J. Microbiol. Biotechnol. (2013), 23(6), 766–770 http://dx.doi.org/10.4014/jmb.1301.01002 First published online April 14, 2013 pISSN 1017-7825 eISSN 1738-8872
The Developmental Regulators, FlbB and FlbE, are Involved in the Virulence of Aspergillus fumigatus
Kim, Sung-Su1, Young Hwan Kim2,3, and Kwang-Soo Shin4*
1Department of Biomedical Laboratory Science, Daejeon University, Daejeon 300-716, Korea
2Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang 363-883, Korea
3Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Korea
4Department of Microbiology and Biotechnology, Daejeon University, Daejeon 300-716, Korea Received: January 2, 2013 / Revised: January 28, 2013 / Accepted: February 22, 2013
Several upstream activators required for proper activation of brlA are involved in the development, vegetative growth, toxin production, and pathogenesis of Aspergillus fumigatus.
In this study, we characterized the roles of two upstream developmental regulators, A. fumigatus flbB (AfuflbB) and flbE (AfuflbE), in toxin production and virulence. The deletion of AfuflbB and AfuflbE resulted in reduction of the expression of AfulaeA. Moreover, only about 8% to 10%
of fumagillin was produced in the two mutants compared with that of wild type, and ∆AfuflbB strain produced 85%
of gliotoxin compared with wild type, whereas none was produced by ∆AfuflbE. Flow-cytometric analysis revealed decreased necrotic and apoptotic polymorphonuclear leukocytes cell death after exposure to supernatants from
∆AfuflbB and ∆AfuflbE strains compared with the wild type.
These results indicate that FlbB and FlbE are necessary for the proper laeA expression, toxin production, and virulence of A. fumigatus.
Key words: Aspergillus fumigatus, FlbB, FlbE, LaeA, PMN death
Aspergillus fumigatus is the most important species causing infective lung diseases in humans and animals [8, 12]. This species has been reported to produce a large number of extrolites, including economically valuable secondary metabolites and toxins [6, 14]. In the present paper, we tried to elucidate the function of FlbB and FlbE in the production of secondary metabolites and virulence.
Several upstream activators (FluG, FlbA, FlbB, FlbC, FlbD, and FlbE) required for proper activation of brlA in A. nidulans have illuminated genetic regulatory cascades
leading to the activation of asexual development (conidiation) [1, 15, 18]. Among these, FlbB, FlbC, FlbD, and FlbE have been defined by the fluffy delayed-conidiation mutants [18]. FlbB, FlbC, and FlbD are putative transcription factors (TFs), containing a basic leucine zipper domain (bZIP), two C2H2 zinc fingers, and a cMyb-DNA binding domain, respectively, and have been shown to be associated with the activation of brlA expression [5, 7, 10, 17]. Recent studies demonstrated that bZIP TF FlbB is necessary for the expression of flbD, and that FlbB and FlbD activate brlA in a supportive manner [7]. A. fumigatus flbB (AfuflbB) is vital for A. fumigatus morphological development and gliotoxin production. Unlike flbB in A. nidulans, AfuflbB produces two transcripts predicted to encode two bZIP polypeptides, AfuFlbBα (420 aa) and AfuFlbBβ (390 aa).
A series of complementation studies indicate that both AfuFlbB polypeptides are necessary for proper asexual and chemical development within A. fumigatus [19]. Our recent studies have demonstrated that A. fumigatus FlbE (AfuFlbE) is crucial for proper conidiation and is functionally conserved in two aspergilli [11]. The predicted FlbE proteins are composed of 222 and 201 aa in A. fumigatus and A. nidulans, respectively. Whereas flbE is transiently expressed during the early phase of growth in A. nidulans, it is somewhat constitutively expressed during the life cycle of A. fumigatus. The deletion of flbE causes reduced conidiation and delayed expression of brlA and vosA in both species. Moreover, FlbE is necessary for salt-induced development in liquid submerged cultures of A. fumigatus.
The A. nidulans flbE null mutation is fully complemented by A. fumigatus flbE, indicating functional conservation of FlbE in Aspergillus. Both the deletion and overexpression of flbE in A. nidulans result in developmental defects, enhanced autolysis, precocious cell death, and delayed expression of brlA/vosA, suggesting that the balanced activity of FlbE is crucial for proper growth and development. In this study,
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we characterized the roles of these two developmental regulators, FlbB and FlbE, in the production of toxins and virulence in A. fumigatus.
The Aspergillus strains used in this study were AF293 (wild- type A. fumigatus), TKSS1.01 (AfuflbB::AnipyrG+; pyrG1), and TKSS6.07(AfuflbE::AnipyrG+; pyrG1). Isolation of genomic DNA and total RNA was carried out as described previously [21]. Five micrograms (5 µg) of total RNA was reverse-transcribed using RNA to cDNA EcoDry Premix (Clontech, USA). Quantitative PCR assays were performed according to the manufacturer’s instruction (Qiagen, USA) using 96-well optical plates and a Rotor-Gene Q (Qiagen, USA). Each run was assayed in triplicates in a total volume of 20 µl containing the DNA template, 2× qPCR SYBR green Mix (Doctor Protein, Korea), and 100 mM of each primer. The primers used for laeA were 5'-TGAAAC GATTCGAAATCTGC-3' (forward) and 5'-AGAGGG AGTCCAACCATTTG-3' (reverse), and for EF1α were 5'- CCATGTGTGTCGAGTCCTTC-3' (forward) and 5'-GAA CGTACAGCAACAGTCTGG-3' (reverse). PCR conditions were 95oC/15 min for one cycle, followed by 95oC/30 s and 55oC/30 s for 40 cycles. Amplification of one single specific target DNA was checked through melting curve analysis (+0.5oC ramping for 10 s, from 55oC to 95oC).
Fig. 1. Expression of the AfulaeA gene during liquid submerged culture.
Gene expression during growth was determined by quantitative real-time PCR. Cultures were incubated in liquid MMG + 0.1% YE and the expression ratios were normalized using the EF1α gene, according to the
∆∆Ct method. Data are the mean ± standard deviation from three independent experiments. Student’s t-test: *p < 0.005; **p < 0.001.
Fig. 2. LCMS-IT-TOF analysis of the culture filtrates from the wild-type, ∆AfuflbB, and ∆AfuflbE strains.
(A) HPLC chromatogram (upper) of the culture filtrates from the wild-type and mutants strains, indicating fumagillin presence with Rt = 12.58; and MS spectrum (lower), indicating fumagillin in the presence of molecular ion with mass of 459.2379 m/z (M+H+). (B) HPLC chromatogram (upper) of the culture filtrates from wild-type and mutants strains, indicating gliotoxin presence with Rt = 7.62; and MS spectrum (lower), indicating gliotoxin in the presence of molecular ion with mass of 327.0466 m/z (M+H+).
LB AND LB ARE NVOLVEDIN FUMIGATUS IRULENCE
The expression ratios were normalized to EF1α expression, and were calculated according to the ∆∆Ct method [13].
To verify the absence of genomic DNA contamination, negative controls, in which reverse transcriptase was omitted, were used for each gene set. Three independent biological replicates were performed. The culture filtrates of each strain were examined for fumagillin and gliotoxin by analyzing them using LCMS-IT-TOF (Shimadzu, Japan).
The monitoring wavelengths of fumagillin and gliotoxin were 351 and 254 nm, respectively. Toxins were identified by their retention time and mass spectra, which should match both the relative intensities of their ions and their respective accurate masses. Polymorphonuclear leukocytes (PMN) were purified from whole blood of healthy adult human donors by Ficoll gradient centrifugation, by following the leukocyte separation protocol (Sigma, USA).
Red blood cells were then lysed in hypotonic solution, and PMNs were separated by centrifugation (800 ×g, 10 min) and were resuspended in RPMI 1640-HEPES. Lyophilized fungal supernatants were resuspended in 4 ml of phosphate- buffered saline (concentrated SN) and were frozen at -70oC for later use. PMNs (2− 106 cells) were exposed to 200µl of SN (test conditions), medium (live controls), or Triton
X-100 (dead controls) for 60 min. Cells were then centrifuged (1,200 rpm, 10 min) and were resuspended in 100µl of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2; pH 7.4) containing either Annexin V (5µl;
BD Biochemicals, USA), propidium iodide (PI; 10µl; Sigma, USA), or both Annexin V and PI. Cells were incubated for 15 min in the dark, and 400µl of 1× binding buffer was added. Four separate experiments were conducted. PI and Annexin V positivity values were measured by a FACS Calibur flow cytometer (BD Biosciences, USA).
LaeA is nuclear protein that acts as a regulator of secondary metabolism in the aspergilli, and the loss of laeA in A. fumigatus results in reduced virulence in a murine model [2-4]. Thus, we assessed the expression of laeA in WT, ∆AfuflbB, and ∆AfuflbE strains grown in liquid media.
As shown in Fig. 1, the deletion of flbB and flbE caused delay in the accumulation of laeA mRNA, and exhibited a low level of laeA expression compared with WT, except
∆AfuflbE strain, at 72 h. Based on these results, we attempted to determine the contents of representative toxins of A.
fumigatus; that is, fumagillin and gliotoxin. LCMS-IT-TOF analysis of the culture filtrates confirmed the presence of the molecular ions with masses of 459.2379 (Fig. 2A) and
Fig. 3. PMN killing as determined by FACS analysis.
(A) Representative FACS data summarizing the FACS results. (B) PMN PI positivity after exposure to concentrated culture supernatants from wild-type,
∆AfuflbB, and ∆AfuflbE strains. (C) PMN apoptosis after exposure to concentrated culture supernatants from wild-type, ∆AfuflbB, and ∆AfuflbE strains.
Percentages of total cells that were Annexin V positive are shown. Data are the mean ± standard deviation from three independent experiments. Student’s t- test: *p < 0.005; **p < 0.001.
327.0466 m/z (M+H+) (Fig. 2B) as fumagillin and gliotoxin, respectively. Fumagillin was eluted at about 12.58 min, and the quantities produced by the two mutants strains were drastically reduced (by about 90%) compared with that of the WT (Fig. 2A). Compared with the WT, about 85% of gliotoxin was produced by the ∆AfuflbB strain but not produced by the ∆AfuflbE strain (Fig. 2B). It has been demonstrated that supernatants (SN) from ∆laeA were less cytotoxic to PMNs than SN from WT [2]. In order to test the hypothesis that LaeA mediates the perceived differences in PMN cytotoxicity, we exposed PMNs to WT, ∆AfuflbB, and ∆AfuflbE strain SNs, and analyzed by flow cytometry (Fig. 3A). Quantification of propidium iodide (PI)-positive cells after 60 min incubations showed equivalent cell death after exposure to the SNs from strains ∆AfuflbB and
∆AfuflbE (Fig. 3B). Although there was a tendency for decreased cell death from the ∆AfuflbB and ∆AfuflbE strain SNs, this decrease was not found to be significant at p < 0.005. As a more sensitive indicator of cellular apoptosis, Annexin V uptake was measured after exposure to the different supernatants. Results of four different experiments from four different donors demonstrated that PMN apoptosis was significantly decreased after exposure to SNs from the
∆AfuflbB and ∆AfuflbE strains compared with the WT (Fig. 3C). There was no statistical difference in PMN PI positivity after exposure to the SNs, although a trend was noted (Fig. 3B), as exposures to ∆AfuflbB and ∆AfuflbE SNs were associated with less PMN apoptosis compared with that of WT SN (Fig. 3C). This may be an important observation, given the fact that neutrophils constitute the primary cellular defense against invasive Aspergillus hyphae.
Many microorganisms, including bacteria and viruses, evade destruction by neutrophils by triggering neutrophil apoptosis, thereby resulting in chronic infections [9, 16, 20].
From these results, we conclude that FlbB and FlbE are necessary for the proper LaeA expression, toxin production, and virulence of A. fumigatus.
Acknowledgments
This work was supported by the Daejeon University Research Grant, as well as by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0007836).
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