Chitin deacetylases Cod4 and Cod7 are involved in polar growth of Aspergillus fumigatus

Abstract Chitin is one of the key components of fungal cell wall, and chitin deacetylases (CDAs) have been found in fungi; however, their functions remain unknown. Aspergillus fumigatus is known to cause fatal invasive aspergillosis (IA) among immunocompromised patients with a high mortality rate. Although the A. fumigatus cell wall has long been taken as a unique target for drug development, its dynamic remodeling is complicated and not well understood. Seven putative CDAs are annotated in the A. fumigatus genome. In this study, we analyzed the function of the putative CDAs, Cod4 and Cod7, in A. fumigatus. Biochemical analysis of recombinant proteins showed that Cod4 preferentially deacetylated (GlcNAc)4 and was less active on chitooligosaccharides with DP > 5, whereas Cod7 was unable to catalyze deacetylation. Simulation of three‐dimensional structure revealed that both Cod4 and Cod7 shared a similar folding pattern with HyPgdA from Helicobacter pylori and, similar to HyPgdA, a substitution of Thr8 by Ala8 in Cod7 abolished its CDA activity. Deletion of the cod4, cod7, or both in A. fumigatus led to polarity abnormality and increased conidiation. Furthermore, the expression level of the genes related to polarity was upregulated in the mutants. Our results demonstrated that Cod4 and Cod7 were involved in polarity, though Cod4 was inactive.

septation, and branching), and conidiation (Barhoom & Sharon, 2004; d 'Enfert, 1997). These morphological events require a dynamic remodeling of the cell wall at the budding site, hyphal tip, and septation site. IA caused by A. fumigatus is featured with the penetration of growing hyphae into lung tissue and blood vessel. Therefore, the polar growth of A. fumigatus is vital for infection. However, it is still not well understood how A. fumigatus modulates the remodeling of cell wall during polar growth.
Chitin is one of the critical components of the A. fumigatus cell wall (Bernard & Latgé, 2001), and the genome of A. fumigatus contains seven putative CDA genes; however, their functions remain unknown. In this study, we cloned the A. fumigatus cod4 and cod7 genes and expressed them in E. coli with an appended short N-terminal His-tag. Biochemical characterization of the recombinant enzymes was carried out. We further deleted the cod4 and cod7 separately as well as both of them to construct the deletion mutants ∆cod4, ∆cod7, and ∆cod4∆cod7. Our results showed that the Cod4 was an active CDA, whereas Cod7 was inactive; however, both of them contributed to polar growth and Cod4 was required for conidiation of A. fumigatus.

| Strains and growth conditions
Aspergillus fumigatus strain YJ-407 (China General Microbiological Culture Collection Center, CGMCC0386) was maintained on potato glucose (2%) agar slant. A. fumigatus strain CEA17 (Weidner, d'Enfert, Koch, Mol, & Brakhage, 1998), a kind gift from C. d'Enfert, Institute of Pasteur, France, was propagated at 37°C on YGA (0.5% yeast extract, 2% glucose, and 1.5% Bacto agar), complete medium, or minimal medium with 0.5 mM sodium glutamate as a nitrogen source (Cove, 1966). 5 mM uridine and uracil were added when CEA17 or revertant strain was grown. Mycelium was grown in complete liquid medium at 37°C with constant shaking at 250 rpm. Mycelia were collected and washed with distilled water, and then frozen in liquid nitrogen and ground by pestles. The powder was then stored at −70°C for DNA, RNA, and protein extraction. Conidiospores were acquired by growing A. fumigatus strains on PDA plates with uridine and uracil for 36 hr at 37°C. The spores were collected first with distilled water, then washed twice with 0.05% Tween-20 in phosphate-buffered saline (PBS), and stored in 0.05% Tween-20 in PBS, and its concentration was confirmed via hemocytometer counting.

| Molecular cloning of the cod4 and cod7
Protein sequences were analyzed using Conserved Domain Search, SignalP3.0, and the TMHMM Server v.2.0. Protein sequence and AFUA number were retrieved from the CADRE Geno. Protein sequences were aligned using ClustalX 2.0.
The putative CDA gene was identified by searching the conserved domain of NodB that is homologous among the members of carbohydrate esterase family 4 in the genome database of A. fumigatus 293 using a Blastp program. A 1,040-bp and a 915-bp genomic DNA fragment were found to contain the entire ORF and named as cod4 and cod7, respectively.

| Expression and purification of recombinant Cod4 and Cod7 in E. coli
The cDNA of the A. fumigatus cod4 or cod7 gene was amplified from Teasy-cod4 or Teasy-cod4 and subcloned into pET-21a (Novagen).

| Activity assay
The assay of CDA was carried out by the acetic acid released from the substrates (Fukushima, Kitajima, & Sekiguchi, 2005). Standard enzyme assay was performed in a mixture containing 20 mM Tris-HCl (pH 7.4), 1 mM ZnCl 2 , and 5 μl of pNP-(GlcNAc) 3 or (GlcNAc) 4 (1 mg/ml) in a total volume of 100 μl. The reaction was carried out at 37°C for 30 min and then stopped in boiled water for 10 min. Acetic acid released by the enzyme was quantified with the K-ACET kit (Megazyme). The amount of enzyme that releases 1 μmol of acetate from ethylene glycol chitin per minute is defined as one unit.

| Construction of the mutants and revertants
To delete the cod4, a knockout vector construction was designed to replace the entire coding region of the cod4 with a pyrG cassette by homologous recombination (d' Enfert et al., 1996). PCR primers were designed to amplify a 1.  Enfert et al., 1996) with HpaI was cloned into the site between the up-and downstream noncoding regions of the cod4 to yield the deletion construct pcod4-pyrG.
To construct deletion mutant of the cod7, primers were designed to generated the upstream noncoding region ( To generate the revertant strains or double mutant, the pyrG gene was first deleted in the Δcod4 or Δcod7 mutant using the method described by d' Enfert et al. (1996) to generate the Δcod4ΔpyrG or Δcod7ΔpyrG strain. Then, the wild-type copy of the cod4 or cod7 was transformed into the Δcod4ΔpyrG or Δcod7ΔpyrG strain to replace pyrG, respectively. The double mutant was constructed by deletion of the cod7 gene in the Δcod4ΔpyrG strain.

| Phenotypic analyses of the mutants
Growth kinetics of A. fumigatus strains was carried out by spotting 1 × 10 6 conidiospores onto the center of a solid CM plate at 37°C or 50°C, respectively. The diameter of the colony was measured intermittently until the stationary phase, and the mean diameter was used to plot against the growth kinetics. This experiment was carried out in triplicate.
To test the sensitivity to antifungal reagents, the same amount of conidiospores freshly collected from the wild type, the mutants, and the revertant strains was spotted on CMU plates in the presence of 250 μg/ml calcofluor white or 250 μg/ml Congo red. After incubation at 37°C or 50°C for 24-48 hr, the plates were taken out and photographed.
To examine spore germination, 10 ml complete liquid medium was inoculated with 10 7 freshly harvested conidia, poured into a petri dish containing a glass coverslip, and incubated at 37°C for the To count the number of germ tubes during spore germination, 10 ml of liquid CM was inoculated with 10 6 spores in a petri plate containing sterilized glass coverslips and incubated at 37°C or 50°C.
The coverslips with adhering germinated conidia were taken out, fixed in PFA solution (3.7% paraformaldehyde, 50 mM phosphate buffer, pH 7.0, and 0.1% Triton X-100), and observed and counted under differential interference contrast microscope.

| Analysis of the cell wall
Cell wall components were isolated and determined as described by Yan et al. (2013). Cell wall chitin was isolated as described by White, Farina, and Fulton (1979). The purification procedures included an alkaline treatment, which followed an acidic environment.

| Real-time PCR
Examination of the expression level of genes by relative real-time RT-PCR analysis was performed as described previously (Yan et al., 2013). The primers used for specific genes in this study are shown in Table 1. First, total RNAs were extracted using TRIzol (Invitrogen).
The cDNA synthesis was carried out from total RNA using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas). Then, the PCR was performed by using SYBR ® Premix Ex Taq™ (Takara). A triplicate of samples was tested in each assay, and each experiment was repeated 3 times. To testify the contamination of fungal genomic DNA, negative controls were set up for each gene.

| Expression and biochemical characterization of recombinant CDAs
Based on the Blast results, seven genes were found to encode Note: Standard enzyme assays were performed in a mixture containing 20 mM Tris-HCl (pH 7.4), 1 mM ZnCl 2 , and 5 μl of 1 mg/ml pNP-(GlcNAc) 1-6 or (GlcNAc) 1-6 . Reaction mixture was incubated at 37°C for 30 min and then boiled at 100°C for 10 min to stop the reaction. Acetic acid released by the enzyme was quantified with the K-ACET kit (Megazyme). The amount of enzyme that releases 1 μmol of acetate from ethylene glycol chitin per minute is defined as one unit.
F I G U R E 2 Effect of pH, temperature, and metal ions on activity of Cod4. Standard activity assay was carried out by adding 10 μg of purified Cod4 to a mixture containing 20 mM Tris-HCl (pH 7.4), 1 mM ZnCl 2 , and 5 μl of 1 mg/ml pNP-(GlcNAc 3 ) in a total volume of 100 μl, and the reaction mixture was incubated at 37°C for 30 min. Reactions were terminated by boiling the mixture at 100°C for 10 min. Acetic acid released by the enzyme was quantified with the K-ACET kit (Megazyme). The amount of enzyme that releases 1 μmol of acetate per minute is defined as one unit. To determine the temperature optimum for activity, reactions were performed at 20-60°C under otherwise standard conditions. To determine the pH optimum for activity, reactions were performed at pH 5.0-9.0 under otherwise standard conditions. protein were purified to homogeneity, respectively ( Figure 1).
Under standard assay conditions, a variety of substrates were tested. As summarized in Table 2 showed the highest activity toward pNP-(GlcNAc) 3 and (GlcNAc) 4 .
As p-nitrophenol ring can be treated as a sugar ring analog at the reducing end, it is reasonable to conclude that Cod4 prefers (GlcNAc) 4 and is less active on chitooligosaccharides with DP > 5.
It is proposed that CDAs have four subsites (−2, −1, 0, and +1). F I G U R E 4 Southern blotting of the mutants and revertants. The mutants and revertant strains were constructed as described under Section 2. After PCR confirmation of the mutants and revertants, the positive strains were further confirmed by Southern blot using a 1-kb fragment amplified from the upstream noncoding region of the cod4 or cod7 gene as a probe and Leu194 that form a hydrophobic pocket, which is important for effective catalysis and preferable deacetylated site. Cod4 contains the His-His-Asp metal-binding triad (H87, H91, D14), a catalytic acid His248 and a catalytic base Asp12 ( Figure 4b). As compared with AnCDA, it seems that Asp12 and His91 are responsible for interaction with the −1 sugar, while at subsite + 1 the counterparts of Leu139 and Leu194 were not found; indeed, they are replaced by hydrophobic amino acids Gly123 and Ile247, respectively (Figure 3b).
On the other hand, in contrast to Cod4, Cod7 did not show any CDA activity toward the substrates tested in this study though Cod7 shares a similarity of 80% with Cod4. We further compared these two proteins with other reported members of the CE4 family (Andrés et al., 2014;Blair & van Aalten, 2004;Blair et al., 2005;Fadouloglou et al., 2017;Shaik, Cendron, Percudani, & Zanotti, 2011), including alignment, secondary structure, and three-dimensional structure.
Blast search revealed that Cod4 and Cod7 shared 67% of similarity with a putative peptidoglycan deacetylase from Helicobacter pylori (HpPgdA; Shaik et al., 2011). As shown in Figure 4a HpPgdA (Figure 3b,c). Like other CDAs, in Cod4 a catalytic triad, T8-D12-H248, is identified to be required for deacetylation and H87-H91-D14 is for Zn 2+ binding. In HpPgdA, the corresponding residues of the catalytic triad Thr-Asp-His are A7, D11, and H247. Previously, F I G U R E 5 Growth rate of the Δcod4, Δcod7, and Δcod4Δcod7 at 37°C and 50°C. A total of 10 6 fresh conidiospores of each strain were spotted onto the center of a solid CM plate and incubated at 37°C or 50°C, respectively. The diameter of the colony was measured intermittently until the stationary phase, and the mean diameter was used to plot against the growth kinetics. This experiment was carried out in triplicate it has been shown that HpPgdA is inactive on peptidoglycans and polyamines. Similarly, Cod7 was inactive on N-acetyl-oligosaccharides. Therefore, it is reasonable to conclude that, as in HpPgdA, Cod7 is unable to catalyze deacetylation due to the substitution of T 8 by A 8 .

| Functional analyses of the cod4 and cod7 gene
To evaluate the physiological function of the cod4 and cod7 in A. fumigatus, the deletion mutants were constructed by replacing the cod4 or cod7 with pyrG as described under Section 2, respectively.
As a result, the Δcod4 and Δcod7 were obtained and confirmed by PCR and Southern blotting analysis ofXbaI-digested genomic DNA, in which the wild-type 7.9-kbXbaI fragment was converted into a 5.4-kbXbaI fragment ( Figure 5). The double mutant Δcod4Δcod7 and revertant strains of the Δcod4 and Δcod7 were constructed and confirmed by Southern blot (Figure 4).
The growth rate was determined on solid complete medium at 37°C and 50°C. As shown in Figure 5, the growth rate of the Δcod4 mutant was higher than that of the wild type or the revertant strain, while the growth rate of the Δcod7 and Δcod4Δcod7 was similar to that of the wild type.
Considering that CDAs are the enzymes that deacetylate fungal cell wall chitin, we further analyzed the cell wall of the mutants. When the Δcod4, Δcod7, and Δcod4Δcod7 mutants were grown on solid complete medium containing calcofluor white or Congo red, all three mutants were similar to the wild type at both 37°C and 50°C ( Figure 6). These observations demonstrate that deletion of the cod4, cod7, or both does not affect the cell wall integrity of A. fumigatus.
We further analyzed the cell wall contents of the mutants. As summarized in Table 3, as compared with the wild type, the Δcod4 mutant showed decreases in glycoprotein (by 5%) and β-glucan (by 10%) and increases in α-glucan (by 8%) and chitin (by 21%).
Meanwhile, glycoprotein, α-glucan, and chitin in the Δcod7decreased by 6%-11%, whereas β-glucan increased by 6%. The Δcod4Δcod7 showed a similar pattern with the Δcod7 mutant but displayed more severe decreases in glycoprotein, α-glucan, and chitin. Although the cell wall integrity was not affected in the mutants, the cell wall contents were changed in all three mutants. Also Cod4 and Cod7 showed different effects on cell wall contents. Deletion of the cod4 led to a significant increase in cell wall chitin, while deletion of the cod4 caused a slight increase in β-glucan, which suggests their functions might be different.

| Morphogenesis of the mutants
The biological functions of fungal CDAs have been extensively studied in plant pathogenic fungi and yeasts El Gueddari et al., 2002;Zhao et al., 2010). Cbp1, a CDA from the rice blast fungus M. oryzae, is confirmed to be critical for appressorium formation, which involves in tip growth and requires accumulation of chitosan at the tips of germ tubes (Kuroki et al., 2017). In S. cerevisiae, conversion of chitin to chitosan by either Cda1 or Cda2 is required for formation of the second layer of the spore cell wall, which is important for spores to retain its structural rigidity and resistance to various stresses (Christodoulidou, Briza, Ellinger, & Bouriotis, 1999). CDA from S. pombeis also required for proper spore formation (Matsuo et al., 2005), whereas in C. neoformans, Cda1, Cda2, and Cda3are confirmed as virulence factorsand responsible for providing cell wall integrity during vegetative growth (Baker et al., 2007;Baker, Specht, & Lodge, 2011;Upadhya et al., 2016).
The developmental process of filamentous fungi is featured with the establishment and maintenance of polarity. The nucleus, meanwhile, undergoes several mitotic divisions, and new nuclei move out into the germ tube. Septation takes place after the third nuclear division by placement of a cross-wall at the basal end of the germ tube (Harris, Hamer, Sharpless, & Hamer, 1997;Harris, Hofmann, Tedford, & Lee, 1999;Momany & Taylor, 2000 Note: Cell wall chitin was isolated as described under Section 2. Degree of deacetylation of chitosan was determined by the IR spectrophotometry. F I G U R E 7 Fluorescent observation of nuclear and cell wall of Δcod4, Δcod7, and Δcod4Δcod7 mutants. Ten milliliters of complete liquid medium was inoculated with 10 7 freshly harvested conidia, poured into a petri dish containing a glass coverslip, and incubated at 37°C for the time indicated in each experiment. At the specified times, the coverslides with adhering germlings were removed and fixed in a fixative solution (4% formaldehyde, 50 mM phosphate buffer, pH 7.0, and 0.2% Triton X-100) for 30 min. The coverslips with geminated spores were then washed with phosphate-buffered saline (PBS), incubated for 15 min with DAPI (1 μg/ml; Sigma), washed with PBS three times, then incubated for 5 min with a 10 μg/ml solution of fluorescent brightener 28 (Sigma), and washed again, and the germlings were photographed using a microscope elongated mostly toward one direction, the second germ tube and the first germ tube showed typically bipolar pattern at an angle of 180 degrees, and the second germ tube and the first septation occurred after four rounds of mitosis (7 hr). The septum formed at the basal area of the first germ tube (Figure 7). In comparison with the wild type, the second germ tube formed earlier in the Δcod4, Δcod7, and double mutant. The second germ tube of all three mutants occurred at a 120° angle only after the second mitosis (5-6 hr), and the third germ tube was found after the third or the fourth nuclear division (6-7 hr). All spores of three mutants germinated at 7 hr and 14%-24% of them had the third germ tube, while all wild-type spores germinated at 8 hr and only 2% of had the third germ tube (Table 5). These observations suggest that all three mutants germinate earlier than the wild type and display abnormal polarized growth.
Besides abnormal polar growth, conidiation was also affected in the mutants. At either 37°C or 50°C, the conidiospores produced by the Δcod4 and Δcod4Δcod7 were dramatically increased, while the conidiospores produced by the Δcod7were similar to those of the wild type (Table 6). Under electron microscope, the conidia of the ∆cod4mutant displayed a thickened cell wall (Figure 8) and displays an increased susceptibility to lysozyme degradation (Shaik et al., 2011).

TA B L E 6 Counting of conidiospores in the mutants
In conclusion, our results suggest that Cod4 is a soluble CDA and involved in polarity and conidiation in A. fumigatus. On the other hand, although Cod7 is unable to deacetylate the N-acetylated polysaccharides recognized by the typical CDAs, it also contributes to polarity of A. fumigatus; however, its mechanism needs further investigation.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation of China (31320103901) and partially supported by Bagui Scholar Program Fund (2016A24) of Guangxi Zhuang Autonomous Region to CJ.

CO N FLI C T O F I NTE R E S T S
None declared.

AUTH O R CO NTR I B UTI O N S
CJ conceived, administered, and supervised the project; validated the data with equal contributions from MX and XZ; carried out visualization experiments; and acquired funding. YL developed the methodology, curated the data, and implemented the software; MX and XZ carried out investigation and analyzed the data with support from YL, and wrote the original draft. All authors reviewed and edited the manuscript, and gave the final approval for publication.

E TH I C A L A PPROVA L
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this F I G U R E 9 Expression level of polarity-related gene in the mutants. A total of 10 8 conidiospores were inoculated into 100 ml of liquid CM medium and incubated at 37°C with shaking (200 rpm). After 4, 6, and 9 hr of cultivation, RNA was extracted and analyzed as described under Section 2. TBP was used as internal standard. The experiments were repeated three times