mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Supplement article
Apoptotic-like phenotype triggered by hydrogen peroxide and amphotericin B in the fungus Rhizopus arrhizus Sibu Wang, Ruoyu Li and Jin Yu Department of Dermatology, Peking University First Hospital, Beijing, China
Summary
Rhizopus is the most common genus of invasive mucormycosis, whose prognosis and outcome was not improved over the past decades. We studied the apoptotic-like phenotype in Rhizopus arrhizus exposed to hydrogen peroxide (H2O2) and amphotericin B (AMB). The strain provided by Fungal Genetic Stock centre was studied about the apoptotic-like phenotype treated with different concentrations of H2O2 and AMB, and then analyzed by fluorescent microscopy (observed by Annexin-V/FITC and TUNEL staining), flow cytometry (stained with DHR123/PI), and DNA agarose gel electrophores. When R. arrhizus was treated with H2O2 and AMB, there was a loss of viability associated with different phenotype of apoptosis makers. Membrane externalization of phosphatidylserine (PS) on the cell surface, DNA fragmentation, chromatin condensation can be induced and observed obviously by Annexin-V/FITC, DAPI and TUNEL staining. DNA smear not DNA ladder was also visible in R. arrhizus. Flowcytometry of R. arrhizus cells revealed not only the increase of apoptosis cell stained with DHR123 under the nonfungicida doses but dead cells stained with PI under the fungicida concentrations.This study indicated that both H2O2 and AMB could induce the apoptotic-like phenotype in R. arrhizus.
Key words: Rhizopus arrhizus, Hydrogen peroxide, Amphotericin B, Apoptosis.
Introduction The incidence of invasive fungal disease has increased over the past two decades due to increasing numbers of immunocompromised individuals. After Candidia and Aspergillus, members of the Mucorales order are the most common clinically relevant pathogens involved in human invasive fungal diseases. Mucormycosis is commonly present in recipients of hematopoietic stem cell/solid organ transplants as well as patients with haematological malignancies, diabetes mellitus, burns, trauma and low birth weight.1–3 Correspondence: Jin Yu, Department of Dermatology and Venereology, Peking University First Hospital, and Research Center for Medical Mycology, Peking University, Beijing, PR China. No. 8 Xi-Shi-Ku St. Xicheng District. Beijing, 100034, China. Tel.: (8610) 83573056. Fax: (8610) 66551216. E-mail: [email protected] Submitted for publication 18 November 2013 Revised 5 February 2014 Accepted for publication 10 February 2014
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Rhizopus spp. is most commonly the root of invasive mucormycosis.2,4 The lung is easily infected because the respiratory tract is the most frequent entry route of sporangiospores. When entering the body, the spores first challenge the innate immune cells (phagocytes, neutrophils). One of the early host responses after a fungal attack is the production of high levels of reactive oxygen species (ROS; including hydrogen peroxide [H2O2] and hydroxyl radicals).5 This so-called oxidative burst might induce apoptosis of the pathogen. This apoptotic-like phenotype has been observed in yeast and Aspergillus fumigatus.6,7 Experimental data indicate that an apoptotic pathway is induced by a host–pathogen interaction. Moreover, amphotericin B (AmB), the most active anti-Mucorales agent, has also been seen as a strong trigger for inducing cell death in the opportunistic pathogen A. fumigatus.7 In this paper, we tried to study whether the apoptotic-like phenotype can be observed in Rhizopus arrhizus induced by H2O2 and AmB.
doi:10.1111/myc.12240
S. Wang et al.
Annexin V analysis
Materials and methods Strains, reagents and growth conditions
Rhizopus arrhizus was provided by the Fungal Genetic Stock Center (Kansas, MO, USA). H2O2 (30%, m/v; Beijing Chemical works, Beijing, China) diluted in water and AmB (Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in dimethyl sulfoxide were stored at 4 and 80 °C, respectively. Media used in this study included potato dextrose agar (PDA) and Yeast peptone glucose medium (YPG, a rich medium containing 0.3% yeast extract, 1% peptone and 2% glucose, PH4.5). Rhizopus arrhizus isolates were grown on PDA for 5 days at 28 °C. Freshly harvested sporangiospores (2.5 9 104 spores ml 1) were inoculated into 100 ml flasks containing 30 ml of YPG liquid medium at 30 °C with constant shaking (200 rpm).
Apoptosis was detected by fluorescence microscopy using Annexin V-FITC (Annexin V-FITC Apoptosis Detection KIt; Merck, Darmstadt, Germany) and propidium iodide (PI) to assess cellular integrity and phosphatidylserine (PS) externalisation as previously described.9 Each assay was repeated for at least three times. TUNEL assay
For terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL), protoplasts were washed twice in PBS and then fixed in 3.6% paraformaldehyde. TUNEL assay was performed according to the manufacturer’s instructions as previously described.10 ROS production
Growth and viability assays 1
Various concentrations of H2O2 (0–25 mmol l ) and AmB (0–8 lg ml 1) from stock solution were added to YPG at the time of spore inoculation (0 h). The growth assay was performed in 96-well plates at 30 °C using microplate reader at OD450 (Model 550, Bio-RAD, Hercules, California, USA). Viability was assessed using the XTT method (XTT, 100 lg ml 1, menadione, 25 lmol l 1) after inoculation.8,9
Cells (2.5 9 106 spores ml 1) were collected by centrifugation, washed once in 1 ml of PBS, resuspended in 1 ml of PBS containing various concentrations of H2O2 or AmB and incubated at 30 °C on a rotary shaker (100 rpm) for 3 h. The cells were stained with dihydrorhodamine123 (DHR123; Merck) at 37 °C for 2 h and then with PI. After staining, cells were analysed using flow cytometry.
Results Agarose gel electrophoresis of DNA
Genomic DNA was extracted from mycelia of the exponential phase after exposure to different concentrations of H2O2 (0, 1.2, 3.6 and 6.0 lmol l 1) and AmB (0, 0.25, 0.5 and 1 lg ml 1) in phosphate-buffered saline (PBS; pH 7.4) for up to 3 h. DNA was examined on a 1.5% (w/v) agarose gel in TAE buffer and visualised after ethidium bromide staining. Protoplast preparation
Rhizopus arrhizus cells (2.5 9 104 spores ml 1) grown firstly in YPG for 6 h to induce sporulation to expose the germinating material to cell wall lysing enzymes. Then they were treated with different concentrations of H2O2 or AmB for 3 h. Protoplast cells of R. arrhizus were prepared in 2 ml of 0.5 mol l 1 glucose (pH 5.8) containing Novozym 234 (5 mg ml 1; Sigma-Aldrich Co.), chitinase (3 mg ml 1; Sigma-Aldrich Co.) and chitosanase (1.5 mg ml 1; Sigma-Aldrich Co.) and incubated at 30 °C for 3 h.
26
Growth and viability of R. arrhizus
As shown in Fig. 1, the minimum fungicidal doses in R. arrhizus were 6 mmol l 1 H2O2 and 2 lg ml 1 AmB, at which point growth ceased and the fungi lost the ability to recover. Growth was not obviously affected below the concentrations of 0.6 mmol l 1 H2O2 and 0.03 lg ml 1 AmB, whereas cell viability was affected above 0.6 mmol l 1 H2O2 and 0.0625 lg ml 1 AmB. At the higher concentrations of 3.0–4.8 mmol l 1 H2O2 and 0.5–1.0 lg ml 1 AmB, growth ceased for more than 6 h and then recovered. Agarose gel electrophoresis of DNA
Incubation of R. arrhizus mycelia with H2O2 and AmB for 3 h resulted in DNA fragmentation, which was visible as a smear using the agarose gel electrophoresis (Fig. 2). Figure 2 shows that DNA fragmentation appeared obviously after treatment with H2O2 (3.6
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Apoptotic-like phenotype in Rhizopus arrhizus
(a)
(b)
(c)
(d)
Figure 1 The growth and viability of
Rhizopus oryzae. Effect of different concentrations of H2O2 (b, d) and amphotericin B (a, c) on the growth (a, b) and viability (c, d) of Rhizopus oryzae.
and 6.0 mmol l 1) and AmB (1 lg ml smears but not ladders were observed.
1
). DNA
Apoptosis phenotypes of R. arrhizus
Apoptosis is characterised by several morphological and biochemical changes, such as membrane externalisation of PS on the cell surface, DNA fragmentation, chromatin condensation, etc.10 This study observed whether these apoptotic-like responses existed in the R. arrhizus induced by 3.6 mmol l 1 H2O2 and 1 lg ml 1 AmB for 3 h. The hallmark of apoptosis is the externalisation of PS from the inner to the outer leaflet of the plasma membrane. Hence, the annexin V-FITC/PI assay was used to examine the PS externalisation in R. arrhizus protoplasts. As shown in Fig. 3, green fluorescence indicating the
binding of annexin V was found in most of the protoplasts from the fungi treated with H2O2 or AmB (Fig. 3a); the red fluorescence of PI represented dead cells (Fig. 3b). Another apoptosis marker is DNA fragmentation detected by the TUNEL assay. As shown in Fig. 3, strong TUNEL staining was observed in the protoplasts from the fungi treated with H2O2 and AmB (Fig. 3c) but was rarely detected in untreated cells. ROS production
Reactive oxygen species production can be monitored using DHR123, which is oxidised to a green fluorescent derivative by intracellular ROS and can stain cells without protoplast preparation. Flow cytometry of R. arrhizus cells incubated in H2O2 and AmB for 3 h and then stained with DHR123 and PI revealed increased numbers of DHR123-positive cells after treatment with non-fungicidal concentrations of the inducers but decreased numbers of DHR123-positive cells after treatment with inducers at greater than minimal fungicidal concentrations. The percentage of PI-stained cells increased as the inducer concentration increased (Fig. 4).
Discussion Figure 2 DNA agarose gel electrophores of Rhizopus oryzae. Agarose gel electrophoresis of genomic DNA extracted from mycelia of the exponential phase after exposure to different concentrations of H2O2 or AmB for 3 h. Lane 1, DNA maker; lane 2, mock stress; lane 3, 1.2 mmol l 1 H2O2; lane 4, 3.6 mmol l 1 H2O2; lane 5, 6.0 mmol l 1 H2O2; lane 6, 0.25 lg ml 1 AmB; lane 7, 0.5 lg ml 1 AmB; lane 8, 1.0 lg ml 1 AmB.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Living cells have the ability to undergo programmed cell death under certain conditions, which is not only restricted to metazoans but also exists in other living organisms including plants, fungi and bacteria.11–14 Apoptosis has great importance in the development and homeostasis of organisms. The
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S. Wang et al.
(a)
(b)
(c)
Figure 3 The fluorescent microscopy of Rhizopus oryzae. After H2O2 or amphotericin B treatment, changes were seen under the microscope. Externalisation of phosphatidylserine and dead cells stained separately by annexin V-FITC and propidium iodide (a, b). Accumulation of DNA breaks (revealed by TUNEL-positive staining) (c).
(a)
(b)
(c)
Figure 4 Flow cytometry of Rhizopus oryzae cells stained with
DHR123 and propidium iodide (PI). Cells after exposure to various concentrations of H2O2 (a) and amphotericin B (AmB; b) for 3 h were stained with DHR123 and PI and then analysed by flow cytometry. The percentages of cells that are apoptotic (blue bars) and necrotic (red bars) are shown. (c) FL1 LOG, DHR123 staining; FL3 LOG, PI staining.
apoptotic-like phenotype has now been described in a range of fungi, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, A. fumigatus, Aspergillus nidulans, Mucor racemosus and R. arrhizus.7,9,15–20 Similarly, our result
28
demonstrated that the apoptotic-like phenotype can also be observed in R. arrhizus. H2O2 and AmB are exogenous triggers that can be provided externally in the form of chemical or physical stress and have been studied in several fungi.7,17 The optimal apoptosis-inducing concentrations of H2O2 and AmB differ in C. albicans and A. fumigatus. Exposure of C. albicans to 5–10 mmol l 1 H2O2 or 4–8 lg ml 1 AmB produced cellular changes reminiscent of mammalian apoptosis.17 However, treated with much lower levels of H2O2 (0.1 mmol l 1) or AmB (0.5 lg ml 1), A. fumigatus showed loss of cell viability and death associated with a number of phenotypic changes characteristic of apoptosis. In our study, the concentrations of H2O2 and AmB that induced R. arrhizus manifestations of the apoptotic-like phenotype were between C. albicans and A. fumigatus. Under 3.6 mmol l 1 of H2O2 and 1 lg ml 1 of AmB, most of the cells expressed the apoptotic-like phenotype. Dose variability of H2O2 and AmB existed among different fungi. We first detected the early marker of apoptosis in R. arrhizus after treatment with these two triggers and used the annexin V-FICT/PI staining assay to distinguish cells in early apoptosis from normal cells or dead PI-positive cells using fluorescence microscopy. The report indicated increased PI staining and decreased annexin V staining at higher concentrations of both triggers, which revealed membrane disintegration and necrotic cell death. A DNA ladder indicating the late stage of apoptosis in many mammalian cells21 and M. racemosus19 was not detected in this study. However, a DNA ladder is not a stable apoptotic characteristic in all fungi. In A. fumigatus, DNA smearing was found after treatment with H2O2 and AmB as well as in A. nidulans after
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Apoptotic-like phenotype in Rhizopus arrhizus
treatment with phytosphingosine.22,23 DNA smearing rather than a ladder was demonstrated by agarose electrophoresis in R. arrhizus after treatment with H2O2 and AmB. The apoptotic-like phenotype of R. arrhizus was also indentified using the TUNEL assay, which is more sensitive than DNA agarose electrophoresis for analysing apoptotic DNA fragmentation. Microscopic images revealed the presence of significant green fluorescence in the cells treated by high but non-fungicidal concentrations of the two triggers, but minimal fluorescence was seen under low concentrations. These phenomena were also reported in many other fungi, such as S. cerevisiae treated with H2O2 and acetic acid, C. albicans treated with farnesol, A. fumigatus treated with H2O2 and AmB and A. fumigatus in the stationary phase.7,9,23,24 DHR123/PI double-staining by flow cytometry can better explain the change of apoptotic or dead cells. In our study, an apoptotic phenotype can be induced by low but toxic concentrations of both triggers through ROS accumulation within cells, whereas dead cells stained with PI increased after treatment with high concentrations of the triggers. These findings indicate that treatment with low concentrations of both triggers can induce an apoptotic-like phenotype through ROS accumulation and ultimately cause death under continued accumulation with increased PI-positive staining. It is well known that ROS plays a major role in signalling and/or effector functions in apoptosis.25 The production of ROS in apoptotic cells has been examined in other fungal cells, including C. albicans, S. cerevisiae and A. nidulans.18,26,27 ROS accumulation has also been demonstrated in many fungal and mammalian cells and played a central role in the induction of apoptosis.6,7,28 This study indicated that both H2O2 and AMB could induce the apoptotic-like phenotype in R. arrhizus, which might be usefully exploited in the search for and design of novel therapies in the future.
Acknowledgments This work was supported by National Natural Science Foundation (81371783) from the National Natural Science Foundation of China.
Reference 1
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Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
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Greenberg RN, Scott LJ, Vaughn HH, Ribes JA. Zygomycosis (mucormycosis): emerging clinical importance and new treatments. Curr Opin Infect Dis 2004; 17: 517–25. Ribes JA, Vanover-Sams CL, Baker DL. Zygomycetes in human disease. Clin Microbiol Rev 2000; 13: 236–301. Spellberg B, Edwards J Jr, Ibrahim A. Novel perspectives on mucormycosis: pathophysiology, presentation, and management. Clin Microbiol Rev 2005; 18: 556–69. Diwakar A, Dewan RK, Chowdhary A, Randhawa HS, Khanna G, Gaur SN. Zygomycosis—a case report and overview of the disease in India. Mycoses 2007; 50: 247–54. Chamilos G, Lewis RE, Lamaris G, Walsh TJ, Kontoyiannis DP. Zygomycetes hyphae trigger an early, robust proinflammatory response in human polymorphonuclear neutrophils through toll-like receptor 2 induction but display relative resistance to oxidative damage. Antimicrob Agents Chemother 2008; 52: 722–4. Madeo F, Fr€ ohlich E, Ligr M et al. Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 1999; 145: 757–67. Mousavi S, Robson GD. Oxidative and amphotericin B-mediated cell death in the opportunistic pathogen Aspergillus fumigatus is associated with an apoptotic-like phenotype. Microbiology 2004; 150: 1937–45. Antachopoulos C, Meletiadis J, Roilides E, Sein T, Walsh TJ. Rapid susceptibility testing of medically important zygomycetes by XTT assay. J Clin Microbiol 2006; 44: 553–60. Mousavi SA, Robson GD. Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus. Fungal Genet Biol 2003; 39: 221–9. Jacquet H, Raux G, Thibaut F et al. PRODH mutations and hyperprolinemia in a subset of schizophrenic patients. Hum Mol Genet 2002; 11: 2243–9. Lewis K. Programmed death in bacteria. Microbiol Mol Biol Rev 2000; 5: 503–14. Golstein P, Aubry L, Levraud JP. Cell-death alternative model organisms: why and which? Nat Rev Mol Cell Biol 2003; 4: 798–807. Robson GD. Programmed cell death in the aspergilli and other filamentous fungi. Med Mycol 2006; 44: S109–14. Ramsdale M. Programmed cell death and apoptosis in fungi. Mycota 2006; 13: 113–45. Madeo F, Herker E, Wissing S, Jungwirth H, Eisenberg T, Fr€ ohlich KU. Apoptosis in yeast. Curr Opin Microbiol 2004; 7: 655–60. Zhang Q, Chieu HK, Low CP, Zhang S, Heng CK, Yang H. Schizosaccharomyces pombe cells deficient in triacylglycerol synthesis undergo apoptosis upon entry into the stationary phase. J Biol Chem 2003; 278: 47145–55. Philips AJ, Sudbery I, Ramsdale M. Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. Proc Natl Acad Sci USA 2003; 100: 14327–32. Semighini CP, Hornby JM, Dumitru R, Nikerson KW, Harris SD. Farnesol-induced apoptosis in Aspergillus nidulans reveals a possible mechanism for antagonistic interactions between fungi. Mol Microbiol 2006; 59: 753–64. Roze LV, Linz JE. Lovastatin triggers an apoptosis-like cell death process in the fungus Mucor racemosus. Fungal Genet Biol 1998; 25: 119–33. Shirazi F, Kontoyiannis DP. Mitochondrial respiratory pathways inhibition in Rhizopus oryzae potentiates activity of posaconazole and intraconazole via apoptosis. PLoS ONE 2013; 8: e62293. Bustamante J, Bersier G, Romero M, Badin RA, Boveris A. Nitric oxide production and mitochondrial dysfunction during rat thymocyte apoptosis. Arch Biochem Biophys 2000; 376: 239–47. Cheng J, Park TS, Chio LC, Fischl S, Ye XS. Induction of apoptosis by sphingoid long-chain bases. Mol Cell Biol 2003; 23: 163–77.
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Shirtliff ME, Krom BP, Meijering RA et al. Farnesol-induced apoptosis in Candida albicans. Antimicrob Agents Chemother 2009; 53: 2392– 401. Ludovico P, Sousa MJ, Silva MT, Leao C, Corte-Real M. Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology 2001; 147: 2409–15. Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiology 2000; 7: 153–63.
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Dai BD, Cao YY, Huang S et al. Baicalein induces programmed cell death in Candida albicans. J Microbiol Biotechnol 2009; 19: 803–9. Madeo F, Engelhardt S, Herker E et al. Apoptosis in yeast: a new model system with applications in cell biology and medicine. Curr Genet 2002; 41: 208–16. Greenlund LJ, Deckwerth TL, Johnson EM. Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 1995; 14: 303–15.
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Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Supplement article
Apoptotic-like phenotype triggered by hydrogen peroxide and amphotericin B in the fungus Rhizopus arrhizus Sibu Wang, Ruoyu Li and Jin Yu Department of Dermatology, Peking University First Hospital, Beijing, China
Summary
Rhizopus is the most common genus of invasive mucormycosis, whose prognosis and outcome was not improved over the past decades. We studied the apoptotic-like phenotype in Rhizopus arrhizus exposed to hydrogen peroxide (H2O2) and amphotericin B (AMB). The strain provided by Fungal Genetic Stock centre was studied about the apoptotic-like phenotype treated with different concentrations of H2O2 and AMB, and then analyzed by fluorescent microscopy (observed by Annexin-V/FITC and TUNEL staining), flow cytometry (stained with DHR123/PI), and DNA agarose gel electrophores. When R. arrhizus was treated with H2O2 and AMB, there was a loss of viability associated with different phenotype of apoptosis makers. Membrane externalization of phosphatidylserine (PS) on the cell surface, DNA fragmentation, chromatin condensation can be induced and observed obviously by Annexin-V/FITC, DAPI and TUNEL staining. DNA smear not DNA ladder was also visible in R. arrhizus. Flowcytometry of R. arrhizus cells revealed not only the increase of apoptosis cell stained with DHR123 under the nonfungicida doses but dead cells stained with PI under the fungicida concentrations.This study indicated that both H2O2 and AMB could induce the apoptotic-like phenotype in R. arrhizus.
Key words: Rhizopus arrhizus, Hydrogen peroxide, Amphotericin B, Apoptosis.
Introduction The incidence of invasive fungal disease has increased over the past two decades due to increasing numbers of immunocompromised individuals. After Candidia and Aspergillus, members of the Mucorales order are the most common clinically relevant pathogens involved in human invasive fungal diseases. Mucormycosis is commonly present in recipients of hematopoietic stem cell/solid organ transplants as well as patients with haematological malignancies, diabetes mellitus, burns, trauma and low birth weight.1–3 Correspondence: Jin Yu, Department of Dermatology and Venereology, Peking University First Hospital, and Research Center for Medical Mycology, Peking University, Beijing, PR China. No. 8 Xi-Shi-Ku St. Xicheng District. Beijing, 100034, China. Tel.: (8610) 83573056. Fax: (8610) 66551216. E-mail: [email protected] Submitted for publication 18 November 2013 Revised 5 February 2014 Accepted for publication 10 February 2014
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Rhizopus spp. is most commonly the root of invasive mucormycosis.2,4 The lung is easily infected because the respiratory tract is the most frequent entry route of sporangiospores. When entering the body, the spores first challenge the innate immune cells (phagocytes, neutrophils). One of the early host responses after a fungal attack is the production of high levels of reactive oxygen species (ROS; including hydrogen peroxide [H2O2] and hydroxyl radicals).5 This so-called oxidative burst might induce apoptosis of the pathogen. This apoptotic-like phenotype has been observed in yeast and Aspergillus fumigatus.6,7 Experimental data indicate that an apoptotic pathway is induced by a host–pathogen interaction. Moreover, amphotericin B (AmB), the most active anti-Mucorales agent, has also been seen as a strong trigger for inducing cell death in the opportunistic pathogen A. fumigatus.7 In this paper, we tried to study whether the apoptotic-like phenotype can be observed in Rhizopus arrhizus induced by H2O2 and AmB.
doi:10.1111/myc.12240
S. Wang et al.
Annexin V analysis
Materials and methods Strains, reagents and growth conditions
Rhizopus arrhizus was provided by the Fungal Genetic Stock Center (Kansas, MO, USA). H2O2 (30%, m/v; Beijing Chemical works, Beijing, China) diluted in water and AmB (Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in dimethyl sulfoxide were stored at 4 and 80 °C, respectively. Media used in this study included potato dextrose agar (PDA) and Yeast peptone glucose medium (YPG, a rich medium containing 0.3% yeast extract, 1% peptone and 2% glucose, PH4.5). Rhizopus arrhizus isolates were grown on PDA for 5 days at 28 °C. Freshly harvested sporangiospores (2.5 9 104 spores ml 1) were inoculated into 100 ml flasks containing 30 ml of YPG liquid medium at 30 °C with constant shaking (200 rpm).
Apoptosis was detected by fluorescence microscopy using Annexin V-FITC (Annexin V-FITC Apoptosis Detection KIt; Merck, Darmstadt, Germany) and propidium iodide (PI) to assess cellular integrity and phosphatidylserine (PS) externalisation as previously described.9 Each assay was repeated for at least three times. TUNEL assay
For terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL), protoplasts were washed twice in PBS and then fixed in 3.6% paraformaldehyde. TUNEL assay was performed according to the manufacturer’s instructions as previously described.10 ROS production
Growth and viability assays 1
Various concentrations of H2O2 (0–25 mmol l ) and AmB (0–8 lg ml 1) from stock solution were added to YPG at the time of spore inoculation (0 h). The growth assay was performed in 96-well plates at 30 °C using microplate reader at OD450 (Model 550, Bio-RAD, Hercules, California, USA). Viability was assessed using the XTT method (XTT, 100 lg ml 1, menadione, 25 lmol l 1) after inoculation.8,9
Cells (2.5 9 106 spores ml 1) were collected by centrifugation, washed once in 1 ml of PBS, resuspended in 1 ml of PBS containing various concentrations of H2O2 or AmB and incubated at 30 °C on a rotary shaker (100 rpm) for 3 h. The cells were stained with dihydrorhodamine123 (DHR123; Merck) at 37 °C for 2 h and then with PI. After staining, cells were analysed using flow cytometry.
Results Agarose gel electrophoresis of DNA
Genomic DNA was extracted from mycelia of the exponential phase after exposure to different concentrations of H2O2 (0, 1.2, 3.6 and 6.0 lmol l 1) and AmB (0, 0.25, 0.5 and 1 lg ml 1) in phosphate-buffered saline (PBS; pH 7.4) for up to 3 h. DNA was examined on a 1.5% (w/v) agarose gel in TAE buffer and visualised after ethidium bromide staining. Protoplast preparation
Rhizopus arrhizus cells (2.5 9 104 spores ml 1) grown firstly in YPG for 6 h to induce sporulation to expose the germinating material to cell wall lysing enzymes. Then they were treated with different concentrations of H2O2 or AmB for 3 h. Protoplast cells of R. arrhizus were prepared in 2 ml of 0.5 mol l 1 glucose (pH 5.8) containing Novozym 234 (5 mg ml 1; Sigma-Aldrich Co.), chitinase (3 mg ml 1; Sigma-Aldrich Co.) and chitosanase (1.5 mg ml 1; Sigma-Aldrich Co.) and incubated at 30 °C for 3 h.
26
Growth and viability of R. arrhizus
As shown in Fig. 1, the minimum fungicidal doses in R. arrhizus were 6 mmol l 1 H2O2 and 2 lg ml 1 AmB, at which point growth ceased and the fungi lost the ability to recover. Growth was not obviously affected below the concentrations of 0.6 mmol l 1 H2O2 and 0.03 lg ml 1 AmB, whereas cell viability was affected above 0.6 mmol l 1 H2O2 and 0.0625 lg ml 1 AmB. At the higher concentrations of 3.0–4.8 mmol l 1 H2O2 and 0.5–1.0 lg ml 1 AmB, growth ceased for more than 6 h and then recovered. Agarose gel electrophoresis of DNA
Incubation of R. arrhizus mycelia with H2O2 and AmB for 3 h resulted in DNA fragmentation, which was visible as a smear using the agarose gel electrophoresis (Fig. 2). Figure 2 shows that DNA fragmentation appeared obviously after treatment with H2O2 (3.6
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Apoptotic-like phenotype in Rhizopus arrhizus
(a)
(b)
(c)
(d)
Figure 1 The growth and viability of
Rhizopus oryzae. Effect of different concentrations of H2O2 (b, d) and amphotericin B (a, c) on the growth (a, b) and viability (c, d) of Rhizopus oryzae.
and 6.0 mmol l 1) and AmB (1 lg ml smears but not ladders were observed.
1
). DNA
Apoptosis phenotypes of R. arrhizus
Apoptosis is characterised by several morphological and biochemical changes, such as membrane externalisation of PS on the cell surface, DNA fragmentation, chromatin condensation, etc.10 This study observed whether these apoptotic-like responses existed in the R. arrhizus induced by 3.6 mmol l 1 H2O2 and 1 lg ml 1 AmB for 3 h. The hallmark of apoptosis is the externalisation of PS from the inner to the outer leaflet of the plasma membrane. Hence, the annexin V-FITC/PI assay was used to examine the PS externalisation in R. arrhizus protoplasts. As shown in Fig. 3, green fluorescence indicating the
binding of annexin V was found in most of the protoplasts from the fungi treated with H2O2 or AmB (Fig. 3a); the red fluorescence of PI represented dead cells (Fig. 3b). Another apoptosis marker is DNA fragmentation detected by the TUNEL assay. As shown in Fig. 3, strong TUNEL staining was observed in the protoplasts from the fungi treated with H2O2 and AmB (Fig. 3c) but was rarely detected in untreated cells. ROS production
Reactive oxygen species production can be monitored using DHR123, which is oxidised to a green fluorescent derivative by intracellular ROS and can stain cells without protoplast preparation. Flow cytometry of R. arrhizus cells incubated in H2O2 and AmB for 3 h and then stained with DHR123 and PI revealed increased numbers of DHR123-positive cells after treatment with non-fungicidal concentrations of the inducers but decreased numbers of DHR123-positive cells after treatment with inducers at greater than minimal fungicidal concentrations. The percentage of PI-stained cells increased as the inducer concentration increased (Fig. 4).
Discussion Figure 2 DNA agarose gel electrophores of Rhizopus oryzae. Agarose gel electrophoresis of genomic DNA extracted from mycelia of the exponential phase after exposure to different concentrations of H2O2 or AmB for 3 h. Lane 1, DNA maker; lane 2, mock stress; lane 3, 1.2 mmol l 1 H2O2; lane 4, 3.6 mmol l 1 H2O2; lane 5, 6.0 mmol l 1 H2O2; lane 6, 0.25 lg ml 1 AmB; lane 7, 0.5 lg ml 1 AmB; lane 8, 1.0 lg ml 1 AmB.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Living cells have the ability to undergo programmed cell death under certain conditions, which is not only restricted to metazoans but also exists in other living organisms including plants, fungi and bacteria.11–14 Apoptosis has great importance in the development and homeostasis of organisms. The
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(a)
(b)
(c)
Figure 3 The fluorescent microscopy of Rhizopus oryzae. After H2O2 or amphotericin B treatment, changes were seen under the microscope. Externalisation of phosphatidylserine and dead cells stained separately by annexin V-FITC and propidium iodide (a, b). Accumulation of DNA breaks (revealed by TUNEL-positive staining) (c).
(a)
(b)
(c)
Figure 4 Flow cytometry of Rhizopus oryzae cells stained with
DHR123 and propidium iodide (PI). Cells after exposure to various concentrations of H2O2 (a) and amphotericin B (AmB; b) for 3 h were stained with DHR123 and PI and then analysed by flow cytometry. The percentages of cells that are apoptotic (blue bars) and necrotic (red bars) are shown. (c) FL1 LOG, DHR123 staining; FL3 LOG, PI staining.
apoptotic-like phenotype has now been described in a range of fungi, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, A. fumigatus, Aspergillus nidulans, Mucor racemosus and R. arrhizus.7,9,15–20 Similarly, our result
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demonstrated that the apoptotic-like phenotype can also be observed in R. arrhizus. H2O2 and AmB are exogenous triggers that can be provided externally in the form of chemical or physical stress and have been studied in several fungi.7,17 The optimal apoptosis-inducing concentrations of H2O2 and AmB differ in C. albicans and A. fumigatus. Exposure of C. albicans to 5–10 mmol l 1 H2O2 or 4–8 lg ml 1 AmB produced cellular changes reminiscent of mammalian apoptosis.17 However, treated with much lower levels of H2O2 (0.1 mmol l 1) or AmB (0.5 lg ml 1), A. fumigatus showed loss of cell viability and death associated with a number of phenotypic changes characteristic of apoptosis. In our study, the concentrations of H2O2 and AmB that induced R. arrhizus manifestations of the apoptotic-like phenotype were between C. albicans and A. fumigatus. Under 3.6 mmol l 1 of H2O2 and 1 lg ml 1 of AmB, most of the cells expressed the apoptotic-like phenotype. Dose variability of H2O2 and AmB existed among different fungi. We first detected the early marker of apoptosis in R. arrhizus after treatment with these two triggers and used the annexin V-FICT/PI staining assay to distinguish cells in early apoptosis from normal cells or dead PI-positive cells using fluorescence microscopy. The report indicated increased PI staining and decreased annexin V staining at higher concentrations of both triggers, which revealed membrane disintegration and necrotic cell death. A DNA ladder indicating the late stage of apoptosis in many mammalian cells21 and M. racemosus19 was not detected in this study. However, a DNA ladder is not a stable apoptotic characteristic in all fungi. In A. fumigatus, DNA smearing was found after treatment with H2O2 and AmB as well as in A. nidulans after
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 25–30
Apoptotic-like phenotype in Rhizopus arrhizus
treatment with phytosphingosine.22,23 DNA smearing rather than a ladder was demonstrated by agarose electrophoresis in R. arrhizus after treatment with H2O2 and AmB. The apoptotic-like phenotype of R. arrhizus was also indentified using the TUNEL assay, which is more sensitive than DNA agarose electrophoresis for analysing apoptotic DNA fragmentation. Microscopic images revealed the presence of significant green fluorescence in the cells treated by high but non-fungicidal concentrations of the two triggers, but minimal fluorescence was seen under low concentrations. These phenomena were also reported in many other fungi, such as S. cerevisiae treated with H2O2 and acetic acid, C. albicans treated with farnesol, A. fumigatus treated with H2O2 and AmB and A. fumigatus in the stationary phase.7,9,23,24 DHR123/PI double-staining by flow cytometry can better explain the change of apoptotic or dead cells. In our study, an apoptotic phenotype can be induced by low but toxic concentrations of both triggers through ROS accumulation within cells, whereas dead cells stained with PI increased after treatment with high concentrations of the triggers. These findings indicate that treatment with low concentrations of both triggers can induce an apoptotic-like phenotype through ROS accumulation and ultimately cause death under continued accumulation with increased PI-positive staining. It is well known that ROS plays a major role in signalling and/or effector functions in apoptosis.25 The production of ROS in apoptotic cells has been examined in other fungal cells, including C. albicans, S. cerevisiae and A. nidulans.18,26,27 ROS accumulation has also been demonstrated in many fungal and mammalian cells and played a central role in the induction of apoptosis.6,7,28 This study indicated that both H2O2 and AMB could induce the apoptotic-like phenotype in R. arrhizus, which might be usefully exploited in the search for and design of novel therapies in the future.
Acknowledgments This work was supported by National Natural Science Foundation (81371783) from the National Natural Science Foundation of China.
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Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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