1

ß-glucan unmasking in some Candida albicans mutants correlates with increases in cell wall 1

surface roughness and decreases in cell wall elasticity 2

Sahar Hasim1, David P. Allison2, 3, Scott T. Retterer2, 4, Alex Hopke5, Robert T. Wheeler5, Mitchel 3

J. Doktycz2, 4, Todd B. Reynolds1# 4

1Dept. Microbiology, University of Tennessee, Knoxville, Tennessee, 37996-0840, USA 5

2Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831-6445, 6

USA. 3Dept. Biochemistry & Cellular & Molecular Biology, University of Tennessee, Knoxville, 7

Tennessee, USA, and 4Center for Nanophase Materials Sciences, Oak Ridge National 8

Laboratory, Oak Ridge, Tennessee, 37831-6488, USA. 5Department of Molecular and 9

Biomedical Sciences, University of Maine, Orono, Maine, USA 10

11

# Corresponding author 12

Todd B. Reynolds 13

Department of Microbiology, University of Tennessee, Knoxville, TN 37996 14

Ph: 865-974-4025, FAX: 865-974-4007 15

Email: treynol6@utk.edu 16

This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-17 00OR22725 with the U.S. Department of Energy. The United States Government retains and the 18 publisher, by accepting the article for publication, acknowledges that the United States 19 Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or 20 reproduce the published form of this manuscript, or allow others to do so, for United States 21 Government purposes. The Department of Energy will provide public access to these results of 22 federally sponsored research in accordance with the DOE Public Access Plan 23 (http://energy.gov/downloads/doe-public-access-plan) 24

IAI Accepted Manuscript Posted Online 14 November 2016Infect. Immun. doi:10.1128/IAI.00601-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Abstract 25

Candida albicans is among the most common human fungal pathogens, causing a broad range of 26

infections including life threatening systemic infections. The cell wall of C. albicans is the 27

interface between the fungus and the innate immune system. The cell wall is composed of an 28

outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in ß-(1, 29

3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for 30

ß-(1, 3)-glucan, is important for the innate immune system to recognize systemic fungal 31

infections. Increased exposure of ß-(1, 3)-glucan to the immune system occurs when the mannan 32

layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall 33

during unmasking were explored in live cells with atomic force microscopy (AFM). Two 34

mutants, cho1∆/∆ and kre5∆/∆, were selected as representatives that exhibit modest and strong 35

unmasking, respectively. Comparisons of cho1∆/∆ and kre5∆/∆ to wild-type reveal 36

morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In 37

addition, AFM tips functionalized with Dectin-1 revealed that the binding force of Dectin-1 to all 38

of the strains was similar, but the frequency of binding was highest for kre5∆/∆, decreased for 39

cho1∆/∆ and rare for wild type. These data show that nanoscale changes in surface topology 40

correlate with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips 41

functionalized with immunologically relevant molecules, can map epitopes of the cell wall and 42

increase our understanding of pathogen recognition by the immune system. 43

44

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Key words 45

Candida albicans, Atomic Force Microscopy (AFM), Gelatin immobilization, Young’s modulus, 46

Elasticity, Adhesion force mapping, Indentation force mapping, Dectin-1, ß-(1, 3)-glucan, 47

Macrophage, Caspofungin. 48

49

Introduction: 50

The polymorphic, commensal yeast Candida albicans is one of the most prevalent fungal 51

pathogens infecting humans. It can infect a broad range of tissues including skin, mucus 52

membranes, gastrointestinal and urogenital tracts, and it can also cause life threatening systemic 53

infections (1, 2). C. albicans presents serious medical challenges especially for 54

immunocompromised patients, including those with HIV, being treated with corticosteroids, 55

undergoing cancer chemotherapy, or having organ transplantations. The most serious of these 56

infections are systemic bloodstream infections, and these can have a mortality rate of 40-60% (3-57

5). Although bacteria are the predominant microorganisms, Candida species have emerged as 58

one of the leading causes of hospital-acquired infections and are responsible for 80% of all 59

nosocomial infections caused by fungi (6). Most drugs used to treat systemic fungal infections 60

fall into three classes. The azoles inhibit synthesis of the essential lipid ergosterol, and the 61

polyene amphotericin B interacts directly with ergosterol to damage the cell membrane (7). The 62

echinocandins inhibit synthesis of the essential cell wall polymer ß(1-3)-glucan (8). However, 63

antifungal resistance is limiting the effectiveness of both azoles and echinocandins (9-11), and 64

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toxicity from treatment with amphotericin B can be a serious problem for patients (12) . 65

Development of new antifungal drugs is urgently needed (13-15). 66

One promising avenue for development of antifungal therapies is to improve the innate 67

immune system’s recognition of the pathogen. A healthy immune system can respond 68

effectively to C. albicans (16), so enhancement of the immune response in immunocompromised 69

patients can potentially be a powerful therapy (17, 18). This requires a comprehensive 70

understanding of the interactions between C. albicans and the human immune system, 71

particularly in the early stages of infection. The C. albicans cell wall consists primarily of 72

polysaccharides and can be divided into three components (Figure 1A) (19-21). The outer surface 73

layer is enriched in mannose polysaccharides linked to protein to form a mannoprotein barrier 74

(mannan) that acts as a filter for large molecular weight materials. The inner layer consists 75

mostly of the glucose polysaccharides β (1, 3)-glucan and a minor amount of β (1, 6)-glucan, 76

which is important for cross–linking other components of the wall. In addition to the glucan 77

polymers, the inner layer contains a small, but crucial amount of chitin, a linear β (1, 4)-linked N-78

acetylglucosamine polymer. 79

Macrophages recognize Candida through fungal-specific pathogen-associated molecular 80

patterns (PAMPs) such as ß (1, 3)-glucan. The Dectin-1 receptor on macrophages specifically 81

recognizes and binds ß (1, 3)-glucan, and is part of the mechanism by which these first responder 82

cells detect fungi (22, 23). Increased exposure of ß (1, 3)-glucan to the immune system can occur 83

when the mannan layer in the cell wall is altered or removed by mutations or antifungal drugs 84

like caspofungin. This “unmasking” can lead to an increase in Dectin-1 binding, and thereby 85

improve macrophage recognition (Figure 1) (24, 25). It is therefore possible that the 86

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development of approaches that increase unmasking in vivo might enhance immune responses 87

and facilitate adjunctive therapies. 88

An understanding of unmasking at the nanoscale level is needed to elucidate this process, 89

and this is at an early stage. Recently, Lin J. et, al. examined ß (1, 3)-glucan distribution during 90

caspofungin-mediated unmasking using direct stochastic optical reconstruction microscopy 91

(dSTORM). This study revealed, when using fluorescently labeled soluble Dectin-1 binding 92

probes to detect unmasked ß-glucan, that single and multiple point exposure sites were observed 93

(26). However, the multiple point sites increased in size and frequency during caspofungin-94

induced unmasking, and the frequency of single point sites also increased. This nanoscale 95

analysis was performed at ~20nm resolution. The dSTORM approach yielded a nanoscale 96

description of unmasking points, and revealed patterns of ß-glucan exposure. However, these 97

studies do not show the distribution of the rest of the cell wall polymers relative to ß-glucan. 98

Complementary studies of the effects of caspofungin on cell wall ultrastructure have been 99

performed using AFM and reveal the three dimensional topology of the wall along with effects 100

on the physical properties of the wall such as elasticity (27-29). By pressing the cantilever tip 101

against the surface of the cell, differences in both surface elasticity (30-33) and indentation (34, 102

35) can be measured. Previous studies reported that wild type C. albicans cells treated with 103

caspofungin has decreased surface elasticity (Young’s modulus) compared to untreated cells 104

(29). However, other work has shown that surface elasticity increases compared to wild-type 105

following treatement with caspofungin (36). Thus, there is some controversy on how caspofungin 106

impacts cell wall elasticity. An open question is whether other conditions that cause unmasking, 107

such as cell wall mutations, will cause similar effects as caspofungin, or differ in some respects. 108

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In addition to measuring changes to surface topology and elasticity, AFM can also spatially 109

identify and measure molecular adhesion between a probe attached to the tip of the cantilever and 110

a cell surface (37-41). Previous studies employing Saccharomyces cerevisiae cells expressing the 111

C. albicans adhesion protein Als5p, attached to the AFM cantilever, have measured adhesion 112

events on hydrophobic and fibronectin coated substrates (42). In a similiar experiment, 113

antibodies to the adhesion protein Als5p attached to the cantilever tip were used to bind to an 114

Als5p adhesion protein on the surface of C. albicans and initiate force induced nanodomains over 115

the entire cell surface (43). In addition, multiple lectins and antibodies were used on 116

functionalized AFM tips to examine the flexibility of outer cell wall mannan polymers and inner 117

cell wall glucan and chitin polymers in C. albicans, C. glabrata, and S. cerevisiae, as well as to 118

compared adhesins and mannans in C. albicans yeast and hyphae (44, 45). Furthermore, 119

functionalized AFM tips have been used to characterize the manner in which macrophages bind 120

to C. albicans cells. Binding was shown to depend on mannose-binding lectins from the 121

macrophage, although the precise lectins and their contributions were not identified, and the 122

binding is impacted by the flexible nature of the macrophage membrane (46). 123

In this study, we employ several mutants with varying levels of unmasking along with 124

AFM to interrogate the impact of ß-glucan exposure on nanoscale topology and cell wall 125

elasticity, as well as the distribution of ß-glucan exposure as seen with a Dectin-1 functionalized 126

AFM probe. We also measure the binding forces of Dectin-1 to masked versus unmasked ß(1,3)-127

glucan. We examine the cell wall topography in wild type and in two mutants, cho1∆/∆ and 128

kre5∆/∆, that exhibit unmasking. The cho1∆/∆ strain is a phosphatidylserine synthase mutant that 129

exhibits increases in chitin and a modest level of ß (1, 3)-glucan unmasking that occurs in a 130

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punctate pattern (47). The kre5∆/∆ mutant exhibits stronger defects in cell wall composition than 131

cho1∆/∆, including increased chitin and ß(1,3)-glucan and decreased ß (1, 6)-glucan and mannan, 132

and stronger unmasking (48, 49). We also compare these mutants to wild type cells treated with 133

caspofungin, such that they are unmasked, and exhibit increased ß (1, 3)-glucan exposure. 134

135

Material and methods: 136

Strains, growth media, and chemicals 137

C. albicans SC5314 (50), cho1∆/∆ (47), and kre5∆/∆ (a GFP expressing version of the kre5∆/∆ 138

mutant carrying the ENO1::PENO1-EGFP-NATr plasmid, derived from KAH3 (48, 51)) were 139

used throughout this study. For all experiments, strains were grown overnight at 30°C in YPD 140

(1% yeast extract, 2% peptone, and 2% dextrose) media under aerobic conditions, and the next 141

day the cells were diluted into fresh YPD at OD600 of 0.1 and grown for 3 hrs at 30 °C. In some 142

experiments, wild type cells were also treated with 50 ng/ml caspofungin (Merck, Kenilworth, 143

NJ, USA) for 3 hrs before imaging with AFM. Three biological replicates were carried out for 144

all experiments. The following media and chemicals were obtained from Sigma-Aldrich, St. 145

Louis, MO; aminopropyltriethoxy silane (APTES), trimethylamine, Acid-PEG25-NHS, EDC (1-146

ethyl-3-(dimethylaminopropyl) carbodiimide, Sulfo-NHS (N-Hydroxysulfosuccinimide. From 147

Thermo Fisher Scientific, Waltham, MA; MES (2-(N-morpholino) ethanesulfonic acid. YPD, 148

bovine serum albumin (BSA). Calcofluor white (Fluka Analytical, #18909), Dectin-1 was 149

produced by Dr. Robert Wheeler’s lab at the University of Maine, ß (1-3)-glucan antibody was 150

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obtained from Biosupplies Australia Pty. Ltd., and goat antimouse antibody-Cy3 or HRP from 151

Jackson ImmunoResearch Laboratories, West Grove, PA, USA. 152

153

Fluorescent microscopy to visualize ß (1, 3)-glucan and chitin 154

We measured the distribution of chitin and ß (1, 3)-glucan on the cell surface using confocal 155

microscopy. For chitin staining we used calcofluor white (CFW), and we used anti-β (1, 3)-156

glucan antibody and a goat anti-mouse Cy3 secondary antibody for staining ß (1, 3)-glucan 157

exposed on the cell wall (25). For each experiment, the staining protocol was identical across all 158

samples, and all samples were imaged under identical conditions and on the same day. Three 159

biological replicates were carried out for all experiments. For fluorescence imaging of ß-glucan 160

and chitin, C. albicans strains were grown overnight in YPD at 30°C. The next day, cells were 161

inoculated at an OD 600nm= 0.1 into fresh YPD medium and grown for 3 more hours to reach log 162

phase. Cells were washed three times with PBS (phosphate buffered saline) and blocked for 30 163

min with PBS+3 % bovine serum albumin (BSA), after which they were incubated on ice with 164

anti-ß-(1, 3)-glucan antibody at a1:600 dilution for 90 min. After rigorous washing in PBS to 165

remove unbound primary antibody, cells were incubated with a secondary goat anti-mouse 166

antibody conjugated to Cy3 in PBS+5 % BSA at room temperature for 20 minutes. Cells were 167

washed in PBS again and the pellet was resuspended in 500 μl of 0.01mg/ml calcofluor white. 168

The tubes were covered with aluminum foil and incubated on the rocker at room temperature for 169

5 min. The cells were washed three times with 1 ml water and resuspended in 100 μl water prior 170

to observation under the confocal microscope. For an antibody negative control, only the 171

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primary or secondary antibody was added. Finally the fluorescence intensities were calculated for 172

~ 32 individual cells/condition by using ImageJ software (NIH ((http://rsb.info.nih.gov/ij)). 173

174

Preparation of Cells for AFM 175

C. albicans cells were grown overnight in YPD media at 30 °C with shaking at 250 RPM. The 176

next day the cells were diluted into a fresh YPD media and incubated at 30 °C with shaking at 177

250 RPM for 3 more hours. Cells in 1.5 ml aliquots were harvested in a microcentrifuge at 5000 178

RPM, resuspended and washed 3 times with 10 mL sodium acetate buffer (29), and 100 µl of 179

cells in buffer (~ 1x10 4 cells /ml) were pipetted onto a freshly cleaved mica wafer coated with 180

gelatin, incubated for 5 minutes, rinsed in a stream of water, and mounted in the AFM wet cell 181

prior to imaging in water (52). The mica surfaces that we used were freshly cleaved by applying 182

clear adhesive tape to each surface and removing multiple layers until the surface appeared to be 183

completely flat. A 0.5% gelatin solution was prepared by dissolving 0.5 g of gelatin (porcine 184

gelatin G6144 Sigma-Aldrich) in 100 ml of water that has been heated to boiling. Gelatin 185

concentrations from 0.5% down to 0.025%, in water, worked well, so cells adhere to the surface 186

and do not move during the scanning. Subsequently, the cleaved mica was dipped into a gelatin 187

solution that is heated to 60 °C, and withdrawn in a single motion. The mica is then placed edge 188

down on filter paper with the upper edge leaning against a surface. After drying overnight these 189

gelatin surfaces are ready to use for at least a month. 190

191

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AFM imaging and force measurements 192

C. albicans cells grown in YPD and suspended in sodium acetate buffer were centrifuged at 193

5,000 RPM in a microcentrifuge, and rinsed 2 times in water. After deposition onto a gelatin 194

coated mica surface, and incubated for 5 minutes, the surface was rinsed with water and placed in 195

the AFM wet cell. The cells were imaged in water using a 5500 PicoPlus AFM (Keysight 196

Technologies Inc., Santa Rosa, CA). The instrument was operated, using the PicoView 197

1.20.2 system, in contact mode for imaging using MLCT probes (Bruker AFM Probes, 198

Camarillo, CA) with either the C or D cantilever with spring constants of 0.01 or 0.03 N/m 199

respectively. Tip radius of 10 nm and Poisson ratio of 0.5 were placed into Pico View software 200

and used to calculate cell elasticity and indentation. The applied force was kept in a range of 3-5 201

nN for both imaging and force spectroscopy. The same cantilever was used for obtaining force 202

volume maps on the wild type and both mutants (34). The force volume map was obtained over a 203

2 x 2 micron scan of the uppermost area of the cell surface with an average of six 32 x 32 force 204

volume maps collected for each sample. Each of the 32 x 32 points of the force volume map is 205

the average of 3 force curves. The data presented is an average of three experiments. Images 206

were taken at a line scan speed of one line per second with either 256 or 512 pixels/per line. 207

Measurements of elasticity (30-33) and indentation (34, 35, 53, 54) were performed on the wild 208

type (SC5314, CIA4), cho1∆∕∆, and kre5∆/∆ cells and on wild type cells treated with 209

caspofungin as follows. Prior to measuring the elasticity of the cell surface, a force curve was 210

generated on a mica sheet covered with gelatin and used as the reference point. After imaging a 211

sample, force curves were generated for each approach, and the slope of each curve was 212

compared to the reference point to calculate force distance curves and elasticity. The force 213

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distance curves were transformed into force indentation by subtracting the cantilever deflection 214

on a mica sheet. The same cantilever was used for obtaining force volume maps on the wild type 215

and both mutants (34, 55). This occurred over a 2 x 2 micron force volume scan of the uppermost 216

area of the cell surface with an average of six 32 x 32 points taken with an average of 3 force 217

curves per point collected for each sample. The data presented is an average of three 218

experiments. 219

220

221

Adhesion force between Dectin-1 bound to the cantilever and β (1, 3)-glucan on the cell 222

surface 223

For adhesion experiments (37-41) AFM tips were functionalized with sDectin-1-Fc to act as a 224

binding probe for interacting with the ß (1, 3)-glucan layer on the C. albicans cell wall (22, 23, 225

25). The cantilevers were functionalized based on a protocol from Dr. Hermann Gruber, 226

Johannes Kepler University (http://www.jku.at/biophysics/content). Briefly cantilevers were 227

cleaned by rinsing in chloroform, dried with nitrogen gas, placed in a desiccator purged with 228

argon gas containing 30µl of APTES and 10µl of triethylamine, in separate trays, for two hours 229

to amino-functionalize the cantilever tips. After removing the trays, the desiccator was purged 230

with argon gas and the tips were left for two days. Next, 1mg of Acid-PEG25-NHS was added to 231

0.5ml of chloroform in a tray, placed in the desiccator and 30µl trimethylamine was added to the 232

tray and mixed, followed by purging the desiccator with argon gas and incubating for 2 hours. 233

For coupling Dectin-1 to the Acid-PEG25 functionalized cantilever tip, 0.4mg of EDC and 234

1.1mg of Sulfo-NHS were dissolved in 1ml of 0.1M MES buffer at pH 6.1. Droplets of 235

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EDC/Sulfo-NHS were then placed on parafilm and cantilevers were placed in contact with the 236

droplets for 15 minutes, followed by rinsing 4 times in PBS. Dectin-1 was diluted to 5.6 x 109 237

particles per ml in PBS and droplets were placed on parafilm. Cantilevers were then placed in 238

contact with the droplets for 2 hours to functionalize the tips with Dectin-1, rinsed 4 times in 239

PBS and stored in PBS. Force volume maps over a 2 x 2 area of cell surfaces were collected as 240

described in the previous section for obtaining elasticity and indentation measurements. 241

242

Results 243

Exposure of ß (1, 3)-glucan on the cell surface of C. albicans 244

In order to further characterize nanoscale effects of unmasking on cell wall architecture, we 245

chose to analyze two mutants that exhibit differential defects in cell wall structure and 246

microscopic unmasking patterns. First, we chose the kre5∆/∆ mutant, because it exhibits 247

extensive defects in cell wall composition, including increased chitin and ß (1, 3)-glucan, and 248

decreased mannan and ß (1, 6) glucan, and appears to have ß(1,3)-glucan unmasking over the 249

whole cell (48, 49). Second, for contrast, we chose the cho1∆/∆ phosphatidylserine (PS) 250

synthase mutant, which also exhibits ß (1, 3)-glucan exposure, but only in punctate regions 251

around yeast-form cells (25). Additionally, this mutant does not have as extensive gross 252

alterations in all cell wall polymers like kre5∆/∆. Only its cell wall chitin level is increased, 253

whereas other cell wall polymers, ß (1, 3)-glucan, ß (1, 6) glucan, and mannan, are unchanged 254

(data not shown) (47). Both mutants exhibit increased recognition by macrophages as evidenced 255

by greater binding with anti-ß (1, 3) glucan antibodies and soluble Dectin-1 (sDectin-1), as well 256

as increased elicitation of TNFα from macrophages (24, 25). 257

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We first directly compared the pattern of unmasking between wild-type (SC5314), kre5∆/∆, 258

and cho1∆/∆ strains using secondary immunofluorescence with a ß (1, 3)-glucan specific primary 259

antibody (Figure 2). As expected, the kre5∆/∆ mutant exhibits whole cell unmasking (Fig 2A) 260

that is quantitatively greater than that of the cho1∆/∆ mutant (Figure 2B). The cho1∆/∆ mutant 261

exhibits punctate regions of unmasking (Figure 2A), and this is significantly higher than the 262

unmasking quantified for wild-type, but significantly lower than kre5∆/∆ (Fig 2B). 263

In addition to examining ß (1, 3)-glucan exposure, we also examined the chitin distribution and 264

levels by staining with calcofluor white (CFW). The cho1∆/∆ and kre5∆/∆ mutants both exhibit 265

higher levels of chitin when compared to wild type cells (Fig 2A). In wild type, CFW stains 266

mainly the bud neck and bud scar while the mutants show major increases in chitin throughout 267

the entire cell wall, and kre5∆/∆ staining is more pronounced than cho1∆/∆, based on both visual 268

inspection (Fig 2A) and quantification with image J (Fig 2B). Wild-type cells treated with 269

caspofungin exhibited increased ß(1,3)-glucan exposure and increased chitin levels that were 270

distributed over the whole cells, more similar to the kre5∆/∆ mutant, but of lower intensity (Fig 271

2A,B). 272

273 C. albicans cells were immobilized and imaged in liquid on mica coated with different 274

percentages of gelatin 275

In order to begin examining the ultrastructure of the C. albicans cell wall we used atomic force 276

microscopy (AFM), as it gives high resolution images of the cell wall in a liquid environment, 277

without damaging the sample. This allows for real time imaging of metabolically active samples 278

(56-59). First, we needed to immobilize the cells to a suitable surface. This was achieved using 279

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gelatin coated mica surfaces. This positively charged surface was developed for imaging 280

bacterial cells in a liquid environment (52, 58, 59) and was extended here for mounting C. 281

albicans. In Figure 3, the cells were immobilized on the surface of a mica sheet covered with 282

gelatin of various percentages (0.5-0.025%) and imaged in water. The cells are dispersed 283

uniformly, making it easier to detect cells and image them by AFM. A lower percentage of 284

gelatin (0.025%), was used as it seems to reduce the level of aggregation, which is greater in 285

higher percentages of gelatin (ie. 0.1-0.5%) (57). 286

287

Cell wall unmasking correlates with increased cell surface roughness 288

Once immobilization was achieved, we examined how the nanoscale topology of unmasked 289

mutants differs from that of wild-type using AFM in deflection mode in water (Figure 4). The 290

cell walls of both mutants appear rougher when compared to the wild type (SC5314), which 291

appears smooth. The CAF2 strain, which is closely related to the background strain of kre5∆/∆ 292

(48), was also examined by AFM, and is similar to SC5314, so all studies reported herein discuss 293

SC5314 as the reference wild-type strain. Of the two mutants, kre5∆/∆ has the roughest 294

appearance followed by cho1∆/∆, which has a rough appearance in specific regions. Roughness 295

can be quantified (black trace below each image), and expressed as the root mean square (RMS) 296

surface roughness, which for our strains are 34.35±5.81 for kre5∆/∆, 29.67±3.21 for cho1∆/∆, 297

and 8.21±2.028 for wild type. The increased rough areas in cho1∆/∆ are more sporadic, which 298

may correlate with the punctate unmasking in this mutant (Figure 2). The rough appearance in 299

these two mutants was similar to that found in wild-type cells that have been treated with 300

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caspofungin (Fig 4). The RMS surface roughness of caspofungin treated wild-type cells was 39.5 301

±6.91. 302

The kre5∆/∆ and cho1∆/∆ mutants’ cell walls exhibit increased rigidity 303

The increased roughness in the cell wall was hypothesized to correlate with changes in cell wall 304

elasticity. To test this, force volume maps, across a 2µm square area on the top of the cell, were 305

recorded to assess cell wall elasticity. Each map contains an array of 32 x 32 points, with each 306

point being an average of 3 force curves. 307

These data were then converted to elasticity and indentation measurements by the PicoPlus 308

AFM software to determine the Young’s modulus for all three strains. The Young’s modulus 309

measures the ability of the material to undergo stress and strain. Lower Young’s modulus values 310

indicate increased elasticity. As a control, caspofungin-treated cells were also tested because this 311

drug inhibits synthesis of cell wall ß (1, 3)-glucan and causes unmasking of ß (1, 3)-glucan and 312

increased roughness (Fig 4) (27, 29). The results of these experiments are shown in Figure 5A, 313

where the X axis records the Young’s modulus and the Y axis displays the elasticity distribution. 314

The wild type has a Young’s modulus of ~9x104 Pa, whereas strains affected by cho1∆/∆ or 315

kre5∆/∆ mutations, or caspofungin (wild-type), have Young’s moduli that increase to ~5x105 Pa, 316

~8x105 Pa, or ~8.2x105 Pa, respectively (Figure 5A). Therefore, the cell walls of these strains are 317

less elastic than the untreated wild type. 318

Figure 5C shows a heat map of the 32 x 32 point display of the 2 µm square cell surface 319

where the transition from green to orange-red corresponds to an increase in the Young’s 320

modulus. It is evident from this, that the Young’s modulus of the wild type is less than either of 321

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the two mutants or wild-type treated with caspofungin. Thus, these cell wall stresses are 322

resulting in decreased elasticity (increased stiffness). 323

A closely related property to the Young’s Modulus is indentation, which measures the 324

depth that the AFM tip can press into the cell wall. Cells with greater indentation exhibit greater 325

elasticity. Like the Young’s modulus, indentation measurements indicated that wild type cells 326

are more elastic than the two mutants or the caspofungin-treated cells (Figure 5B). 327

Nanoindentation measurements were performed on the 2µm square area by recording force 328

curves on 32 x 32 points for 10 different cells (n=10). Statistical evaluation of the results using 329

Pico View 1.20 software shows that the indentation depth for the wild type cell surface is larger 330

(~7x10^2 nm±34.16 nm) than the indentation depth for kre5∆/∆ (~3x10^2 nm±31.02nm) and 331

cho1∆/∆ (~2x10^2nm±33.04nm) cells and caspofungin (~6x10^2nm±72.61) treated cell surfaces 332

(Figure 5B). Thus, the conditions tested here all lead to decreased elasticity compared to wild-333

type. Compared to the other conditions tested, the kre5∆/∆ mutant exhibits a broader distribution 334

of elasticity suggesting that it has a more uneven and perhaps a more disturbed surface. 335

336 The unmasked mutants show nanoscale increases in Dectin-1 binding 337

As indicated above, AFM images and measurements of elasticity and indentation show that the 338

kre5∆/∆ and cho1∆/∆ mutants have disorganized cell walls that lead to distinguishable physical 339

properties. We wanted to analyze the nanoscale spatial organization of the immunologically 340

relevant PAMP ß (1, 3)-glucan in the cell wall of unmasked mutant cells. Therefore, AFM tips 341

were functionalized with sDectin-1-Fc (design of this experiment represented in Figure 1). This 342

C-type signaling lectin is specific for ß (1,3)-glucan, and therefore adhesion force measurements 343

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using Dectin-1 functionalized AFM tips can reveal the presence and nanoscale distribution of 344

exposed ß (1,3)-glucan in wild-type, mutant, and caspofungin-treated cells. 345

Using the sDectin-1-Fc-functionalized AFM tip to measure the nanoscale distribution of 346

adhesion forces, an adhesion force volume map was generated for a 32 x 32 pixel array of a 2x2 347

µm sample area on the surface of C. albicans wildtype ± caspofungin, cho1∆/∆, and kre5∆/∆ 348

cells. As a control we measured adhesion between the gelatin surface and the functionalized tip, 349

and there was no interaction, indicating the gelatin did not interfere with this experiment. The 350

heat map clearly shows a progression from the wild type (blue-green), showing lowest adhesion, 351

to cho1∆/∆ shows intermediate adhesion (mixture of green, yellow, orange, and red), to kre5∆/∆ 352

and caspofungin-treated wild-type cells (mostly orange-red) that show the most adhesion (Figure 353

6A). 354

A histogram of the adhesion force frequency versus rupture strength was generated for the 355

different C. albicans strains and caspofungin-treated wild-type cells. Rupture strength, in this 356

case, is the force required to break adhesion between the Dectin-1 functionalized tips and ß (1, 357

3)-glucan in the cell wall of the fungus (Figure 6B). There is some interaction between the 358

Dectin-1 coated cantilever tip and the surface of wild type C. albicans, but it is less than that 359

observed for the mutants. It is likely that wild type cells possess some gaps in the mannan layer, 360

or that the surface coatings are dynamically organized, which leads to infrequent exposure of ß 361

(1, 3)-glucan and subsequent interaction with the Dectin-1 coated tip. For the wild type, the peak 362

adhesion frequency was ~1% while the peak adhesion frequency for the cho1∆∕∆ and kre5∆/∆ 363

mutants increase to ~8% and ~11% respectively. Peak adhesion frequency for caspofungin 364

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treated cells is at ~10%. These values indicate that the frequency with which the tip interacts with 365

the ß (1, 3)-glucan surface is due to increased exposure of the ß (1, 3)-glucan layer. 366

Peak adhesion rupture strength of wild type, cho1∆∕∆, and caspofungin treated cells are all 367

about the same, which indicates that the same target molecule, ß (1, 3)-glucan, is being 368

recognized, but the frequency is greater during unmasking. However, for kre5∆/∆, the peak 369

adhesion rupture strength is lower. The kre5∆/∆ mutant has diminished ß (1, 6)-glucan synthesis 370

(48), and ß (1, 6)-glucan serves as a cross linker for the ß (1, 3)-glucan layer and lower 371

crosslinking may affect the structure of the ß (1, 3)-glucan layer and subsequently the force 372

required to rupture the adhesion. 373

In control experiments to show that binding of the functionalized tip to the cell surfaces is 374

dependent on sDectin-1-Fc, soluble ß (1, 3)-glucan (laminarin) was added to the imaging buffer. 375

The solid purple line in the Figure 6B shows that sDectin-1-Fc coated tip is effectively blocked 376

by laminarin, so no interaction between the tip and cell surface occurs under these conditions. 377

Thus, the force curves are only due to Dectin-1-dependent adhesion to ß (1, 3)-glucan on the cell 378

surface. 379

380

Discussion 381

This study presents a nanoscale map of ß (1, 3)-glucan epitope exposure and cell wall 382

morphology in mutants that exhibit unmasking, using AFM. Features common to both mutants, 383

in addition to the increased ß (1, 3)-glucan exposure (Figure 2), is increased overall surface 384

roughness (Figure 4), and decreased elasticity (Figure 5). It was of interest to notice that the 385

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pattern of ß (1, 3)-glucan exposure in the cho1∆/∆ mutant, which was punctate in appearance, 386

correlate with intermittent regions of surface roughness (Figure 4). Furthermore, kre5∆/∆, which 387

has global ß (1, 3)-glucan exposure, had a more uniform increase in surface roughness. 388

It was of interest to us that these rough places on the wall are similar in appearance to that 389

generated by caspofungin in our work (Fig 4) and that from El-Kirat-Chatel and Dufrene (27) and 390

Formosa et al (29). In addition, we also observed that our mutants exhibited decreased 391

elasticity, which again is similar to what has been observed for caspofungin treatment by 392

Formosa et al (29). However, it contrasts with what was observed by El-Kirat-Chatel and 393

Dufrene (27). We do not fully understand the reasons for these contradictory results. We suspect 394

that in the El-Kirat-Chatel and Dufrene study (27) the deformation of the cell they observed 395

indicates that the cell wall is being more heavily damaged than in our study, and as a result, the 396

elasticity was decreased because of a loss of structural integrity. In our case, we were attempting 397

to use sublethal doses of caspofungin, and observed a loss of elasticity. We think that our results 398

showing increased rigidity of the wall reveal what happens in a sublethal treatment that causes 399

increased chitin synthesis and unmasking. We were ostensibly using the same concentrations of 400

caspofungin as El-Kirat-Chatel and Dufrene (27), so the precise reasons for such a difference 401

remain unclear. 402

Previous work (60) indicated that loss of chitin leads to increased elasticity and increased chitin 403

has the opposite effect. Both mutants analyzed in this study also show increased chitin levels 404

(47-49). It is of interest to determine if the enhanced surface roughness observed in this study is 405

caused by increased chitin levels. One can envision a model in which increased chitin synthesis 406

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deforms the overall structure of the wall, creating bulges that result in surface roughness 407

increases. This leads to other questions. Do these bulges influence unmasking and/or how well 408

mutants are detected by the immune system? Observations of unmasking using dSTORM 409

revealed clusters of unmasking foci (26). Do the regions of clustered ß(1,3)-glucan exposure 410

revealed by dSTORM correlate with peaks of roughness observed by AFM in the cell wall? How 411

often does surface roughness correlate with unmasking, and does this impact the epitope 412

exposure to the immune system in unique ways? This again, will be better addressed by 413

expanding this analysis to study additional mutants and strains. 414

In addition to these topological observations of the wall, a functionalized AFM tip was used to 415

measure the specific binding force between Dectin-1 and ß (1-3)-glucan residues on the surface 416

of the living C. albicans mutant and wild-type cells. It was of interest that the binding force 417

between wild-type and the mutants was very similar, but the binding frequency of the 418

functionalized tips to the kre5∆/∆ and cho1∆/∆ mutant cells and wild-type cells treated with 419

caspofungin is higher than the wild type cells (Figure 6). This indicates that unmasking leads to 420

greater frequencies of interactions rather than increased binding strength. This also demonstrates 421

a novel approach to mapping surface epitopes using immunologically relevant lectins. 422

Finally, in this study, we used gelatin coated mica to immobilize cells, allowing for a versatile 423

approach to immobilization that can be expanded to other strains, morphologies, and 424

functionalizations of the tip. In combination with other work on imaging and immobilization of 425

the cells (28, 57, 61), our approach to analyzing interactions between host lectins and pathogens 426

using functionalized tips provides another method to address nanoscale interactions in fungal 427

immunology. Exposure of ß (1, 3)-glucan plays an important role in detection of C. albicans by 428

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macrophage cells and therapies to enhance exposure could potentially aid in control and 429

elimination of C. albicans infections. In addition, future efforts to identify site-specific molecular 430

tags that, when attached to the AFM cantilever, interact with sufficient binding strength and 431

frequency to the surface of C. albicans should be pursued. These tags conjugated with antifungal 432

agents would offer an additional approach for treating C. albicans infections. 433

434

Acknowledgements 435

We are grateful to Merck, Kenilworth, NJ, USA for the contribution of caspofungin. DPA, STR 436

and MJD acknowledge support from the U.S. DOE Office of Biological and Environmental 437

Research, Genomic Science Program under the Plant-Microbe Interfaces Scientific Focus Area at 438

Oak Ridge National Laboratory. Oak Ridge National Laboratory is managed by UT-Battelle, 439

LLC, for the US Department of Energy under Contract no. DEAC0500OR22725. We gratefully 440

acknowledge the support of the University of Tennessee-Oak Ridge National Lab (UT-ORNL) 441

Joint Institute for Biological Sciences for this project. 442

443 References 444

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607

608

Figure legends 609

Figure 1. Schematic of AFM ligand-receptor interactions during unmasking of ß(1,3)-610

glucan. In these cartoons, the AFM tip has been functionalized by attaching Dectin-1 to the 611

cantilever tip. Dectin-1 can access the ß 1,3 glucan layer if the mannan layer is disturbed. (A) In 612

untreated wild-type cells, the wall consists of two main layers, with mannan in the outer layer and 613

ß(1,3)-glucan and chitin in the inner layer. As the cantilever, with Dectin-1 attached, approaches 614

the cell wall surface of wild-type cells, it rarely interacts with ß 1,3 glucan due to the overlying 615

mannan layer. Therefore, the cantilever approaches the surface of the cell (red line in graph) 616

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touches the surface and withdraws (blue line) without making contact with the ß 1,3 glucan layer. 617

(B) In cells where ß(1,3)-glucan is more exposed through the mannan layer, the cantilever, with 618

Dectin-1 attached, contacts the ß(1,3)-glucan (1 ). As the cantilever begins to withdraw, strain 619

appears on the cantilever, and the cantilever bends (2). As the cantilever continues to be 620

withdrawn, the adhesion force builds until it ruptures and the cantilever snaps off the surface (3). 621

622

Figure 2. ß-(1, 3)-glucan exposure and chitin levels increase in cho1∆/∆, kre5∆/∆, and 623

caspofungin-treated wild-type cells compared to untreated wild-type. (A) Cells were stained 624

with anti-β-glucan primary antibodies and Cy3 labeled secondary antibodies and calcofluor white 625

(CFW). Three biological replicates were carried out for all experiments. (B) Graph of the relative 626

mean fluorescent intensity of each strain, as quantified by Image J. These data reveal that the 627

kre5∆/∆, caspofungin treated cells and cho1∆/∆mutants have greater exposure of ß (1, 3)-glucan 628

and greater chitin than the wild type. (*=p<0.0001, ø=p<0.0026). 629

630

Figure 3. Immobilization of C. albicans cells onto mica surfaces coated with different 631

percentages of gelatin solution. Mica sheets were first coated with (0.5 - 0.025%) gelatin and 632

allowed to dry overnight. Then 100μL (~ 1x10 4 cells /ml) of Candida albicans cells in sodium 633

acetate buffer were pipetted onto the gelatin-coated mica sheet. After 5 minutes of incubation, the 634

surfaces were washed with water and placed in the AFM for imaging in distilled water. 635

636

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Figure 4. Topographic 2 X 2 micron images of the cell wall surface of C. albicans wild-type 637

± caspofungin, cho1∆/∆, and kre5∆/∆ strains. Cells were mounted on gelatin coated mica, and 638

imaged in water in contact mode with cantilevers having spring constants of 0.01 or 0.03 N/m 639

using a 5500 PicoPlus AFM. The PicoPlus AFM software was used to measure surface 640

roughness, which is shown in the plot below each corresponding image. The plot for each strain 641

was the average from measurements made from 30 cells. 642

643

Figure 5. Elasticity of cho1∆/∆, kre5∆/∆, and caspofungin treated cells decreases compared 644

to untreated wild-type. Force volume maps of the surface of wild type, cho1∆/∆, and kre5∆/∆ 645

mutants along with caspofungin treated wild type C. albicans, were taken by scanning a 2µm 646

square area on the top of a cell and recording 32 X 32 points, with each point being an average of 647

3 force curves. This data was converted into elasticity and indentation maps using the PicoPlus 648

AFM software. (A) Percentage distributions of Young’s modulus values corresponding to the 649

elasticity maps of SC5314, cho1∆/∆, kre5∆/∆ strains along with caspofungin treated cells. (B) 650

Representative histogram of the indentation values for the different strains and the caspofungin 651

treated cells. (C) Shows the heat map for the different strains and the caspofungin treated cells. 652

The color map indicates the modulus of elasticity (in Pa) using force curves generated at each 653

point on the cell surface. The more intense the red color, the less elastic (stiffer) the surface. 654

655

Figure 6. Adhesion force volume maps using Dectin-1 coated cantilevers recorded on wild-656

type, cho1∆/∆, kre5∆/∆, and caspofungin-treated wild-type strains. (A) The heat map force 657

curves recorded on the wild type strain show low frequency adhesion to the surface of the cell. 658

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Force curves collected on kre5∆/∆, cho1∆/∆, and caspofungin-treated cell surfaces indicate 659

higher frequency adhesion between the cell surface and the tip. Increased adhesion is indicated 660

by the progression from the blue and green to the orange and red colors. (B) Corresponding 661

histograms of the force curves between a Dectin-1 functionalized tip and ß (1, 3)-glucan on the 662

surface of wild type, kre5∆/∆, cho1∆/∆, and caspofungin-treated cells. The histogram data are 663

derived from 4096 force curves for each of the surfaces tested. In order to show that the 664

interaction of Dectin-1 on the tip is specific for ß (1, 3)-glucan on the surface of the mutants 665

(kre5∆/∆ or cho1∆/∆), ß (1, 3)-glucan was injected into the media. This treatment blocks the 666

Dectin-1 bound to the tip and prevents tip adhesion to the cell surface (purple line). 667

668

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