Detailed Materials and Methods

Candida Strains

C. albicans K1 was used for all studies. The organism is a clinical isolate from a systemic

biofilm Candida infection (1). The organism was routinely maintained and grown on yeast

peptone dextrose (YPD) agar plates.

Biofilm Formation and Matrix Isolation

A large scale rolling bottle system was used to generate matrix material for detailed

biochemical and functional analyses. Briefly, aliquots (25 ml) of C. albicans overnight cultures

grown in RPMI (Roswell Park Memorial Institute medium 1640 buffered with MOPS) at 37°C,

200 RPM, were used to inoculate 100 ml of RPMI into a polystyrene roller bottle with a surface

area of 850 cm2 (Corning #3970). One hundred bottles were placed on a roller apparatus

(Wheaton Science Products, Millville, NJ), rolling at the rate of 20 RPM at 37°C. After 24 h, the

biofilm culture medium was replaced and the bottles were incubated for another 24 h. The media

were carefully removed from the bottles and the biofilms washed with sterile water + 0.1% SDS

to facilitate removal of biofilm from the device wall. To isolate the matrix, the intact C. albicans

biofilms were scrapped into an Erlenmeyer flask and then gently sonicated at 42 kHz for 20 min

(Branson 1510 Ultrasonic Cleaner sonicator) and then sonicated with a 1 cm × 5 cm probe in an

Intrasonic Processor (Cole Parmer, Vernon Hills, IL) at amplitude of 70 for 10 min. The

aggregate biofilm was then centrifuged at 9165×g for 20 min in order to separate fungal cells and

matrix. The supernatant containing matrix was then collected and lyophilized. The sample was

resuspended in 100 ml of distilled sterile water and dialyzed (3-kDa molecular weight cut off

dialysis membrane tubing, Spectrum® Laboratories Inc., Rancho Dominguez, CA) at a ratio of 1

1

to 100 (by vol) at 4°C for 5 consecutive days with twice daily water changes. The dialyzed

matrix was next lyophilized yielding the ‘crude’ C. albicans biofilm matrix. Overall, a total

number of 700 bottles of the matrix corresponding to the biofilm area of 59.5 m2 were collected

for biochemical analysis. A similar cell mass of planktonic (non-biofilm), C. albicans was

collected and similarly processed to discern the impact of matrix processing on the cell wall.

Carbohydrate Analysis

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The carbohydrate concentration of crude matrix was determined colorimetrically (492

nm) using the phenol-sulfuric acid method with D-Glc as standard (2). Subsequent structural

analysis was performed after a series of purification and fractionation steps. The matrix sample

was dissolved in 3 ml of 20 mM bis-Tris/HCl (pH 6.5) loading buffer. The initial matrix

fractionation was performed on a HiPrep™ 26/10 Desalting column prepacked with Sephadex™

G-25 Fine (GE Healthcare Life Sciences, Uppsala, Sweden), which yielded two major pools:

high molecular weight polymeric materials and low molecular weight materials co-eluted with

salts. High molecular weight material was next separated on an anion exchanger HiPrep™ 16/10

DEAE FF column (GE Healthcare Life Sciences) equilibrated with 20 mM bis-Tris/HCl (pH

6.5). Elution was carried out in a 20 mM bis-Tris/HCl (pH 6.4)/0.5 M NaCl buffer system at a

flow rate of 1 ml/min in a linear gradient of salt from 0 to 100% in 20 column volumes. Neutral

free carbohydrates were found in flow-through fractions, which were then pooled together,

lyophilized, dissolved in 2 ml of 150 mM NH4HCO3, and applied to gel filtration on a HighPrep

16/60 Sephacryl™ S-300 HR column (GE Healthcare Life Sciences). Matrix components were

eluted at a flow rate of 0.5 ml/min and 1 ml fractions were collected. All chromatographic

separation steps were performed at room temperature on the high-performance liquid

chromatography ÄKTA-Purifier 10 system (GE Healthcare Life Sciences). All buffers used were

filtered through 0.2 μm nylon membrane filters (Nalgene, Rochester, NY) and degassed prior to

use. Isolated carbohydrate fractions were lyophilized, resuspended in a small volume of water,

and incubated at 55C overnight in order to decompose and remove any remaining ammonium

bicarbonate. These steps were repeated until all of the salt was removed and the isolated

carbohydrates appeared as an anamorphous cotton-like material after the final lyophilization. The

calculation of molecular weight of biofilm matrix neutral carbohydrates was done using size

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exclusion column calibrated with a set of Leuconostoc spp. dextran standards, which included

100 kDa, 70 kDa, 40 kDa, 25 kDa, and 6 kDa polymers.

Carbohydrate fractions were subjected to detailed structural and biochemical analyses as

described below. Matrix monosugar composition and quantification was determined as alditol

acetate derivatives by GLC-FID (Shimadzu GC-2010 system, Shimadzu Co., Kyoto, Japan)

using a 50% cyanopropylphenyl methylpolysiloxane column (#007-225; 30 m × 0.25 mm with

0.25 µm film thickness, Quadrex Co., Woodbridge, CT) as previously described (3). The GLC

conditions were as follows: injector at 220 °C, detector at 240 °C, and a temperature program of

215°C for 2 min, then 4°C/min up to 230°C before holding for 11.25 min run at constant linear

velocity of 33.4 cm/sec and split ratio of 25:1.

NMR spectroscopy, 1D and 2D, was performed to complement monosugar assessment

and to provide linkage assessment for six carbohydrate polymers from the Sephacryl S-300

fractionation (F2, F5, F10, F14, F16, and F17). Choice of these fractions was based upon

relative abundance and size range.

All data were collected at 70°C on a Bruker Biospin Avance III 500 MHz NMR

spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a 5 mm triple

resonance, cryogenic probe, CPTXI 500 H-C/N-D. One dimensional spectra were collected with

32 acquisitions using a standard one pulse experiment. The spectral width was 10 ppm centered

at 4.7 ppm. The relaxation delay time was 2 s with an acquisition time of 3.3 s (32768 data

points). 32 acquisitions were collected.

Multiplicity edited, phase sensitive, echo-antiecho 1H{13C} HSQC spectra were obtained

using 4 acquisitions per indirect time point with 1H decoupling during acquisition. Matched

swept adiabatic 13C inversion pulses were used. The raw data matrix size was 2048 × 128 blocks.

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Spectra were collected with a relaxation time delay of 2 s and an acquisition time of 0.2 s with

sweep widths of 10 ppm (1H) and 65 ppm (13C), respectively. The center of the spectrum was 4.7

ppm (1H) and 82 ppm (13C).

Phase sensitive 1H{1H} NOESY spectra were obtained using 16 acquisitions per indirect

time point. The NOE mixing time was 300 ms. The raw data matrix size was 2048 × 128 blocks.

Spectra were collected with a relaxation time delay of 2 s and an acquisition time of 0.2 s with

sweep widths of 10 ppm in both 1H dimensions. The center of the spectrum was 4.7 ppm (1H) in

both dimensions.

Phase sensitive, echo-antiecho 1H{13C} HSQC-TOCSY spectra were obtained using 8

acquisitions per indirect time point with 13C decoupling during acquisition. Matched swept

adiabatic 13C inversion pulses were used and a DIPSI2 sequence was used for the TOCSY

mixing time of 300 ms. The raw data matrix size was 2048 × 128 blocks. Spectra were collected

with a relaxation time delay of 2 s and an acquisition time of 0.2 s with sweep widths of 10 ppm

(1H) and 65 ppm (13C). The center of the spectrum was 4.7 ppm (1H) and 82 ppm (13C).

Phase sensitive, echo-antiecho 1H{13C} HMBC spectra were obtained using 4

acquisitions per indirect time point with no 13C decoupling during acquisition. A three-fold, low-

pass J-filter was applied to suppress one-bond correlations and the delay to maximize long range

coupling was set to 8 Hz. The raw data matrix size was 4096 × 128 blocks. Spectra were

collected with a relaxation time delay of 2 s and an acquisition time of 0.4 s with sweep widths

of 10 ppm (1H) and 65 ppm (13C). The center of the spectrum was 4.7 ppm (1H) and 82 ppm

(13C).

Phase sensitive 1H{1H} gradient selected DQFCOSY spectra were obtained using 4

acquisitions per indirect time point. The raw data matrix size was 2048 × 128 blocks. Spectra

5

were collected with a relaxation time delay of 2 s and an acquisition time of 0.2 s with sweep

widths of 10 ppm (1H) in both dimensions. The center of the spectrum was 4.7 ppm (1H) in both

dimensions.

Complementary carbohydrate linkage analysis was determined from partially o-

methylated alditol acetate derivatives by GC/MS on a Varian 3300 gas chromatograph linked to

a Finningan Ion-Trap 810 R-12 mass spectrometer using a Zebron™ ZB-1 column (30 m × 0.25

mm with 0.25 µm film thickness) (Phenomenex, Torrance, CA) (4). Helium was used as carrier

gas at the constant pressure of 8.8 psi. The GC/MS conditions were as follows: injector at 275

°C, ion source at 260 °C, split ratio of 50:1, and a temperature program of 150°C for 2 min, then

4°C/min up to 250°C before holding for 6 min run. Additionally, quantitative measurement of

matrix β-1,3 glucan was also assessed for each carbohydrate chromatographic fraction using

Limulus lystate based Glucatell detection kit (Associates of Cape Cod, MA) as previously

described (5,6).

Small-angle xray scattering (SAXS) was used to gain further insight into the molecular

size and shape of the most abundant mannan polysaccharide using a Bruker Nanostar bench top

system. X-rays were generated with a rotating Cu anode Turbo X-ray Source and detected with a

Vantec-2000 (2048 x 2048 pixel) detector. The sample to detector distance was 67 cm allowing

for detection of a q-range spanning 0.012 to 384 Å-1. Sample and buffer scattering patterns were

collected for 2 hours with frames recorded every hour. Each frame was compared to check for

radiation damage and none was detected. Carbohydrate samples were extensively dialyzed for 48

hours in a 20 mM TRIS, 100 mM NaCl, pH 7 buffer to assure buffer matching for buffer

subtraction. Samples and buffer were run through a 0.2 µm filter preceding SAXS experiments

to remove any potential small particles. Three sample concentrations ranging between 0.07 and

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3.0 mg/ml were run for each carbohydrate to check for inter-particle interactions. No

concentration dependent differences were detected in the scattering patterns. Data was processed

using the ATSAS software suite (7). GNOM was used to obtain pairwise distance distribution

functions for each carbohydrate (8-10). Dmax was varied in increments of 2 Å-1 until the pairwise

distance distribution function dropped smoothly to zero. The output from GNOM was then used

in conjunction with DAMMIF to generate 20 independent ab inito dummy atom models of the

average carbohydrate molecular shape (11,12). The 20 models of each carbohydrate had

excellent agreement with the experimental scattering curve (2 < 1) and were also very similar

with a normalized spatial discrepancy (NSD) of less than 0.6. The dummy atom models were

then averaged with DAMAVER. Atomic carbohydrate models were energetically minimized

using the MMFF94 force field implementation in ChemBio3D version 13 (PerkinElmer).

To explore the location of matrix carbohydrates, biofilms were labeled with purified

antibodies and imaged by confocal and electron microscopy. Biofilms were grown on sterile

coverslips (Thermanox) for 24 h as previously described (13). Matrix glucomannans were used

to raise mAbs in mice. Individual clones recognizing branched mannan and β 1,6 glucan were

selected for subsequent qualitative and quantitative matrix analyses. Commercially available β

1,3 glucan antibodies similarly used. Briefly, purified components were injected intraperitoneally

into mice followed by spleen collection, cell isolation and myeloma cell fusion. Monoclonal cell

lines and antibodies were collected from hybridomas, purified, and selective performance tested

as previously described (14). For confocal imaging, the individual mannan and β 1,6 glucan

antibodies were fluorescently labeled with rhodamine and FITC (Pierce), respectively, and

imaged on a Nikon A1R confocal microscope. Confocal images for FITC (excitation 494 nm,

emission 517 nm) and rhodamine (excitation 552 nm, emission 575 nm) were conceived

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simultaneously using the Z-stack mode. Depth measurements were taken at regular intervals

across the biofilm. Images were processed for display using Nikon NIS-Elements Viewer V3.2.

Lipid Analysis

Lipids were extracted from the desalted lyophilized matrix powder with a mixture of

CHCl3/MeOH (2:1, by vol.) containing 0.1 g/l BHT and processed as described elsewhere (15).

All steps were performed in organic solvent-washed glassware. The sample was vigorously

vortexed for 5 min at full speed, incubated in the darkness for 2 h at room temperature and then

centrifuged at 1200×g for 5 min. The separated organic solvent layer was removed and the pellet

was washed with 2 ml of CHCl3/MeOH (2:1, by vol.) and centrifuged again using the same

parameters. The collected lipid extracts were combined and dried under a stream of nitrogen.

After drying, the lipid sample was reconstituted in 0.5 ml of CHCl3 and subjected to TLC

separation on 20 cm 20 cm silica gel Si60 plates (Merck KGaA, Darmstadt, Germany).

Neutral lipids were separated in hexane/ethyl ether/AcOH (90:20:1, by vol.), which yielded

triacylglycerols, sterol esters, free fatty acids, and a pool of immobile phospholipids. The latter

group was scrapped off the plate, extracted from the silica gel and subjected to another TLC

separation in CHCl3/MeOH/AcOH/H2O (50:37.5:3.5:2, by vol.). This step yielded four classes of

glycerolipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and

phosphatidylinositol) and one class of sphingolipids. Lipids were visualized under UV light after

spraying plates evenly with a 0.2% solution of fluorescein in EtOH. All isolated lipid classes

were scraped off their silica gel plates and re-extracted with CHCl3/MeOH (4:1, by vol.)

containing 0.1g/l BHT. Samples were vortexed, incubated overnight at room temperature and

then centrifuged at 1200×g for 1 min to remove silica gel particles. The internal standard,

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pentadecanoic acid (100 µl of 0.05 mg/ml in ethanol) was added to each sample and the organic

solvents were evaporated under nitrogen. Next, isolated lipids were subjected to methylation in

the presence of 0.5 ml of 14% BF3 in MeOH. Vials containing the processed lipids were tightly

screwed and boiled for 30 min at 100C. After cooling, the samples were mixed with 1 ml hexane

and 0.5 ml H2O, vortexed extensively for 30 s, and centrifuged at room temperature at 1200×g

for 5 min. The top hexane layer containing methyl ester derivatives was transferred to a new

clean glass tube, dried under nitrogen, resuspended in 100 l hexane and transferred to GC vials.

Methylation of fatty acids was performed using 0.5 ml of 14% BF3 in MeOH and methyl esters

were recovered with hexane (16). Fatty acid methyl esters were analyzed by gas chromatography

using a Hewlett-Packard 5890 (Hewlett Packard, Palo Alto, CA) equipped with a capillary

column coated with DB-225 (30 m × 0.25 mm, internal diameter of 0.25 µm) (Agilent

Technologies Inc., Wilmington, DE). Column temperature was kept at 70°C for 1 min, increased

to 180°C at a rate of 20°C/min and then to 220°C at a rate of 3°C/min. The temperature was kept

at 220°C for 15 min. Injector and detector temperatures were set at 250ºC and the injection port

temperature was set at 300ºC. Peaks were identified by a comparison of retention times with FA

standards (Supelco, Bellefonte, PA). The abundance of FAs was calculated from the relative

peak areas as described elsewhere (16).

Prostaglandins were isolated with MeOH from the initial CHCl3/MeOH lipid extract,

which was separated after adding 1.25 ml distilled water, followed by vortexing and

centrifugation 1200×g for 1 min. The content of MeOH was adjusted to 15% in the upper layer

and acidified to pH 3.0. The extract was loaded on a preactivated SepPak Classic C18 cartridge

(Waters Corp., Milford, MA), which was first washed with 20 ml 15% MeOH, followed by 20

ml distilled water, and dried with a syringe. Prostaglandins were eluted from the solid phase

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extraction cartridge in 10 ml methyl formate, which was further removed under nitrogen and the

remaining extract was reconstituted in 100 l CHCl3/MeOH (2:1, by vol.). The obtained samples

were diluted 1:2 [vol:vol] with 0.1% formic acid and chromatographically resolved with Agilent

ZORBAX 300SB-C18 1.8µm 2.1x50mm column on an Agilent 1200 HPLC (Agilent, Palo Alto,

CA) with an autosampler held at 5ºC, using linear gradient of 70% water : 30% acetonitrile :

0.1% Formic acid to 95% acetonitrile : 5% water : 0.1% formic acid over 30 min. The flow rate

was 0.25 ml/min. The mass spectrometer used in conjunction with chromatographic separation

was an Agilent LC/MSD TOF with electrospray ionization used in negative ion mode

(http://www.biotech.wisc.edu/facilities/massspec/instrumentationoverview/LCMSDTOF). The

following instrumental parameters were used to generate the most optimum deprotonated ions

[M-H+] in negative mode: capillary voltage 3500 V; drying gas 12.0 l/min; nebulizer 30 psig; gas

temperature 350°C; Oct DC1 -41.5 V; fragmentor 130 V; Oct RF 250 V; skimmer 60V. Internal

calibration was achieved with assisted spray of two reference masses, 112.9856 m/z and

1033.9881 m/z. Acquired data was processed using the Analyst QS 1.1 build:9865 software

(Agilent, Palo Alto, CA) to extract parent masses observed in the range from 100 to 3200 amu,

while formulas were generated with an integrated molecular mass calculator and the Agilent

Mass Hunater Qualitative Analysis software [version B.01.03, build: 1.3.157.0].

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Sterols (STs) were extracted and analyzed according to a previously described protocol

(17). Briefly, sterols from the matrix lipids were saponified with 1 ml of 6% (w/v) KOH in

MeOH for 1 h at 90 °C. After addition of 1 vol of H2O, the unsaponifiable fraction was extracted

three times with 3 vol of C6H6. The sterols were identified and quantified by Fast GC–MS. The

separation was performed on a Zebron ZB-5MS capillary column (10 m × 0.1 mm i.d., 0.1 μm

film thickness), (Phenomemex) and spectroscopy was performed on a Shimadzu 2010 Plus

system equipped with a Shimadzu QP 2010 Ultra mass spectrometer detector. The system was

operated in constant linear velocity (70 cm/s) using helium as a carrier gas and the sample was

injected in the split mode (Split ratio 20:1). The GC oven temperature was started at 80 °C,

increased to 220 °C at a rate of 60 °C min−1, then to 300 °C at a rate of 20 °C min−1 and finally

held to 300 °C for 1.5 min. The injection port, transfer line and ion source temperature were

maintained at 285, 300 and 250 °C, respectively. The MS was run in the mode of electron impact

(EI) mode with an electron energy of 70 eV and the spectra were scanned in the range of m/z 50–

500. The sterols were identified by comparing their mass spectra with the standard mass spectra

in the NIST MS library. An additional FID detector allowed the quantitative analysis of sterol.

The FID temperature was 320 °C and the oven temperature program was the same as to the GC–

MS operation. Cholesterol was used as an internal standard.

Protein Analysis

Protein concentrations from the crude matrix sample were assessed colorimetrically at

562 nm using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL), with bovine

serum albumin as standard as previously described (18). Enzymatic “in liquid” digestion and

mass spectrometric analysis was done at the Mass Spectrometry Facility, Biotechnology Center,

11

University of Wisconsin-Madison. About 200 g of matrix proteins were extracted by

precipitation with 15% TCA/60% acetone and then incubated at -20°C for 30 min. The treated

sample was centrifuged at 16,000g for 10 min and the resulting pellet was washed twice with

ice-cold acetone followed by an ice-cold MeOH wash. Pelleted proteins were re-solubilized and

denatured in 10 μl of 8 M urea in 100 mM NH4HCO3 for 10 min, then diluted to 60 μl for tryptic

digestion with the following reagents: 3 μl of 25 mM DTT, 4.5 μl of acetonitrile, 36.2 μl of 25

mM NH4HCO3, 0.3 μl of 1M Tris-HCl and 6 μl of 100 ng/μl Trypsin Gold solution in 25 mM

NH4HCO3 (Promega Co., Madison, WI). Digestion was conducted in two stages, first overnight

at 37°C, then an additional 4 μl of trypsin solution was added and the mixture was incubated at

42°C for additional 2 h. The reaction was terminated by acidification with 2.5% TFA to a final

concentration of 0.3%, and then centrifuged at 16,000g for 10 min. An 8-μl aliquot of the

digested sample was subjected to spectrometry analysis. Trypsin-generated peptides were

analyzed by nanoLC-MS/MS using an Agilent 1100 nanoflow system (Agilent, Palo Alto, CA)

connected to a hybrid linear ion trap-orbitrap mass spectrometer (LTQ-Orbitrap, Thermo Fisher

Scientific, San Jose, CA) equipped with a nanoelectrospray ion source. Capillary HPLC was

performed using an in-house fabricated column with an integrated electrospray emitter

essentially as described elsewhere (19), except that 360 µm 75 µm fused silica tubing was

used. The column was packed with 5-µm C18 particles (Column Engineering, Ontario, Canada) to

approximately 12 cm. Sample loading and desalting were achieved using a trapping column in

line with the autosampler (Zorbax 300SB-C18, 5 µm, 5 0.3 mm, Agilent). HPLC solvents

were as follows: loading solvent: 1% (v/v) acetonitrile, 0.1 M acetic acid; solvent A: 0.1M acetic

acid in water; and solvent B: 95% (v/v) acetonitrile, 0.1 M acetic acid in water. Sample loading

and desalting were done at a rate of 10 l/min with the loading solvent delivered from an

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isocratic pump. Gradient elution was performed at 200 l/min with the % of B in A increasing

from 0 to 40 in 200 min, 40 to 60 in 20 min, and 60 to 100 in 5 min. The LTQ-Orbitrap was set

to acquire MS/MS spectra in a data-dependent mode as follows: MS survey scans from m/z 300

to 2000 were collected in profile mode with a resolving power of 100,000. MS/MS spectra were

collected on the 5 most-abundant signals in each survey scan. Dynamic exclusion was employed

to increase the dynamic range and maximize peptide identifications. This feature excluded

precursors that were up to 0.55 m/z below or 1.05 m/z above previously selected precursors.

Precursors remained on the exclusion list for 15 sec. Singly-charged ions and ions for which the

charge state could not be assigned were rejected from consideration for MS/MS. Raw MS/MS

data were searched against a concatenated C. albicans amino acid sequence database using an in-

house MASCOT search engine (20), with methionine oxidation, asparagine and glutamine

deamidation as variable modifications; peptide mass tolerance was set at 15 ppm and fragment

mass at 0.6 Da. Identified proteins were further annotated and filtered to 1.5% peptide and 0.1%

protein false-discovery-rate with Scaffold Q+ version 3.0 (Proteome Software Inc., Portland,

OR) using the protein prophet algorithm (21).

Functional mapping of the matrix proteome was performed using the Kyoto

Encyclopedia of Genes and Genomes (KEGG) (22,23). Each protein predicted from the C.

albicans genome assigned a KEGG Ontology ID (KOID) was obtained, and the specific pathway

and super-pathway membership information retained. Upon correlation with the experimental

proteome data, the number of proteins expressed within a given pathway was determined.

Tabulated proteins were presented as a percentage of the total number of proteins predicted to

belong to a given pathway from the C. albicans genome, as determined by KEGG. The

visualization of relative quantities of biofilm matrix proteins was also performed using KEGG

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protein functional categorization. On the basis of this hierarchical classification scheme Voronoi

treemaps have been constructed as previously described (24). This approach divides screen space

according hierarchy levels where main functional categories determine screen sections on the

first level, subsidiary categories on the second level and so forth. The polygonic cells of the

deepest level represented functionally classified matrix proteins and were colored according to

relative abundance of each protein that was determined based on total counts of corresponding

trypsin-digested peptides.

Nucleic Acid Analysis

Nucleic acid concentrations were measured either spectrophotometrically (260/280 nm)

with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, San Jose, CA) or by

fluorescence (excitation at 350 nm, emission 460 nm) using the Hoechst 33342 solution (Pierce

Biotechnology) (25). To determine if the nucleic acid represented distinct coding regions, the C.

albicans biofilm matrix nucleic acid was cloned and sequenced. Briefly, extracellular DNA was

isolated by a two-step protocol, utilizing the MasterPure™ Yeast DNA Purification Kit

(Epicentre Biotechnologies, Madison, WI) followed by the QIAquick PCR Purification Kit

(Qiagen Inc., Valencia, CA). The sample was digested either with EcoRI or HindIII and

generated eDNA fragments were subsequently cloned into a pGEM®-T Easy vector (Promega)

predigested with EcoRI or HindIII, respectively, and transformed into the RapidTrans™ E. coli

TAM-1 strain (Active Motif, Carlsbad, CA). Plasmids were isolated from 40 randomly selected

transformants per sequence-driven metagenomic library and the introduced eDNA inserts were

sequenced with T7 and SP6 primers using the ABI BigDye™ Terminator v1.1 Cycle Sequencing

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Kit (Applied Biosystems, Foster City, CA). Nucleotide sequences of eDNA were analyzed using

the Candida Genome Database BLAST (26).

In vivo Matrix Analysis

The clinical relevance of select matrix components was explored using rodent central

venous catheter and denture biofilm models (27,28). Specific-pathogen-free male Sprague-

Dawley rats at weights of 350 g (Harlan Sprague-Dawley, Indianapolis, IN) were used for all

studies as described earlier (27,28). Animals were maintained in accordance with the American

Association for Accreditation of Laboratory Care criteria, and all studies were approved by the

Institutional Animal Care Committee. Following a 48 hour biofilm formation phase, the

implanted medical devices were removed for processing. Two samples from each model were

collected for gel-free proteomics using the protocol described above. An additional two samples

from the venous catheter model was also utilized for microscopy to analyze and localize matrix

carbohydrates. Monoclonal antibodies to each of the three major matrix polysaccharides, α 1,2

branched α 1,6 mannan, β 1,6 glucan were isolated as described above. The fluorescently

labeled β 1,6 glucan and α 1,2 branched α 1,6 mannan antibodies described above were used for

confocal microscopy of intact in vivo catheter associated biofilms. The distal 2 cm of catheter

was cut perpendicular to the catheter length with an 11-blade scalpel into three 2- to 3-mm-long

“doughnut” segments. Stained catheter segments were transferred to a glass-bottom petri dish

(coverslip 1.5, 35-mm disk P325G 1.5-14C; MatTek, Ashland, Mass.) in an on-end orientation.

Biofilms were observed with confocal microscopy as described above. For transmission electron

microscopy (TEM), β 1,3 glucan antibodies were linked to nano-gold particles and used for

labeling intact in vivo biofilms. After catheter removal, biofilms were collected by centrifugation

15

and prepared for immuno-transmission electron microscopy. Briefly, biofilms scrapped from in

vivo catheters were immersion fixed in a mixture of 4% paraformaldehyde/0.1% glutaraldehyde

in a 0.1M phosphate buffer (PB), pH 7.4 overnight at 4oC. The following day, the samples were

rinsed several times 0.1M PB, dehydrated through a graded ethanol series and embedded in LR

White embedding media (PolySciences Cat #17411) and polymerized at 50oC for 48 hrs.

Following polymerization, the samples were sliced into 70nm sections with a Leica EM UC6

Ultramicrotome and placed on 300 mesh pioloform (Ted Pella, Inc. #19224) coated nickel TEM

grids. TEM grids were floated on reagent drops, specimen side down, and transferred from drop

to drop with fine forceps. All procedures were carried out at room temperature unless otherwise

noted. Samples were incubated for 15 minutes with 0.05M glycine in PBS (10 mM Phosphate

buffer, 150mM NaCl) pH 7.4 to inactivate residual aldehyde groups. Next, the samples were

incubated in Aurion rabbit-gold conjugate blocking solution (Aurion Immuno Gold Reagents,

The Netherlands) for 30 minutes. After blocking, the grids were rinsed 3 x 5 minutes in

incubation buffer (PBS with 0.2% BSA-c) and then incubated in the primary β-1,3 glucan

antibody diluted to 5ug/ml in incubation buffer, overnight in a moist chamber at 4°C. For

immuno-labeling control samples, the primary antibody was omitted. The following day the

samples were rinsed 6 x 5 minutes in incubation buffer and then incubated in a secondary

antibody for 2 hours using Aurion rabbit anti-goat IgG gold conjugate (diluted 1:25 in incubation

buffer). Next, the grids were washed 6 x 5 minutes in incubation buffer; 3 x 5 minutes in PBS;

and then postfixed in 2% glutaraldehyde in 0.1M phosphate buffer for 5 minutes. Samples were

observed with a Philips CM120 Transmission Electron Microscope and images were collected

for analysis with a SIS MEGA-VIEW III digital camera using SIS analysis software.

Functional Matrix Analysis

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Previous studies have linked matrix drug sequestration to the biofilm drug resistance

phenotype (29-31). We further explored the relevance of this matrix function using two

complementary assays. First, we utilized 3H-labeled fluconazole to assess biofilm and matrix

sequestration before and after matrix removal. Briefly, biofilms were grown in 6-well plates for

48 h, washed and the matrix was removed from half of the culture wells by gentle sonication

followed by washing as described above. Fluconazole was prepared by adding 30 μCi to 18 ml

of RPMI/MOPS (1,686,500 dpm/ml), and 0.6 ml was added to each well containing either intact

biofilms or matrix-deprived biofilms, followed by chases with non-radioactive drug. Plates were

incubated with shaking at a rate of 50 rpm for 30 min at 37°C. Biofilms were washed, dislodged,

and collected. After biofilm disruption by scrapping and vortexing, cells were pelleted (4500g

for 20 min) and the matrix containing supernatant was collected. The cell pellet was disrupted

with glass beads in order to fractionate biofilm cells into cell wall and intracellular fractions. The

quantity of radiolabeled drug in the total and each biofilm component was measured by liquid

scintillation counting (TRI-CARB 2100TR, Packard). Data were normalized for biofilm dry cell

weight. Four biologic replicates were performed.

Interactions between the biofilm matrix and fluconazole were also probed using one-

dimensional 1H-NMR. Data were collected at 37°C on a

Bruker Biospin Avance III 600 MHz NMR spectrometer (Bruker BioSpin GmbH)

equipped with a 1.7 mm triple resonance, cryogenic probe, CPTXI 500 H-C/N-D

(32). One-dimensional spectra were collected with 16 acquisitions using a one pulse

sequence experiment with water suppression and excitation sculpting with gradients (zgesgp)

(33). The spectral width was 16 ppm centered at 4.7 ppm. The relaxation delay time was 2 s with

an acquisition time of 1.7 s (32768 data points). The approach was based on monitoring chemical

17

shifts of fluconazole-specific protons in the absence and presence of the biofilm matrix under pH

controlled conditions (34). In this study, all tested reactions were prepared in PBS (pH 7.2) and

fluconazole was used at a constant concentration of 0.653 mM. In this drug/matrix system,

interactions were represented by broadening of the chemical shift peaks of protons present in

fluconazole.

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