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1 Department of Human Microbiology, Sackler School of Medicine, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel
2 Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel
Correspondence
Nir Osherov
nosherov{at}post.tau.ac.il
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ABSTRACT |
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Figures showing sequence alignments and a phylogenetic tree are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Invasive pulmonary aspergillosis is caused by inhalation of A. fumigatus spores and growth of the fungus inside the lungs, which often spreads from the initial site of infection to attack various organs in the body. It is the leading cause of death in leukaemia and bone-marrow-transplant patients, with between 10 and 20 % of patients contracting this disease. Left untreated, mortality rates from this disease are extremely high (>90 %), and even following aggressive antifungal treatment, fatality rates of 50 to 70 % are common (Steinbach & Stevens, 2003). Therefore, there is an urgent need for a deeper understanding at the molecular level of the interaction of A. fumigatus with the infected host.
The fungal cell wall plays a crucial role in infection. In A. fumigatus, as in other pathogenic fungi, the cell wall is in continuous contact with the host, acting as a reservoir for displayed and secreted antigens and enzymes. The cell wall protects the fungus and interacts directly with the host immune system. It is an elastic, dynamic and highly regulated structure, and is essential for growth, viability and infection. The cell wall of A. fumigatus is composed of a polysaccharide skeleton interlaced and coated with cell wall proteins (CWPs). The main building blocks of the polysaccharide skeleton are an interconnected network of glucan, chitin and galactomannan polymers (Latgé et al., 2005). The cell wall of A. fumigatus differs considerably from that of yeast such as Saccharomyces cerevisiae and Candida albicans. It contains a much higher level of chitin and unique polysaccharides such as 1,3-
-glucan, (1,3)(1,4)-
-glucan and galactomannan (Latgé et al., 2005).
The major class of fungal CWPs is the glycophosphatidylinositol (GPI)-modified proteins (de Groot et al., 2003; Eisenhaber et al., 2004). They contain an N-terminal hydrophobic signal peptide sequence that targets them to the endoplasmic reticulum (ER), and a C-terminal hydrophobic domain that is cleaved off in the ER and replaced with a GPI anchor. The GPI anchor directs the attachment of these proteins to the plasma membrane. Subsequently, the GPI anchor may be processed and attached to 1,6--glucan in the cell wall. ECM33/SPS2-family proteins have the typical features of GPI-anchored proteins, with a signal peptide, a serine and threonine-rich region and a potential C-terminal domain for GPI-anchor attachment (Percival-Smith & Segall, 1987; Caro et al., 1997; Tougan et al., 2002; Terashima et al., 2003). They play an important role in fungal cell wall organization. Deletion of S. cerevisiae ECM33 results in a weakened and disorganized cell wall, defects in glycosylation, and activation of the cell wall integrity pathway (Pardo et al., 2004). The C. albicans CaEcm33 protein is required for normal cell wall architecture and expression of cell-surface proteins. CaEcm33-deleted mutants display a reduced ability to invade and damage epithelial cells, and show decreased virulence in a murine model of systemic candidosis (Martinez-Lopez et al., 2004, 2006).
Proteomic analysis of A. fumigatus membrane preparations identified nine GPI-anchored proteins, including the protein encoded by AfuEcm33 (Afu4g06820), the homologue of S. cerevisiae ECM33 (Bruneau et al., 2001). In light of the importance of the ECM33/SPS2-family proteins in cell wall organization and virulence, we have undertaken the characterization of the A. fumigatus ECM33 homologue, AfuEcm33 (Afu4g06820). This is believed to be the first time that such a characterization has been undertaken in a pathogenic filamentous fungus. Interestingly, we show that disruption of AfuEcm33 in A. fumigatus results in rapid conidial germination, increased cell–cell adhesion, resistance to the antifungal caspofungin and increased virulence in an immunocompromised mouse model for disseminated aspergillosis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), were used throughout this study. The AF293.1 strain is deficient in pyrG (encoding OMP-decarboxylase) and, consequently, is auxotrophic for uridine/uracil (Osherov et al., 2001). AF293.1 was grown on YAG UU medium, which consists of 0.5 % (w/v) yeast extract, 1 % (w/v) glucose, 10 mM MgCl2, 10 mM uracil and 5 mM uridine, supplemented with trace elements, vitamins and 1.5 % (w/v) agar when needed (Bainbridge, 1971). For phenotypic analysis, mutants were grown on minimal medium (MM) containing 70 mM NaNO3, 1 % glucose, 12 mM potassium phosphate pH 6.8, 4 mM MgSO4, 7 mM KCl, trace elements and 1.5 % agar (for MM agar plates). Conidia were harvested in 0.2 % (v/v) Tween 80, resuspended in double-distilled water (DDW) and counted with a haemocytometer. Escherichia coli strain DH10B (Invitrogen) was used to clone and replicate the genes in the pGEM-T/A and pGPS1/pyr4 transposon-containing vectors (Jadoun et al., 2004).
RNA analysis.
AF293 and disrupted strains AfuEcm33-D1–4 were grown for the indicated time in liquid MM at 37 °C. Total RNA was extracted by the ‘hot SDS’ protocol (May & Morris, 1988). Northern blot analysis was performed as described previously (Osherov et al., 2002). Briefly, 5 µg fungal total RNA was run on a 1 % (w/v) agarose gel under denaturing conditions, transferred to a Nytran N nylon membrane (Schleicher & Schuell) and hybridized with an [-32P]dCTP radiolabelled AfuEcm33 full-length probe at 47 °C. For RT-PCR, total RNA was treated with DNase (Ambion) according to the manufacturer's instructions. The RNA concentration was assessed and 3 µg were taken for the RT reaction using PowerScript reverse transcriptase (Clontech). PCR was performed using ReddyMix PCR master mix (ABgene) with the following designed primer pairs (Table 1): AfuEcm33 forward and reverse primers were used to identify the AfuEcm33 transcript, and AfugpdA forward and reverse primers were used as a loading control. The PCR was carried out according to the manufacturer's instructions. PCR products were analysed by gel electrophoresis.
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). Briefly, this approach utilizes a modified transposon containing the Neurospora crassa pyr4 gene, which is randomly inserted in vitro into a target sequence of interest. Clones in which the gene of interest has been disrupted are identified by PCR and used to transform a pyrG-deficient strain of A. fumigatus. Primers were designed to contain an AscI restriction site at their 5' end (Table 1
) and generated a 
4 kb fragment using the Expand high-fidelity PCR system (Roche Diagnostics). The primer pairs used to amplify the fragments for the AfuEcm33 gene were AfuEcm33+AscI site forward and AfuEcm33+AscI site reverse (Table 1). A. fumigatus AF293 CsCl2-purified genomic DNA (1 µg per reaction) was used as a template in the reactions. PCR conditions were as recommended by the manufacturer. The PCR fragment produced was gel purified by the Wizard SV gel and clean-up system (Promega), and cloned into the pGEM T/A cloning vector (Promega), according to the manufacturer's instructions, to obtain pGEM-AfuEcm33. Inactivation of the AfuEcm33 gene was performed using the GPS-1 genome priming system (New England Biolabs) with the modified GPS-1/pyr4 transposon (Jadoun et al., 2004) according to the manufacturer's instructions. Clones carrying transposon-disrupted genes were identified by PCR using AfuEcm33 forward and reverse primers (Table 1). Sequencing indicated that the transposon inserted 624 bp downstream of the AfuEcm33 gene start codon. The gene fragment with the transprimer 1-pyr4 disruption, and its flanking sequence, was released by cleavage with AscI, purified with the Wizard SV gel and clean-up system and used to transform A. fumigatus strain AF293.1 (Jadoun et al., 2004). Rapid initial screening for positive disrupted mutants was performed by PCR amplification of crude conidial genomic DNA. Conidia from transformed colonies were collected and transferred to 500 µl DDW to give a conidial concentration of 107 conidia ml–1. The tubes were snap frozen in liquid nitrogen for 10 min, heated at 95 °C for 5 min and used for PCR with the AfuEcm33 forward and reverse primers (Table 1). Amplification of the housekeeping gpdA gene was used as a positive control. Colonies negative for gene amplification but positive for gpdA amplification were chosen for further analysis. For further Southern blot analysis, A. fumigatus genomic DNA was extracted with a MasterPure yeast DNA purification kit (Epicenter), with modifications for A. fumigatus as described by Jin et al. (2004). Southern hybridization analysis was performed as described previously (Jadoun et al., 2004). Briefly, 10 µg fungal genomic DNA were digested with HindIII and run on a 1 % (w/v) agarose gel. The cleaved DNA was transferred to a Nytran N nylon membrane (Schleicher & Schuell) and hybridized with an 32[dCTP] radiolabelled N. crassa pyr4 probe at 65 °C. The AF293.1 pyr4 complemented strain was obtained by transforming the auxotrophic AF293.1 strain with the N. crassa pyr4 gene. The AfuEcm33 KI strain was prepared by complementing the AfuEcm33-D1 strain with plasmid pGEM-AfuEcm33 containing the pTrpC-hyg cassette (Punt et al., 1987). Three AfuEcm33 KI strains were obtained and verified for AfuEcm33 mRNA expression by RT-PCR. All three strains were phenotypically identical to the control wild-type strain AF293 as assessed by clumping, aerosolization and germination experiments (data not shown).
Phenotypic analysis of the AfuEcm33 disrupted mutant
Growth assay.
A. fumigatus AF293 and disrupted AfuEcm33 mutant isolates were grown at a concentration of 104 conidia ml–1 in MM without glucose supplemented with 1 % (w/v) raffinose, sucrose, glycerol, ethanol or BSA as sole carbon sources. For growth experiments at 37 °C and 42 °C, MM containing 1 % glucose (w/v) was used. For growth analysis at different pH values, conidia were grown in MM buffered to pH 6 and pH 9 with 50 mM BIS or citrate buffers, respectively. For analysis of growth at reduced osmolarity, conidia were grown in MM containing 7 mM NaNO3, 0.4 mM MgSO4 and 0.7 mM KCl. For growth experiments in a high osmolarity environment, MM containing 1.0 M NaCl was used.
Qualitative assay for conidial clumping and aerosolization.
For assessment of conidial clumping, freshly harvested A. fumigatus AF293 and disrupted AfuEcm33 mutant conidia grown for 3 days on YAG agar plates were harvested in 0.2 % (v/v) Tween 80, resuspended in DDW and counted with a haemocytometer. A total of 2x108 conidia ml–1 were transferred to a sterile glass tube, vortexed for 30 s, allowed to stand for 15 min and photographed. For qualitative assessment of conidial aerosolization, strains were grown on YAG agar plates for 3 days. During harvesting of conidia, four sterile YAG agar plates were placed uncovered around and immediately adjacent to the harvested plate. The four plates were subsequently incubated for 2 days at 37 °C to allow growth of aerosolized conidia. The extent of colonization, correlating to the degree of conidial aerosolization during harvesting, was assessed visually.
Microscopic analysis.
Conidia at a concentration of 103 conidia ml–1 were grown in 1 ml liquid MM on glass disks in a stationary 24-well plate at 37 °C. Before microscopic examination, the conidia were stained with 8 µl Fluorescent Brightener 28 (0.5 µg ml–1) (Sigma-Aldrich) and observed under an Olympus IX50 fluorescent microscope at a magnification of x400. Hyphal growth rate and germination studies were performed by plating 103–105 freshly harvested spores ml–1 onto 96-well plates in 200 µl liquid YAG at 37 °C. At various time points, growth was observed under a grid-mounted Olympus CK inverted microscope at a magnification of x200. The percentage of germinated conidia (n=200) was assessed, and the lengths of the germlings (n=50) were measured in microns.
Sensitivity to reagents and antifungals.
A. fumigatus AF293 and disrupted AfuEcm33 mutant isolates were grown in 96-well plates at a concentration of 104 conidia ml–1 in MM supplemented with reagents and antifungals in 96-well plates. MICs (the lowest drug concentrations to completely arrest germination and growth) were evaluated after 24 h incubation at 37 °C. Unless otherwise specified, all reagents were from Sigma-Aldrich. The concentration ranges of the reagents and antifungals were: Congo red 1–160 µg ml–1; caspofungin (Merck) 1–160 µg ml–1; Calcofluor white 10–320 µg ml–1; hygromycin B 5–320 µg ml–1; amphotericin B 1–80 µg ml–1; itraconazole 0.25–8 µg ml–1; tunicamycin 1–80 µg ml–1; trifluoroperazine 10–160µg ml–1.
For sensitivity testing on agar plates, conidia from the mutant and AF293 wild-type strain (108 conidia ml–1) were point inoculated on MM plates containing either 80 µg Congo red ml–1 or 40 µg caspofungin ml–1 or no drug control. The strains were grown for 48 h at 37 °C.
Murine model for systemic aspergillosis.
Six-week-old female ICR mice were injected intraperitoneally with 200 mg cyclophosphamide kg–1 at 3 days prior to conidial infection. Mice were inoculated intravenously via the tail vein with a 2.5x105 conidia per mouse inoculum of freshly harvested AF293 wild-type, AfuEcm33-D1 disrupted, AfuEcm33 KI (complemented) or AF293.1 pyr4 (pyr4 complemented) conidia. To prolong neutropenia, additional cyclophosphamide (70 mg kg–1) was administered 2 and 5 days after infection. Systemic aspergillosis was followed up for 30 days. Statistical analysis of mouse survival was performed with GraphPad Prism 4 software (GraphPad Software). P values of <0.05 were considered significant in this analysis. Animal studies were performed in accordance with Tel-Aviv University institutional policies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-site of AfuEcm33p is predicted to be at sequence position ASN372, with the typical consensus of two alanines (+1, +2) and serine (–1) (Eisenhaber et al., 2004). The most likely signal peptide cleavage site is predicted to be between ALA19 and ALA20. Based on multiple sequence alignment, AfuEcm33p has significant sequence identity to Aspergillus nidulans hypothetical protein AN4390.2 (60 % identity in 398 amino acids), Magnaporthe grisea Ecm33-like AAX07654 (36 % identity in 326 amino acids), N. crassa Sps2-like CAD70996 (32 % identity in 333 amino acids), C. albicans CaEcm33p (31 % identity in 318 amino acids), S. cerevisiae Ecm33p (31 % identity in 319 amino acids) and S. cerevisiae Sps2p (28 % identity in 304 amino acids). A search of the A. fumigatus database yielded a single additional predicted protein sequence with a low but significant degree of similarity to AfuEcm33p, the gene product of Afu6g10290 (29 % identity in 141 amino acids). Afu6g10290 exhibits significant similarity to S. cerevisiae PstIp (31 % identity in 172 amino acids, probability value 2x10–7), a protein secreted by regenerating protoplasts and a member of the ECM33/SPS2 family of proteins (Pardo et al., 1999).
A complete evolutionary analysis of AfuEcm33p using 25 sequence homologues with the ConSeq web server (http://conseq.bioinfo.tau.ac.il/) (Berezin et al., 2004) revealed a significant conservation of large amino acid blocks between the Aspergillus species, and a low conservation towards other fungal species (see the supplementary figures available with the online journal). This is characteristic of serine/threonine-rich CWPs containing large numbers of nucleotide repeat units.
We showed by RT-PCR, using primers AfuEcm33 forward and reverse (Table 1), that AfuEcm33 is not significantly expressed in dormant conidia, but is expressed throughout germination and hyphal growth (Fig. 1).
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). After transformation of pAfuEcm33-D into AF293.1, 20 pyrG+ transformants were purified and screened by PCR for putative insertion mutants. Four putative mutants were identified and further characterized by Southern blotting (Fig. 2b). Based on this analysis, all four transformants were disrupted in the AfuEcm33 gene alone (AfuEcm33-D1–4). The indication based on Northern blot analysis is that a truncated AfuEcm33 mRNA is expressed in all four disrupted strains (1.4 kb in size as compared to 2.5 kb in the wild-type, Fig. 2c). RT-PCR indicated that this mRNA contains the first 624 bp of AfuEcm33, a short portion of the transposon, but not the 676 bp of coding sequence downstream of the transposon (our unpublished results). Although it cannot be formally ruled out that a truncated form of AfuEcm33p protein is expressed, this form would lack the C-terminal GPI-anchor motif targeting it to the cell membrane, and therefore it is highly unlikely that it would remain functional (Terashima et al., 2003). However, to formally rule out the possibility that a phenotype is resulting from expression of a truncated form of AfuEcm33p protein, complete gene deletion mutants are being generated and their phenotype will be compared to that of the disrupted mutants described in this report.
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). A representative strain, AfuEcm33-D1 was chosen for further characterization. The AfuEcm33-D1 mutant strain grew normally on rich YAG medium or defined MM agar plates, indicating that the AfuEcm33 gene is not essential for growth (Fig. 3a). Freshly harvested AfuEcm33-disrupted conidia formed large aggregates in DDW, suggesting an increase in cell–cell adhesive properties (Fig. 3b). AfuEcm33-disrupted conidia showed a greatly decreased ability to aerosolize during harvesting. Sterile YAG agar plates left uncovered in the direct vicinity of harvested plates showed very high levels of colonization by conidia from wild-type plates as compared to plates containing the mutant AfuEcm33-D1 strain (Fig. 3c). Microscopic analysis of freshly harvested conidia indicated that there is no statistically significant difference in size or shape between the mutant and wild-type conidia (AF293 conidial diameter 3.2±0.5 microns, AfuEcm33-D1 conidial diameter 4±0.6 microns, n=100). Microscopic analysis in liquid MM demonstrated that the AfuEcm33-D1 strain germinates earlier than the wild-type strain and forms large ‘star-shaped’ clusters of germinating conidia, in which the conidial cell bodies adhere to each other and the germ tubes grow outwards, suggestive of an alteration in cell–cell adherence (Fig. 3d). The time needed for 50 % of conidia to germinate was 3 h 45 min for the mutant AfuEcm33-D1 strain and 6 h 15 min for the AF293 control wild-type strain (Fig. 3e). However, the growth rates of the AF293 wild-type and mutant AfuEcm33-D1 strain were similar (compare the slopes in Fig. 3e). After 12 to 24 h there was no significant difference in hyphal length, colony size or morphology of the AfuEcm33-D1 strain compared to the wild-type AF293 parental strain.
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). The MIC of both agents was eightfold higher in the AfuEcm33-D1 strain as compared to the AF293 control strain and was specific for these agents alone (Table 2). This result suggests that changes in the composition of the cell wall in the AfuEcm33-D1 strain may render it more resistant to damage by these agents.
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shows survival curves obtained during the course of the experiment. By day 7, all 14 mice injected with the AfuEcm33-D1 (n=10) and AfuEcm33-D2 (n=4) disrupted strains had died [mean survival time (MST)=4.1 days]. In contrast, 60 % of the mice injected with the wild-type AF293 strain or AF293.1 pyr4 strains were still alive after 30 days (MST=21 days, n=10) while 30 % of mice infected with the AfuEcm33 KI strain remained alive after 30 days (MST=13.2 days, n=10). Statistical analysis of these data by the Wilcoxon rank sum test showed a highly significant survival difference between the mouse groups infected with the wild-type AF293, control AF293.1 pyr4, AfuEcm33 KI strains and the AfuEcm33 disrupted A. fumigatus strains (P<0.0005), supporting a conclusion that the AfuEcm33 disrupted strains exhibit hypervirulence in the tested mouse model. The survival difference between the wild-type AF293 strain and control AfuEcm33 KI strain was not statistically significant (P=0.19).
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DISCUSSION |
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; Pardo et al., 2004), (ii) biochemical studies had already identified AfuEcm33p as a genuine GPI-anchored protein in A. fumigatus (Bruneau et al., 2001) and (iii) ECM33-family proteins have not been previously characterized in the filamentous fungi.
AfuEcm33 is involved in conidial adherence and morphogenesis
Disruption of the AfuEcm33 gene in A. fumigatus resulted in subtle and unusual morphological changes, including rapid germination and conidial clustering during harvesting and germination. C. albicans CaECM33-deleted cells also exhibit a marked tendency to flocculate (cluster) extensively (Martinez-Lopez et al., 2004). We hypothesize that cell wall alteration in the AfuEcm33 disrupted mutant may lead to greater exposure of the cell-surface proteins involved in adherence, leading to increased cell clustering. However, the AfuEcm33 disrupted strains exhibited no substantial differences in adherence to polystyrene (a measure of changes in cell wall hydrophobicity), laminin (a component of the extracellular matrix) or A549 lung-cell extracellular matrix as compared to the wild-type AF293 strain using either dormant or germinating conidia (our unpublished observations). This indicates that the increased cell–cell clustering we observed in the mutant is distinct from its ability to interact with the matrix.
The precocious germination of the AfuEcm33 disrupted mutant is intriguing. S. cerevisiae ECM33 deleted cells exhibit marked disorganization of the cell wall, and in particular the mannoprotein outer layer (Pardo et al., 2004). Disruption of MEU10, an ECM33/SPS2-like gene in Schizosaccharomyces pombe results in the formation of fragile spores containing a weakened cell wall (Tougan et al., 2002). Perhaps disruption of AfuEcm33 leads to the formation of a softer, more pliable cell wall, enabling germination to proceed more rapidly.
Disruption of AfuEcm33 leads to increased resistance to cell wall-disrupting agents
Surprisingly, we found that the AfuEcm33 disrupted strain is more resistant to the cell wall-disrupting agents Congo red and caspofungin (Fig. 4). This result is in contrast to that found in S. cerevisiae and C. albicans ECM33 deletion mutants, which display increased sensitivity to Congo red and Calcofluor white (Martinez-Lopez et al., 2004; Pardo et al., 2004). This could be a consequence of the considerable differences in polymer organization and cell wall content between S. cerevisiae or C. albicans and A. fumigatus (Bernard & Latgé, 2001; Latgé et al., 2005). Resistance to the antifungal drug caspofungin typically results from mutations in its target, (1,3)--glucan synthase (Kartsonis et al., 2003). Our work is believed to be the first to demonstrate a connection between caspofungin resistance in A. fumigatus and the loss of function of a defined gene. A plausible explanation for our finding is that disruption of AfuEcm33 may lead to increased levels of chitin and (1,3)--glucan in the cell wall, thereby compensating for the reduced synthesis of (1,3)--glucan by glucan synthase (Reinoso-Martin et al., 2003).
The AfuEcm33 disrupted A. fumigatus mutant is hypervirulent in a murine model of disseminated aspergillosis
We demonstrate that disruption of AfuEcm33 in A. fumigatus leads to hypervirulence in a mouse model of disseminated aspergillosis. This is in contrast to the results obtained using the C. albicans CaECM33 disrupted mutant, which exhibited decreased virulence in a similar model system. A number of mechanisms may explain the increased virulence of the AfuEcm33 mutant, including physical occlusion of blood vessels by clumps of conidia, enhanced resistance to phagocytes due to rapid germination and hyphal growth, or hyperstimulation of the immune system, leading to septic shock. Very few examples of fungal hypervirulence resulting from gene knockout have been described to date. Partial silencing of the A. fumigatus AfppoA, AfppoB and AfppoC genes encoding fatty acid dioxygenases resulted in increased resistance to oxidative stress and hypervirulence in mice (Tsitsigiannis et al., 2005). It was proposed that Ppo reaction products (prostaglandins and other oxylipins) may serve as activators of host immune defences. A decrease in prostaglandin production by the AfppoA–C silenced mutant might lead to a weaker host response resulting in hypervirulence. Disruption of the Candida glabrata ACE2 gene encoding a transcription factor and the S. cerevisiae SSD1 gene encoding a CWP of unknown function leads to hypervirulence in mice by inducing severe septic shock in infected animals (Wheeler et al., 2003; Kamran et al., 2004). This has been tentatively attributed to an increase in the exposure of fungal cell-surface antigens, which hyperstimulate the immune system of the infected animals (Wheeler et al., 2003). We are now performing experiments to determine whether similar mechanisms can explain the hypervirulence of the AfuEcm33 disrupted mutant.
In summary, our findings suggest that the A. fumigatus AfuEcm33 gene is involved in key aspects of cell wall architecture. The increased conidial aggregation, precocious germination, resistance to cell wall-destabilizing drugs and increased virulence resulting from the disruption of AfuEcm33 suggest that significant changes in the cell wall have occurred. Further elucidation of the mechanisms responsible for these changes may shed new light on the pathogenesis of A. fumigatus at the molecular level.
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ACKNOWLEDGEMENTS |
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Received 19 February 2006;
revised 20 March 2006;
accepted 22 March 2006.
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, AF293; 
, AfuEcm33-D1.
) (n=10), pyr4 complemented AF293.1 pyr4 (
) (n=10), AfuEcm33-D1 (
) (n=10 mice) or AfuEcm33-D2 (
) (n=4 mice) disrupted strains. Percentage survival was monitored daily over the 30 day study period.