This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schöbel, F.
Right arrow Articles by Brock, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schöbel, F.
Right arrow Articles by Brock, M.

 Previous Article  |  Next Article 

Infection and Immunity, March 2007, p. 1237-1244, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01416-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Aspergillus fumigatus Does Not Require Fatty Acid Metabolism via Isocitrate Lyase for Development of Invasive Aspergillosis{triangledown}

Felicitas Schöbel,1 Oumaïma Ibrahim-Granet,2 Patrick Avé,3 Jean-Paul Latgé,2 Axel A. Brakhage,1 and Matthias Brock1*

Leibniz Institute for Natural Product Research and Infection Biology (HKI) and Friedrich-Schiller University Jena, Beutenbergstr. 11a, 07745 Jena, Germany,1 Aspergillus Unit, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris, France,2 Histopathology Unit, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris, France3

Received 3 September 2006/ Returned for modification 10 October 2006/ Accepted 10 December 2006


arrow
ABSTRACT
 
Aspergillus fumigatus is the most prevalent airborne filamentous fungus causing invasive aspergillosis in immunocompromised individuals. Only a limited number of determinants directly associated with virulence are known, and the metabolic requirements of the fungus to grow inside a host have not yet been investigated. Previous studies on pathogenic microorganisms, i.e., the bacterium Mycobacterium tuberculosis and the yeast Candida albicans, have revealed an essential role for isocitrate lyase in pathogenicity. In this study, we generated an isocitrate lyase deletion strain to test whether this strain shows attenuation in virulence. Results have revealed that isocitrate lyase from A. fumigatus is not required for the development of invasive aspergillosis. In a murine model of invasive aspergillosis, the wild-type strain, an isocitrate lyase deletion strain, and a complemented mutant strain were similarly effective in killing mice. Moreover, thin sections demonstrated invasive growth of all strains. Additionally, thin sections of lung tissue from patients with invasive aspergillosis stained with anti-isocitrate lyase antibodies remained negative. From these results, we cannot exclude the use of lipids or fatty acids as a carbon source for A. fumigatus during invasive growth. Nevertheless, test results do imply that the glyoxylate cycle from A. fumigatus is not required for the anaplerotic synthesis of oxaloacetate under infectious conditions. Therefore, an antifungal drug inhibiting fungal isocitrate lyases, postulated to act against Candida infections, is assumed to be ineffective against A. fumigatus.


arrow
INTRODUCTION
 
Aspergillus fumigatus is a ubiquitous airborne filamentous fungus. Aberrant immune responses to A. fumigatus result in a spectrum of human diseases that include allergic bronchopulmonary aspergillosis. In severe cases, this leads to pulmonary hypersensitivity and compromised lung function, which is called farmer's lung (26). A mild form of infection is the colonization of cavities within the lung, known as aspergilloma or a fungal ball, and is often found in immunocompetent people who have recovered from a Mycobacterium tuberculosis infection (14). Growth of the fungus is restricted to a specific area in the tissue, and the mycelium does not spread. The most severe infection is invasive aspergillosis, in which the fungus vigorously spreads through the tissue and can also disseminate to other organs. This infection generally occurs in immunocompromised individuals (6). Due to the increasing numbers of organ transplant recipients and illnesses like leukemia and AIDS, which are accompanied by a suppression of the immune system (20), the number of individuals developing invasive aspergillosis is rising. Treatment of invasive aspergillosis is hampered by several problems. Diagnostic tools are effective only when the aspergillosis has already manifested. Furthermore, the progression of the illness from onset to death occurs within a few months (25). Different antifungals, which target either the cell membrane (like azoles or amphotericin B) or the cell wall (like caspofungin) of growing hyphae, are used, but their effectiveness varies greatly from person to person. The mortality rate, therefore, is between 60 and 90% (3). This dismal success rate demands the search for new and more-effective antifungals which do not allow the fungus to develop resistance against treatment. One hypothesized route to achieve this goal is the specific inhibition of primary fungal metabolism.

Investigations of glyoxylate cycle mutants of human-pathogenic microorganisms, i.e., both the bacterium Mycobacterium tuberculosis and the yeast Candida albicans, which carried a defect in the isocitrate lyase coding region, revealed a strong attenuation in virulence (15-17, 19). Therefore, it was concluded that fatty acids and lipids account for the major carbon sources during growth of these microorganisms in infected tissues (2). It was also assumed that blocking of the lipid metabolism might be a suitable goal for new antimicrobial compounds.

In a recent study, we investigated the isocitrate lyase from A. fumigatus in order to study the presence of the enzyme under various growth conditions (9). It was shown that isocitrate lyase is already present in conidia but is rapidly lost when the fungus grows on media which do not require the glyoxylate cycle for anaplerotic synthesis of oxaloacetate. Coincubation of conidia and macrophages showed a strong specific immunofluorescence for isocitrate lyase in the hyphae of early germlings inside the macrophages. Additionally, the use of 3-nitropropionate, a specific inhibitor of the isocitrate lyase, inhibited germination of conidia on media containing acetate as the sole carbon source, which confirms the essential role of isocitrate lyase to support growth on C2-generating carbon sources. We therefore concluded that an A. fumigatus mutant carrying a deletion of the isocitrate lyase coding region would also display attenuation in virulence in cases in which lipids or fatty acids provide a major carbon source during invasive aspergillosis (9).

To prove this assumption, we generated a specific isocitrate lyase knockout mutant of A. fumigatus and studied its phenotype under various in vivo and in vitro conditions.


arrow
MATERIALS AND METHODS
 
Media and growth conditions. A. fumigatus strains were grown at 37°C on Aspergillus minimal medium with glucose as the carbon source and hygromycin B (Roche Diagnostics, Mannheim, Germany) at a concentration of 180 µg/ml when required. For transformation of the {Delta}akuB wild-type strain, agar plates contained 0.6 M KCl and 240 µg/ml hygromycin B. For animal experiments, malt extract agar was inoculated with conidia and incubated at room temperature for 7 days. The conidial suspensions used in all experiments were filtered through a 40-µm cell strainer (BD Bioscience, Heidelberg, Germany), and the number of conidia was counted prior to use. Concentrations of carbon sources utilized in phenotypic analysis were used as described for the single experiments. Analysis of conidial germination was performed with four-well LabTek chamber slides (Nalge Nunc International, New York) containing 500 µl of medium. The following media were used: Sabouraud-2% dextrose broth (Merck KGaA, Darmstadt, Germany) and Aspergillus minimal medium (http://www.fgsc.net/methods/anidmed.html) with 50 mM glucose, 10% cell culture qualified fetal calf serum (Invitrogen), 100 mM glycerol, or 100 mM acetate as the carbon source. Additionally, germination was monitored in Aspergillus minimal medium with 50 mM acetate and various concentrations of peptone (Applichem GmbH, Darmstadt, Germany) ranging from 0.1 to 1%. The peptone was free of glucose as determined by a glucose oxidase-peroxidase-ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid]-based assay as described previously (4). The media were inoculated with 50,000 conidia and incubated at 37°C for the times indicated (see Fig. 4). Bright-field microscopy was carried out with a Leica DM4500 B fluorescence microscope which was equipped with a Leica DFC 480 camera. For determination of isocitrate lyase activity from peptide- and lipid-containing media, 1% peptone, 0.5% olive oil (Fluka, Steinheim, Germany), or a mixture of both was used. Replicates of 100 ml of medium in 250-ml flasks were inoculated with a final concentration of 1 x 106 conidia/ml and were incubated for at least 20 h.


Figure 4
View larger version (96K):
[in this window]
[in a new window]

  		FIG. 4. Growth analysis of the wild type and an isocitrate lyase deletion mutant on different carbon sources. (A) Representative sections from chamber slide wells are shown. Carbon sources and the times of incubation are indicated on the left. (B) Growth analysis of the wild type and a deletion strain in the presence of 50 mM acetate (A 50) and different concentrations of peptone (P). The growth time was fixed to 7.5 h. (C) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses of the wild type grown on 1% (wt/vol) peptone (lanes 1), 1% (wt/vol) peptone plus 0.5% (wt/vol) olive oil (lanes 2), and 0.5% (wt/vol) olive oil (lanes 3). Lanes C, purified isocitrate lyase taken as a control. The values (mU/mg) mark the isocitrate lyase activities determined from the cell extracts.

Generation of deletion mutants and complemented strains. For deletion of the isocitrate lyase coding region, a 908-bp upstream fragment was amplified from genomic DNA of A. fumigatus ATCC 46645 using the oligonucleotides BglAFIclup (5'-AGA TCT GGT GTT TGA GAA CCG ATC-3'; the BglII restriction site is underlined) and DelSalupIclAf (5'-GAT CCT CTG TCG ACC AGG CCT TGA CGG CTT GG-3'; the SalI restriction site is underlined, and the complementary sequence to the downstream fragment is in italics). Additionally, a 782-bp downstream fragment was amplified using the oligonucleotides BglAFIcldown (5'-AGA TCT CTT CCC TTC ATC CAT GG-3'; the BglII restriction site is underlined) and DelSalDoIclAf (5'-GCC TGG TCG ACA GAG GAT CAG TTC AAG C-3'; the SalI restriction site is underlined, and the complementary sequence to the upstream fragment is in italics). After gel purification, aliquots of both fragments were mixed, denatured, annealed, and elongated by Taq polymerase. The oligonucleotides BglAFIclup and BglAFIcldown were added, and the fusion of the up- and downstream fragments was amplified. The resulting PCR product was cloned into the PCR 2.1 vector (Invitrogen GmbH, Karlsruhe, Germany) and restricted with SalI. Sticky ends were filled with a Klenow fragment as described by the manufacturer (New England Biolabs GmbH, Frankfurt/Main, Germany). A 3-kb fragment, containing the hygromycin B resistance cassette under control of the gpdA promoter from Aspergillus nidulans, was restricted with EcoRV and SmaI from plasmid pUChph (14a), and the blunt ends were ligated to the Klenow-treated vector containing the up- and downstream fragments. The deletion fragment containing the hygromycin B resistance cassette flanked by the up- and downstream regions was cut from independent plasmids by BglII restriction, gel purified, and used for transformation of the A. fumigatus {Delta}akuB strain. Transformation of protoplasts was performed by standard procedures (11). Transformants were preanalyzed based on their inability to grow on ethanol as the sole carbon source and further checked by Southern analysis (22). Genomic DNA was isolated by use of the MasterPure yeast DNA purification kit (Epicenter Biotechnologies, Wisconsin) and restricted with EcoRI. A digoxigenin-labeled probe on the upstream region was amplified from the plasmid carrying the deletion construct with the oligonucleotides BglAFIclup and AfPICLATG_Bam (5'-GGA TCC CAT TGT GAC AGG TAT G-3') and used in hybridization. Hybridized DNA fragments were visualized by CDP-Star as recommended by the manufacturer (Roche Diagnostics).

For complementation of the {Delta}acuD Af8B1 and {Delta}acuD Af12C1 independent deletion strains, a PCR fragment was amplified from genomic DNA of the {Delta}akuB strain using the oligonucleotides AfPICL_upst_Bam (5'-GGA TCC GAA GGA CAG GAA C-3') and BglAFIcldown and directly used for transformation of protoplasts. The resulting transformants were selected by plating the protoplasts on media containing ethanol as the sole carbon source. As described above, genomic DNA was isolated, restricted with EcoRI, and analyzed by Southern blotting with the probe against the isocitrate lyase upstream region.

Enzyme activity and Western blot analysis. Isocitrate lyase activity was monitored by a phenylhydrazine-based assay, as described earlier (9), using a millimolar extinction coefficient for glyoxylate-phenylhydrazone of 16.8 mM–1 cm–1. The cultures were grown in duplicate, and the enzyme activities were determined with two separate amounts of enzyme from each extract. All activities were in a range of ±5% from the mean values, which are presented in Results. Western blot analysis of crude cell extracts from different A. fumigatus strains was performed by running 12.5 µg total protein of each strain on a precast NuPAGE 4 to 12% Bis-Tris gel (Invitrogen) and subsequent blotting on a polyvinylidene difluoride membrane (Millipore, Schwalbach, Germany). A specific anti-isocitrate lyase antibody, E30-F8, was used and, as a secondary antibody, an alkaline phosphatase-conjugated anti-mouse immunoglobulin G from a rabbit was employed. Staining was done with a mixture of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate, as described by the manufacturer (Pierce, Bonn, Germany).

Animal model and histopathology. For investigation of the virulence of the isocitrate lyase mutant, mouse survival experiments were carried out almost exactly as described earlier (12). In brief, the virulence levels of the {Delta}akuB wild-type strain and the complemented strain were compared to that of a {Delta}acuD strain. Cohorts of 10 male Swiss OF1 mice (6 to 8 weeks old) were immunosuppressed by intraperitoneal injection of cortisone acetate (25 mg/mouse) 3 days prior to and on the day of infection. For intranasal infection, mice were anesthetized with 0.1 ml of a mixture of ketamine (10 µg/ml; Merial) and xylazine (2 µg/ml; Bayer) by intramuscular injection. A 20-µl suspension containing 1 x 105 conidia in phosphate-buffered saline (PBS)-0.1% Tween 80 was applied to the nares of each mouse, and survival was monitored for 14 days. For controls, 10 mice were immunosuppressed and infected with PBS without conidia. The lungs from the mice which died from infection on day 5 were removed in order to prepare tissue thin sections. Control mice, which did not receive conidia, were sacrificed, and the lungs were removed. After being stained with hematoxylin and eosin stain and with Gomori-Grocott methenamine silver nitrate (1, 24), tissue sections were analyzed by using bright-field microscopy at a 40-fold magnification. From these sections, the inflammation indices and fungal burdens were evaluated. The inflammatory and fungal invasion indices were determined by comparing the number of inflammatory foci with the number of foci of the whole lung. The index of inflammation was classified between 0 and 5, with 5 being the classification for the more-severe lesions (1, ≤20%; 2, 20 to 40%; 3, 40 to 60%; 4, 60 to 80%; 5, 100%). As done for the inflammatory index, the fungal invasion index was graduated between 1 and 5, with the following classification: 1, few hyphae around the bronchi; 2, several foci of hyphae limited to the periphery of the bronchi and blood vessels; 3, invasive aspergillosis in which the hyphae were observed to cross the vascular wall and extend to the alveolae; and 4 to 5, severe invasive aspergillosis with massive hyphal invasion resulting in the necrosis of the whole lung.


arrow
RESULTS
 
Deletion and reintroduction of the isocitrate lyase coding region (acuD) from the A. fumigatus {Delta}akuB strain. For deletion of the acuD gene from A. fumigatus, the {Delta}akuB strain was used; this strain contains a deletion in the KU80 gene, which is responsible for the nonhomologous end-joining repair mechanism. Deletion of the KU80 gene results in a strain that preferably integrates DNA homologously into the genome. Because virulence of the {Delta}akuB strain is not attenuated, it was used as a wild-type control strain in subsequent investigations (5).

The hygromycin B resistance cassette flanked by a 908-bp 5' upstream and a 782-bp 3' downstream fragment of the acuD gene was used for transformation of protoplasts of the {Delta}akuB strain. Transformation yielded several hundred colonies, and 36 transformants were tested for their ability to grow on ethanol or glucose when they were used as the sole carbon and energy sources. Neither of the transformant strains grew on ethanol, but all strains grew without a phenotype when glucose was used. Seven strains were selected for Southern analysis (Fig. 1A). All of these strains showed a deletion of the acuD gene. Two of these strains contained an additional copy of the deletion construct and were not used for further studies (Fig. 1B). Complementation of two independent isocitrate lyase deletion strains was performed by reintroducing the acuD gene as well as the promoter and the downstream region. Due to the {Delta}akuB background of the isocitrate lyase mutant, we expected a homologous integration into the original acuD locus to be accompanied by a loss of hygromycin B resistance. Therefore, transformants were selected by their ability to grow on ethanol when it was used as the sole carbon and energy source. Again, several hundred colonies were obtained. The possibility of contamination during the transformation by wild-type strains was excluded by the fact that the negative control, which contained water instead of DNA, yielded no colonies. Three strains from each transformation were selected for Southern analysis. All strains showed a single integration of the PCR fragment into the acuD locus (Fig. 1C). A comparison of the phenotypes for a selected isocitrate lyase mutant, corresponding complemented mutant strains, and the wild-type {Delta}akuB strain is shown in Fig. 1D. The isocitrate lyase deletion strains were unable to grow on ethanol, but these were the only strains which grew on glucose in the presence of hygromycin B. When glucose was used as the sole carbon source, no phenotypes were observed, and all complemented strains behaved exactly like the wild type behaved.


Figure 1
View larger version (60K):
[in this window]
[in a new window]

  		FIG. 1. Deletion and reintroduction of the isocitrate lyase gene. (A) Scheme of the genomic situation at the acuD locus in the wild type, a complemented deletion mutant, and a deletion mutant. Restriction with EcoRI results in a 7.96-kb fragment for both the wild-type and the complemented strains and a 4.24-kb fragment for the mutant when the probe is hybridized to the upstream region. (B) Southern blot analysis of genomic DNA of the wild-type strain and the independent acuD deletion mutants. The shift of the fragment is indicated by an arrow. (C) Southern blot analysis of the deletion strains complemented with the acuD gene. Bands characteristic of the acuD deletion strain and the wild type are indicated by arrows. (D) Growth analysis of the wild type, an isocitrate lyase mutant, and four complemented strains. WT, wild type; {Delta}, isocitrate lyase deletion strain; R, complemented strains.

Enzymatic determination of isocitrate lyase activity and Western blot analysis. Isocitrate lyase activity is hardly detectable when glucose is used as the sole carbon and energy source but strongly induced when cells are grown on acetate, ethanol, or fatty acids (9). Due to the inability of the isocitrate lyase deletion strain to grow on these carbon sources, a mixture of 50 mM glucose and 100 mM acetate was used. This medium composition leads to an intermediate production of isocitrate lyase, as shown earlier (9). Media were inoculated with 1 x 106 conidia/ml and incubated at 37°C under vigorous shaking for 22 h. The mycelium was harvested, pressed dry, ground to a fine powder, and suspended in a buffer for determination of isocitrate lyase activity. The protein content was determined, and equal amounts of protein from the crude extracts were separated on a polyacrylamide gel. One gel was stained with Coomassie blue, and proteins from a second gel were blotted on a polyvinylidene difluoride membrane. Detection of isocitrate lyase was achieved with the specific anti-isocitrate lyase antibody E30F8. For a control, purified isocitrate lyase from A. fumigatus was loaded onto the gel. Specific activities and results from the Western blot analysis of the wild type, acuD deletion strains, and complemented mutant strains are shown in Fig. 2. All the isocitrate lyase mutants, which showed a deletion of the acuD gene by Southern analysis, remained negative when stained specifically for isocitrate lyase. In addition, activity was below the detection limit (Fig. 2A). All complemented strains showed both a band for isocitrate lyase in Western blot analysis and enzyme activity, although the level of activity varied slightly between different complemented strains (Fig. 2B). The 12C1RC2 strain, which showed the same activity as the wild type, was taken as a complemented reference strain in the subsequent virulence tests.


Figure 2
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 2. Western blot analysis and determination of isocitrate lyase activity. (A) The upper panel shows a Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of cell extracts of isocitrate lyase deletion mutants and the wild type ({Delta}akuB WT) grown on a glucose-acetate medium. The lower panel shows the respective Western blot analysis performed by use of the specific anti-isocitrate lyase antibody E30F8. As a control, purified isocitrate lyase was loaded (Icl control). Numbers below the panels indicate the specific isocitrate lyase activities which were determined for each extract. (B) Same as for panel A, but in addition to the wild-type and deletion strains, several complemented strains were investigated.

Murine infection model. In order to test the possible attenuation in virulence of the isocitrate lyase mutant, a murine infection model of invasive aspergillosis was used. Mice were immunosuppressed with cortisone acetate. As a result of this treatment, the ability of alveolar macrophages and neutrophils to kill A. fumigatus conidia was strongly reduced, which enabled conidia to germinate and grow invasively throughout the lung tissue (7, 23).

Conidial suspensions from the fifth generation of the isocitrate lyase {Delta}acuD Af12C1 deletion mutant, the fourth generation of the complemented 12C1RC2 strain, and the {Delta}akuB wild-type strain were freshly prepared and applied using intranasal infection. Early generations of the mutant and the complemented strain were used for this analysis in order to avoid secondary mutations, which could occur in a {Delta}akuB background. Figure 3A shows the survival curves recorded over a period of 14 days. A comparison of the curves revealed that the isocitrate lyase mutant showed at least the same ability to kill the mice as the wild type or the complemented strain did. Additionally, 5 days after infection, the lung sections from mice infected with the wild type, the mutant, and the complemented strain revealed indices of inflammation and of fungal invasion of 3.5 to 4, with a hemorrhagic necrosis and strong fungal invasion of the lung tissues (Fig. 3B). Therefore, we conclude that a deletion of isocitrate lyase from A. fumigatus does not reduce its ability to manifest as invasive aspergillosis.


Figure 3
View larger version (76K):
[in this window]
[in a new window]

  		FIG. 3. Animal model for invasive aspergillosis. (A) Survival curves of cortisone acetate-treated Swiss OF1 mice (in cohorts of 10 mice each) infected with 1 x 105 conidia from the wild type (WT), with the {Delta}acuD 12C1 deletion strain, with the complemented 12C1RC2 strain, and with PBS as a control. (B) Histopathology of lung tissue sections 5 days after infection. All lung sections shown in panel B derive from the animal experiment described for panel A.

Analysis of germination on different carbon sources. The ability of conidia from the wild type and the isocitrate lyase mutant to germinate in the presence of different carbon sources was investigated. As carbon sources, a complete medium (Sabouraud) and minimal media with different supplementations (fetal calf serum, glucose, glycerol, and acetate) were studied. Additionally, 50 mM acetate was taken as the main carbon source but supplemented with different concentrations of peptone (in the range of 0.1 to 0.5%). Figure 4A shows that growth and germination on Sabouraud, fetal calf serum, and glucose were very homogenous for both strains. On glycerol, germination was not synchronized, which means that some conidia were swollen, whereas others had already germinated, but this result was the same for both the wild type and the mutant strain. The use of acetate allowed germination and growth of the wild type, whereas the conidia from the mutant stayed in a resting state, confirming the essential role of isocitrate lyase for growth on acetate. However, when the acetate medium was supplemented with peptone (Fig. 4B), a concentration of 0.1% (wt/vol) was sufficient to restore the ability of the mutant to germinate, although the germination was somewhat delayed in comparison to that of the wild type. By increasing the amount of peptone, the phenotype of the mutant was even less pronounced. This indicates that in the cometabolism of acetate and proteins, the isocitrate lyase is of minor importance.

Large amounts of acetate, as used in this experiment, may be easily taken up by the mycelium and may, therefore, serve as the major carbon source. This assumption could explain the slight growth inhibition which is visible even in the presence of peptone. However, although the presence of large amounts of acetate in infected lung tissues is unlikely to occur, it is likely that a mixture of proteins and lipids or fatty acids is present. Therefore, we were interested in the isocitrate lyase activity of the wild-type strain when grown on peptone with or without the addition of olive oil as a source of lipids and fatty acids. Western blot analysis and specific isocitrate lyase activities are shown in Fig. 4C. However, the basal activity with peptone used as the carbon source, at 12 mU/mg, was higher than that with glucose (<1 mU/mg) (9). Activity increased only slightly in the presence of lipids (34 mU/mg). In the absence of peptone, the isocitrate lyase activity was high (223 mU/mg), indicating that isocitrate lyase is important for the metabolism of lipids when applied as the sole carbon source. This finding was confirmed by growing the isocitrate lyase mutant on peptone, peptone-olive oil, and olive oil as the sole carbon source. The deletion strain did not grow when olive oil was used as the sole carbon and energy source (not even after 144 h), whereas no phenotypes were observed on peptone alone or on the mixture of peptone and olive oil. These results show that the presence of both peptone and olive oil does not necessarily require isocitrate lyase, because peptone seems to be sufficient for replenishing the oxaloacetate pool.


arrow
DISCUSSION
 
Our results clearly demonstrate that isocitrate lyase is essential for growth only when C2-generating carbon sources are present. This confirms a previous study in which specific inhibition of isocitrate lyase by 3-nitropropionate inhibited germination on an acetate medium (9). However, during cometabolism with other carbon sources, which do not require the glyoxylate bypass, isocitrate lyase seems to play only a minor role.

Most interestingly, we were able to show that isocitrate lyase is not important for the development of invasive aspergillosis in a murine infection model. This result was rather unexpected, because it was generally believed that lipids and fatty acids serve as one of the major carbon sources during the infection process and that the glyoxylate bypass is needed to avoid a shortage of oxaloacetate. This assumption was supported by the results obtained with isocitrate lyase mutants of various microorganisms, including both human and plant pathogens. Deletion of the two isocitrate lyases present in M. tuberculosis, which can substitute for each other, led to the rapid elimination of bacteria from the lungs and to the impairment of intracellular replication (19). Deletion of the single isocitrate lyase from C. albicans led to a strong attenuation in virulence in a murine infection model (15, 16), and furthermore, the plant-pathogenic fungi Leptosphaeria maculans (13) and Magnaporthe grisea (28) displayed significantly reduced virulence levels when the isocitrate lyase gene was deleted.

In our study, we used a corticosteroid-based immunosuppression model in order to test the A. fumigatus isocitrate lyase mutant for its virulence. Although this system does not cause neutropenia, it is suitable as a model for invasive aspergillosis. In our cortisone acetate immunosuppression system, mice infected with wild-type, isocitrate lyase mutant, or isocitrate lyase-complemented strains died from invasive aspergillosis. Although a severe inflammatory response accompanied by a massive recruitment of neutrophils to the lung tissue occurs under cortisone acetate immunosuppression, a dramatic decrease in the production of tumor necrosis factor alpha (TNF-{alpha}) has been observed (8). TNF-{alpha} is supposedly essential for the induction of protective immunity against A. fumigatus (18). However, in a neutropenic model, although large amounts of TNF-{alpha} have been found in the bronchoalveolar lavage fluids of infected mice, this cytokine was not efficient to prevent the development of invasive aspergillosis (1). Therefore, in both corticosteroid-based and neutropenic immunsuppression models, extensive pulmonary invasion by growing hyphae occurs, and this results in the death of mice, caused by an acute cardiorespiratory insufficiency. Despite the differences in immune response, both models seem to be suitable to test the attenuation in virulence of Aspergillus mutants.

Due to the strong hyphal growth of our isocitrate lyase mutant, which was comparable to that of the wild type and the complemented mutant, we conclude that isocitrate lyase is not required for invasive growth of A. fumigatus. In agreement with this finding is the finding that isocitrate lyase from another pathogenic fungus, Cryptococcus neoformans, was shown not to be required for pathogenesis (21). In C. neoformans, isocitrate lyase was first thought to represent a putative target for antifungal drugs, because the gene was found to be up-regulated when reisolated from a rabbit infection model 7 days postinfection. This was explained by the fact that immune effector cells, like macrophages, at that time acquire their maximum activation state for fungicidal activity, which leads to phagocytosis of yeast cells and subsequent activation of isocitrate lyase because of lipids which are present in macrophages. Disruption of the isocitrate lyase coding region, however, revealed that this had no effect on virulence when the mutant strain was tested in a murine inhalation model for cryptococcosis.

Therefore, we can conclude that, although lipid-specific Nile red staining of the macrophages confirmed the presence of large lipid loads (9), isocitrate lyase is also not required for development of invasive aspergillosis. This could be explained by the fact that the macrophages may contain not only lipids but also proteins or carbohydrates. Furthermore, germinating conidia grow for only a short time within the macrophages, because the elongating hyphae destroy them (27). This makes available alternative carbon sources like proteins, which are released from the surrounding tissue during invasive growth, and leads to the assumption that isocitrate lyase may, in the macrophages, be required only for long-term persistence. This is also supported by investigations of the pathogenic bacterium Salmonella enterica serovar Typhimurium (10). An isocitrate lyase mutant of this bacterium shows no reduction in acute virulence. However, when long-term persistence in the macrophages was examined, the wild-type strain was able to persist in the macrophages, whereas the isocitrate lyase mutant was progressively cleared. Whether the persistence of A. fumigatus conidia also plays a role in the infection of humans has not yet been investigated. It therefore remains unclear whether all cases of invasive aspergillosis derive from a "de novo infection" with conidia taken up from the environment or from persisting conidia which start to germinate when the immune system becomes suppressed.

We showed that during cometabolism of proteins and lipids, isocitrate lyase is hardly induced and, therefore, may not be required for anaplerosis of oxaloacetate. Additionally, immunostaining of isocitrate lyase in the tissues of patients suffering from an invasive fungal infection revealed only negative results (Frank Ebel, Max von Pettenkofer Institute, Munich, Germany, personal communication). Theoretically, the possibility that the antibodies were unable to detect isocitrate lyase in the tissue sections after they were fixed and embedded in paraffin cannot be excluded. However, the fact that the antibodies worked well on infected and formaldehyde-fixed macrophages, and that they clearly showed the presence of isocitrate lyase (9), seems to exclude this possibility.

We conclude that isocitrate lyase, and hence the glyoxylate cycle, is not required for the virulence of A. fumigatus in the murine infection model. Therefore, from our results, isocitrate lyase does not represent an antifungal drug target. Furthermore, results show that even among different fungal species infecting human tissues, diverse metabolic pathways are required. Although lipids may be consumed during invasive growth of A. fumigatus, they do not provide the major carbon source. Hence, it seems very likely that proteins released from the host tissue represent at least one additional carbon source supporting the growth of A. fumigatus during invasive aspergillosis. This hypothesis is currently being tested.


arrow
ACKNOWLEDGMENTS
 
We thank Frank Ebel (Max von Pettenkofer Institut, Munich, Germany) for providing information on immunofluorescence data of infected tissues and helpful discussions on the manuscript. Additionally, we thank Michel Huerre for analyzing the tissue sections.

This work was supported by grant BR 2216/1-4 from the Deutsche Forschungsgemeinschaft to M.B. and by grants from the Aspergillus Unit of the Institute Pasteur and from the Programme Transversal de Recherche: Aspergillus fumigatus and the Alveolar Macrophage to O.I.-G.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Leibniz Institute for Natural Products Research and Infection Biology, Beutenbergstr. 11a, 07745 Jena, Germany. Phone: 49 (0)3641 656865. Fax: 49 (0)3641 656825. E-mail: Matthias.brock{at}hki-jena.de. Back

{triangledown} Published ahead of print on 18 December 2006. Back

Editor: A. Casadevall


arrow
REFERENCES
 
    1
  1. Balloy, V., M. Huerre, J.-P. Latgé, and M. Chignard. 2005. Differences in patterns of infection and inflammation for corticosteroid treatment and chemotherapy in experimental invasive pulmonary aspergillosis. Infect. Immun. 73:494-503.[Abstract/Free Full Text]
    2. 2
  2. Bishai, W. 2000. Lipid lunch for persistent pathogen. Nature 406:683-685.[CrossRef][Medline]
    3. 3
  3. Brakhage, A. A. 2005. Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Curr. Drug Targets 6:875-886.[CrossRef][Medline]
    4. 4
  4. Brock, M., and W. Buckel. 2004. On the mechanism of action of the antifungal agent propionate. Eur. J. Biochem. 271:3227-3241.[Medline]
    5. 5
  5. da Silva Ferreira, M. E., M. R. V. Z. Kress, M. Savoldi, M. H. S. Goldman, A. Härtl, T. Heinekamp, A. A. Brakhage, and G. H. Goldman. 2006. The akuBKU80 mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5:207-211.[Abstract/Free Full Text]
    6. 6
  6. Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781-805.[Medline]
    7. 7
  7. Dixon, D. M., A. Polak, and T. J. Walsh. 1989. Fungus dose-dependent primary pulmonary aspergillosis in immunosuppressed mice. Infect. Immun. 57:1452-1456.[Abstract/Free Full Text]
    8. 8
  8. Dubourdeau, M., R. Athman, V. Balloy, M. Huerre, M. Chignard, D. J. Philpott, J. P. Latgé, and O. Ibrahim-Granet. 2006. Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4. J. Immunol. 177:3994-4001.[Abstract/Free Full Text]
    9. 9
  9. Ebel, F., M. Schwienbacher, J. Beyer, J. Heesemann, A. A. Brakhage, and M. Brock. 2006. Analysis of the regulation, expression, and localisation of the isocitrate lyase from Aspergillus fumigatus, a potential target for antifungal drug development. Fungal Genet. Biol. 43:476-489.[CrossRef][Medline]
    10. 10
  10. Fang, F. C., S. J. Libby, M. E. Castor, and A. M. Fung. 2005. Isocitrate lyase (AceA) is required for salmonella persistence but not for acute lethal infection in mice. Infect. Immun. 73:2547-2549.[Abstract/Free Full Text]
    11. 11
  11. Fincham, J. R. S. 1989. Transformation in fungi. Microbiol. Rev. 53:148-170.[Abstract/Free Full Text]
    12. 12
  12. Ibrahim-Granet, O., B. Philippe, H. Boleti, E. Boisvieux-Ulrich, D. Grenet, M. Stern, and J. P. Latgé. 2003. Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages. Infect. Immun. 71:891-903.[Abstract/Free Full Text]
    13. 13
  13. Idnurm, A., and B. J. Howlett. 2002. Isocitrate lyase is essential for pathogenicity of the fungus Leptosphaeria maculans to canola (Brassica napus). Eukaryot. Cell 1:719-724.[Abstract/Free Full Text]
    14. 14
  14. Kim, H. Y., K. S. Song, J. M. Goo, J. S. Lee, K. S. Lee, and T. H. Lim. 2001. Thoracic sequelae and complications of tuberculosis. Radiographics 21:839-858.[Abstract/Free Full Text]
    15. 14a
  15. Liebmann, B., M. Müller, A. Braun, and A. A. Brakhage. 2004. The cyclic AMP-dependent protein kinase. A network regulates development and virulence in Aspergillus fumigatus. Infect. Immun. 72:5193-5203.[Abstract/Free Full Text]
    16. 15
  16. Lorenz, M. C., and G. R. Fink. 2001. The glyoxylate cycle is required for fungal virulence. Nature 412:83-86.[CrossRef][Medline]
    17. 16
  17. Lorenz, M. C., and G. R. Fink. 2002. Life and death in a macrophage: role of the glyoxylate cycle in virulence. Eukaryot. Cell 1:657-662.[Free Full Text]
    18. 17
  18. McKinney, J. D., K. Honer zu Bentrup, E. J. Munoz-Elias, A. Miczak, B. Chen, W. T. Chan, D. Swenson, J. C. Sacchettini, W. R. Jacobs, Jr., and D. G. Russell. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735-738.[CrossRef][Medline]
    19. 18
  19. Mehrad, B., R. M. Strieter, and T. J. Standiford. 1999. Role of TNF-alpha in pulmonary host defense in murine invasive aspergillosis. J. Immunol. 162:1633-1640.[Abstract/Free Full Text]
    20. 19
  20. Munoz-Elias, E. J., and J. D. McKinney. 2005. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11:638-644.[CrossRef][Medline]
    21. 20
  21. Oren, I., and N. Goldstein. 2002. Invasive pulmonary aspergillosis. Curr. Opin. Pulm. Med. 8:195-200.[CrossRef][Medline]
    22. 21
  22. Rude, T. H., D. L. Toffaletti, G. M. Cox, and J. R. Perfect. 2002. Relationship of the glyoxylate pathway to the pathogenesis of Cryptococcus neoformans. Infect. Immun. 70:5684-5694.[Abstract/Free Full Text]
    23. 22
  23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    24. 23
  24. Schaffner, A. 2002. Host-parasite relation in invasive aspergillosis. Nippon Ishinkin Gakkai Zasshi 43:161.[Medline]
    25. 24
  25. Sinha, B. K., D. P. Monga, and S. Prasad. 1988. A combination of Gomori-Grocott methenamine silver nitrate and hematoxylene and eosin staining technique for the demonstration of Candida albicans in tissue. Quad. Sclavo Diagn. 24:129-132.[Medline]
    26. 25
  26. Subira, M., R. Martino, T. Franquet, C. Puzo, A. Altes, A. Sureda, S. Brunet, and J. Sierra. 2002. Invasive pulmonary aspergillosis in patients with hematologic malignancies: survival and prognostic factors. Haematologica 87:528-534.[Medline]
    27. 26
  27. Tillie-Leblond, I., and A. B. Tonnel. 2005. Allergic bronchopulmonary aspergillosis. Allergy 60:1004-1013.[CrossRef][Medline]
    28. 27
  28. Waldorf, A. R. 1989. Pulmonary defense mechanisms against opportunistic fungal pathogens. Immunol. Ser. 47:243-271.[Medline]
    29. 28
  29. Wang, Z. Y., C. R. Thornton, M. J. Kershaw, L. Debao, and N. J. Talbot. 2003. The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Mol. Microbiol. 47:1601-1612.[CrossRef][Medline]

Infection and Immunity, March 2007, p. 1237-1244, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01416-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zaugg, C., Monod, M., Weber, J., Harshman, K., Pradervand, S., Thomas, J., Bueno, M., Giddey, K., Staib, P. (2009). Gene Expression Profiling in the Human Pathogenic Dermatophyte Trichophyton rubrum during Growth on Proteins. Eukaryot Cell 8: 241-250 [Abstract] [Full Text]  
  • Brock, M., Jouvion, G., Droin-Bergere, S., Dussurget, O., Nicola, M.-A., Ibrahim-Granet, O. (2008). Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis. Appl. Environ. Microbiol. 74: 7023-7035 [Abstract] [Full Text]  
  • Hynes, M. J., Murray, S. L., Khew, G. S., Davis, M. A. (2008). Genetic Analysis of the Role of Peroxisomes in the Utilization of Acetate and Fatty Acids in Aspergillus nidulans. Genetics 178: 1355-1369 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schöbel, F.
Right arrow Articles by Brock, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schöbel, F.
Right arrow Articles by Brock, M.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»