Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density

ABSTRACT Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

4, The strategy that described in this manuscript based on a Δags1Δgtb3 mutant strain. As the authors and many other publications noted, such mutant strain has very different cell wall architecture, which means it responds differently as a wildtype strain. In addition, mutant A. fumigatus strains that lack alpha-glucan and GAG are both less virulent. Therefore, it would be hard to tell if the outcomes from this strategy could also be useful for the clinical important strains, which should not be alpha-glucan and GAG defective strains.
5 Other strategies have also be used for testing drug sensitivity of filamentous fungi, such as testing colony growth on drug containing solid medium, or testing the pellets diameter in a shaken liquid culture. To validate the application of the proposed strategy in this manuscript, authors should compare these different methods and discuss how the proposed method may overwhelm other methods.
Minor point: 1 The title of the manuscript was not straightforward to represent the work in this study. 2 line 208, gene names are not italic 3 there is a typo in line 322, "he" should be "the" 4 Did author test the GAG content in gtb3 single deletion strain? It would be very helpful to show that gtb3 directly regulated GAG formation in A. fumigatus. 5 In their previous work, authors have generated other alpha-glucan and GAG defective A nidulans and A oryzae strains. Authors may consider to test their strategy with more strains to validate this idea.
Reviewer #2 (Comments for the Author): incorporate all the comments indicated in the manuscript.
Staff Comments:

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Thank you for submitting your paper to Microbiology Spectrum. α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute 23 to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus 24 fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, 25 similar to that of yeasts. We constructed a double disruptant mutant of both the primary 26 α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. 27 fumigatus AfS35, and found that the hyphae of this mutant were fully dispersed. 28 Although the mutant completely lost α-1,3-glucan and GAG, its growth and 29 susceptibility to antifungal agents were not significantly different from those of the 30 parental strain. Biomass of the mutant in shake-flask cultures was proportional to 31 optical density for at least 18 h. We were also able to quantify the dose response of 32 hyphal growth to antifungal agents by measuring optical density. is quantified by chitin content (2). The amount of ergosterol, a unique component of 46 fungal cells, is also useful (3,4). Quantitative PCR can be used to quantify fungal cells 47 in soil and infected hosts. Banerjee et al. have measured turbidity of ground hyphal cells 48 (5). A method for measurement of fluorescence of formazan produced by living cells 49 from 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-tetrazolium-5-carboxanilide (XTT) has 50 been developed (6). Recently, several methods based on image analysis have been 51 reported (7-10). 52 In filamentous fungi, the surface structure differs between conidia and hyphae. 53 The outer layer of conidia, termed the rodlet layer, is composed of polymerized 54 4 hydrophobin (11,12) and is underlaid by melanin (13). Below the melanin is the cell 55 wall composed mainly of polysaccharides, i.e. glucan, chitin, and mannan (13,14). 56 When hyphae extend from conidia, the polysaccharide layer is exposed to the surface, 57 and the structure of the polysaccharide network is continuously modified (15). In 58 Aspergillus species, hyphae have α-glucan in the outermost layer, which covers the 59 β-1, 3-glucan and chitin layers (16 The sequences of all primers are listed in Table S1. Transformation of A. fumigatus was 96 performed as described previously (30) with some modifications. Briefly, conidia were 97 inoculated into YG medium (10 mL) and incubated at 37°C with shaking at 150 rpm. (2-fold dilution). By repeating this procedure, the dilution series were prepared; 200 µL 204 of each dilution was dispensed into 5 wells of a 96-well plate, and OD 600 was measured. 205 The remaining mycelial suspension (4 mL) was freeze-dried and weighed. inhibition of growth were 0.125 µg/mL for VRC and 0.0313 µg/mL for ITC (Fig. 3). 296 Growth was completely inhibited at 2 µg/mL of VRC and 1 µg/mL of ITC (Fig. 3), 297 which was in agreement with MICs determined by CLSI M38-A2 ( Table 2). The 298 concentrations that caused 50% growth inhibition were 0.0625 µg/mL for AMB and 4 299 µg/mL for 5FC (Fig. 3). Growth was completely inhibited at 0.5 µg/mL of AMB and 300 256 µg/mL of 5FC (Fig. 3). Growth inhibition by MCFG was hard to evaluate ( In the present study, we constructed a double disruptant of the ags1 and gtb3 genes, 308 which have roles in α-1,3-glucan and GAG biosynthesis, respectively, and observed that 309 the hyphae of the mutant were fully dispersed in shake-flask culture (Fig. 1A). Using here showed no biofilm formation (Fig. S4A). GalN was hardly detected in the gtb3 327 disruptant (Fig. 1C), suggesting that Gtb3 is essential for GAG biosynthesis. Although

Reviewer #1
In this manuscript, Ken and colleagues constructed a mutant strain of A. fumigatus that lacks the alpha-glucan and galactosaminogalactan in the cell wall. Based on this mutant strain, they developed a strategy to assess the cell growth by measuring optical density.
As the authors proposed, this new strain may be applied for high throughput anti-fungal drug screening in shaken liquid growth condition. However, there are several concerns for the high throughput screening strategy. Substantially more experiment should be performed before it could be validated strategy.
Major points: 1, Growth of unicellular organism and filamentous fungi (multicellular) are fundamentally different. Use of optical density for measuring the growth of unicellular organism is based on their uniform distribution. In contrast, filamentous fungi grow in a way to elongate the existing mycelia, as the authors show in Fig. 2D. The conidia and mycelia of filamentous fungi tend to cluster together and form pellets in a shaken liquid culture. Even the Δags1Δgtb3 mutant strain, the mycelia were not totally separated from each other. They still form small but visible pellets in the shaken liquid medium, especially in the YG medium. If so, it is hard to agree with the concept that a simple measurement of optical density could faithfully represent the cell density.
Our experiments suggest that AMM and RPMI medium are suitable for the evaluation of the growth of the A. fumigatusΔags1Δgtb3 strain by optical density. To gain insight into why small but visible pellets were formed in YG medium, we labelled the Δags1Δgtb3 mutant grown in YG medium for 24 h with alpha-1,3-glucanase alpha-1,3-glucan-binding domain (AGBD) fused with GFP (AGBD-GFP), and observed clear labelling of the septa and the outline of the cell. As A. fumigatus has three alpha-1,3-glucan synthase genes (ags1-3), alpha-1,3-glucan in labeled hyphae of the Δags1Δgtb3 mutant was likely synthesized by Ags2 and/or Ags3. In A. oryzae ΔagsAΔagsBΔagsCΔsphZΔugeZ (AGΔ-GAGΔ) all the three genes encoding alpha-1,3-glucan synthases were disrupted, and this mutant had fully dispersed hyphae in all the media tested including YG medium. Although the data on A. oryzae AGΔ-GAGΔ are consistent with the above data on A. fumigatus Δags1Δgtb3, further experiments are needed to prove the effect of alpha-1,3-glucan on pellet formation in the Δags1Δgtb3 mutant. We have revised the manuscript in lines 354-370.
2, Materials and Methods section. For the measurement of optical density, authors described that "100µL was mixed with 100 µL of 100 mM sodium phosphate buffer (pH 7.0) containing 4% paraformaldehyde in a 96-well plate". However, this statement was not clear. Especially when the pellets of the colonies become larger in late time points, the transfer of such colony suspension cannot be performed by simple pipetting (not sure if it is a problem for Δags1Δgtb3 mutant strain, but it must be a problem for a wildtype strain). Authors should add more details for these steps. Otherwise the result could be quite inconsistent due to different operations.
We have added more details as follows.
Line 196-199: The culture (2 mL) was withdrawn with wide-bore tips at each sampling point, and 100 µL of the culture was mixed by pipetting with wide-bore tips with 100 µL of 100 mM sodium phosphate buffer (pH 7.0) containing 4% paraformaldehyde in a 96-well plate.
3, Regarding to the data consistency, optical density measurements were performed in different medium cultures, such as AMM, YG and RPMI. The time course of OD600 results were shown in Fig2C and FigS5B. Comparing these data, the growth rate was different in each medium and data reproducibility was also quite different. Especially in YG medium the data reproducibility was the worst. As Fig. S5A showed, Δags1Δgtb3 mutant strain formed more visible pellets in YG medium than that in RPMI and AMM.
Authors should compare such data and draw a conclusion for the best growth condition (medium selection and time for growth) of the proposed strategy. Otherwise, such variations will greatly limit the use of this strategy.
We have revised the text to describe the best conditions for the proposed strategy in lines 354-376.
4, The strategy that described in this manuscript based on a Δags1Δgtb3 mutant strain.
As the authors and many other publications noted, such mutant strain has very different cell wall architecture, which means it responds differently as a wildtype strain. In addition, mutant A. fumigatus strains that lack alpha-glucan and GAG are both less virulent. Therefore, it would be hard to tell if the outcomes from this strategy could also be useful for the clinical important strains, which should not be alpha-glucan and GAG defective strains.
We have explained the outcomes of this strategy as follows. At present, we are investigating culture conditions that prevent pellet formation in the parental strain, which would be useful for testing clinical isolates. The perspectives are described in the manuscript as follows.
Line 407-410: Establishing culture conditions that prevent pellet formation of a strain with an intact cell wall structure could expand the application of growth monitoring by optical density. Understanding the biochemical and physicochemical properties of α-1,3-glucan and GAG will contribute to finding suitable culture conditions. 5 Other strategies have also be used for testing drug sensitivity of filamentous fungi, such as testing colony growth on drug containing solid medium, or testing the pellets diameter in a shaken liquid culture. To validate the application of the proposed strategy in this manuscript, authors should compare these different methods and discuss how the proposed method may overwhelm other methods.
We have revised the manuscript to compare our method with conventional methods as follows.
Line 377-389: Monitoring growth by optical density is superior to that by conventional methods for several reasons: 1) growth monitoring is quantitative and continuous. During drug testing based on CLSI M38-A2, growth has to be observed visually. The mutant with dispersed hyphae would allow establishment of automated drug screening for Aspergillus. 2) Fungicidal and fungistatic drugs could be selected using our strategy. We propose to screen anti-Aspergillus drugs from a drug library, although drugs that do not inhibit germination but disorder hyphal extension, such as echinocandin, might be hard to select using our method. Recently, Beattie and Krysan reported that the release of intracellular adenylate kinase from hyphal cells is a sensitive readout to detect cell lysis and is useful for screening antifungal reagents against A.
fumigatus. In combination with the adenylate kinase method, our strategy may allow selection of anti-Aspergillus drugs with various spectra by monitoring optical density of fungal culture.
Minor point: 1 The title of the manuscript was not straightforward to represent the work in this study.
We have changed the title as follows: Quantitative monitoring of mycelial growth of Aspergillus fumigatus in liquid culture by optical density 2 line 208, gene names are not italic Plain text is correct in this case because the whole heading is in italics.
3 there is a typo in line 322, "he" should be "the" Corrected.
4 Did author test the GAG content in gtb3 single deletion strain? It would be very helpful to show that gtb3 directly regulated GAG formation in A. fumigatus.
We have added the data on GalN content of Δgtb3 to Fig. 1C.
5 In their previous work, authors have generated other alpha-glucan and GAG defective A nidulans and A oryzae strains. Authors may consider to test their strategy with more strains to validate this idea.
We have monitored the growth of the Aspergillus oryzae mutant lacking both alpha-1,3-glucan and GAG, and added Figure S7. As expected, our strategy was applicable to this A. oryzae mutant. We have revised the text (lines 202-205 in the Materials and Methods, lines 292-297 in the Results, and Table 1). Unfortunately, the Aspergillus nidulans mutant is not available to me at my current institute.

Reviewer #2
Incorporate all the comments indicated in the manuscript.
Line 92: The abbreviation should be indicated in bracket as it is for the 1 st time.
We have added the names of YG and RPMI media in full on lines 92-94.
To simply quantify growth, we cultured the fungi at 37°C. To evaluate drug sensitivity, we cultured them at 35°C according to the protocols of CLSI M38-A2.
Line 233: Why here? This should be in the discussion part unless otherwise the Results and Discussion parts merged together.
We have moved the sentence to the Discussion section (lines 340-343).
Line 256-259: More evidences from previous researchers that support the current finding should be required in the discussion part.
We have explained the supposed mechanism of the increase in AI-Glc and AI-GlcN in Δags1Δgtb3 and cited appropriate references (lines 347-353).
Line 314: The discussion part is very short and not well expressed. In paragraph 1, 2 and 4 you only present the finding and not well interpreted and do not compared with previous findings. Generally the discussion part require major revision We have substantially revised the Discussion. Cell wall rearrangements were shown to lead to differences in antifungal susceptibility. Both reviewers suggested to perform additional tests using common cell wall stress agents and conditions (T, pH, etc.) or different medium. Furthermore, the a-1,3 beta glucan labeling with GFP should be added to the manuscript, as suggested by reviewer 1. Visualization of the labeling under different conditions (different medium or cell wall stress) would further strengthen the manuscript findings. Please include the requested modifications to the revised version.
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The authors have addressed most of my concerns during the revision. Especially the elaborated Discussion pointed out the potential advantages and drawbacks of the proposed strategy. I have only one concern left regarding the α-1,3-glucan compensation in YG medium. It is unclear why the result of α-1,3-glucan labeling with GFP was not shown in the manuscript. Author should add such data in the revised manuscript. Moreover, if re-formation of α-1,3-glucan was the reason for the pellet in YG medium. This result suggested that other factors, especially the medium composition, may challenge the proposed strategy. Beauvais has previously generated a triple A. fumigatus deletion stain that had no α-1,3-glucan at all. And there should be no compensation of α-1,3-glucan in this strain. I would suggest the authors to further construct a new mutant stain based on this triple deletion strain. This would further warranty the compatibility of the proposed strategy.

Response to reviewers
Reviewer #1 (Comments for the Author): The authors have addressed most of my concerns during the revision.
Especially the elaborated Discussion pointed out the potential advantages and drawbacks of the proposed strategy. I have only one concern left regarding the α-1,3-glucan compensation in YG medium. It is unclear why the result of α-1,3-glucan labeling with GFP was not shown in the manuscript. Author should add such data in the revised manuscript.
Moreover, if re-formation of α-1,3-glucan was the reason for the pellet in YG medium. This result suggested that other factors, especially the medium composition, may challenge the proposed strategy. Beauvais has previously generated a triple A. fumigatus deletion stain that had no α-1,3-glucan at all. And there should be no compensation of α-1,3-glucan in this strain. I would suggest the authors to further construct a new mutant stain based on this triple deletion strain. This would further warranty the compatibility of the proposed strategy.
We have added images of mycelial cells labeled with AGBD-GFP. The AfS35 strain was clearly labeled with AGBD-GFP along the outline of the cells. The Δags1Δgtb3 strain cultured in YG medium (pellet formed) was clearly labeled with AGBD-GFP in the septa and along the hyphal outline. These results suggest that hyphal pellet formation in Δags1Δgtb3 cultured in YG medium depended on α-1,3-glucan synthesized by ags2 and/or ags3. We regret not having constructed the mutant with a triple deletion of α-1,3-glucan synthase genes because of the need for several transformations and the tight revision age1 and gtb3 double mutant using absorbance OD600 as an indicator. This is also the first study to demonstrate that Gtb3 is involved in biosynthesizing glycosaminoglycans (GAGs). The method was used to assess the effects of antifungal drugs used in clinical treatment, and the results were consistent with those based on the method described in CLSI M38-A2. Monitoring growth by absorbance allows for rapid screening of antifungal drugs. Additionally, this study was robustly conducted and had no technical problems. However, I think this study needs to be revised on several points before publication.
1. I feel uncomfortable with the choice of the word "biomass." I think the term is inappropriate to describe the tiny weight of fungus.
2. The cell wall of age1 and gtb3 double mutant may be thinner than the parental strain, making it easier for antifungal agents to penetrate it. Considering such influence, I think the number of antifungal agents' examples in the experiment is too small. Therefore, please present additional data that have been widely verified, such as the effects of chemical compounds (except the antifungal agents shown here) and the effects of some stress conditions (temperature, osmotic pressure and pH etc).
We have evaluated the growth of the A. fumigatus AfS35, Δags1, Δgtb3, and