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MODEL
Microbes and Infection xx (2014) 1e11 www.elsevier.com/locate/micinf
Original article
Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation Xiaodong Jia a,1, Fangyan Chen a,1, Weihua Pan b,1, Rentao Yu a,c, Shuguang Tian a, Gaige Han a, Haiqin Fang a, Shuo Wang a, Jingya Zhao a, Xianping Li a, Dongyu Zheng a, Sha Tao a, Wanqing Liao b, Xuelin Han a,*, Li Han a,* a b
Department for Hospital Infection Control & Research, Institute of Disease Control & Prevention of PLA, Academy of Military Medical Sciences, Beijing, China Shanghai Key Laboratory of Molecular Mycology, Department of Dermatology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China c Patent Examination Cooperation Center of the Patent Office, Beijing, China Received 10 September 2013; accepted 4 March 2014
Abstract The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement. Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
Keywords: Gliotoxin; Aspergillus fumigatus; Internalization; Phospholipase D
1. Introduction Aspergillus fumigatus is one of the major fungal pathogens which cause allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis [1]. Its airborne conidia are inhaled from the environment and then colonize within the * Corresponding authors. E-mail addresses: [email protected] (X. Han), [email protected], [email protected] (L. Han). 1 These authors contribute equally to this article.
lung alveoli. Like many intracellular bacterial pathogens [2], A. fumigatus conidia are able to bind to and internalize into lung type II alveolar epithelium cells. They can then survive within these normally non-phagocytic host cells. This brings the possibility of immune evasion as well as the possibility of dissemination [3e5]. To date, the mechanism associated with A. fumigatus internalization into type II lung epithelial cells remains unclear. Gliotoxin is one of the well-known members of the epipolythiodioxopiperazine class of metabolites produced by A. fumigatus. It is characterized by a disulfide bridge across a
http://dx.doi.org/10.1016/j.micinf.2014.03.001 1286-4579/Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Jia X, et al., Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.001
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piperazine ring which is essential for its toxicity [6]. It should be noted that gliotoxin possesses multiple immuno-suppressive activities [7]. In murine models of invasive aspergillosis (IA), gliotoxin has been shown to inhibit specifically the nuclear transcription factor NF-kB [8,9], which consequently induces host cell apoptosis [10,11] and suppresses the cytotoxic T-cell response [12,13]. Moreover, gliotoxin is also able to inhibit macrophage and polymorphonuclear cell function, including phagocytosis [14] and respiratory bursts [15,16]. It has been demonstrated that gliotoxin from A. fumigatus affects the process of phagocytosis and actin cytoskeleton rearrangement of human neutrophils through distinct signaling pathways, which involve cAMP and arachidonic acid signals, respectively [17]. Recently, an immune-active role of gliotoxin was reported that gliotoxin can activate the platelets and its antifungal function [18]. For epithelial cells, it has been shown that gliotoxin can reduce the ciliary movement of epithelial cells and alter membrane permeability leading to damage of the epithelial cells [14,19,20]. Gliotoxin may also reduce TGFb1, IL-6 and IL-8 levels in A549 lung epithelial cells at lower concentrations (below 50 ng/ml) [20]. However, much less is known about the effect of gliotoxin on A. fumigatus internalization into lung epithelial cells. It has been generally accepted that the internalization of A. fumigatus conidia into type II A549 lung epithelial cells is closely related to host actin dynamics including invagination of the membrane and formation of the pseudopods that engulf the conidia [3,5,21]. Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of the most abundant membrane phospholipids, phosphatidylcholine (PC) to produce phosphatidic acid（PA）and choline [22]. In mammalian cells PLD activity has been found to be closely related to actin dynamics [23e25]. In our previous study, it was demonstrated that the internalization of A. fumigatus conidia into epithelial cells was regulated by b-1,3-glucan-induced PLD activation in epithelial cells [26]. However, block of dectin-1, the receptor on lung epithelial cells for b-1,3-glucan, could not fully inhibit the internalization of conidia or conidia-induced PLD activation. This implied that some other factors may also be involved in the regulation of A. fumigatus internalization into lung epithelial cells. Therefore, we hypothesized that gliotoxin from A. fumigatus may be involved in regulating the conidial internalization into lung epithelial cells. In present study, we discovered that gliotoxin facilitates the internalization of A. fumigatus conidia by stimulating PLD activity in A549 cells. 2. Materials and methods 2.1. A. fumigatus strains, cell lines A. fumigatus wild type strain B5233, its mutant strain gliPD (deletion of the peptide synthetase gliP), and the gliP reconstituted strain gliPR were generously provided by Dr. KJ. Kwon-Chung (National Institute of Health, Bethesda, Maryland) [27]. Except where indicated, all of the A. fumigatus strains were propagated on Sabouraud dextrose agar slants
(10 g/l peptone, 10 g/l glucose, 15 g/l agar, pH6.0) for 5e8 days at 37 C. The type II human pneumocyte cell line A549 and murine macrophage cell line J774 were obtained from the ATCC and the 16HBE14o human bronchial epithelial cell line was purchased from shanghai Beinuo life science company. These three kinds of cells were cultured in DMEM supplemented with a 10% fetal calf serum (GIBCO, Germany), 100 mg/l streptomycin, 16 mg/l penicillin at 37 C in 5% CO2. The human monocytic leukemia cell line THP-1 was gift from Beijing Proteome Research Center and cultured in suspension cultures in RPMI-1640 (GIBCO, Germany) supplemented with 10% fetal calf serum (GIBCO, Germany), 100 mg/l streptomycin, 16 mg/l penicillin at 37 C in 5% CO2. THP1 cells were differentiated into macrophages by 100 ng/ml phorbol myristate acetate (PMA) for 60 h. Afterwards, the macrophages were cultured in DMEM/F12 1:1 medium (GIBCO, Germany) supplemented with 10% fetal calf serum (GIBCO, Germany), 100 mg/l streptomycin, 16 mg/l penicillin at 37 C in 5% CO2. 2.2. Preparation of the conidia A. fumigatus conidia were harvested and prepared in the same way as described in a previous study [28]. After 5e8 days of cultivation, A. fumigatus conidia were dislodged from agar plates with gentle washing. They were then re-suspended in sterile phosphate-buffered saline supplemented with 0.1% Tween 20 (PBST). The conidia were passed through 8 layers of sterile gauze in order to remove hyphal fragments. At this point, they were enumerated on a hemacytometer and the resting conidia were washed twice and stored at 4 C to be used within 48 h. For preparation of swollen conidia, the resting conidia were incubated at 37 C in DMEM for the indicated periods. The preparations which were incubated for 5 h contained swollen conidia and early germlings with 96%) of both environmental and clinical A. fumigatus has the ability to produce gliotoxin [31]. In the case of cystic fibrosis (CF), A. fumigatus colonization is associated with increased gliotoxin in the bronchoalveolar lavage fluid (BALF) [33]. On the other hand, it has been proven that gliotoxin is produced in experimental animal aspergillosis as well as in human IA, with serum concentrations of 166e785 ng/ml in 80% of patients with IA [30,34]. Thus, concerning that the internalization of A. fumigatus into the lung epithelium allows them to protect themselves from the phagocytosis by macrophages or neutrophils, it seemed physiologically feasible that a low amount of gliotoxin produced by A. fumigatus from mouldy environments or lung secretions may help A. fumigatus internalization and evade further immune attacks at the initial step of infection. Another critical finding in the present study was that gliotoxin is able to stimulate the PLD activity of A549 lung epithelial cells. In this way, it can promote the efficient internalization of A. fumigatus into the cells. This seemed to be complementary to another PLD signal pathway, b-1,3-glucandectin-1 cascade which was demonstrated in our previous report [26]. To some extent, this finding was surprising because gliotoxin is a well-known immunosuppressive agent in many immune cells including neutrophils, macrophages, and thymocytes [14,35]. It inhibits several important enzymes, such as NADPH oxidase of neutrophils [15,16], and the nuclear transcription factor NF-kB and 20S proteasome activity [35]. However, gliotoxin was recently shown to activate the platelets [18]. It was also reported that gliotoxin can increase the intracellular cAMP concentration [11], and induce Ca2þ release from the mitochondria of intact rat livers [36,37]. Both cAMP and Ca2þ are the putative agonists for PLD stimulation in several signaling events [38,39], which may also be involved
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in a gliotoxin-induced PLD activation pathway. Therefore, PLD may be a novel intracellular target of gliotoxin. For this reason, the detailed mechanism for PLD activation by gliotoxin in epithelial cells should be investigated further. Furthermore, the control of PLD activity on the gliotoxininduced A. fumigatus internalization was in line with the previous findings that PLD is a critical regulator in many phagocytosis or internalization processes in response to a large variety of agonists. The regulation of PLD on pathogen internalization mostly resulted from its close interaction with host actin dynamic induced by the pathogens. In mammalian cells, elements of the actin dynamic system interact directly or indirectly with PLD enzymes and its related phospholipid metabolism [23,29,40]. Inversely, the phosphatidic acid produced by PLD catalysis could induce stress fiber formation and deliberately regulate the formation of phagocytic cups [41]. In the present study, gliotoxin induced significant actin cytoskeleton rearrangement (Fig. 5) along with the promotion of A. fumigatus internalization. Therefore, the stimulated PLD activity by gliotoxin may interact with actin cytoskeleton rearrangement in order to control A. fumigatus internalization. Compared to PLD2, PLD1 played a more important role in gliotoxin-induced A. fumigatus internalization into epithelial cells. This discrepancy might be gliotoxin-specific because it has been previously reported that both PLD1 and PLD2 contribute coordinately to phagosome formation and development during the internalization of A. fumigatus into A549 lung epithelial cells [24,26]. Moreover, since the subcellular distribution of PLD1 was not significantly altered by gliotoxin in A549 cells (Fig. 5), the gliotoxin-induced PLD activation may not be associated with the subcellular translocation of PLD1. Therefore, it is interesting to analyze the precise function and distribution of PLD isotypes during gliotoxin-induced A. fumigatus internalization. Finally, gliotoxin significantly blocked the ROS production in A549 epithelial cells at low concentrations (from 10 to 100 ng/ ml). In contrast, in human neutrophils, the inhibition of ROS production by gliotoxin has been reported only at higher concentrations (above 1 mg/ml [15,16]) and no inhibition of NADPH-oxidase activity was observed at the low concentrations of gliotoxin (from 10 to 100 ng/ml) [17]. As NADPH-oxidase is one of major modulators responsing to oxidative stress in the cell, it can be deduced that NADPH oxidase complex of epithelial cells might be more sensitive to gliotoxin than that of J774 macrophages and neutrophils. In addition, as the epithelial cells constitute the first barrier against A. fumigatus, the low threshold value of gliotoxin concentration required for enough inhibition on ROS response in lung epithelial cells might facilitate the internalization and intracellular living of A. fumigatus. In addition, concerning that the nystatin protection assay is an indirect and error-prone approach, we carefully repeated the internalization experiments, even for several times in order to get the reliable and stable results. We had also certified the correlation of internalization results between nystatin protection assay and immunofluorescence assay (Fig. S3). Certainly, more accurate assay would be expected for further interpretation on the exact role of gliotoxin on A. fumigatus internalization into lung epithelium.
Please cite this article in press as: Jia X, et al., Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.001
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In conclusion, we reported an important finding that gliotoxin is able to regulate A. fumigatus internalization into epithelial cells by inducing host PLD activation. However, the detailed function and regulatory mechanism of gliotoxin on A. fumigatus entry into epithelial cells need to be further clarified. Conflict of interest The authors have no conflict of interest. Acknowledgments We would like to thank Dr. KJ. Kwon-Chung for kindly providing the various A. fumigatus strains. This project was supported by grants from 973 Program (No. 2013CB531606) and from the Chinese National Scientific Foundation Committee (No. 30772029 and No. 81172801). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.micinf.2014.03.001. References [1] Segal BH. Aspergillosis. N Engl J Med 2009;360:1870e84. [2] Bonazzi M, Vasudevan L, Mallet A, Sachse M, Sartori A, Prevost MC, et al. Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization. J Cell Biol 2011;195:525e36. [3] Osherov N. Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells. Front Microbiol 2012;3:346. [4] Li X, Gao M, Han X, Tao S, Zheng D, Cheng Y, et al. Disruption of the phospholipase D gene attenuates the virulence of Aspergillus fumigatus. Infect Immun 2012;80:429e40. [5] Wasylnka JA, Moore MM. Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein. Infect Immun 2002;70:3156e63. [6] Gardiner DM, Waring P, Howlett BJ. The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis. Microbiology 2005;151:1021e32. [7] Scharf DH, Heinekamp T, Remme N, Hortschansky P, Brakhage AA, Hertweck C. Biosynthesis and function of gliotoxin in Aspergillus fumigatus. Appl Microbiol Biotechnol 2012;93:467e72. [8] Rashmi R, Schnulle PM, Maddox AC, Armbrecht ES, Koenig JM. Flice inhibitory protein is associated with the survival of neonatal neutrophils. Pediatr Res 2011;70:327e31. [9] Spikes S, Xu R, Nguyen CK, Chamilos G, Kontoyiannis DP, Jacobson RH, et al. Gliotoxin production in Aspergillus fumigatus contributes to host-specific differences in virulence. J Infect Dis 2008;197:479e86. [10] Hagens WI, Beljaars L, Mann DA, Wright MC, Julien B, Lotersztajn S, et al. Cellular targeting of the apoptosis-inducing compound gliotoxin to fibrotic rat livers. J Pharmacol Exp Ther 2008;324:902e10. [11] Waring P, Khan T, Sjaarda A. Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion. J Biol Chem 1997;272:17929e36. [12] Stanzani M, Orciuolo E, Lewis R, Kontoyiannis DP, Martins SL, St John LS, et al. Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes. Blood 2005;105:2258e65.
[13] Wichmann G, Herbarth O, Lehmann I. The mycotoxins citrinin, gliotoxin, and patulin affect interferon-gamma rather than interleukin-4 production in human blood cells. Environ Toxicol 2002;17:211e8. [14] Peterson DE, Collier JM, Katterman ME, Turner RA, Riley MR. Cytotoxicity of bacterial-derived toxins to immortal lung epithelial and macrophage cells. Appl Biochem Biotechnol 2010;160:751e63. [15] Nishida S, Yoshida LS, Shimoyama T, Nunoi H, Kobayashi T, Tsunawaki S. Fungal metabolite gliotoxin targets flavocytochrome b558 in the activation of the human neutrophil NADPH oxidase. Infect Immun 2005;73:235e44. [16] Tsunawaki S, Yoshida LS, Nishida S, Kobayashi T, Shimoyama T. Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase. Infect Immun 2004;72:3373e82. [17] Comera C, Andre K, Laffitte J, Collet X, Galtier P, Maridonneau-Parini I. Gliotoxin from Aspergillus fumigatus affects phagocytosis and the organization of the actin cytoskeleton by distinct signalling pathways in human neutrophils. Microbes Infect 2007;9:47e54. [18] Speth C, Hagleitner M, Ott HW, Wurzner R, Lass-Florl C, Rambach G. Aspergillus fumigatus activates thrombocytes by secretion of soluble compounds. J Infect Dis 2013;207:823e33. [19] Amitani R, Taylor G, Elezis EN, Llewellyn-Jones C, Mitchell J, Kuze F, et al. Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium. Infect Immun 1995;63:3266e71. [20] Johannessen LN, Nilsen AM, Lovik M. Mycotoxin-induced depletion of intracellular glutathione and altered cytokine production in the human alveolar epithelial cell line A549. Toxicol Lett 2007;168:103e12. [21] Paris S, Boisvieux-Ulrich E, Crestani B, Houcine O, Taramelli D, Lombardi L, et al. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells. Infect Immun 1997;65:1510e4. [22] Peng X, Frohman MA. Mammalian phospholipase D physiological and pathological roles. Acta Physiol 2012;204:219e26. [23] Han L, Stope MB, de Jesus ML, Oude Weernink PA, Urban M, Wieland T, et al. Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin. EMBO J 2007;26:4189e202. [24] Han X, Yu R, Ji L, Zhen D, Tao S, Li S, et al. InlB-mediated Listeria monocytogenes internalization requires a balanced phospholipase D activity maintained through phospho-cofilin. Mol Microbiol 2011;81:860e80. [25] Kusner DJ, Barton JA, Wen KK, Wang X, Rubenstein PA, Iyer SS. Regulation of phospholipase D activity by actin. Actin exerts bidirectional modulation of Mammalian phospholipase D activity in a polymerization-dependent, isoform-specific manner. J Biol Chem 2002;277:50683e92. [26] Han X, Yu R, Zhen D, Tao S, Schmidt M, Han L. beta-1,3-Glucaninduced host phospholipase D activation is involved in Aspergillus fumigatus internalization into type II human pneumocyte A549 cells. PLoS ONE 2011;6:e21468. [27] Sugui JA, Pardo J, Chang YC, Zarember KA, Nardone G, Galvez EM, et al. Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone. Eukaryot Cell 2007;6:1562e9. [28] Hohl TM, Van Epps HL, Rivera A, Morgan LA, Chen PL, Feldmesser M, et al. Aspergillus fumigatus triggers inflammatory responses by stagespecific beta-glucan display. PLoS Pathog 2005;1:e30. [29] Huang P, Frohman MA. The potential for phospholipase D as a new therapeutic target. Expert Opin Ther Targets 2007;11:707e16. [30] Speth C, Kupfahl C, Pfaller K, Hagleitner M, Deutinger M, Wurzner R, et al. Gliotoxin as putative virulence factor and immunotherapeutic target in a cell culture model of cerebral aspergillosis. Mol Immun 2011;48:2122e9. [31] Kupfahl C, Michalka A, Lass-Florl C, Fischer G, Haase G, Ruppert T, et al. Gliotoxin production by clinical and environmental Aspergillus fumigatus strains. Int J Med Microbiol 2008;298:319e27. [32] Thrasher JD, Crawley S. The biocontaminants and complexity of damp indoor spaces: more than what meets the eyes. Toxicol Ind Health 2009;25:583e615. [33] Coughlan CA, Chotirmall SH, Renwick J, Hassan T, Boon Low T, Bergsson G, et al. The effect of Aspergillus fumigatus infection on
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Vitamin d receptor expression in cystic fibrosis. Am J Respir Crit Care Med 2012;186:999e1007. Lewis RE, Wiederhold NP, Chi J, Han XY, Komanduri KV, Kontoyiannis DP, et al. Detection of gliotoxin in experimental and human aspergillosis. Infect Immun 2005;73:635e7. Pahl HL, Krauss B, Schulze-Osthoff K, Decker T, Traenckner EB, Vogt M, et al. The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-kappaB. J Exp Med 1996;183:1829e40. Hurne AM, Chai CL, Moerman K, Waring P. Influx of calcium through a redox-sensitive plasma membrane channel in thymocytes causes early necrotic cell death induced by the epipolythiodioxopiperazine toxins. J Biol Chem 2002;277:31631e8. Boesewetter DE, Collier JL, Kim AM, Riley MR. Alterations of A549 lung cell gene expression in response to biochemical toxins. Cell Biol Toxicol 2006;22:101e18.
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[38] Cho JH, Yoon MS, Koo JB, Kim YS, Lee KS, Lee JH, et al. The progesterone receptor as a transcription factor regulates phospholipase D1 expression through independent activation of protein kinase A and Ras during 8-Br-cAMP-induced decidualization in human endometrial stromal cells. Biochem J 2011;436:181e91. [39] Yoon MS, Koo JB, Hwang JH, Lee KS, Han JS. Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells. FEBS Lett 2005;579:5635e42. [40] Komati H, Naro F, Mebarek S, De Arcangelis V, Adamo S, Lagarde M, et al. Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton. Mol Biol Cell 2005;16:1232e44. [41] Yeung T, Grinstein S. Lipid signaling and the modulation of surface charge during phagocytosis. Immunol Rev 2007;219:17e36.
Please cite this article in press as: Jia X, et al., Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.001
MODEL
Microbes and Infection xx (2014) 1e11 www.elsevier.com/locate/micinf
Original article
Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation Xiaodong Jia a,1, Fangyan Chen a,1, Weihua Pan b,1, Rentao Yu a,c, Shuguang Tian a, Gaige Han a, Haiqin Fang a, Shuo Wang a, Jingya Zhao a, Xianping Li a, Dongyu Zheng a, Sha Tao a, Wanqing Liao b, Xuelin Han a,*, Li Han a,* a b
Department for Hospital Infection Control & Research, Institute of Disease Control & Prevention of PLA, Academy of Military Medical Sciences, Beijing, China Shanghai Key Laboratory of Molecular Mycology, Department of Dermatology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China c Patent Examination Cooperation Center of the Patent Office, Beijing, China Received 10 September 2013; accepted 4 March 2014
Abstract The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement. Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
Keywords: Gliotoxin; Aspergillus fumigatus; Internalization; Phospholipase D
1. Introduction Aspergillus fumigatus is one of the major fungal pathogens which cause allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis [1]. Its airborne conidia are inhaled from the environment and then colonize within the * Corresponding authors. E-mail addresses: [email protected] (X. Han), [email protected], [email protected] (L. Han). 1 These authors contribute equally to this article.
lung alveoli. Like many intracellular bacterial pathogens [2], A. fumigatus conidia are able to bind to and internalize into lung type II alveolar epithelium cells. They can then survive within these normally non-phagocytic host cells. This brings the possibility of immune evasion as well as the possibility of dissemination [3e5]. To date, the mechanism associated with A. fumigatus internalization into type II lung epithelial cells remains unclear. Gliotoxin is one of the well-known members of the epipolythiodioxopiperazine class of metabolites produced by A. fumigatus. It is characterized by a disulfide bridge across a
http://dx.doi.org/10.1016/j.micinf.2014.03.001 1286-4579/Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Jia X, et al., Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.001
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piperazine ring which is essential for its toxicity [6]. It should be noted that gliotoxin possesses multiple immuno-suppressive activities [7]. In murine models of invasive aspergillosis (IA), gliotoxin has been shown to inhibit specifically the nuclear transcription factor NF-kB [8,9], which consequently induces host cell apoptosis [10,11] and suppresses the cytotoxic T-cell response [12,13]. Moreover, gliotoxin is also able to inhibit macrophage and polymorphonuclear cell function, including phagocytosis [14] and respiratory bursts [15,16]. It has been demonstrated that gliotoxin from A. fumigatus affects the process of phagocytosis and actin cytoskeleton rearrangement of human neutrophils through distinct signaling pathways, which involve cAMP and arachidonic acid signals, respectively [17]. Recently, an immune-active role of gliotoxin was reported that gliotoxin can activate the platelets and its antifungal function [18]. For epithelial cells, it has been shown that gliotoxin can reduce the ciliary movement of epithelial cells and alter membrane permeability leading to damage of the epithelial cells [14,19,20]. Gliotoxin may also reduce TGFb1, IL-6 and IL-8 levels in A549 lung epithelial cells at lower concentrations (below 50 ng/ml) [20]. However, much less is known about the effect of gliotoxin on A. fumigatus internalization into lung epithelial cells. It has been generally accepted that the internalization of A. fumigatus conidia into type II A549 lung epithelial cells is closely related to host actin dynamics including invagination of the membrane and formation of the pseudopods that engulf the conidia [3,5,21]. Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of the most abundant membrane phospholipids, phosphatidylcholine (PC) to produce phosphatidic acid（PA）and choline [22]. In mammalian cells PLD activity has been found to be closely related to actin dynamics [23e25]. In our previous study, it was demonstrated that the internalization of A. fumigatus conidia into epithelial cells was regulated by b-1,3-glucan-induced PLD activation in epithelial cells [26]. However, block of dectin-1, the receptor on lung epithelial cells for b-1,3-glucan, could not fully inhibit the internalization of conidia or conidia-induced PLD activation. This implied that some other factors may also be involved in the regulation of A. fumigatus internalization into lung epithelial cells. Therefore, we hypothesized that gliotoxin from A. fumigatus may be involved in regulating the conidial internalization into lung epithelial cells. In present study, we discovered that gliotoxin facilitates the internalization of A. fumigatus conidia by stimulating PLD activity in A549 cells. 2. Materials and methods 2.1. A. fumigatus strains, cell lines A. fumigatus wild type strain B5233, its mutant strain gliPD (deletion of the peptide synthetase gliP), and the gliP reconstituted strain gliPR were generously provided by Dr. KJ. Kwon-Chung (National Institute of Health, Bethesda, Maryland) [27]. Except where indicated, all of the A. fumigatus strains were propagated on Sabouraud dextrose agar slants
(10 g/l peptone, 10 g/l glucose, 15 g/l agar, pH6.0) for 5e8 days at 37 C. The type II human pneumocyte cell line A549 and murine macrophage cell line J774 were obtained from the ATCC and the 16HBE14o human bronchial epithelial cell line was purchased from shanghai Beinuo life science company. These three kinds of cells were cultured in DMEM supplemented with a 10% fetal calf serum (GIBCO, Germany), 100 mg/l streptomycin, 16 mg/l penicillin at 37 C in 5% CO2. The human monocytic leukemia cell line THP-1 was gift from Beijing Proteome Research Center and cultured in suspension cultures in RPMI-1640 (GIBCO, Germany) supplemented with 10% fetal calf serum (GIBCO, Germany), 100 mg/l streptomycin, 16 mg/l penicillin at 37 C in 5% CO2. THP1 cells were differentiated into macrophages by 100 ng/ml phorbol myristate acetate (PMA) for 60 h. Afterwards, the macrophages were cultured in DMEM/F12 1:1 medium (GIBCO, Germany) supplemented with 10% fetal calf serum (GIBCO, Germany), 100 mg/l streptomycin, 16 mg/l penicillin at 37 C in 5% CO2. 2.2. Preparation of the conidia A. fumigatus conidia were harvested and prepared in the same way as described in a previous study [28]. After 5e8 days of cultivation, A. fumigatus conidia were dislodged from agar plates with gentle washing. They were then re-suspended in sterile phosphate-buffered saline supplemented with 0.1% Tween 20 (PBST). The conidia were passed through 8 layers of sterile gauze in order to remove hyphal fragments. At this point, they were enumerated on a hemacytometer and the resting conidia were washed twice and stored at 4 C to be used within 48 h. For preparation of swollen conidia, the resting conidia were incubated at 37 C in DMEM for the indicated periods. The preparations which were incubated for 5 h contained swollen conidia and early germlings with 96%) of both environmental and clinical A. fumigatus has the ability to produce gliotoxin [31]. In the case of cystic fibrosis (CF), A. fumigatus colonization is associated with increased gliotoxin in the bronchoalveolar lavage fluid (BALF) [33]. On the other hand, it has been proven that gliotoxin is produced in experimental animal aspergillosis as well as in human IA, with serum concentrations of 166e785 ng/ml in 80% of patients with IA [30,34]. Thus, concerning that the internalization of A. fumigatus into the lung epithelium allows them to protect themselves from the phagocytosis by macrophages or neutrophils, it seemed physiologically feasible that a low amount of gliotoxin produced by A. fumigatus from mouldy environments or lung secretions may help A. fumigatus internalization and evade further immune attacks at the initial step of infection. Another critical finding in the present study was that gliotoxin is able to stimulate the PLD activity of A549 lung epithelial cells. In this way, it can promote the efficient internalization of A. fumigatus into the cells. This seemed to be complementary to another PLD signal pathway, b-1,3-glucandectin-1 cascade which was demonstrated in our previous report [26]. To some extent, this finding was surprising because gliotoxin is a well-known immunosuppressive agent in many immune cells including neutrophils, macrophages, and thymocytes [14,35]. It inhibits several important enzymes, such as NADPH oxidase of neutrophils [15,16], and the nuclear transcription factor NF-kB and 20S proteasome activity [35]. However, gliotoxin was recently shown to activate the platelets [18]. It was also reported that gliotoxin can increase the intracellular cAMP concentration [11], and induce Ca2þ release from the mitochondria of intact rat livers [36,37]. Both cAMP and Ca2þ are the putative agonists for PLD stimulation in several signaling events [38,39], which may also be involved
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in a gliotoxin-induced PLD activation pathway. Therefore, PLD may be a novel intracellular target of gliotoxin. For this reason, the detailed mechanism for PLD activation by gliotoxin in epithelial cells should be investigated further. Furthermore, the control of PLD activity on the gliotoxininduced A. fumigatus internalization was in line with the previous findings that PLD is a critical regulator in many phagocytosis or internalization processes in response to a large variety of agonists. The regulation of PLD on pathogen internalization mostly resulted from its close interaction with host actin dynamic induced by the pathogens. In mammalian cells, elements of the actin dynamic system interact directly or indirectly with PLD enzymes and its related phospholipid metabolism [23,29,40]. Inversely, the phosphatidic acid produced by PLD catalysis could induce stress fiber formation and deliberately regulate the formation of phagocytic cups [41]. In the present study, gliotoxin induced significant actin cytoskeleton rearrangement (Fig. 5) along with the promotion of A. fumigatus internalization. Therefore, the stimulated PLD activity by gliotoxin may interact with actin cytoskeleton rearrangement in order to control A. fumigatus internalization. Compared to PLD2, PLD1 played a more important role in gliotoxin-induced A. fumigatus internalization into epithelial cells. This discrepancy might be gliotoxin-specific because it has been previously reported that both PLD1 and PLD2 contribute coordinately to phagosome formation and development during the internalization of A. fumigatus into A549 lung epithelial cells [24,26]. Moreover, since the subcellular distribution of PLD1 was not significantly altered by gliotoxin in A549 cells (Fig. 5), the gliotoxin-induced PLD activation may not be associated with the subcellular translocation of PLD1. Therefore, it is interesting to analyze the precise function and distribution of PLD isotypes during gliotoxin-induced A. fumigatus internalization. Finally, gliotoxin significantly blocked the ROS production in A549 epithelial cells at low concentrations (from 10 to 100 ng/ ml). In contrast, in human neutrophils, the inhibition of ROS production by gliotoxin has been reported only at higher concentrations (above 1 mg/ml [15,16]) and no inhibition of NADPH-oxidase activity was observed at the low concentrations of gliotoxin (from 10 to 100 ng/ml) [17]. As NADPH-oxidase is one of major modulators responsing to oxidative stress in the cell, it can be deduced that NADPH oxidase complex of epithelial cells might be more sensitive to gliotoxin than that of J774 macrophages and neutrophils. In addition, as the epithelial cells constitute the first barrier against A. fumigatus, the low threshold value of gliotoxin concentration required for enough inhibition on ROS response in lung epithelial cells might facilitate the internalization and intracellular living of A. fumigatus. In addition, concerning that the nystatin protection assay is an indirect and error-prone approach, we carefully repeated the internalization experiments, even for several times in order to get the reliable and stable results. We had also certified the correlation of internalization results between nystatin protection assay and immunofluorescence assay (Fig. S3). Certainly, more accurate assay would be expected for further interpretation on the exact role of gliotoxin on A. fumigatus internalization into lung epithelium.
Please cite this article in press as: Jia X, et al., Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.001
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In conclusion, we reported an important finding that gliotoxin is able to regulate A. fumigatus internalization into epithelial cells by inducing host PLD activation. However, the detailed function and regulatory mechanism of gliotoxin on A. fumigatus entry into epithelial cells need to be further clarified. Conflict of interest The authors have no conflict of interest. Acknowledgments We would like to thank Dr. KJ. Kwon-Chung for kindly providing the various A. fumigatus strains. This project was supported by grants from 973 Program (No. 2013CB531606) and from the Chinese National Scientific Foundation Committee (No. 30772029 and No. 81172801). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.micinf.2014.03.001. References [1] Segal BH. Aspergillosis. N Engl J Med 2009;360:1870e84. [2] Bonazzi M, Vasudevan L, Mallet A, Sachse M, Sartori A, Prevost MC, et al. Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization. J Cell Biol 2011;195:525e36. [3] Osherov N. Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells. Front Microbiol 2012;3:346. [4] Li X, Gao M, Han X, Tao S, Zheng D, Cheng Y, et al. Disruption of the phospholipase D gene attenuates the virulence of Aspergillus fumigatus. Infect Immun 2012;80:429e40. [5] Wasylnka JA, Moore MM. Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein. Infect Immun 2002;70:3156e63. [6] Gardiner DM, Waring P, Howlett BJ. The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis. Microbiology 2005;151:1021e32. [7] Scharf DH, Heinekamp T, Remme N, Hortschansky P, Brakhage AA, Hertweck C. Biosynthesis and function of gliotoxin in Aspergillus fumigatus. Appl Microbiol Biotechnol 2012;93:467e72. [8] Rashmi R, Schnulle PM, Maddox AC, Armbrecht ES, Koenig JM. Flice inhibitory protein is associated with the survival of neonatal neutrophils. Pediatr Res 2011;70:327e31. [9] Spikes S, Xu R, Nguyen CK, Chamilos G, Kontoyiannis DP, Jacobson RH, et al. Gliotoxin production in Aspergillus fumigatus contributes to host-specific differences in virulence. J Infect Dis 2008;197:479e86. [10] Hagens WI, Beljaars L, Mann DA, Wright MC, Julien B, Lotersztajn S, et al. Cellular targeting of the apoptosis-inducing compound gliotoxin to fibrotic rat livers. J Pharmacol Exp Ther 2008;324:902e10. [11] Waring P, Khan T, Sjaarda A. Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion. J Biol Chem 1997;272:17929e36. [12] Stanzani M, Orciuolo E, Lewis R, Kontoyiannis DP, Martins SL, St John LS, et al. Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes. Blood 2005;105:2258e65.
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