# cspA Influences Biofilm Formation and Drug Resistance in Pathogenic Fungus Aspergillus fumigatus

Authors: Zhongqi Fan; Zhe Li; Zongge Xu; Hongyan Li; Lixiang Li; Cong Ning; Lin Ma; Xiangli Xie; Guangyi Wang; Huimei Yu
Journal: BioMed Research International
 (2015)

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## Abstract

The microbial cell wall plays a crucial role in biofilm formation and drug resistance.cspA encodes a repeat-rich glycophosphatidylinositol-anchored cell wall protein in the pathogenic fungus Aspergillus fumigatus. To determine whether cspA has a significant impact on biofilm development and sensitivity to antifungal drugs in A. fumigatus, a ΔcspA mutant was constructed by targeted gene disruption, and we then reconstituted the mutant to wild type by homologous recombination of a functional cspA gene. Deletion of cspA resulted in a rougher conidial surface, reduced biofilm formation, decreased resistance to antifungal agents, and increased internalization by A549 human lung epithelial cells, suggesting that cspA not only participates in maintaining the integrity of the cell wall, but also affects biofilm establishment, drug response, and invasiveness of A. fumigatus.


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## Body

## 1. Introduction

Aspergillus fumigatus is a major cause of infection in individuals with a compromised immune system, including patients undergoing treatment for leukemia, HIV, or organ transplant, and in those suffering from an underlying disease such as cystic fibrosis [1, 2]. Aspergilloma and invasive aspergillosis are the main forms ofA. fumigatus infection, both of which are characterized by high mortality rates (50–95%) and the germination of conidia and subsequent invasion of mycelia into tissues such as human pulmonary alveoli [3].Under natural conditions, such as in human bodies, most fungal and bacterial pathogens are present as part of a biofilm, which contributes to their decreased response to antibiotics and host immune defenses compared with bacteria in the planktonic state [4]. Severalin vitro studies have shown thatA. fumigatus biofilms usually contain parallel-packed hyphae and that some cultures even contain self-produced extracellular matrix (ECM) [5–7]. Recently, two aspergilloma specimens were dissected [8] and observed to contain hyphae surrounded by ECM. This presentation is clinically recognized as the primary evidence of biofilm formation byA. fumigatus.Despite ample evidence of biofilm formation byA. fumigatus, there is very little research on the mechanisms involved in this process. Among fungi,Candida albicans biofilm formation has been the most intensively studied system. Proteins localized on the cell wall, including Hwp1 and Als3, appear to be closely linked to biofilm formation in this species [9]. It is reasonable to suppose that cell wall proteins also participate in the formation of biofilm byA. fumigatus. Glycophosphatidylinositol- (GPI-) anchored proteins are the main cell wall proteins. The antifungal agent E1210 interrupts GPI biosynthesis and suppresses biofilm formation byC. albicans [10].cspA encodes cell surface protein A, a 433-aa protein containing a putative leader sequence and a specific GPI modification site inA. fumigatus. A previous study also found thatcspA lacked recognizable catalytic domains, and the only homologous gene regions were inAspergillus species. Deletion ofcspA resulted in reduced adhesion to ECM, along with an increase in exposed chitin on the cell wall inA. fumigatus [11]. Therefore, we hypothesized that the GPI-anchored proteincspA may influence biofilm formation through its effects on the cell wall.In this study, we constructed a ΔcspA A. fumigatus strain by targeted gene disruption mediated byAgrobacterium tumefaciens, along with a complemented mutant strain generated by homologous introduction of an intactcspA sequence. Deletion ofcspA changed colony and conidia morphology, reduced biofilm formation, decreased resistance to antifungal agents, and increased internalization by A549 human lung epithelial cells. These findings suggested thatcspA not only participates in maintaining the integrity of the cell wall, but also plays an important role in biofilm establishment, drug resistance, and invasiveness ofA. fumigatus.


## 2. Materials and Methods

### 2.1. Strains and Culture Conditions

A. fumigatus wild type (WT) strain Af293 was used in this study. The WT strain, the ΔcspA mutant, and the complementation strain (cspAC) were cultured in potato dextrose broth (PDB; BD Biosciences) medium and incubated at 37°C. Conidia were harvested from strains grown on potato dextrose agar (PDA) plates.


### 2.2. Construction of the ΔcspA andcspAC Strains ofA. fumigatus

The plasmid used to generate the ΔcspA mutant strain was constructed using theA. fumigatus cspA sequence (locus tag AFUA 3G08990 in GenBank accession NC_007196.1). Based on this sequence, we designed primers to amplify thecspA LB and RB regions. The primers forcspA LB were 5′-GCG-GTA-TTG-TTG-TAA-GGT-CG-3′ and 5′-GTG-GAG-TCG-CTT-GAT-GTT-T-3′. The primers forcspA RB were 5′-GCT-GGT-ATC-TGG-GTT-GTC-AT-3′ and 5′-ACT-TTG-AGC-GTC-TCC-TCT-G-3′. To construct thecspA gene deletion plasmid, thecspA LB and RB regions were amplified fromA. fumigatus genomic DNA. ThecspA LB andcspA RB fragments were ligated into the upstream and downstream regions of the hygromycin B phosphotransferase resistance gene (hph) in the pXEH vector, respectively, generating plasmid pXEH-ΔcspA. AnA. fumigatus Af 293 ΔcspA mutant strain was then generated using the pXEH-ΔcspA vector byA. tumefaciens-mediated transformation (ATMT), as described previously [12].To ensure that the mutant phenotype obtained could be attributed to the desired deletion, thecspA deletion was complemented by integration of the Af 293cspA gene to generate a complementation strain,cspAC. Briefly, a DNA fragment containing thecspA promoter, open reading frame, and terminator was cloned and inserted into the pCB1532 vector containing the phleomycin resistance gene (phl), generating complementation vector pCB1532-cspAC. ThecspAC strain was then generated by ATMT using the complementation vector.


### 2.3. Growth Rate and Morphological Observation

Strains were incubated at 37°C. Conidia were harvested from strains grown on potato dextrose agar (PDA) plates and resuspended in sterilized dH2O to a final concentration of 1 × 107 spores/mL. To measure growth rates, 105 conidia in 10 μL dH2O were spotted onto the center of a plate of PDA and incubated at 37°C for 4 days; then the changes of the color, shape, and diameter of colonies were observed at various time points. In order to compare the microscopic morphology among the stains, they were inoculated on the PDA agar block, after the emergence of the pending hyphae and conidia (about 30 h at 37°C), whose morphology was observed under microscope at 400x magnification.


### 2.4. Crystal Violet Assay

The 96-well plates were inoculated with 100μL of PDB containing 105, 104, and 103 per well and incubated for 20 h at 37°C, and at least 5 replicates of each parameter were adapted. The residual medium was abandoned carefully, and biofilms were washed 3 times with PBS to remove mycelia or conidia floating in the medium. After the extra PBS was aspirated, the biofilms were fixed in formaldehyde for 15 min and washed 3 times with PBS. Biofilms were stained by 0.5% (w/v, methanol as solution) crystal violet for 30 min at room temperature. The solution was discarded. The microplates were soaked in dH2O until the floating color faded. After the rinsing 3 times, 96-well plates dried naturally. Then 100 μL of 95% (v/v) ethanol was added into each well and slightly shaken for 10 min until the crystal violet dissolved uniformly. The biofilm quantification was performed by Microplate Reader (Molecular Devices), and absorbance at a wavelength of 570 nm (A
570) was reported.


### 2.5. Biofilm Formation and Fluorescence Microscopy

To generate anA. fumigatus biofilm, 200 μL of eachA. fumigatus strain culture grown in PDB was inoculated onto sterilized 1 cm2 coverslips arranged in 24-well plates (Wuxi NEST Biotechnology Co., Ltd.). The concentration ofA. fumigatus conidia was 105/mL. Following incubation for 16 h at 37°C, the supernatant was carefully removed and the biofilms were washed three times with PBS. Each well was stained with 150 μL of 20 μM probe FUN1 (Life Technologies), which stains the cytoplasm with a diffusely distributed green fluorescence, for 30 min at room temperature in the dark. Biofilms were washed three times with PBS and then observed using a fluorescence microscope (Olympus, IX71) at 100x magnification.


### 2.6. Scanning Electron Microscopy (SEM)

Biofilm samples of the WT, ΔcspA, andcspAC strains were prepared as for fluorescence microscopy. The coverslips were then prefixed with 2% (wt/vol) glutaraldehyde at 4°C for 10 h and then metalized with gold and observed by scanning electron microscopy.


### 2.7. Quantitative Real-Time PCR (qRT-PCR)

A total of 108 conidia of the WT, ΔcspA, andcspAC strains were incubated in 10 mL of PDB in a rotary shaker at 37°C for 24 h. The mycelia were collected and total RNA was extracted according to the Trizol (Invitrogen) method. cDNA was generated using a Transgen reverse transcription kit according to the manufacturer’s instructions. Levels ofcspA mRNA transcript were then investigated in each of the strains by real-time PCR using the fluorescent reporter SYBR Green (ABI, 4385612) and an ABI 7300 thermocycler (Applied Biosystems). Thegapdh gene was used as an internal control. The primers forcspA were 5′-ATG-ATG-CTC-CAC-CTG-ACC-T-3′, 5′-AAG-TCG-GAA-CCA-GAG-GAT-3′. The primers for GAPDH were 5′-ATT-CCT-TCT-CTC-AAC-GGC-3′, 5′-ACA-ACA-TCG-TCC-TCA-GTG-3′.


### 2.8. Internalization Assay

Human A549 lung epithelial cells were incubated in microwell plates and co-incubated for 48 h at 37°C under 5% CO2 (approximately 8000 cells per well) supplemented with 10% foetal bovine serum (FBS, Hyclone) and RMPI 1640 (GIBCO). Then the medium was aspirated, followed by thorough rinsing three times using sterile PBS. Subsequently, 1 × 105 conidia resuspended were cocultured with A549 to induce internalization for 4 h under the same cultured conditions. Each experimental parameter was repeated three times. Afterwards, the cell monolayers were washed three times with PBS. 1640 plus 10% FBS containing 20 μg/mL nystatin was used to dispose each well for 3.5 h to kill noninternalized conidia. Then the cell monolayers were treated with 100 μL liquid comprising PBS and 0.1% Triton X-100 for 15 min at 37°C to induce cell lysis and the release of internalized conidia. The released conidia were diluted and incubated on PDA at 37°C for 24~36 h. The internalization rate was determined to be the percentage of monocolonies compared to the initial inoculum of conidia.


### 2.9. Antifungal Agents and End-Point Susceptibility Testing

Planktonic cell minimum inhibitory concentrations (PMICs) were examined according to the National Committee for Clinical Laboratory Standards M38-A standard methodology (NCCLS, 2002). The antifungal agents itraconazole (ICZ), amphotericin B (AmB), and 5-fluorocytosine (5-FC) were prepared as stock solutions and diluted in PDB to working concentrations. ICZ and AmB were serially diluted twofold to produce a concentration range of 0.0156–16μg/mL, while the concentration of 5-FC was 0.0625–64 μg/mL. The pathogen was challenged for 24 h at 37°C. Following incubation, all wells were treated with 100 μL of PBS containing 1 μM 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide and 1 μM menadione for 3 h. Absorbance was examined at a wavelength of 490 nm (A
490). Sessile cell minimum inhibitory concentrations (SMICs) were determined as 50% and 90% reduction in A
490 compared with the untreated control.


### 2.10. Statistical Analysis

All experiments were repeated at least three times. The means ± standard deviations of the colony diameters, crystal violet density, and the internalized rate were determined using GraphPad Prism 5 software. Data were analyzed using the repeated measures of SPSS 16.0.P
<
0.05 was considered statistically significant.


## 2.1. Strains and Culture Conditions

A. fumigatus wild type (WT) strain Af293 was used in this study. The WT strain, the ΔcspA mutant, and the complementation strain (cspAC) were cultured in potato dextrose broth (PDB; BD Biosciences) medium and incubated at 37°C. Conidia were harvested from strains grown on potato dextrose agar (PDA) plates.


## 2.2. Construction of the ΔcspA andcspAC Strains ofA. fumigatus

The plasmid used to generate the ΔcspA mutant strain was constructed using theA. fumigatus cspA sequence (locus tag AFUA 3G08990 in GenBank accession NC_007196.1). Based on this sequence, we designed primers to amplify thecspA LB and RB regions. The primers forcspA LB were 5′-GCG-GTA-TTG-TTG-TAA-GGT-CG-3′ and 5′-GTG-GAG-TCG-CTT-GAT-GTT-T-3′. The primers forcspA RB were 5′-GCT-GGT-ATC-TGG-GTT-GTC-AT-3′ and 5′-ACT-TTG-AGC-GTC-TCC-TCT-G-3′. To construct thecspA gene deletion plasmid, thecspA LB and RB regions were amplified fromA. fumigatus genomic DNA. ThecspA LB andcspA RB fragments were ligated into the upstream and downstream regions of the hygromycin B phosphotransferase resistance gene (hph) in the pXEH vector, respectively, generating plasmid pXEH-ΔcspA. AnA. fumigatus Af 293 ΔcspA mutant strain was then generated using the pXEH-ΔcspA vector byA. tumefaciens-mediated transformation (ATMT), as described previously [12].To ensure that the mutant phenotype obtained could be attributed to the desired deletion, thecspA deletion was complemented by integration of the Af 293cspA gene to generate a complementation strain,cspAC. Briefly, a DNA fragment containing thecspA promoter, open reading frame, and terminator was cloned and inserted into the pCB1532 vector containing the phleomycin resistance gene (phl), generating complementation vector pCB1532-cspAC. ThecspAC strain was then generated by ATMT using the complementation vector.


## 2.3. Growth Rate and Morphological Observation

Strains were incubated at 37°C. Conidia were harvested from strains grown on potato dextrose agar (PDA) plates and resuspended in sterilized dH2O to a final concentration of 1 × 107 spores/mL. To measure growth rates, 105 conidia in 10 μL dH2O were spotted onto the center of a plate of PDA and incubated at 37°C for 4 days; then the changes of the color, shape, and diameter of colonies were observed at various time points. In order to compare the microscopic morphology among the stains, they were inoculated on the PDA agar block, after the emergence of the pending hyphae and conidia (about 30 h at 37°C), whose morphology was observed under microscope at 400x magnification.


## 2.4. Crystal Violet Assay

The 96-well plates were inoculated with 100μL of PDB containing 105, 104, and 103 per well and incubated for 20 h at 37°C, and at least 5 replicates of each parameter were adapted. The residual medium was abandoned carefully, and biofilms were washed 3 times with PBS to remove mycelia or conidia floating in the medium. After the extra PBS was aspirated, the biofilms were fixed in formaldehyde for 15 min and washed 3 times with PBS. Biofilms were stained by 0.5% (w/v, methanol as solution) crystal violet for 30 min at room temperature. The solution was discarded. The microplates were soaked in dH2O until the floating color faded. After the rinsing 3 times, 96-well plates dried naturally. Then 100 μL of 95% (v/v) ethanol was added into each well and slightly shaken for 10 min until the crystal violet dissolved uniformly. The biofilm quantification was performed by Microplate Reader (Molecular Devices), and absorbance at a wavelength of 570 nm (A
570) was reported.


## 2.5. Biofilm Formation and Fluorescence Microscopy

To generate anA. fumigatus biofilm, 200 μL of eachA. fumigatus strain culture grown in PDB was inoculated onto sterilized 1 cm2 coverslips arranged in 24-well plates (Wuxi NEST Biotechnology Co., Ltd.). The concentration ofA. fumigatus conidia was 105/mL. Following incubation for 16 h at 37°C, the supernatant was carefully removed and the biofilms were washed three times with PBS. Each well was stained with 150 μL of 20 μM probe FUN1 (Life Technologies), which stains the cytoplasm with a diffusely distributed green fluorescence, for 30 min at room temperature in the dark. Biofilms were washed three times with PBS and then observed using a fluorescence microscope (Olympus, IX71) at 100x magnification.


## 2.6. Scanning Electron Microscopy (SEM)

Biofilm samples of the WT, ΔcspA, andcspAC strains were prepared as for fluorescence microscopy. The coverslips were then prefixed with 2% (wt/vol) glutaraldehyde at 4°C for 10 h and then metalized with gold and observed by scanning electron microscopy.


## 2.7. Quantitative Real-Time PCR (qRT-PCR)

A total of 108 conidia of the WT, ΔcspA, andcspAC strains were incubated in 10 mL of PDB in a rotary shaker at 37°C for 24 h. The mycelia were collected and total RNA was extracted according to the Trizol (Invitrogen) method. cDNA was generated using a Transgen reverse transcription kit according to the manufacturer’s instructions. Levels ofcspA mRNA transcript were then investigated in each of the strains by real-time PCR using the fluorescent reporter SYBR Green (ABI, 4385612) and an ABI 7300 thermocycler (Applied Biosystems). Thegapdh gene was used as an internal control. The primers forcspA were 5′-ATG-ATG-CTC-CAC-CTG-ACC-T-3′, 5′-AAG-TCG-GAA-CCA-GAG-GAT-3′. The primers for GAPDH were 5′-ATT-CCT-TCT-CTC-AAC-GGC-3′, 5′-ACA-ACA-TCG-TCC-TCA-GTG-3′.


## 2.8. Internalization Assay

Human A549 lung epithelial cells were incubated in microwell plates and co-incubated for 48 h at 37°C under 5% CO2 (approximately 8000 cells per well) supplemented with 10% foetal bovine serum (FBS, Hyclone) and RMPI 1640 (GIBCO). Then the medium was aspirated, followed by thorough rinsing three times using sterile PBS. Subsequently, 1 × 105 conidia resuspended were cocultured with A549 to induce internalization for 4 h under the same cultured conditions. Each experimental parameter was repeated three times. Afterwards, the cell monolayers were washed three times with PBS. 1640 plus 10% FBS containing 20 μg/mL nystatin was used to dispose each well for 3.5 h to kill noninternalized conidia. Then the cell monolayers were treated with 100 μL liquid comprising PBS and 0.1% Triton X-100 for 15 min at 37°C to induce cell lysis and the release of internalized conidia. The released conidia were diluted and incubated on PDA at 37°C for 24~36 h. The internalization rate was determined to be the percentage of monocolonies compared to the initial inoculum of conidia.


## 2.9. Antifungal Agents and End-Point Susceptibility Testing

Planktonic cell minimum inhibitory concentrations (PMICs) were examined according to the National Committee for Clinical Laboratory Standards M38-A standard methodology (NCCLS, 2002). The antifungal agents itraconazole (ICZ), amphotericin B (AmB), and 5-fluorocytosine (5-FC) were prepared as stock solutions and diluted in PDB to working concentrations. ICZ and AmB were serially diluted twofold to produce a concentration range of 0.0156–16μg/mL, while the concentration of 5-FC was 0.0625–64 μg/mL. The pathogen was challenged for 24 h at 37°C. Following incubation, all wells were treated with 100 μL of PBS containing 1 μM 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide and 1 μM menadione for 3 h. Absorbance was examined at a wavelength of 490 nm (A
490). Sessile cell minimum inhibitory concentrations (SMICs) were determined as 50% and 90% reduction in A
490 compared with the untreated control.


## 2.10. Statistical Analysis

All experiments were repeated at least three times. The means ± standard deviations of the colony diameters, crystal violet density, and the internalized rate were determined using GraphPad Prism 5 software. Data were analyzed using the repeated measures of SPSS 16.0.P
<
0.05 was considered statistically significant.


## 3. Results

### 3.1. Disruption and Complementation of theA. fumigatus cspA Gene

The construction of the plasmid for generation of the ΔcspA strain is outlined in Figure 1(a), while construction of the complementation plasmid is outlined in Figure 1(b). At 4 days after transformation of the respective plasmids by ATMT, we observed colonies on the selection plates. The downregulated expression ofcspA in the mutant strain and restored expression in the complementation strain were confirmed by real-time PCR. Compared with the WT, there was a 4.7-fold decrease in transcription ofcspA in the ΔcspA strain, while transcription increased 2-fold in thecspAC strain (Figure 2).The constructed schematic of ΔcspA andcspAC inA. fumigatus mutant. (a) The constructed schematic of ΔcspA vector. ThecspA gene was replaced by hph in the pXEH vector. (b) The constructed schematic ofcspAC vector. The fragment containing thecspA promoter and ORF was cloned and inserted into the pCB1532 vector with phl.
(a)
(b)Figure 2
cspA gene disruption changes the morphology ofA. fumigatus. A total of 108 conidia were incubated in PDB in a rotatory shaker for 24 h. Total RNA was extracted based on Trizol method, and it was reversed to cDNA. The transcription level ofcspA was evaluated by real-time PCR.


### 3.2. Disruption ofcspA Changes Colony and Conidial Morphology ofA. fumigatus

Differences in morphology between the WT, ΔcspA mutant, andcspAC strains were examined by inoculating conidia from each strain onto PDA and observing their morphology by light microscopy and SEM. There was a significant color difference between the WT and ΔcspA mutant, with the mutant strain showing reduced pigmentation compared with the WT strain (Figure 3(a)). ThecspAC strain was similar in appearance to the WT (Figure 3(a)). However, there was no obvious difference in morphology of the conidia and hyphae of the three strains under light microscopy (Figure 3(b)). Under SEM, the conidia of the ΔcspA strain had a rougher surface with more ornaments compared with the smooth appearance of the WT andcspAC strains (Figure 3(c)).cspA gene disruption changes the morphology ofA.fumigatus. (a) Conidia ofA. fumigatus 293T wild type (WT), ΔcspA, andcspAC were inoculated and cultured in 37°C. 105 conidia in 10 μL were spotted in the center of PDA plates and cultured for 4 d. The colonial morphology was observed when cultured for 72 h. (b) The coverslips were also stained by lactophenol cotton-blue and observed by light microscope immediately. The data exhibited here are the mean ± SE (n
=
3 for every experiment). The statistical differences existing between mutants and WT are analyzed with t-test. *
P
<
0.05. (c) The coverslips covered on PDA cubes with mycelia were transferred and prefixed with 2% glutaraldehyde at 4°C for 10 h and then metalized with gold and investigated by scanning electron microscope.
(a)
(b)
(c)


### 3.3. Disruption ofcspA Slightly Inhibits Growth ofA. fumigatus

To investigate whethercspA is associated with the growth ofA. fumigatus, a known amount of conidia was spotted onto PDA, and colony diameters were measured daily. A growth curve was established based on the diameters. Over the first 72 h, no obvious differences in growth were observed for the three strains (Figure 4). However, the diameter of the ΔcspA colony (73.2
±
2.06 mm) was significantly (P
<
0.05) smaller than that of the WT (76.8
±
0.74 mm) when measured at 96 h after inoculation. The diameter ofcspAC was akin to WT, with a colony diameter of 79.4
±
2.04 mm at 96 h after inoculation (P
>
0.05) (Figure 4).Figure 4
cspA gene disruption changes the growth ofA. fumigatus. Conidia ofA. fumigatus 293T (WT), ΔcspA, andcspAC were inoculated and cultured in 37°C. 105 conidia in 10 μL were spotted in the center of PDA plates and cultured for 4 d. The colonial diameter was measured each day, and the data was used to prepare a growth rate curve.


### 3.4. Disruption ofcspA Contributes to Reduced Biofilm Formation

#### 3.4.1. Crystal Violet Assay

Crystal violet can bind to the components of cell walls and to the extracellular polysaccharide secreted during biofilm formation. At final spore concentrations of 106, 105, and 104 spores/mL, the A
570 values for the WT strain were 3.16
±
0.20, 2.80
±
0.18, and 1.81
±
0.26, respectively. At the corresponding spore concentrations, the A
570 readings for ΔcspA were 2.51
±
0.47, 1.77
±
0.20, and 0.64
±
0.06, respectively. These observed decreases in the amount of bound crystal violet were significant (P
<
0.05) (Figure 5). An increase in biofilm formation compared with the WT was observed for the complementation strain, with A
570 values of 3.16
±
0.26, 2.87
±
0.28, and 1.61
±
0.27 for the respective spore concentrations (Figure 5).Figure 5
cspA gene disruption postponesA. fumigatus biofilm development. Each of 96-well plates was inoculated with 100 μL of PDB containing different concentrations of conidia of WT or mutants per well and incubated for 20 h at 37°C. Then they were stained by 0.5% crystal violet for 30 min at room temperature, and absorbance at a wavelength of 570 nm (A
570) was examined. The data exhibited here are the mean ± SE (n
=
5
-
6 for every experiment). The statistical differences existing between mutants and WT are analyzed with t-test. *
P
<
0.05.


#### 3.4.2. Fluorescence Microscopy and SEM

Probe FUN1 was used to observe the morphology of the filaments under a fluorescence microscope. Disruption ofcspA contributed to the sparse, irregular arrangement of hyphae, which formed almost circle-like connections (Figure 6). Conversely, WT hyphae formed several clusters of parallel interwoven filaments forming an acute angle (Figure 6). When analyzed by SEM, a similar hyphal morphology was observed (Figure 7). Interestingly, nodules appeared at the intersections of overlapping hyphae in the ΔcspA mutant. The WT strain was rich in a membrane-like substance surrounding hyphae, which did not appear to be present in the ΔcspA strain (Figure 7).Figure 6
cspA gene disruption rendersA. fumigatus different hyphal crosslinking. 200 μL PDB containing 2
×
10
4 conidia was inoculated per well of 24-well plate and cultured for 16 h at 37°C. Then each sample was stained by 20 μM FUN1 probe. Fluorescence microscope was adopted.Figure 7
cspA gene disruption reducesA. fumigatus biofilm architecture. 2
×
10
4 conidia were inoculated per well of 24-well plate containing 200 μL PDB and cultured for 16 h at 37°C. Then samples were desiccated naturally and observed by SEM.


### 3.5. Disruption ofcspA SensitizesA. fumigatus to Antifungal Agents

PMICs and SMICs were examined by end-point susceptibility testing. Of the three antifungal agents examined, the most significant differences in susceptibility of the three strains were observed in response to exposure to 5-FC. The PMICs for this agent for the WT, ΔcspA, andcspAC strains were 0.5 μg/mL, <0.125 μg/mL, and 0.25 μg/mL, respectively. However, no differences were observed between the strains when treated with AmB or ICZ. In contrast to the planktonic cells, sessile hyphae were much more sensitive to AmB and ICZ. Using AmB, SMIC50 results were 32, 16, and 32 μg/mL for the wild type, ΔcspA, and ΔcspAC strains, respectively, while the SMIC90 values were 64, 32, and 64 μg/mL, respectively. Meanwhile, in response to ICZ, the SMIC50 results were 8, 1, and >32 μg/mL for the WT, ΔcspA, and ΔcspAC strains, respectively, while the SMIC90 values were 16, 2, and >32 μg/mL for the three strains, respectively. However, no difference was observed between the strains in response to 5-FC treatment (>256 μg/mL) (Table 1).Table 1
Minimal inhibitory concentration (µg/mL) of the antifungal agents against A. fumigatus strains after 24 hours of treatment in vitro.
Strains
Itraconazole
Amphotericin B
5-Fluorocytosine
PMIC
SMIC50
SMIC90
PMIC
SMIC50
SMIC90
PMIC
SMIC50
SMIC90
WT
0.5
8
16
16
32
64
0.5
>256
>256
ΔcspA
0.5
1
2
16
16
32
<0.125
>256
>256
cspAC
0.25
>32
>32
>16
32
64
0.5
>256
>256


### 3.6. Disruption ofcspA PromotesA. fumigatus Internalization into A549 Cells

We used an A594 cell-conidia coculturing technique to examine the invasiveness ofA. fumigatus and to observe its ability to escape from host immune cells. The internalization rate of the WT strain was 0.29
±
0.02, and that of the ΔcspA mutant was 0.70
±
0.09 (P
<
0.05).


## 3.1. Disruption and Complementation of theA. fumigatus cspA Gene

The construction of the plasmid for generation of the ΔcspA strain is outlined in Figure 1(a), while construction of the complementation plasmid is outlined in Figure 1(b). At 4 days after transformation of the respective plasmids by ATMT, we observed colonies on the selection plates. The downregulated expression ofcspA in the mutant strain and restored expression in the complementation strain were confirmed by real-time PCR. Compared with the WT, there was a 4.7-fold decrease in transcription ofcspA in the ΔcspA strain, while transcription increased 2-fold in thecspAC strain (Figure 2).The constructed schematic of ΔcspA andcspAC inA. fumigatus mutant. (a) The constructed schematic of ΔcspA vector. ThecspA gene was replaced by hph in the pXEH vector. (b) The constructed schematic ofcspAC vector. The fragment containing thecspA promoter and ORF was cloned and inserted into the pCB1532 vector with phl.
(a)
(b)Figure 2
cspA gene disruption changes the morphology ofA. fumigatus. A total of 108 conidia were incubated in PDB in a rotatory shaker for 24 h. Total RNA was extracted based on Trizol method, and it was reversed to cDNA. The transcription level ofcspA was evaluated by real-time PCR.


## 3.2. Disruption ofcspA Changes Colony and Conidial Morphology ofA. fumigatus

Differences in morphology between the WT, ΔcspA mutant, andcspAC strains were examined by inoculating conidia from each strain onto PDA and observing their morphology by light microscopy and SEM. There was a significant color difference between the WT and ΔcspA mutant, with the mutant strain showing reduced pigmentation compared with the WT strain (Figure 3(a)). ThecspAC strain was similar in appearance to the WT (Figure 3(a)). However, there was no obvious difference in morphology of the conidia and hyphae of the three strains under light microscopy (Figure 3(b)). Under SEM, the conidia of the ΔcspA strain had a rougher surface with more ornaments compared with the smooth appearance of the WT andcspAC strains (Figure 3(c)).cspA gene disruption changes the morphology ofA.fumigatus. (a) Conidia ofA. fumigatus 293T wild type (WT), ΔcspA, andcspAC were inoculated and cultured in 37°C. 105 conidia in 10 μL were spotted in the center of PDA plates and cultured for 4 d. The colonial morphology was observed when cultured for 72 h. (b) The coverslips were also stained by lactophenol cotton-blue and observed by light microscope immediately. The data exhibited here are the mean ± SE (n
=
3 for every experiment). The statistical differences existing between mutants and WT are analyzed with t-test. *
P
<
0.05. (c) The coverslips covered on PDA cubes with mycelia were transferred and prefixed with 2% glutaraldehyde at 4°C for 10 h and then metalized with gold and investigated by scanning electron microscope.
(a)
(b)
(c)


## 3.3. Disruption ofcspA Slightly Inhibits Growth ofA. fumigatus

To investigate whethercspA is associated with the growth ofA. fumigatus, a known amount of conidia was spotted onto PDA, and colony diameters were measured daily. A growth curve was established based on the diameters. Over the first 72 h, no obvious differences in growth were observed for the three strains (Figure 4). However, the diameter of the ΔcspA colony (73.2
±
2.06 mm) was significantly (P
<
0.05) smaller than that of the WT (76.8
±
0.74 mm) when measured at 96 h after inoculation. The diameter ofcspAC was akin to WT, with a colony diameter of 79.4
±
2.04 mm at 96 h after inoculation (P
>
0.05) (Figure 4).Figure 4
cspA gene disruption changes the growth ofA. fumigatus. Conidia ofA. fumigatus 293T (WT), ΔcspA, andcspAC were inoculated and cultured in 37°C. 105 conidia in 10 μL were spotted in the center of PDA plates and cultured for 4 d. The colonial diameter was measured each day, and the data was used to prepare a growth rate curve.


## 3.4. Disruption ofcspA Contributes to Reduced Biofilm Formation

### 3.4.1. Crystal Violet Assay

Crystal violet can bind to the components of cell walls and to the extracellular polysaccharide secreted during biofilm formation. At final spore concentrations of 106, 105, and 104 spores/mL, the A
570 values for the WT strain were 3.16
±
0.20, 2.80
±
0.18, and 1.81
±
0.26, respectively. At the corresponding spore concentrations, the A
570 readings for ΔcspA were 2.51
±
0.47, 1.77
±
0.20, and 0.64
±
0.06, respectively. These observed decreases in the amount of bound crystal violet were significant (P
<
0.05) (Figure 5). An increase in biofilm formation compared with the WT was observed for the complementation strain, with A
570 values of 3.16
±
0.26, 2.87
±
0.28, and 1.61
±
0.27 for the respective spore concentrations (Figure 5).Figure 5
cspA gene disruption postponesA. fumigatus biofilm development. Each of 96-well plates was inoculated with 100 μL of PDB containing different concentrations of conidia of WT or mutants per well and incubated for 20 h at 37°C. Then they were stained by 0.5% crystal violet for 30 min at room temperature, and absorbance at a wavelength of 570 nm (A
570) was examined. The data exhibited here are the mean ± SE (n
=
5
-
6 for every experiment). The statistical differences existing between mutants and WT are analyzed with t-test. *
P
<
0.05.


### 3.4.2. Fluorescence Microscopy and SEM

Probe FUN1 was used to observe the morphology of the filaments under a fluorescence microscope. Disruption ofcspA contributed to the sparse, irregular arrangement of hyphae, which formed almost circle-like connections (Figure 6). Conversely, WT hyphae formed several clusters of parallel interwoven filaments forming an acute angle (Figure 6). When analyzed by SEM, a similar hyphal morphology was observed (Figure 7). Interestingly, nodules appeared at the intersections of overlapping hyphae in the ΔcspA mutant. The WT strain was rich in a membrane-like substance surrounding hyphae, which did not appear to be present in the ΔcspA strain (Figure 7).Figure 6
cspA gene disruption rendersA. fumigatus different hyphal crosslinking. 200 μL PDB containing 2
×
10
4 conidia was inoculated per well of 24-well plate and cultured for 16 h at 37°C. Then each sample was stained by 20 μM FUN1 probe. Fluorescence microscope was adopted.Figure 7
cspA gene disruption reducesA. fumigatus biofilm architecture. 2
×
10
4 conidia were inoculated per well of 24-well plate containing 200 μL PDB and cultured for 16 h at 37°C. Then samples were desiccated naturally and observed by SEM.


## 3.4.1. Crystal Violet Assay

Crystal violet can bind to the components of cell walls and to the extracellular polysaccharide secreted during biofilm formation. At final spore concentrations of 106, 105, and 104 spores/mL, the A
570 values for the WT strain were 3.16
±
0.20, 2.80
±
0.18, and 1.81
±
0.26, respectively. At the corresponding spore concentrations, the A
570 readings for ΔcspA were 2.51
±
0.47, 1.77
±
0.20, and 0.64
±
0.06, respectively. These observed decreases in the amount of bound crystal violet were significant (P
<
0.05) (Figure 5). An increase in biofilm formation compared with the WT was observed for the complementation strain, with A
570 values of 3.16
±
0.26, 2.87
±
0.28, and 1.61
±
0.27 for the respective spore concentrations (Figure 5).Figure 5
cspA gene disruption postponesA. fumigatus biofilm development. Each of 96-well plates was inoculated with 100 μL of PDB containing different concentrations of conidia of WT or mutants per well and incubated for 20 h at 37°C. Then they were stained by 0.5% crystal violet for 30 min at room temperature, and absorbance at a wavelength of 570 nm (A
570) was examined. The data exhibited here are the mean ± SE (n
=
5
-
6 for every experiment). The statistical differences existing between mutants and WT are analyzed with t-test. *
P
<
0.05.


## 3.4.2. Fluorescence Microscopy and SEM

Probe FUN1 was used to observe the morphology of the filaments under a fluorescence microscope. Disruption ofcspA contributed to the sparse, irregular arrangement of hyphae, which formed almost circle-like connections (Figure 6). Conversely, WT hyphae formed several clusters of parallel interwoven filaments forming an acute angle (Figure 6). When analyzed by SEM, a similar hyphal morphology was observed (Figure 7). Interestingly, nodules appeared at the intersections of overlapping hyphae in the ΔcspA mutant. The WT strain was rich in a membrane-like substance surrounding hyphae, which did not appear to be present in the ΔcspA strain (Figure 7).Figure 6
cspA gene disruption rendersA. fumigatus different hyphal crosslinking. 200 μL PDB containing 2
×
10
4 conidia was inoculated per well of 24-well plate and cultured for 16 h at 37°C. Then each sample was stained by 20 μM FUN1 probe. Fluorescence microscope was adopted.Figure 7
cspA gene disruption reducesA. fumigatus biofilm architecture. 2
×
10
4 conidia were inoculated per well of 24-well plate containing 200 μL PDB and cultured for 16 h at 37°C. Then samples were desiccated naturally and observed by SEM.


## 3.5. Disruption ofcspA SensitizesA. fumigatus to Antifungal Agents

PMICs and SMICs were examined by end-point susceptibility testing. Of the three antifungal agents examined, the most significant differences in susceptibility of the three strains were observed in response to exposure to 5-FC. The PMICs for this agent for the WT, ΔcspA, andcspAC strains were 0.5 μg/mL, <0.125 μg/mL, and 0.25 μg/mL, respectively. However, no differences were observed between the strains when treated with AmB or ICZ. In contrast to the planktonic cells, sessile hyphae were much more sensitive to AmB and ICZ. Using AmB, SMIC50 results were 32, 16, and 32 μg/mL for the wild type, ΔcspA, and ΔcspAC strains, respectively, while the SMIC90 values were 64, 32, and 64 μg/mL, respectively. Meanwhile, in response to ICZ, the SMIC50 results were 8, 1, and >32 μg/mL for the WT, ΔcspA, and ΔcspAC strains, respectively, while the SMIC90 values were 16, 2, and >32 μg/mL for the three strains, respectively. However, no difference was observed between the strains in response to 5-FC treatment (>256 μg/mL) (Table 1).Table 1
Minimal inhibitory concentration (µg/mL) of the antifungal agents against A. fumigatus strains after 24 hours of treatment in vitro.
Strains
Itraconazole
Amphotericin B
5-Fluorocytosine
PMIC
SMIC50
SMIC90
PMIC
SMIC50
SMIC90
PMIC
SMIC50
SMIC90
WT
0.5
8
16
16
32
64
0.5
>256
>256
ΔcspA
0.5
1
2
16
16
32
<0.125
>256
>256
cspAC
0.25
>32
>32
>16
32
64
0.5
>256
>256


## 3.6. Disruption ofcspA PromotesA. fumigatus Internalization into A549 Cells

We used an A594 cell-conidia coculturing technique to examine the invasiveness ofA. fumigatus and to observe its ability to escape from host immune cells. The internalization rate of the WT strain was 0.29
±
0.02, and that of the ΔcspA mutant was 0.70
±
0.09 (P
<
0.05).


## 4. Discussion

A. fumigatus is an opportunistic fungus capable of surviving under various environmental conditions, which contributes to it being the primary cause of invasive aspergillosis in liver transplant recipients amongst theAspergillus species [2].CspA, containing a GPI-associated site, is a cell wall protein found only inAspergillus species and is present in multiple copies in the genome ofA. fumigatus. Als proteins, also characterized as GPI-anchored proteins inC. albicans, play a critical role in biofilm formation. Thus, to unravel the relationship betweencspA and biofilm formation inA. fumigatus, we constructed a ΔcspA mutant strain by targeted gene disruption, along with a complementedcspA strain (cspAC) by homologous introduction of a complementation plasmid. The ΔcspA mutant strain was examined by real-time PCR, and a 5-fold reduction in expression ofcspA was identified. This finding confirmed the validity of this method for achieving targeted gene disruption of filamentous fungi, which is a prerequisite for studying fungal gene function.Compared with the WT strain, the ΔcspA mutant showed a reduction in the grey-green pigmentation that is common to this species, with more ornaments on the conidial cell surface observed by SEM. The latter feature was consistent with a previous study, providing direct evidence of the function ofcspA in the cell wall. However, almost no alteration of pigment was observed in the previous work [11]. This discrepancy is likely caused by differences in the strains used and the culture conditions. A recent study showed that two kinds of pigment are generated byA. fumigatus: pyomelanin, which binds to the cell wall of hyphae and imparts a brown coloration, and DHN melanin, which has an affinity for conidia, rendering them gray-greenish [13]. There are two major factors that might account for the observed decrease in pigment. The first is a decrease in the number of conidia; however, this can be excluded by the results of both culturing and SEM. The second reason is loss or poor production of melanin. The amount of melanin and the roughness of the conidial surface appear to be closely related [14–16]. Rather than being formed as a solid layer anchored to the cell wall, the melanin of fungi is produced as granules that are held in place by scaffolds constructed out of cell wall components, such as chitin [17]. InCryptococcus neoformans, deletion of chitin synthase (Chs3) and its regulator (Csr2) contributed to a decrease in melanin retention [18]. Furthermore, disruption of chitin synthase resulted in the failure to produce melanin byC. albicans [19]. Together, these findings suggest thatcspA might affect cellular melanin levels either by influencing melanin retention or moderating its synthesis. Thus, further research is necessary to elucidate the link betweencspA and melanin inA. fumigatus.Obvious differences in biofilm density existed between the WT, ΔcspA mutant, andcspAC complementation strains, confirming a role forcspA inA. fumigatus biofilm formation. Biofilms are involved in 65–80% of chronic clinical infections in humans and represent the sessile stage of most microorganisms. During biofilm formation, three landmark events should be taken into consideration: adherence to the surrounding substances, correct hyphal cross-linking, and sufficient ECM production [20, 21].Emerging evidence suggests that certain cell wall proteins function as adhesins as well as effectors in biofilm formation. The disruption of RodA resulted in decreased adherence ofA. fumigatus to epithelial cells and material, such as collagen and albumin, with higher levels ofrodA transcription recorded in biofilms than in conidia. Meanwhile, GPI proteins play an important role in the adherence to epithelial and endothelial cell lines ofC. albicans [9, 22]. Based on this evidence, we predicted thatcspA, characterized as a cell wall GPI protein, was a potential adhesin. Our findings showed that the biofilm formed by the ΔcspA strain was more easily detached by washing than that of the WT strain and that the mutant took longer to attach to the bottom of the microwell plates (data not shown). Reduced adherence to the ECM by a ΔcspA mutant was also confirmed by another study [11].Furthermore, we used Fun1 labeling of the hyphal wall to observe filament morphology. Disruption ofcspA contributed to a sparse, irregular arrangement of hyphae, which formed almost circle-like connections instead of the classic bundles of parallel filaments intersecting and forming acute angles [5]. This abnormal hyphal arrangement might be the cause of inferior biofilm construction.The production and components of the ECM ofA. fumigatus have already been described in detail under bothin vitro andin vivo conditions [5, 8]. ECM acts like a “glue,” packing the hyphae and enhancing biofilm formation by attaching to the host cells and substrates [23]. ECM can be observed using SEM whenA. fumigatus is cultured in a medium with 10% fetal calf serum. SEM analysis showed that WT hyphae were surrounded with a membrane-like substance, which was not present in ΔcspA cultures. This substance has been predicted to be ECM [5]. Therefore, it is reasonable to suggest thatcspA is associated with ECM production, and deletion of this gene severely affects biofilm formation.As a growing number of patients are infected with pan-drug-resistantA. fumigatus strains, it is imperative to understand the resistance mechanisms and uncover novel drug targets [24]. Mowat et al. studied the effects of various antifungal drugs on severalA. fumigatus strains and showed that biofilm formation produced a significant increase (>500-fold) in tolerance to these agents compared with planktonic cells [6]. In our study, 5-FC, which interferes with DNA synthesis, was the most effective antifungal agent for planktonic cells (PMIC 0.5 μg/mL for WT). However, it had little effect once a biofilm was established, even at a 512-fold increase in concentration over the PMIC. On the other hand, AmB, which increases the permeability of the cell wall, inhibited biofilm development at a concentration only 2-fold higher than the PMIC. Therefore, a combination of 5-FC and AmB would be most effective for the treatment ofA. fumigatus infection [25].The ΔcspA strain showed increased susceptibility to 5-FC when in the planktonic state, while the sessile hyphae were more susceptible to AmB and ICZ compared with the WT strain. In WT cells, the ECM may prevent these molecules from penetrating into mycelia [26], meaning that the ΔcspA mutant, with its decreased production of ECM, may be more vulnerable to these antifungal agents.Internalization by A549 lung alveolar epithelial cells is a model representing the invasiveness of fungi towards host cells and their ability to escape the immune system. Herein, we found that the ΔcspA mutant strain showed a 2-fold increase in internalization of conidia into lung alveolar epithelial cells compared with the WT strain. Little is known about the interaction between fungi and host nonimmunocytes. However, changes in the physical properties of conidia, such as hydrophobicity, surface roughness, and electrostatic charge, significantly affect cellular uptake [21]. Most cell lines, including A549, tend to absorb nanoparticles with a cationic charge and a rougher surface [27]. Previous studies have shown that carbohydrates with a negative charge can interfere with the adherence of conidia to connective components of pulmonary epithelial cells. The ΔcspA strain generated in the current study showed a lack of pigmentation (putatively a lack of melanin) and a rough surface and may have more chitin exposure (based on previous work [11]). Melanin confers a negative charge to conidia, while a chitin carries a cationic charge. These factors might indicate that the mutant strain carries a more positive charge than the WT [17, 28]. In addition, aC. albicans strain with increased exposure of chitin exhibited a stronger interaction with normal human gingival fibroblasts [29].


## 5. Conclusion

In this study, a ΔcspA mutant strain was achieved by targeted gene disruption mediated byAgrobacterium tumefaciens, and a reconstitutedcspA strain was acquired by homologous introduction. Our results showed that deletion ofcspA resulted in a lighter gray-green colony, rougher conidia surface, incomplete biofilm formation, and increased internalization into A549 cells, as well as enhanced sensitivity to some antifungal drugs, which suggests thatcspA participates in maintaining the integrity of cell wall, plays an important role in biofilm formation, and may become a breakthrough point to understand the drug resistance mechanism ofA. fumigatus.



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*Source: 960357-2015-03-03.xml*