International Journal of
Molecular Sciences Article
Deletion of afpab1 Causes Increased Sensitivity to Oxidative Stress and Hypovirulence in Aspergillus fumigatus Dongyang Wang, Shunan Wang, Dan He, Song Gao, Baiji Xue and Li Wang * Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China; [email protected] (D.W.); [email protected] (S.W.); [email protected] (D.H.) [email protected] (S.G.); [email protected] (B.X.) * Correspondence: [email protected]; Tel.: +86-431-8561-9486 Academic Editors: Charles A. Collyer and Patrick C. Y. Woo Received: 31 May 2016; Accepted: 25 October 2016; Published: 29 October 2016
Abstract: Aspergillus fumigatus AFPAB1 is the ortholog of the Aspergillus oryzae cytoplasmic messenger ribonucleoprotein granules AOPAB1 that function to depress the initiation of translation during stress. A. fumigatus can regulate its cellular physiology in response to environmental stresses, but there has been no research on Pab1 in A. fumigatus. The associated gene afpab1 was replaced with a hygromycin-selectable marker to generate the strain ∆afpab1. Phenotypic analysis showed that the ∆afpab1 grew more weakly than the wild-type strain. Also the germination rate of ∆afpab1 was decreased when exposed to oxidative stress. The morphology of ∆afpab1 spores also showed great changes. The killing rate of ∆afpab1 by RAW264.7 murine macrophage cells was increased, and the reactive oxygen species (ROS) scavenging ability of ∆afpab1 was decreased. Pathogenicity testing showed that the deletion strain had decreased virulence. Therefore, we conclude that afpab1 activity is correlated with susceptibility to oxidative stress, and deletion of afpab1 from A. fumigatus possibly leads to observed hypovirulence in an immunosuppressed mouse model. Keywords: Aspergillus fumigatus; Agrobacterium tumefaciens; afpab1; oxidative stress; survival factor
1. Introduction Aspergillus fumigatus (A. fumigatus) is a widespread saprophytic fungus that produces large numbers of asexual spores. It is an opportunistic pathogen with a worldwide distribution. Cases of A. fumigatus infection have increased markedly over recent decades. It has become the most important aerial fungal pathogen of humans, causing disorders dependent on the host immune response [1]. In healthy hosts, spores are usually harmless because they are eliminated by the human immune system [2]. However, in immunocompromised individuals, A. fumigatus conidia can cause aspergillosis. Research into A. fumigatus virulence has therefore become increasingly important. The external environment of A. fumigatus in infection conditions is different to that in its normal growth niche [3]. The ability to sense external stress and respond appropriately is vital for the survival of fungal cells in stress conditions, and helps A. fumigatus to establish successful invasive aspergillosis. Global translational inhibition is one of the main mechanisms by which eukaryotic cells resist external stress [4]. Eukaryotic cells shut down the transcription process and accumulate stalled translation components in cytoplasmic messenger ribonucleoprotein granules, which typically occur when cells are exposed to stress and form in response to the inhibition of translation initiation [5]. Non-translated mRNA and related proteins are collected from polysomes into cytoplasmic messenger ribonucleoprotein granules when cells encounter stress conditions [6,7]. It has been speculated that
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speculated messenger that cytoplasmic messengergranules ribonucleoprotein granules function as transcription cytoplasmic ribonucleoprotein function as transcription repositories that regulate repositories that regulate the translatability stability of mRNAs the translatability and stability of mRNAs inand stress conditions [8,9]. in stress conditions [8,9]. Pab1 is is aa cytoplasmic cytoplasmic messenger messenger ribonucleoprotein ribonucleoproteingranule granulethat thathas has been been widely widely studied studied in in yeast yeast Pab1 cells and is conserved in eukaryotes [10]. A recent study has shown that Pab1 exists in the filamentous cells and is conserved in eukaryotes [10]. A recent study has shown that Pab1 exists in the filamentous fungus Aspergillus Aspergillus.oryzae oryzae[5]. [5].The Thefunction function of of Pab1 Pab1 in in A. A. oryzae oryzae is is to to resist resist external external stresses stresses including including fungus oxidative stress, temperature stress and endoplasmic reticulum (ER) stress (protein folding in the the ER ER oxidative stress, temperature stress and endoplasmic reticulum (ER) stress (protein folding in is impaired impaired resulting resulting in in the the accumulation accumulation of of misfolded misfolded proteins) proteins) [11]. [11]. However, However, there there has has been been no no is research on Pab1 in A. fumigatus, and the ability to cope with external stress is one of the most research on Pab1 in A. fumigatus, and the ability to cope with external stress is one of the most important important survival of A. fumigatus. Therefore, in thewe present study we A. knocked outgene the survival factors of A.factors fumigatus. Therefore, in the present study knocked out the fumigatus A. fumigatus gene of afpab1, a homolog of investigate A. oryzae aopab1, itsof role in the response of afpab1, a homolog A. oryzae aopab1, to its roletoininvestigate the response A. fumigatus to stress, A. fumigatus to stress, and to determine whether it has any effect on pathogenicity. and to determine whether it has any effect on pathogenicity. 2. Results 2. Results 2.1. 2.1. Isolation and Sequence Analysis Analysis of of afpab1 afpab1 Homolog Homolog from from A. A. fumigatus fumigatus A A BLAST BLAST search search with with A. A. oryzae aopab1 as the query identified a predicted cytoplasmic messenger messenger ribonucleoprotein ribonucleoprotein granule protein afpab1 afpab1 in in the the A. A. fumigatus fumigatus genome. genome. The coding coding sequence sequence was was identified cloning and andsequencing sequencingcDNAs cDNAsusing usingRT-PCR. RT-PCR.The Theputative putative afpab1 gene was located identified by cloning afpab1 gene was located on on chromosome 1, and consisted of 2396 bp (with 134 bp in two introns and 2262 bp in three exons), chromosome 1, and consisted of 2396 bp (with 134 bp in two introns and 2262 bp in corresponding corresponding to 753 753 amino amino acid acid residues. residues. The The phylogenetic phylogenetic relationships relationships among pab1 genes genes from from different fungi were analyzed and the results suggest that the pab1 gene is present in a variety of fungi different fungi were analyzed and the results suggest that the pab1 gene is present in a variety of of different species species (Figure (Figure 1). These1).results that the afpab1 gene had significant homology fungi of different Thesedemonstrate results demonstrate that the afpab1 gene had significant with that ofwith A. clavatus, and minimal similarity the gene to from graminearum. homology that of A. clavatus, and minimaltosimilarity theFusarium gene from Fusarium graminearum.
Figure 1. 1. In silico analysis of of the the afpab1 afpab1 in in A. A. fumigatus. fumigatus. Protein alignment alignment was was performed performed with with Figure ClustalX 2 and the phylogenetic tree was created via MEGA 5.2 to examine afpab1 homologs from ClustalX and the phylogenetic tree was created via MEGA 5.2 to examine afpab1 homologs different fungifungi (Aspergillus. fumigatus, AO090003000927; from different (Aspergillus fumigatus,AFUA_1G04190; AFUA_1G04190;Aspergillus. Aspergillus oryzae, oryzae, AO090003000927; ACLA_030250 ; Aspergillus. flavus, AFLA_028910A; niger, Aspergillus.clavatus, clavatus, Aspergillus ACLA_030250; Aspergillus flavus, AFLA_028910A; AspergillusAspergillus. niger, An01c0080; An01c0080;terreus, Aspergillus. terreus, ATEG_03789; Aspergillus. nidulans, AN4000.2; Penicillium. marneffei, Aspergillus ATEG_03789; Aspergillus nidulans, AN4000.2; Penicillium marneffei, PMAA_054700; PMAA_054700; Fusarium. graminearum, FGRRES_08421M). Fusarium graminearum, FGRRES_08421M).
2.2. Deletion Deletion and and Complementation Complementation of of afpab1 afpab1 in in A. A. fumigatus fumigatus 2.2. Todetermine determinethe the function function of of Afpab1 Afpab1 from from A. A. fumigatus, fumigatus, aa ∆afpab1 Δafpab1 deletion deletion strain strain was was constructed constructed To by replacing the entire gene with hph (Figure 2a). The hph gene could be amplified from Δafpab1 using by replacing the entire gene with hph (Figure 2a). The hph gene could be amplified from ∆afpab1 using the primers Hphf/Hphr, but could not be amplified from the WT. Conversely, the afpab1 gene could the primers Hphf/Hphr, but could not be amplified from the WT. Conversely, the afpab1 gene could be amplified amplifiedfrom fromthe theWT, WT,but but from Δafpab1, using specific primers (Figure A schematic be notnot from ∆afpab1, using specific primers (Figure 2b). 2b). A schematic and and the results of Southern hybridization are shown in Figure 2b and c. The Δafpab1 strain showed the results of Southern hybridization are shown in Figure 2b,c. The ∆afpab1 strain showed a 3.4 kb a 3.4 kb hybridization was notin present in complementation WT and complementation hybridization band thatband wasthat not present WT and strain. strain.
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Figure 2. Deletion and reconstitution of afpab1of A. fumigatus. (a) The constructed schematic of Δafpab1
Figure 2. Deletion and reconstitution of afpab1 of A. fumigatus. (a) The constructed schematic of and afpab1C in A. fumigatus; (b) Molecular analysis of WT, Δafpab1 and afpab1C. Hph gene can be ∆afpab1 and afpab1C in A. fumigatus; (b) Molecular analysis of WT, ∆afpab1 and afpab1C. Hph gene can amplified from Δafpab1 (I) but cannot be amplified from WT or afpab1C, andschematic afpba1 gene can be Figure 2. Deletion and reconstitution of afpab1of A. fumigatus. (a) The constructed of Δafpab1 be amplified from ∆afpab1 (I) but cannot be amplified from WT or afpab1C, and afpba1 gene can be amplified from WTfumigatus; and afpab1C which cannot be amplified from Δafpab1. Genomic DNA digested and afpab1C in A. (b)(II) Molecular analysis of WT, Δafpab1 and afpab1C. Hph gene can be amplified fromwas WTprobed and afpab1Can(II) which cannotofbeafpab1 amplified from ∆afpab1. Genomic DNA digested with Pst I from to WT detect Δafpab1, WT, and afpab1C (III); amplified Δafpab1with (I) butupstream cannot beregion amplified from orthe afpab1C, and afpba1 gene can be withamplified PstSouthern I wasfrom probed withafpab1C anschematic upstream region of afpab1 to detect WT, DNA and afpab1C (c) hybridization diagram. M: DNA molecular size∆afpab1, marker Trans 2K plus II; 1:(III); WT and (II) which cannot be amplified from the Δafpab1. Genomic digested (c) Southern schematic diagram. M:ofDNA size marker Trans 2K plus II; 1: WT; WT; Pst 2: Δafpab1; 3: afpab1C. (Green: upstream fragment ofmolecular afpab1 gene, Red: hph gene, Blue: downstream with Ihybridization was probed with an upstream region afpab1 to detect the Δafpab1, WT, and afpab1C (III); 2: ∆afpab1; 3: afpab1C. (Green:schematic upstream fragment afpab1 gene, Red: hph gene, Blue: fragment of afpab1 gene). (c) Southern hybridization diagram. M: of DNA molecular size marker Trans 2K downstream plus II; 1: fragment afpab1 3: gene). WT; 2:of Δafpab1; afpab1C. (Green: upstream fragment of afpab1 gene, Red: hph gene, Blue: downstream 2.3. Growth Phenotype fragmentand of afpab1 gene).of the Δafpab1 Strain
2.3. Growth Phenotype of the disruption ∆afpab1 Strain Toand determine whether of afpab1 contributed to any growth defects, the growth of
2.3. Growth and Phenotype of the Δafpab1 Strain Δafpab1 was examined. were noof significant variations intothe growth of the deletion strain. In of To determine whetherThere disruption afpab1 contributed any growth defects, the growth addition, there was no difference in colony morphology by direct observation or under light To determine whether afpab1 contributed to in any growth defects, growth strain. of ∆afpab1 was examined. Theredisruption were no of significant variations the growth of thethe deletion microscopy S1–S3). Δafpab1 was (Figures examined. There were no significant variations in the growth of the deletion strain. In In addition, there was no difference in colony morphology by direct observation or under light addition, there was no difference in colony morphology by direct observation or under light microscopy (Figures 2.4. Δafpab1 ShowsS1–S3). Increased microscopy (Figures S1–S3). Sensitivity to Oxidative Stress NoShows differences wereSensitivity detected between Δafpab1 and the WT under osmotic, ER or temperature 2.4. ∆afpab1 Increased to Oxidative Stress
2.4. Δafpab1 Shows(Shown IncreasedinSensitivity toInterestingly, Oxidative Stress stress conditions Figure S4). the Δafpab1 showed greater sensitivity to H2O2 No differences were detected between the WT under osmotic, ER or temperature and No menadione than thedetected WT andbetween afpab1C ∆afpab1 (Figuresand 3–5).the We also found under ER scanning electron differences were Δafpab1 and WT under osmotic, or temperature stress conditions (Shown in Figure S4). Interestingly, the ∆afpab1 showed greater sensitivity to2O H2 2 O2 microscope (SEM) that the were smoother (fewer showed ornaments), depressed, deformed, stress conditions (Shown in spore Figuresurfaces S4). Interestingly, the Δafpab1 greater sensitivity to H and and menadione than thethe WT and 3–5). We also6). foundunder under scanning electron and menadione irregularly shaped compared with the (Figures WT and afpab1C (Figure than WT andafpab1C afpab1C (Figures 3–5). We also found scanning electron
microscope (SEM) that thethe spore (fewerornaments), ornaments), depressed, deformed, microscope (SEM) that sporesurfaces surfaceswere were smoother smoother (fewer depressed, deformed, and and irregularly shaped compared with (Figure6). 6). irregularly shaped compared withthe theWT WTand and afpab1C afpab1C (Figure
Figure 3. Plates assay. The A. fumigatus WT, Δafpab1 and afpab1C strains were dot inoculated onto PDA at a gradient of 10-fold dilution for 36–48 h at 28 °C. (a) Medium containing 1.5 mM H2O2; (b) Medium containing 10 µM menadione. Figure 3. Plates assay. The A. fumigatus WT, Δafpab1 and afpab1C strains were dot inoculated onto Figure 3. Plates assay. The A. fumigatus WT, ∆afpab1 and afpab1C strains were dot inoculated onto PDA PDA at a gradient of 10-fold dilution for 36–48 h at 28 °C. (a) Medium containing 1.5 mM H2O2; at a gradient of 10-fold dilution for 36–48 h at 28 ◦ C. (a) Medium containing 1.5 mM H2 O2 ; (b) Medium (b) Medium containing 10 µM menadione.
containing 10 µM menadione.
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Figure 4. Sensitivity of WT, Δafpab1 Δafpab1 and and afpab1C afpab1C to to H H22O O2 stress conditions. conditions. Five microliters microliters of conidial Figure 4. Sensitivity of WT, ∆afpab1 and afpab1C to H stress conditions. Five Five microliters of conidial 2 22 stress 6 cells/mL) were inoculated onto PDA. Cultures were incubated at 28 °C for saline suspensions (1 × 10 6 6 suspensions (1 (1 × × 10 saline suspensions 10 cells/mL) cells/mL)were wereinoculated inoculatedonto ontoPDA. PDA.Cultures Cultures were were incubated at 28 ◦°C C for Figure 4. Sensitivity of WT, Δafpab1 and afpab1C to H2O2 stress conditions. Five microliters of conidial 4 days. 4 days. 6 saline suspensions (1 × 10 cells/mL) were inoculated onto PDA. Cultures were incubated at 28 °C for 4 days.
Figure 5. 5. Sensitivity Sensitivity of of WT, WT, Δafpab1 Δafpab1 and and afpab1C afpab1C to to menadione menadione stress stress conditions. conditions. Five Five microliters microliters of of Figure Figure 5. Sensitivity of of WT, ∆afpab1 and afpab1C to menadione stressconditions. conditions. Five microliters of Figure 5. Sensitivity WT, Δafpab1 and afpab1C to menadione stress Five microliters of 6 cells/mL) were inoculated onto PDA. Cultures were incubated at conidial saline suspensions (1 × 10 were onto PDA. Cultures were incubated at conidial saline saline suspensions suspensions (1 (1 × × 1066 6cells/mL) conidial wereinoculated inoculatedonto ontoPDA. PDA. Cultures were incubated cells/mL) were inoculated Cultures were incubated at at conidial saline suspensions (1 ×10 10 cells/mL) 28 °C for 4 days. 28 ◦°C 44 days. 28 C for for days. 28 °C for 4 days.
Figure 6. The coverslips covered on PDA cubes with spores were transferred and prefixed with 2%
Figure 6. The Thecoverslips coverslipscovered coveredononPDA PDA cubes with spores were transferred prefixed Figure 6. 6. cubes with spores were transferred andand prefixed withwith 2% Figure The coverslips covered on PDA with spores were transferred and prefixed with glutaraldehyde at room temperature for cubes 12 h and then metalized with gold and investigated by 2% 2% glutaraldehyde at room temperature for 12 h and then metalized with gold and investigated glutaraldehyde at room temperature for 12 h and then metalized with gold and investigated by glutaraldehyde at room temperature for 12containing h and then metalized with gold and investigated Medium containing 10 µM by scanning electron microscope. (a) Medium 1.5 mM H2O2; (b) by scanning electron microscope. (a) Medium containing 1.5 H mM containing 2 OMedium 2 ; (b) Medium 2O2; H (b) containing 10 µM scanning electron microscope. (a) Medium containing 1.5 mM scanning electron microscope. (a) Medium containing 1.5 mM H2O2; (b) Medium containing 10 µM menadione. 10 µM menadione. menadione. menadione. As shown in Figure 7, Δafpab1 displayed a significantly reduced rate of germination under Δafpab1 displayed As in Figure ∆afpab1 of under oxidative stress. 127, h incubation, there wasa difference in reduced the controlrate group. The germination As shown shown in After Figure 7, Δafpab1 displayed anosignificantly significantly reduced rate of germination germination under After 12 h incubation, there was no difference oxidative stress. in the control group. The germination rates of WT, Δafpab1, and afpab1C were 77%, 56%, and 76% when medium contained 3 mM H 2O2. oxidative stress. After 12 h incubation, there was no difference in the control group. The germination Under 15 ∆afpab1, µM menadione, the were germination ratesand of 76% thesewhen strains were 74%, 50%, and 75%, Δafpab1, and afpab1C afpab1C were 77%, 56%, 56%, and 76% when medium contained mM H222O rates of and 77%, medium contained 33 mM H O222... rates of WT, WT, Δafpab1, and afpab1C were 77%, 56%, and 76% when medium contained mM H respectively. 15µM µM menadione, germination rates ofstrains these were strains were 50%,respectively. and 75%, Under 15 menadione, the the germination rates of these 74%, 50%,74%, and 75%,
Under 15 µM menadione, the germination rates of these strains were 74%, 50%, and 75%, respectively. respectively.
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Figure 7. Germination rates of of afpab1 afpab1 deletion deletion strain Figure strain compared compared with with WT WT and and afpab1C afpab1C under under control, control, Figure 7. 7. Germination Germination rates mM H 2O 2 or 15 µM menadione. Data shown (means ± SD) are from three independent experiments 333 mM H O or 15 µM menadione. Data shown (means ± SD) are from three independent experiments mM H22 2 or 15 µM ± SD) are from three experiments for each strain. (a) Control; Control; (b) H for (c) Menadione. Menadione. for each each strain. strain. (a) Control; (b) (b) H H222O O222;;; (c) (c) Menadione.
2.5. ROS Production Production of of Δafpab1 Is Increased When Exposed to H O22 2.5. ROS of ∆afpab1 Δafpab1 Is Is Increased Increased When When Exposed Exposed to to H H222O 2.5. 2 ROS can can convert DCFH into 2,7-dichlorofluorescein (DCF), and and DCF DCF is is fluorescent. fluorescent. can convert convert DCFH DCFH into into 2,7-dichlorofluorescein 2,7-dichlorofluorescein (DCF), fluorescent. However, However, ROS DCFH-DA are often used in animal cells or tissues, but are rarely used in fungi cells [12]. Animal cells DCFH-DA are are often often used used in in animal animal cells cells or or tissues, tissues, but but are are rarely rarely used used in in fungi fungi cells cells [12]. [12]. Animal Animal cells cells DCFH-DA do not have cell walls, but fungal cells do. We therefore prepared protoplasts to measure the not have have cell cell walls, walls, but but fungal fungal cells cells do. do. We measure the the do not We therefore prepared protoplasts to measure fluorescence of each strain. Enzymatic hydrolysis was carried out under optimum conditions with each strain. strain. Enzymatic hydrolysis was carried carried out out under under optimum optimum conditions conditions with with fluorescence of each mg/mL ◦ Cfor mg/mL lallzyme, lallzyme, mg/mL cellulase, cellulase, and mg/mL helicase at 28 °C for 150 min. Green fluorescence 555 mg/mL lallzyme, 11 mg/mL mg/mL cellulase, and and 11 mg/mL mg/mLhelicase helicaseat at28 28 °C for150 150 min. min. Green Green fluorescence was observed with confocal laser micro-scopy. The Δafpab1 deletion strain had stronger green was observed observed with with confocal confocal laser laser micro-scopy. micro-scopy. The Δafpab1 stronger green green was ∆afpab1 deletion strain had stronger fluorescence than the WT and afpab1C when treated with 3 mM H 2O2 for 1 h (Figure 8). Analysis with fluorescence than the WT and afpab1C when treated with 3 mM H 2 O 2 for 1 h (Figure 8). Analysis with fluorescence than the WT and afpab1C when treated with 3 mM H2 O2 for (Figure 8). with Image-Pro Plus 6.0 showed that the Δafpab1 fluorescence intensity increased nearly twice as much as Image-Pro Plus Plus 6.0 6.0 showed showed that that the ∆afpab1 Δafpab1 fluorescence fluorescence intensity intensity increased increased nearly nearly twice twice as as much much as as Image-Pro that of WT and afpab1C under treatment with 3 mM H 2O2 (Figure 9). that of of WT WT and and afpab1C afpab1C under under treatment treatment with with 33 mM mM H H22O22 (Figure 9). that
Figure 8. DCFH-DA staining used to determine the ROS levels in the protoplasts treated with Figure 8. 8. DCFH-DA DCFH-DA staining staining was was used used to to determine determine the the ROS ROS levels levels in in the the protoplasts protoplasts treated treated with with Figure was 33 mM H 2O2 for 1 h. The conidia were observed using fluorescence microscopy to determine the ROS mM H 2O2 for 1 h. The conidia were observed using fluorescence microscopy to determine the ROS 3 mM H2 O2 for 1 h. The conidia were observed using fluorescence microscopy to determine the ROS levels; Control group without H O levels; Control Control group group without without H H22O O22.. Bar, Bar, 10 10 µm. µm. levels; 2 2 . Bar, 10 µm.
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Figure 9. The ROS fluorescence intensity was analyzed by software Image-Pro Plus 6.0. The Figure 9. The ROS fluorescence intensity was analyzed by software Image-Pro Plus 6.0. Figure 9. The ROS fluorescence was group, analyzed Image-Pro Plusof6.0. The fluorescence intensity was equal inintensity the control butbythesoftware fluorescence intensity Δafpab1 Thefluorescence fluorescenceintensity intensitywas was equalininthe thecontrol controlgroup, group, but thefluorescence fluorescence intensity of ∆afpab1 intensity Δafpab1 increased nearly two fold to equal that of WT and afpab1C afterbut thethe treatment of 3 mM H2O2.of Data were increased nearly two fold to to that of33mM mMHH . Data were 2 2O increased nearly fold thatofofWT WTand andafpab1C afpab1C after after the the treatment treatment of 2O . 2Data were shown as the meantwo ± SD, p < 0.05. shown as the mean ± SD, p < 0.05. shown as the mean ± SD, p < 0.05.
2.6. Killing Rate for Δafpab1 Is Increased in RAW264.7 Cells Killing Rate Δafpab1 IncreasedininRAW264.7 RAW264.7 Cells Cells 2.6. 2.6. Killing Rate for for ∆afpab1 Is Is Increased Immune cells secrete ROS to kill conidia. A macrophage–conidia co-culturing method was used Immune cells secrete killconidia. conidia. A macrophage–conidia method used cells secrete totokill macrophage–conidia co-culturing method was used to Immune examine the ability of ROS A.ROS fumigatus to resistA immune cell oxidative co-culturing stress. As shown in was Figure 10, to examine the ability of A. fumigatus to resist immune cell oxidative stress. As shown in Figure 10, 10, to examine the ability of A. fumigatus to resist immune cell oxidative stress. As shown in Figure the killing rate for Δafpab1 was 45%. By contrast, only 23% and 25% of conidia from WT and afpab1C the killing Δafpab1 was 45%.By Bycontrast, contrast, only only 23% from WT and afpab1C thewere killing raterate forfor ∆afpab1 was 45%. 23% and and25% 25%ofofconidia conidia from WT and afpab1C killed. were killed. were killed.
Figure of by of conidia obtained from WT, Δafpab1, Δafpab1, and Figure 10.Percentage Percentage of killing killing by macrophage macrophage cells of conidia obtained WT, and Figure 10.10. Percentage of killing by macrophage cellscells of conidia obtained fromfrom WT, ∆afpab1, and afpab1C. afpab1C. The killing rate for each strain was determined from three independent experiments. Data Thefor killing for was eachdetermined strain was determined three independent experiments. The afpab1C. killing rate eachrate strain from three from independent experiments. Data wereData shown were shown were shown asthe the mean SD,pp
Molecular Sciences Article
Deletion of afpab1 Causes Increased Sensitivity to Oxidative Stress and Hypovirulence in Aspergillus fumigatus Dongyang Wang, Shunan Wang, Dan He, Song Gao, Baiji Xue and Li Wang * Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China; [email protected] (D.W.); [email protected] (S.W.); [email protected] (D.H.) [email protected] (S.G.); [email protected] (B.X.) * Correspondence: [email protected]; Tel.: +86-431-8561-9486 Academic Editors: Charles A. Collyer and Patrick C. Y. Woo Received: 31 May 2016; Accepted: 25 October 2016; Published: 29 October 2016
Abstract: Aspergillus fumigatus AFPAB1 is the ortholog of the Aspergillus oryzae cytoplasmic messenger ribonucleoprotein granules AOPAB1 that function to depress the initiation of translation during stress. A. fumigatus can regulate its cellular physiology in response to environmental stresses, but there has been no research on Pab1 in A. fumigatus. The associated gene afpab1 was replaced with a hygromycin-selectable marker to generate the strain ∆afpab1. Phenotypic analysis showed that the ∆afpab1 grew more weakly than the wild-type strain. Also the germination rate of ∆afpab1 was decreased when exposed to oxidative stress. The morphology of ∆afpab1 spores also showed great changes. The killing rate of ∆afpab1 by RAW264.7 murine macrophage cells was increased, and the reactive oxygen species (ROS) scavenging ability of ∆afpab1 was decreased. Pathogenicity testing showed that the deletion strain had decreased virulence. Therefore, we conclude that afpab1 activity is correlated with susceptibility to oxidative stress, and deletion of afpab1 from A. fumigatus possibly leads to observed hypovirulence in an immunosuppressed mouse model. Keywords: Aspergillus fumigatus; Agrobacterium tumefaciens; afpab1; oxidative stress; survival factor
1. Introduction Aspergillus fumigatus (A. fumigatus) is a widespread saprophytic fungus that produces large numbers of asexual spores. It is an opportunistic pathogen with a worldwide distribution. Cases of A. fumigatus infection have increased markedly over recent decades. It has become the most important aerial fungal pathogen of humans, causing disorders dependent on the host immune response [1]. In healthy hosts, spores are usually harmless because they are eliminated by the human immune system [2]. However, in immunocompromised individuals, A. fumigatus conidia can cause aspergillosis. Research into A. fumigatus virulence has therefore become increasingly important. The external environment of A. fumigatus in infection conditions is different to that in its normal growth niche [3]. The ability to sense external stress and respond appropriately is vital for the survival of fungal cells in stress conditions, and helps A. fumigatus to establish successful invasive aspergillosis. Global translational inhibition is one of the main mechanisms by which eukaryotic cells resist external stress [4]. Eukaryotic cells shut down the transcription process and accumulate stalled translation components in cytoplasmic messenger ribonucleoprotein granules, which typically occur when cells are exposed to stress and form in response to the inhibition of translation initiation [5]. Non-translated mRNA and related proteins are collected from polysomes into cytoplasmic messenger ribonucleoprotein granules when cells encounter stress conditions [6,7]. It has been speculated that
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speculated messenger that cytoplasmic messengergranules ribonucleoprotein granules function as transcription cytoplasmic ribonucleoprotein function as transcription repositories that regulate repositories that regulate the translatability stability of mRNAs the translatability and stability of mRNAs inand stress conditions [8,9]. in stress conditions [8,9]. Pab1 is is aa cytoplasmic cytoplasmic messenger messenger ribonucleoprotein ribonucleoproteingranule granulethat thathas has been been widely widely studied studied in in yeast yeast Pab1 cells and is conserved in eukaryotes [10]. A recent study has shown that Pab1 exists in the filamentous cells and is conserved in eukaryotes [10]. A recent study has shown that Pab1 exists in the filamentous fungus Aspergillus Aspergillus.oryzae oryzae[5]. [5].The Thefunction function of of Pab1 Pab1 in in A. A. oryzae oryzae is is to to resist resist external external stresses stresses including including fungus oxidative stress, temperature stress and endoplasmic reticulum (ER) stress (protein folding in the the ER ER oxidative stress, temperature stress and endoplasmic reticulum (ER) stress (protein folding in is impaired impaired resulting resulting in in the the accumulation accumulation of of misfolded misfolded proteins) proteins) [11]. [11]. However, However, there there has has been been no no is research on Pab1 in A. fumigatus, and the ability to cope with external stress is one of the most research on Pab1 in A. fumigatus, and the ability to cope with external stress is one of the most important important survival of A. fumigatus. Therefore, in thewe present study we A. knocked outgene the survival factors of A.factors fumigatus. Therefore, in the present study knocked out the fumigatus A. fumigatus gene of afpab1, a homolog of investigate A. oryzae aopab1, itsof role in the response of afpab1, a homolog A. oryzae aopab1, to its roletoininvestigate the response A. fumigatus to stress, A. fumigatus to stress, and to determine whether it has any effect on pathogenicity. and to determine whether it has any effect on pathogenicity. 2. Results 2. Results 2.1. 2.1. Isolation and Sequence Analysis Analysis of of afpab1 afpab1 Homolog Homolog from from A. A. fumigatus fumigatus A A BLAST BLAST search search with with A. A. oryzae aopab1 as the query identified a predicted cytoplasmic messenger messenger ribonucleoprotein ribonucleoprotein granule protein afpab1 afpab1 in in the the A. A. fumigatus fumigatus genome. genome. The coding coding sequence sequence was was identified cloning and andsequencing sequencingcDNAs cDNAsusing usingRT-PCR. RT-PCR.The Theputative putative afpab1 gene was located identified by cloning afpab1 gene was located on on chromosome 1, and consisted of 2396 bp (with 134 bp in two introns and 2262 bp in three exons), chromosome 1, and consisted of 2396 bp (with 134 bp in two introns and 2262 bp in corresponding corresponding to 753 753 amino amino acid acid residues. residues. The The phylogenetic phylogenetic relationships relationships among pab1 genes genes from from different fungi were analyzed and the results suggest that the pab1 gene is present in a variety of fungi different fungi were analyzed and the results suggest that the pab1 gene is present in a variety of of different species species (Figure (Figure 1). These1).results that the afpab1 gene had significant homology fungi of different Thesedemonstrate results demonstrate that the afpab1 gene had significant with that ofwith A. clavatus, and minimal similarity the gene to from graminearum. homology that of A. clavatus, and minimaltosimilarity theFusarium gene from Fusarium graminearum.
Figure 1. 1. In silico analysis of of the the afpab1 afpab1 in in A. A. fumigatus. fumigatus. Protein alignment alignment was was performed performed with with Figure ClustalX 2 and the phylogenetic tree was created via MEGA 5.2 to examine afpab1 homologs from ClustalX and the phylogenetic tree was created via MEGA 5.2 to examine afpab1 homologs different fungifungi (Aspergillus. fumigatus, AO090003000927; from different (Aspergillus fumigatus,AFUA_1G04190; AFUA_1G04190;Aspergillus. Aspergillus oryzae, oryzae, AO090003000927; ACLA_030250 ; Aspergillus. flavus, AFLA_028910A; niger, Aspergillus.clavatus, clavatus, Aspergillus ACLA_030250; Aspergillus flavus, AFLA_028910A; AspergillusAspergillus. niger, An01c0080; An01c0080;terreus, Aspergillus. terreus, ATEG_03789; Aspergillus. nidulans, AN4000.2; Penicillium. marneffei, Aspergillus ATEG_03789; Aspergillus nidulans, AN4000.2; Penicillium marneffei, PMAA_054700; PMAA_054700; Fusarium. graminearum, FGRRES_08421M). Fusarium graminearum, FGRRES_08421M).
2.2. Deletion Deletion and and Complementation Complementation of of afpab1 afpab1 in in A. A. fumigatus fumigatus 2.2. Todetermine determinethe the function function of of Afpab1 Afpab1 from from A. A. fumigatus, fumigatus, aa ∆afpab1 Δafpab1 deletion deletion strain strain was was constructed constructed To by replacing the entire gene with hph (Figure 2a). The hph gene could be amplified from Δafpab1 using by replacing the entire gene with hph (Figure 2a). The hph gene could be amplified from ∆afpab1 using the primers Hphf/Hphr, but could not be amplified from the WT. Conversely, the afpab1 gene could the primers Hphf/Hphr, but could not be amplified from the WT. Conversely, the afpab1 gene could be amplified amplifiedfrom fromthe theWT, WT,but but from Δafpab1, using specific primers (Figure A schematic be notnot from ∆afpab1, using specific primers (Figure 2b). 2b). A schematic and and the results of Southern hybridization are shown in Figure 2b and c. The Δafpab1 strain showed the results of Southern hybridization are shown in Figure 2b,c. The ∆afpab1 strain showed a 3.4 kb a 3.4 kb hybridization was notin present in complementation WT and complementation hybridization band thatband wasthat not present WT and strain. strain.
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Figure 2. Deletion and reconstitution of afpab1of A. fumigatus. (a) The constructed schematic of Δafpab1
Figure 2. Deletion and reconstitution of afpab1 of A. fumigatus. (a) The constructed schematic of and afpab1C in A. fumigatus; (b) Molecular analysis of WT, Δafpab1 and afpab1C. Hph gene can be ∆afpab1 and afpab1C in A. fumigatus; (b) Molecular analysis of WT, ∆afpab1 and afpab1C. Hph gene can amplified from Δafpab1 (I) but cannot be amplified from WT or afpab1C, andschematic afpba1 gene can be Figure 2. Deletion and reconstitution of afpab1of A. fumigatus. (a) The constructed of Δafpab1 be amplified from ∆afpab1 (I) but cannot be amplified from WT or afpab1C, and afpba1 gene can be amplified from WTfumigatus; and afpab1C which cannot be amplified from Δafpab1. Genomic DNA digested and afpab1C in A. (b)(II) Molecular analysis of WT, Δafpab1 and afpab1C. Hph gene can be amplified fromwas WTprobed and afpab1Can(II) which cannotofbeafpab1 amplified from ∆afpab1. Genomic DNA digested with Pst I from to WT detect Δafpab1, WT, and afpab1C (III); amplified Δafpab1with (I) butupstream cannot beregion amplified from orthe afpab1C, and afpba1 gene can be withamplified PstSouthern I wasfrom probed withafpab1C anschematic upstream region of afpab1 to detect WT, DNA and afpab1C (c) hybridization diagram. M: DNA molecular size∆afpab1, marker Trans 2K plus II; 1:(III); WT and (II) which cannot be amplified from the Δafpab1. Genomic digested (c) Southern schematic diagram. M:ofDNA size marker Trans 2K plus II; 1: WT; WT; Pst 2: Δafpab1; 3: afpab1C. (Green: upstream fragment ofmolecular afpab1 gene, Red: hph gene, Blue: downstream with Ihybridization was probed with an upstream region afpab1 to detect the Δafpab1, WT, and afpab1C (III); 2: ∆afpab1; 3: afpab1C. (Green:schematic upstream fragment afpab1 gene, Red: hph gene, Blue: fragment of afpab1 gene). (c) Southern hybridization diagram. M: of DNA molecular size marker Trans 2K downstream plus II; 1: fragment afpab1 3: gene). WT; 2:of Δafpab1; afpab1C. (Green: upstream fragment of afpab1 gene, Red: hph gene, Blue: downstream 2.3. Growth Phenotype fragmentand of afpab1 gene).of the Δafpab1 Strain
2.3. Growth Phenotype of the disruption ∆afpab1 Strain Toand determine whether of afpab1 contributed to any growth defects, the growth of
2.3. Growth and Phenotype of the Δafpab1 Strain Δafpab1 was examined. were noof significant variations intothe growth of the deletion strain. In of To determine whetherThere disruption afpab1 contributed any growth defects, the growth addition, there was no difference in colony morphology by direct observation or under light To determine whether afpab1 contributed to in any growth defects, growth strain. of ∆afpab1 was examined. Theredisruption were no of significant variations the growth of thethe deletion microscopy S1–S3). Δafpab1 was (Figures examined. There were no significant variations in the growth of the deletion strain. In In addition, there was no difference in colony morphology by direct observation or under light addition, there was no difference in colony morphology by direct observation or under light microscopy (Figures 2.4. Δafpab1 ShowsS1–S3). Increased microscopy (Figures S1–S3). Sensitivity to Oxidative Stress NoShows differences wereSensitivity detected between Δafpab1 and the WT under osmotic, ER or temperature 2.4. ∆afpab1 Increased to Oxidative Stress
2.4. Δafpab1 Shows(Shown IncreasedinSensitivity toInterestingly, Oxidative Stress stress conditions Figure S4). the Δafpab1 showed greater sensitivity to H2O2 No differences were detected between the WT under osmotic, ER or temperature and No menadione than thedetected WT andbetween afpab1C ∆afpab1 (Figuresand 3–5).the We also found under ER scanning electron differences were Δafpab1 and WT under osmotic, or temperature stress conditions (Shown in Figure S4). Interestingly, the ∆afpab1 showed greater sensitivity to2O H2 2 O2 microscope (SEM) that the were smoother (fewer showed ornaments), depressed, deformed, stress conditions (Shown in spore Figuresurfaces S4). Interestingly, the Δafpab1 greater sensitivity to H and and menadione than thethe WT and 3–5). We also6). foundunder under scanning electron and menadione irregularly shaped compared with the (Figures WT and afpab1C (Figure than WT andafpab1C afpab1C (Figures 3–5). We also found scanning electron
microscope (SEM) that thethe spore (fewerornaments), ornaments), depressed, deformed, microscope (SEM) that sporesurfaces surfaceswere were smoother smoother (fewer depressed, deformed, and and irregularly shaped compared with (Figure6). 6). irregularly shaped compared withthe theWT WTand and afpab1C afpab1C (Figure
Figure 3. Plates assay. The A. fumigatus WT, Δafpab1 and afpab1C strains were dot inoculated onto PDA at a gradient of 10-fold dilution for 36–48 h at 28 °C. (a) Medium containing 1.5 mM H2O2; (b) Medium containing 10 µM menadione. Figure 3. Plates assay. The A. fumigatus WT, Δafpab1 and afpab1C strains were dot inoculated onto Figure 3. Plates assay. The A. fumigatus WT, ∆afpab1 and afpab1C strains were dot inoculated onto PDA PDA at a gradient of 10-fold dilution for 36–48 h at 28 °C. (a) Medium containing 1.5 mM H2O2; at a gradient of 10-fold dilution for 36–48 h at 28 ◦ C. (a) Medium containing 1.5 mM H2 O2 ; (b) Medium (b) Medium containing 10 µM menadione.
containing 10 µM menadione.
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Figure 4. Sensitivity of WT, Δafpab1 Δafpab1 and and afpab1C afpab1C to to H H22O O2 stress conditions. conditions. Five microliters microliters of conidial Figure 4. Sensitivity of WT, ∆afpab1 and afpab1C to H stress conditions. Five Five microliters of conidial 2 22 stress 6 cells/mL) were inoculated onto PDA. Cultures were incubated at 28 °C for saline suspensions (1 × 10 6 6 suspensions (1 (1 × × 10 saline suspensions 10 cells/mL) cells/mL)were wereinoculated inoculatedonto ontoPDA. PDA.Cultures Cultures were were incubated at 28 ◦°C C for Figure 4. Sensitivity of WT, Δafpab1 and afpab1C to H2O2 stress conditions. Five microliters of conidial 4 days. 4 days. 6 saline suspensions (1 × 10 cells/mL) were inoculated onto PDA. Cultures were incubated at 28 °C for 4 days.
Figure 5. 5. Sensitivity Sensitivity of of WT, WT, Δafpab1 Δafpab1 and and afpab1C afpab1C to to menadione menadione stress stress conditions. conditions. Five Five microliters microliters of of Figure Figure 5. Sensitivity of of WT, ∆afpab1 and afpab1C to menadione stressconditions. conditions. Five microliters of Figure 5. Sensitivity WT, Δafpab1 and afpab1C to menadione stress Five microliters of 6 cells/mL) were inoculated onto PDA. Cultures were incubated at conidial saline suspensions (1 × 10 were onto PDA. Cultures were incubated at conidial saline saline suspensions suspensions (1 (1 × × 1066 6cells/mL) conidial wereinoculated inoculatedonto ontoPDA. PDA. Cultures were incubated cells/mL) were inoculated Cultures were incubated at at conidial saline suspensions (1 ×10 10 cells/mL) 28 °C for 4 days. 28 ◦°C 44 days. 28 C for for days. 28 °C for 4 days.
Figure 6. The coverslips covered on PDA cubes with spores were transferred and prefixed with 2%
Figure 6. The Thecoverslips coverslipscovered coveredononPDA PDA cubes with spores were transferred prefixed Figure 6. 6. cubes with spores were transferred andand prefixed withwith 2% Figure The coverslips covered on PDA with spores were transferred and prefixed with glutaraldehyde at room temperature for cubes 12 h and then metalized with gold and investigated by 2% 2% glutaraldehyde at room temperature for 12 h and then metalized with gold and investigated glutaraldehyde at room temperature for 12 h and then metalized with gold and investigated by glutaraldehyde at room temperature for 12containing h and then metalized with gold and investigated Medium containing 10 µM by scanning electron microscope. (a) Medium 1.5 mM H2O2; (b) by scanning electron microscope. (a) Medium containing 1.5 H mM containing 2 OMedium 2 ; (b) Medium 2O2; H (b) containing 10 µM scanning electron microscope. (a) Medium containing 1.5 mM scanning electron microscope. (a) Medium containing 1.5 mM H2O2; (b) Medium containing 10 µM menadione. 10 µM menadione. menadione. menadione. As shown in Figure 7, Δafpab1 displayed a significantly reduced rate of germination under Δafpab1 displayed As in Figure ∆afpab1 of under oxidative stress. 127, h incubation, there wasa difference in reduced the controlrate group. The germination As shown shown in After Figure 7, Δafpab1 displayed anosignificantly significantly reduced rate of germination germination under After 12 h incubation, there was no difference oxidative stress. in the control group. The germination rates of WT, Δafpab1, and afpab1C were 77%, 56%, and 76% when medium contained 3 mM H 2O2. oxidative stress. After 12 h incubation, there was no difference in the control group. The germination Under 15 ∆afpab1, µM menadione, the were germination ratesand of 76% thesewhen strains were 74%, 50%, and 75%, Δafpab1, and afpab1C afpab1C were 77%, 56%, 56%, and 76% when medium contained mM H222O rates of and 77%, medium contained 33 mM H O222... rates of WT, WT, Δafpab1, and afpab1C were 77%, 56%, and 76% when medium contained mM H respectively. 15µM µM menadione, germination rates ofstrains these were strains were 50%,respectively. and 75%, Under 15 menadione, the the germination rates of these 74%, 50%,74%, and 75%,
Under 15 µM menadione, the germination rates of these strains were 74%, 50%, and 75%, respectively. respectively.
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Figure 7. Germination rates of of afpab1 afpab1 deletion deletion strain Figure strain compared compared with with WT WT and and afpab1C afpab1C under under control, control, Figure 7. 7. Germination Germination rates mM H 2O 2 or 15 µM menadione. Data shown (means ± SD) are from three independent experiments 333 mM H O or 15 µM menadione. Data shown (means ± SD) are from three independent experiments mM H22 2 or 15 µM ± SD) are from three experiments for each strain. (a) Control; Control; (b) H for (c) Menadione. Menadione. for each each strain. strain. (a) Control; (b) (b) H H222O O222;;; (c) (c) Menadione.
2.5. ROS Production Production of of Δafpab1 Is Increased When Exposed to H O22 2.5. ROS of ∆afpab1 Δafpab1 Is Is Increased Increased When When Exposed Exposed to to H H222O 2.5. 2 ROS can can convert DCFH into 2,7-dichlorofluorescein (DCF), and and DCF DCF is is fluorescent. fluorescent. can convert convert DCFH DCFH into into 2,7-dichlorofluorescein 2,7-dichlorofluorescein (DCF), fluorescent. However, However, ROS DCFH-DA are often used in animal cells or tissues, but are rarely used in fungi cells [12]. Animal cells DCFH-DA are are often often used used in in animal animal cells cells or or tissues, tissues, but but are are rarely rarely used used in in fungi fungi cells cells [12]. [12]. Animal Animal cells cells DCFH-DA do not have cell walls, but fungal cells do. We therefore prepared protoplasts to measure the not have have cell cell walls, walls, but but fungal fungal cells cells do. do. We measure the the do not We therefore prepared protoplasts to measure fluorescence of each strain. Enzymatic hydrolysis was carried out under optimum conditions with each strain. strain. Enzymatic hydrolysis was carried carried out out under under optimum optimum conditions conditions with with fluorescence of each mg/mL ◦ Cfor mg/mL lallzyme, lallzyme, mg/mL cellulase, cellulase, and mg/mL helicase at 28 °C for 150 min. Green fluorescence 555 mg/mL lallzyme, 11 mg/mL mg/mL cellulase, and and 11 mg/mL mg/mLhelicase helicaseat at28 28 °C for150 150 min. min. Green Green fluorescence was observed with confocal laser micro-scopy. The Δafpab1 deletion strain had stronger green was observed observed with with confocal confocal laser laser micro-scopy. micro-scopy. The Δafpab1 stronger green green was ∆afpab1 deletion strain had stronger fluorescence than the WT and afpab1C when treated with 3 mM H 2O2 for 1 h (Figure 8). Analysis with fluorescence than the WT and afpab1C when treated with 3 mM H 2 O 2 for 1 h (Figure 8). Analysis with fluorescence than the WT and afpab1C when treated with 3 mM H2 O2 for (Figure 8). with Image-Pro Plus 6.0 showed that the Δafpab1 fluorescence intensity increased nearly twice as much as Image-Pro Plus Plus 6.0 6.0 showed showed that that the ∆afpab1 Δafpab1 fluorescence fluorescence intensity intensity increased increased nearly nearly twice twice as as much much as as Image-Pro that of WT and afpab1C under treatment with 3 mM H 2O2 (Figure 9). that of of WT WT and and afpab1C afpab1C under under treatment treatment with with 33 mM mM H H22O22 (Figure 9). that
Figure 8. DCFH-DA staining used to determine the ROS levels in the protoplasts treated with Figure 8. 8. DCFH-DA DCFH-DA staining staining was was used used to to determine determine the the ROS ROS levels levels in in the the protoplasts protoplasts treated treated with with Figure was 33 mM H 2O2 for 1 h. The conidia were observed using fluorescence microscopy to determine the ROS mM H 2O2 for 1 h. The conidia were observed using fluorescence microscopy to determine the ROS 3 mM H2 O2 for 1 h. The conidia were observed using fluorescence microscopy to determine the ROS levels; Control group without H O levels; Control Control group group without without H H22O O22.. Bar, Bar, 10 10 µm. µm. levels; 2 2 . Bar, 10 µm.
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Figure 9. The ROS fluorescence intensity was analyzed by software Image-Pro Plus 6.0. The Figure 9. The ROS fluorescence intensity was analyzed by software Image-Pro Plus 6.0. Figure 9. The ROS fluorescence was group, analyzed Image-Pro Plusof6.0. The fluorescence intensity was equal inintensity the control butbythesoftware fluorescence intensity Δafpab1 Thefluorescence fluorescenceintensity intensitywas was equalininthe thecontrol controlgroup, group, but thefluorescence fluorescence intensity of ∆afpab1 intensity Δafpab1 increased nearly two fold to equal that of WT and afpab1C afterbut thethe treatment of 3 mM H2O2.of Data were increased nearly two fold to to that of33mM mMHH . Data were 2 2O increased nearly fold thatofofWT WTand andafpab1C afpab1C after after the the treatment treatment of 2O . 2Data were shown as the meantwo ± SD, p < 0.05. shown as the mean ± SD, p < 0.05. shown as the mean ± SD, p < 0.05.
2.6. Killing Rate for Δafpab1 Is Increased in RAW264.7 Cells Killing Rate Δafpab1 IncreasedininRAW264.7 RAW264.7 Cells Cells 2.6. 2.6. Killing Rate for for ∆afpab1 Is Is Increased Immune cells secrete ROS to kill conidia. A macrophage–conidia co-culturing method was used Immune cells secrete killconidia. conidia. A macrophage–conidia method used cells secrete totokill macrophage–conidia co-culturing method was used to Immune examine the ability of ROS A.ROS fumigatus to resistA immune cell oxidative co-culturing stress. As shown in was Figure 10, to examine the ability of A. fumigatus to resist immune cell oxidative stress. As shown in Figure 10, 10, to examine the ability of A. fumigatus to resist immune cell oxidative stress. As shown in Figure the killing rate for Δafpab1 was 45%. By contrast, only 23% and 25% of conidia from WT and afpab1C the killing Δafpab1 was 45%.By Bycontrast, contrast, only only 23% from WT and afpab1C thewere killing raterate forfor ∆afpab1 was 45%. 23% and and25% 25%ofofconidia conidia from WT and afpab1C killed. were killed. were killed.
Figure of by of conidia obtained from WT, Δafpab1, Δafpab1, and Figure 10.Percentage Percentage of killing killing by macrophage macrophage cells of conidia obtained WT, and Figure 10.10. Percentage of killing by macrophage cellscells of conidia obtained fromfrom WT, ∆afpab1, and afpab1C. afpab1C. The killing rate for each strain was determined from three independent experiments. Data Thefor killing for was eachdetermined strain was determined three independent experiments. The afpab1C. killing rate eachrate strain from three from independent experiments. Data wereData shown were shown were shown asthe the mean SD,pp
