A rho1 mutant specifically defective in activation of the glucan synthase Fks1/2 does not internalize alpha-factor efficiently, and fks1Delta cells exhibit a similar phenotype. Removal of the cell wall does not inhibit internalization, suggesting that the function of Rho1 and Fks1 in endocytosis is not through cell wall synthesis or structural integrity. These findings reveal a novel function for the Tor2-Rho1 pathway in controlling endocytosis in yeast, a function that is mediated in part through the plasma membrane protein Fks1.     

Mechanism of synthesis of D-glucans by D-glucosyltransferases from Streptococcus mutans 6715

Two glucosyltransferases from Streptococcus mutans 6715 were purified and separated. One of the glucosyltransferases synthesized an insoluble glucan, and the other, a soluble glucan. The enzymes were immobilized on Bio-Gel P-2 beads, and the mechanism of glucan synthesis was studied by pulse and chase techniques with 14C-sucrose. Label was associated with the immobilized enzymes. The label could be quantitatively released by heating at pH 2. Analysis of the labeled products from the pulse experiment showed labeled glucose and labeled glucan; the chase experiment showed labeled glucan and a significant decrease in labeled glucose. The glucans from the pulse and the chase experiments were separated from glucose by chromatography on Bio-Gel P-6. They were reduced with sodium borohydride, and the products hydrolyzed with acid. Analysis of the labeled products from the reduced and hydrolyzed, pulsed glucans showed labeled glucose and labeled glucitol; label in the glucitol was greatly decreased in the chase experiment. These experiments showed that glucose and glucan were covalently attached to the active site of the enzymes during synthesis, and that the glucose was being transferred to the reducing end of the glucan chain. A mechanism for the synthesis of the glucans is proposed in which there are two catalytic groups on each enzyme that holds glucosyl and glucanosyl units. During synthesis, the glucosyl and glucanosyl units alternate between the two sites, giving elongation of the glucans from the reducing end. The addition of increasing amounts of B-512F dextran to the insoluble-glucan-forming glucosyltransferase produced a decrease in the proportion of insoluble glucan formed and a concomitant increase in a soluble glucan. The total amount of glucan synthesized (soluble plus insoluble) was increased 1.6 times over the amount of insoluble glucan formed when no exogenous dextran was added. It is shown that the addition of B-512F dextran affects the solubility of the synthesized alpha-(1 to 3)-glucan by accepting alpha-(1-3)-glucan chains at various positions along the dextran chain, to give a soluble, graft polymer.     

The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell.

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.     

Multiple glucan-binding proteins of Streptococcus sobrinus.

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.     

Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase.     

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein.

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of approximately 200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched approximately 300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.     

Cell wall α1-3glucans induce the aggregation of germinating conidia of Aspergillus fumigatus

The germination of Aspergillus fumigatus conidia can be divided into four stages: breaking of dormancy, isotropic swelling, establishment of cell polarity, and formation of a germ tube. Swelling of conidia is associated in liquid medium with a multi-cellular aggregation that produced large clumps of conidia. Conidial aggregation can be specifically prevented by the addition of α1-3glucanase. Swollen conidia specifically adhere to insoluble α1-3glucan chains. Electron microscopy studies showed that cell wall α1-3glucan chains became exposed at the cell surface during the swelling. These results demonstrate that cell wall α1-3glucans play an essential role in the aggregation between swollen conidia. Experiments with α1-3glucan coated latex beads show that α1-3glucan chains interacted between them without the requirement of any other cell wall component suggesting that biophysical properties of α1-3glucans are solely responsible for conidial aggregation.     

Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides.

The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a Ki of 5.3 x 10(-6) M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a Ki of 3 x 10(-9) M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion.     

<Emphasis Type="Italic">Neurospora crassa</Emphasis> FKS Protein Binds to the (1,3)β-Glucan Synthase Substrate,UDP-Glucose

The essential fungal cell-wall polymer (1,3)β-glucan is synthesized by the enzyme (1,3)β-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)β-glucan synthase. We have purified (1,3)β-glucan synthase from Neurospora crassa ∼400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[β-³²P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H⁺-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)β-glucan synthase. Received: 31 May 2002 / Accepted: 27 July 2002     

Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPI-anchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Manalpha1-3Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN.     

Farnesol misplaces tip‐localized Rho proteins and inhibits cell wall integrity signalling in Aspergillus fumigatus

Farnesol is known for inducing apoptosis in some fungi and mammalian cells. To evaluate its potential role as an antifungal agent, we studied its impact on the human pathogen Aspergillus fumigatus. We found that growth of A. fumigatus wild type is inhibited, but two cell wall mutants, Δmnt1 andΔglfA, are much more susceptible to farnesol. This susceptibility is partially rescued by osmotic stabilization, suggesting that farnesol is a cell wall perturbing agent. However, farnesol does not activate but inhibit the cell wall integrity (CWI) pathway. Remarkably, mutants lacking AfMkk2 or AfMpkA, two kinases essential for CWI signalling, are also highly susceptible to farnesol, suggesting that its mode of action goes beyond inhibition of CWI signalling. Farnesyl derivatives are known for interfering with the function of prenylated proteins. We analysed the subcellular localization of two prenylated Rho family GTPases, AfRho1 and AfRho3, which are implicated in controlling CWI and the cytoskeleton. We found that under normal growth conditions AfRho1 and AfRho3 predominantly localize to the hyphal tip. After farnesol treatment this localization is rapidly lost, which is accompanied by swelling of the hyphal tips. Parallel displacement of tropomyosin from the tips suggests a concomitant disorganization of the apical actin cytoskeleton.     

The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus

Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3-glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1alpha (MIP-1alpha), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte-CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan-initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense.