Analysis of Candida albicans Biofilm Mechanisms Causing

Antifungal Drug Resistance

Research by: Harini Shah

Chadwick Class of 2016

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Analysis of Candida albicans Biofilm Mechanisms Causing Antifungal Drug Resistance

Abstract:

C. albicans is an opportunistic human pathogen causing life threatening diseases in

immunocompromised individuals. Typically, these infections are associated with the formation of

biofilms on both host tissues and implanted biomaterials. Biofilms are defined as complex

microbial communities attached to a surface and encased in an expolymeric matrix. The ability to

form biofilms is important from a clinical perspective because these structures show an increased

resistance to antifungal therapy and the cells within such structures are able to withstand host

immune defenses. Candida albicans biofilms are completely resistant to most major classes of

antifungal drugs, including azoles and polyenes. However, this work has indicated excellent

activity of caspofungin, an antifungal drug belonging to the class of echinocandins, against C.

albicans biofilms. In a sequential antifungal drug therapy regimen, treatment of fluconazole first

followed by caspofungin leads to a significant decrease in efficacy of caspofungin. This study

points to the role of cell membrane, extracellular matrix, and cell wall stress response mechanisms

that are triggered by the addition of both caspofungin and fluconazole. Global gene expression

analyses and quantification data signify the synergistic role that chitins, sterols, and glucans have

in antifungal drug resistance.

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Introduction: Candida and candidiasis. C. albicans, a yeast similar in structure to the common brewer’s yeast

Saccharomyces cerevisiae, is part of the normal human microbiota. The fungus is usually acquired

early in neonatal life, and as a commensal organism, it produces little to no damage to the host. As

an opportunistic pathogen, the fungus is capable of causing a myriad of infections, which usually

occur in hosts with defective immunity (32). In such individuals, the fungus typically infects target

organs through overgrowth or hematogenous spread from a colonized site within the body (8).

Most common prompting factors for candidiasis include immunosuppressive therapy, antibiotic

therapy, cytotoxic therapy, surgery, intravenous catheters and indwelling devices, very low birth

weight, AIDS, diabetes, transplantation medicine, drug abuse, as well as others (6). Candidiasis

now represents the third or fourth most frequent nosocomial infection in hospitals in the U.S. and

worldwide (6). The incidence of systemic candidiasis in the US is approximately 20 cases per

100,000 people (or about 60,000 cases per year) and in high risk hospitalized patients this

incidence increases by a factor of 50 (28). Of note, these rates represent a 20-fold increase

compared with just two decades ago mostly as a result of an expanding population of

immunocompromised patients (33). Disseminated candidiasis carries unacceptably high mortality

rates (about 40-60%) even with treatment using available antifungal agents (6). In fact, in a study

designed specifically to identify microbiological factors influencing the outcome of infection,

among the top ten pathogens, Candida was associated with the overall highest attributable

mortality and was the only pathogen identified as an independent determinant of the risk of death

(7). The total estimated direct cost of candidiasis to the US health care system was approximately

$2-4 billion annually in the year 2000 (6).

C. albicans biofilms and their clinical impact. Many microbes in their natural habitats are found

in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Thus,

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biofilms are defined as structured microbial communities that are attached to a surface and encased

in a matrix of exopolymeric material (Figure 1). This fact is particularly important since it is now

estimated that a significant proportion of all human microbial infections involve biofilm formation.

C. albicans remains the fungal species most commonly associated with the formation of biofilms,

and most clinical manifestations of candidiasis are associated with biofilm formation (9). Candida

biofilms carries important clinical repercussions because of their increased resistance to antifungal

therapy and the ability of cells within biofilms to withstand host immune defenses.

C. albicans biofilm drug resistance. Biofilms are known to display very high levels of resistance

to most of the major classes of antifungal agents (12, 22). An example of such resistance is that to

fluconazole – a drug belonging to the azole class of antifungals. The cellular target of fluconazole

in C. albicans is a cytochrome P-450 hemoprotein involved in the ergosterol biosynthetic pathway.

Many studies have also indicated the target of fluconazole to be drug efflux pumps (20, 22).

Historically, fluconazole has been considered the drug of choice for candidiasis. Ironically, this

drug is completely inert towards C. albicans biofilms. Published reports indicate that resistance of

biofilms to fluconazole maybe multifactorial, involving cell aging, multidrug efflux pumps, and

differences in sterol synthesis (2, 20, 24). As mentioned earlier, drug resistance in biofilms is not

limited to azoles. Biofilms are resistant to almost all other major classes of antifungal drugs

including polyenes, polymyxin B, tri-azoles etc. So far, the only class of drugs active against

Candida biofilms is echinocandins, and more specifically, the drug caspofungin. Caspofungin

inhibits the synthesis of β-1,3-glucan, a major component of the fungal cell wall, that also is a

component of the extracellular matrices in biofilms (27, 30). Echinocandin drugs create enormous

stress on the cell by shutting down glucan synthase (23). Studies have shown that β-glucans

account for approximately 60% of C. albicans cell wall weight (3). Only recently has caspofungin

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been advocated as the first line drug, especially for patients suffering with catheter associated

infections.

Problem under investigation. It has been found that, under conditions where C. albicans biofilms

are first treated with fluconazole, these biofilms exhibit enhanced resistance to caspofungin. This

is of great concern, especially under clinical settings, in which biofilms are growing on catheterized

patients. The treatment of such patients with the first line of therapy for candidiasis – fluconazole,

has the potential to completely mitigate the anti-biofilm potency of the only drug that actually

works on such resistant biofilms – caspofungin. The molecular basis underlying this cross

resistance phenomenon was studied. Although past literature identifies the clinical relevance of

these findings, the molecular basis underlying this peculiar resistance phenomenon had yet to be

identified. So what could be happening when the biofilm encounters antifungal drugs? Various

theories assert a relationship between sterols, lipids, and chitin and their contributions individuallu

or in concert, to promoting resistance to drugs (1, 2, 13). Studies have also shown the relevance of

drug-sequestering glucans present on the biofilm matrix that perhaps renders fluconazole inert.

Biofilm matrix production may also govern antifungal resistance by obstructing drug diffusion

into cells (17). The hypothesis of this study is upon fluconazole treatment, C. albicans cells

experience cellular stresses that lead to extensive structural and gene expression changes in the

cell wall. To unravel why fluconazole and caspofungin are antagonistic, the impact of individual

drugs on biofilms must first be understood. To investigate this, the approach of global gene

expression profiling was taken to discover the genomic fingerprint during antifungal drug

exposure. Close attention was paid to the gene classes belonging to the cell membrane (target of

fluconazole) and cell wall (target of caspofungin). These findings will further clarify the

mechanisms by which drug resistance to caspofungin, one of the very few effective drugs on

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biofilms available as treatment at the moment, occurs with initial exposure to fluconazole in C.

albicans biofilms.

Materials and Methods:

The mechanisms of resistance to sequential antifungal treatment were studied through a

variety of lenses. All experiments were conducted in vitro. Since in vitro biofilms are known to

grow with similar phenotypic and molecular properties as those in vivo, sufficient information can

be provided through such methods (10, 28). First, results displaying the phenomenon that

sequential treatment of fluconazole followed by caspofungin creates resistance were replicated and

confirmed. To provide further insight into the sterol, chitin, and glucan compositions as well as

cell responses, microarray, RT-RTPCR, TEM, and sterol analyses were conducted.

Strains. For this study, C. albicans SC5314 was used in all experiments. The strains were

routinely cultivated from cells growing on yeast extract-peptone dextrose (YPD) agar plates (1%

yeast extract, 2% bacteriological peptone and 2% D-glucose) and sub-cultured into liquid YPD

medium for 24 hours at 30 °C. Candida albicans cells grew in the budding yeast phase under these

conditions, and were then grown under static conditions for further biofilm development.

Biofilm Formation. Biofilms were formed in 96-well or 24-well polystyrene flat-well plates as

described by Ramage et al (21). Cell suspensions of 1×106 cells ml-1 were prepared for all strains

by counting in sterile phosphate-buffered saline. Suspensions were added to either RPMI 1640

(with glutamine and phenol red, without bicarbonate and pH adjusted to 7.0) buffered with MOPS

(Sigma), or in YNB (Yeast Nitrogen Base with amino acids and ammonium sulfate supplemented

with 1% D-glucose, pH adjusted to 7.0). The plates were incubated at 37 °C to ensure filamentation

and mature growth of the biofilms for 24 hours before treatment of any drug was applied.

Antifungal Treatment. All drugs were treated as described by Sarkar, et al. (25). The drugs were

exposed to the biofilms for a minimum of 24 h to ensure that the most accurate outcome is

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observed. Biofilms were grown for 24 h and then either exposed to fluconazole (1 mg/ml),

caspofungin (0.25 µg/ml) or a sequential treatment of fluconazole followed by caspofungin. The

biofilms exposed to each of the drugs were incubated at 37oC for 24 h. Control biofilms without

exposure to drugs were also included in every assay run. At the end of the experiment, metabolic

activity of the biofilms were measured by using the XTT assay.

XTT Assay to Determine Metabolic Activity and Absorbance Calculation. A semi-

quantitative measure of biofilm formation was calculated by using an XTT [2,3-bis(2-methoxy-4-

nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide]-reduction assay, adapted from Ramage, et

al and Taff, et al. (21, 29). XTT (Sigma) was prepared in a saturated solution at 0.5 g/L in

phosphate-buffered saline (PBS). The solution was filter sterilized through a 0.22-µm-pore-size

filter, aliquoted, and stored at -80°C. Prior to each assay, an aliquot of stock XTT was thawed, and

menadione (Sigma; 10 mM prepared in acetone) was added to a final concentration of 1µM. An

aliquot (volume depending on size of the plate used, 100 µl for a 96-well plate and 1 ml for a 24-

well plate) of the XTT-menadione solution was then added to each prewashed biofilm and to

control wells (for the measurement of background XTT-reduction levels). The plates were then

incubated in the dark for up to 2 h at 37°C or in light for up to 20 min at 37°C. A colorimetric

change in the XTT-reduction assay, a direct correlation of the metabolic plate activity of the

biofilm, was then measured in a microtiter plate reader (Benchmark Microplate Reader; Bio-Rad,

Hercules, California) at 490 nm.

Transmission electron microscopy (TEM). C. albicans biofilms grown in the presence and

absence of FLC were fixed in 4% formaldehyde and 1% glutaraldehyde, post-fixed with 1%

osmium tetroxide and 1% potassium ferricyanide, stained with 1% uranyl acetate, dehydrated in a

graded series of ethanol solutions, and embedded in Spurr's resin. Sections (70 nm) were cut and

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placed on copper grids, post stained with 8% uranyl acetate in 50% methanol and Reynolds' lead

citrate, and analyzed with a transmission electron microscope (JEOL 1230). Cell wall thickness

was assessed for 50 cells from treated and untreated biofilm using AMT image software.

Ergosterol Quantification. The procedure was conducted as described by Skaggs, et al (2).

Stationary-phase cells were harvested by centrifugation at 3,000 rpm for 5 min and washed once

with sterile distilled water. The net wight of the cell pellet was determined. 3 mL of 25% alcoholic

potassium hydroxide solution (25 g of KOH and 35 ml of sterile distilled water, brought to 100 ml

with 100% ethanol), was added to each pellet and vortexed for 1 min. Cell suspensions were

transferred to 16 x 100 mm sterile borosilicate glass screw-cap tubes and incubated in an 85°C

water bath for 1 h. Following incubation, tubes were allowed to cool to room temperature. Sterols

were then extracted by addition of petroleum ether followed by vigorous vortex mixing for 1 h.

The heptane layer was transferred to a clean borosilicate class screw-cap tube and stored at -20°C

for as long as 24 h. 1 ml aliquot of the sterol extract was scanned spectrophotometrically at 260

nm with a Gilford Response Spectrophotometer (Ciba Corning Diagnostics Corp., Gilford

Systems, Oberlin, Ohio). The total ergosterol content was determined for each isolate with relation

to absorbance values. The wavelength value of 280 nm was used to determine the total absorbance

and the values were compared among all experimental groups.

Real time reverse transcriptase polymerase chain reaction (RT-RTPCR). A total of 1 μg of

RNA was treated with amplification-grade DNase I (Invitrogen, Carlsbad, CA) and used for cDNA

synthesis with the Superscript III cDNA synthesis kit (Invitrogen) as per the manufacturer's

instructions. The genes and respective primer sets were used in conjunction with SYBR Green

PCR master mix (Applied Biosystems, Foster City, CA) and Twin.tec real-time 96-well PCR

plates (Eppendorf AG, Hamburg, Germany) in an ABI 7300 real-time PCR system (Applied

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Biosystems FosterCity,CA). Parameters for primer design were set according to the

recommendations of Applied Biosystems. The primer sizes were between 20 and 25 bases in

length, and the Tm of each primer was 58°C. The amplicons were between 90 and 110 base pairs

in size. Each reaction mixture was set up in triplicate as 25 μl with 25 ng of cDNA for 40 cycles

(thermal cycling conditions were: initial steps of 50°C for 2 min and 95°C for 10 min, followed

by 40 cycles of 95°C for 15 s and 60°C for 1 min). Relative gene expression was quantified using

the threshold cycle (Ct) method with the 7300 System sequence detection software and the RQ

study application from Applied Biosystems. The target genes were normalized to a gene encoding

for C. albicans 18S ribosomal RNA. The change was calculated for each sample by using the

equation, 2−ΔΔCT, and results from the different replicates were averaged.

RNA Extraction (for Micrarray). RNA was extracted from 24 h biofilms grown under static

conditions in six-well microtiter plates. For both DNA microarray and Real Time PCR, total RNA

was prepared using hot acid phenol as described previously by Ausubel et al. (3). Briefly, the

biofilms from each condition (no drug control, fluconazole treated, caspofungin treated and

sequentially treated first with fluconazole followed by caspofungin) were resuspended in acid

phenol, lysis buffer, and SDS buffer and transferred to Eppendorf tubes. The tubes were incubated

at 650C with intermittent vortexing after every 5 min. The aqueous phase was then transferred to

a clean Eppendorf tube, and equal volumes of acid phenol were added to it. This tube was then

vortexed and the aqueous phase was transferred to a pre-spun phase lock tube. Chloroform was

then added and centrifuged for 5 mins at 40C.The aqueous phase was again transferred to a new

Eppendorf tube and ice cold 100% ethanol as well as 3 M Sodium acetate was added. The tubes

were kept for overnight precipitation at -800 C. The tubes were them centrifuged at 4 degrees and

the RNA pellet was resuspended in 75% ethanol and centrifuged again. The supernatant ethanol

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was aspirated and the pellet was dried in a speed vacuum centrifuge. The RNA pellet was finally

resuspended in RNAase free water and quantified in a Nanodrop spectrophotometer. The integrity

of the RNA samples was verified by gel electrophoresis.

Microarray. The Agilent EArray system was used to design microarrays from the C. albicans

(Assembly 21) genome. The help of the Microarray core facility at the Genome Technology

Center, Washington University St. Louis (WUSTL), MO, for probe design, microarray chip

design, hybridizations and bioinformatics was taken. A FASTA file containing sequence

information of 6205 C. albicans genes was retrieved from the Candida Genome Database and

forwarded to the core facility to design 6205 probes. These probes were placed on a glass chip in

an 8 x 15 K format, whereby allowing for comparisons between 8 different conditions, in a single

slide. Each block of 15 K genes included duplicates of the C. albicans genes and several spots

representing negative control (non-C. albicans genes). At least two biological replicates of the

following four conditions were compared to each other on the Agilent microarray slide.

1. C. albicans biofilms (48 h) without any drug treatment

2. C. albicans biofilms (24 h) treated with 1000 µg fluconazole (24 h)

3. C. albicans biofilms (24 h) treated with 0.1 µg caspofungin (24 h)

4. C. albicans biofilms (24 h) treated sequentially with 250 µg fluconazole first (24 h) and

then 0.25 µg caspofungin (24 h)

Total RNA was extracted from these conditions and sent to the WUSTL core facility for cDNA

synthesis, microarray slide hybridizations and statistical analyses of the raw data. For

bioinformatics, Agilent feature extraction files were imported in Partek GS and rProcessedSignal

values were quantile normalized and control features were filtered out. A principal Component

Analysis (PCA) was performed to check the sample quality. PCA applies dimensionality reduction

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using the signal intensity data to visualize the samples in a plane. Analysis of Variance (ANOVA)

was performed to generate the list of differentially expressed (DE) genes, while a p-value cut-off

of 0.05 was used to filter down the genes.

Results:

Pre-treatment with fluconazole render the C. albicans biofilms resistant to caspofungin.

C. albicans biofilms were totally inert to 1000 µg of fluconazole. On the other hand, 0.25 µg of

caspofungin decimated the biofilms. It was found that pretreatment of biofilms with 1000 µg of

fluconazole rendered the biofilm cells resistant to caspofungin. Thus, in a sequential antifungal

drug therapy process in which treatment of mature C. albicans biofilms by fluconazole for 24 h is

followed by another 24 h of caspofungin treatment, a significant decrease in the efficacy of this

echinocandin was observed; therefore, the otherwise excellent in vitro antibiofilm activity was

significantly weakened. Results illustrated in figure 2.

Transmission Electron Microscopy. The goal was to find out whether fluconazole brings about

structural changes in the cell walls of C. albicans biofilm cells and whether this phenomenon could

be a reason behind the resistance towards caspofungin. Transmission electron microscopy was

used to screen for structural differences between the cell walls of fluconazole-treated and untreated

SC5314 biofilms. Imaging of the cell walls from the two conditions demonstrated quantitative

differences. The cell walls of the fluconazole-treated biofilm cells were about 30% thicker than

untreated biofilm cells (Figure 3). This indicates that fluconazole induced cell membrane stress

might cause structural changes in the cell wall.

Quantitation of ergosterol levels. Since ergosterols are the target of fluconazole, the study aimed

to investigate if treatment with the azole might influence the levels of sterols in the fungal

membrane. Previous literature indicates treatment of fluconazole causes the cells to reduce their

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ergosterol content. This was validated by the results. Interestingly it was found that treatment of

caspofungin alone induced the sterol levels to greater than two fold. When caspofungin was added

into the biofilms following fluconazole treatment, the sterol levels rose back to the levels of the

control and remained higher than the sterol content of the fluconazole exposed biofilms (Figure

4). The explanation behind this trend is deliberated in the discussion section.

Microarrays. To gain a better insight on the genes and processes that may have an impact on the

C.albicans genome after treatment with antifungal drugs, especially after sequential, fluconazole,

and, caspofungin treatments, microarrays were performed. Biofilm conditions treated with

fluconazole and caspofungin individually or sequentially were compared to the untreated biofilm

control conditions. A total of two technical and two separate biological replicates for each

condition were performed in the study. Table 1 tabulates the numbers of genes that were

significantly (p< 0.05, ANOVA) and differentially regulated between each treatment condition

versus the control. As has been reported by previous transcriptional analysis studies (REF, David

rogers paper and Christoph de Enferts papers), we found that addition of fluconazole to C. albicans

biofilms only had a modest effect on the C. albicans transcriptome. As expected, several genes

involved in the ergosterol biosynthetic process, such as ERG 1,ERG11, ERG2, ERG24, ERG3,

ERG6, ERG25, ERG5 and UPC2 were found upregulated more than 1.5 fold. Several cell wall

organization genes, as well as genes encoding GPI anchor proteins were found induced. Of the

downregulated genes, several genes had functions in RNA metabolism and ribosome biogenesis.

Compared to cells treated by fluconazole, greater differences in the C. albicans transcriptome were

observed when biofilm cells were treated with caspofungin. Largest categories of genes

upregulated greater than 1.5 fold were involved in metal transport (zinc, potassium, iron),

comprising of ~ 24% of the total number of induced genes. Genes encoding for β-1,3-glucans

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(FKS1 and FKS2), the targets of caspofungin in C. albicans, were overexpressed greater than 1.5

fold. Interestingly, a large number of genes with roles in vesicle mediated transport, endosomal

transport and vacuole mediated transport were found lowered in caspofungin treated biofilms.

Differences in gene expression in biofilms treated sequentially with fluconazole and

caspofungin compared to the untreated controls were then analyzed. Treatment of biofilm with

fluconazole, followed by caspofungin induced robust induction of cell wall associated genes. The

GPI anchor metabolic process was found induced and so was the lipid metabolic process, including

almost all the ergosterol genes being upregulated > 2 fold. Some of the important genes

downregulated were chitinase genes (CHT3 and CHT3) and hyphal associated genes (HWP1,

HGC1, ALS3, ALS1). Additionally, several overlaps in the genes and processes were found when

the sequential treatment condition was compared to the either drug condition alone. Overall,

between 25-35% of the genes were found to have unknown or uncharacterized functions in all

comparisons tested.

From the gene expression results it was obvious that fluconazole caused a disturbance in

the C. albicans cell membranes (indicated by increases in ergosterol genes), which was found

exaggerated on subsequent caspofungin treatment (increase in almost all the ergosterol genes). The

sequential caspofungin treatment also had an impact on the C. albicans cytoskeleton, and cell wall

GPI anchor proteins. Since there were several overlaps in the subsets of genes between the three

treatment conditions, the study proceeded to identify genes that were either up or down regulated

exclusively in one condition compared to the other. For this analysis, a Venn diagram was

formulated (as shown in Figures 5 and 6) and the genes associated with the cell wall and cell

membrane in each category were highlighted.

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Interestingly, only about 63 genes were found to be common between the three conditions. The

identity of the genes exclusively regulated in the sequential treatment condition was further

investigated. It appeared that fluconazole treatment followed by caspofungin induced the cell wall

salvage pathway genes in C. albicans biofilms. This was indicated by increases in a number of

genes regulated by HOG1 (DAP1, MET10, DDR48, RHR2, DAP2, UGP1, PIR1, AHP1, ECM331,

PHO84, MET1), an important MAP kinase of high osmolality and core stress response and a key

player in the cell wall salvage pathway. Other genes with functions in cell wall integrity (IRS4),

cell wall biosynthesis (PMI1) and β-1,3-glucan genes PHR2, PIR32, SSR1 were also induced in

the sequential condition. A cell wall integrity gene with important roles in the cell salvage pathway,

MKC1, was found unchanged. There has been elaboration on the selective regulation of this kinase

specifically in planktonic cells and not the biofilm conditions (5). Finally, large numbers of cell

membrane genes were upregulated exclusively in the sequential condition.

Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The levels of gene

expression known to be associated with antifungal drug resistance were examined. Expression

levels of these genes in the absence of drugs were compared to those treated with fluconazole,

caspofungin, and sequential treatment with caspofungin then fluconazole. It was found that ERG3

and ERG6 expression was highest after exposure to fluconazole. ERG11 showed a greater than 5

fold increase after caspofungin treatment and sequential treatment. The β-1,3-glucan gene GSC1

showed a greater than 5 fold increase under sequential treatment. The chitin gene CHS1 did not

change significantly. CHS3 was upregulated by about 1.9 fold under sequential treatment

conditions while under fluconazole and caspofungin treatment the gene expression did not change.

CHS8 was most upregulated under sequential conditions (greater than 6 fold) followed by

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caspofungin and fluconazole. CHS2 expression was under maximum conditions after fluconazole

treatment while its expression did not change significantly under other two conditions (Table 2).

Exogenous Ergosterol: The study discovered a spike in the ergosterol levels when biofilms were

treated with caspofungin. Also, we found a large number of ergosterol genes being upregulated in

the microarray results, on exposure to caspofungin. We wondered if this increase may have

anything to do with caspofungin resistance. To test this, biofilm cells with caspofungin in the

presence or absence of ergosterol were treated. Interestingly, the study found that ergosterol alone

was capable of inducing biofilm resistance to caspofungin (Figure 7).

Statistical Analysis and Contributions. Data sets were considered statistically different using the

two-tailed Student’s t-test; a p-value greater than 0.05 represented significant results. Analyses

were performed using Microsoft Excel.

Discussion

C. albicans is an opportunistic pathogen that develops highly drug resistant biofilms on

indwelling medical devices. The first line of treatment against candidiasis, fluconazole, is useless

against these resilient entities. Fortunately, biofilms are hypersensitive to caspofungin – one of the

only few drugs in the rapidly shrinking antifungal drug armamentarium. It was demonstrated that

pretreatment with fluconazole renders biofilms completely resistant to caspofungin. This was

dependent on the levels of ergosterol that was produced during cell wall stress.

Sterol Levels of Biofilms in Each Treatment are Significantly Altered. Maintenance of cell

wall and membrane integrity is imperative, as fungi cannot survive without this structure or any

other drastic modifications. Many structural components of the cell wall, extracellular matrix, and

cell membrane contribute to successful resistance for C. albicans biofilms. One of these

components includes ergosterol, a lipid that is present in the cell membrane of C. albicans and

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contributes to cell membrane fluidity (16, 18). The presence or lack of sterols in the cell membrane

of the fungus has specifically been shown to mediate resistance. Sterol metabolism is the primary

cellular process affected by the most widely employed antifungal drugs. Past works of literature

reveal that ergosterol levels are significantly decreased in the intermediate and mature phases of

biofilm growth compared to those in the early phases of development, and hypothesize that

lowered levels of ergosterol present in sessile C. albicans may reflect a physiological state more

conducive to fluconazole resistance (18). As such, sterol analyses in the present work parallel these

predictions. C. albicans is an astute fungus. On fluconazole treatment, C. albicans deliberately

reduce their sterol levels within the cell, so as to escape from the ergosterol inhibitory effects of

fluconazole (14). This successfully allows the cells to combat the drug. Under caspofungin

exposure, C. albicans experiences extreme cell wall stress, and tries to compensate it by

upregulating ergosterol to maintain cell structure and rigidity (26). It is possible that excess of

ergosterol in the membrane leads to cell structure remodeling that renders biofilm cells resistant

to caspofungin. This is seen from the microarray results that reveal upregulation of chitin levels in

the sequential treatment condition. Perhaps along with membrane sterols, the cells also upregulate

synthesis of chitin in their cell walls, as a response to the cell wall stress. High levels of chitin in

the cell wall are known to make cells resistant to caspofungin.

Chitin Levels and β-1,3-Glucan Expression Are Altered Due to Cell-Stress Response

Mechanisms. The different cell wall components interact with each other to give rise to the overall

architecture of the cell wall. The molecular organization of the cell wall of C. albicans is to be

believed as follows: the skeletal inner layer is a 3-dimentional network of branched β-1,3-glucan

molecules that are locally aligned and kept together by hydrogen bonding (11, 31). This network

acts as a scaffold for the attachment of the other macromolecules in the cell wall, including chitin

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and proteins (16). Chitin, a glycoprotein that is located on the cell wall of C. albicans, has been

known to contribute immensely to antifungal resistance (15, 19). Multiple studies have shown the

negligible effect that fluconazole has on chitin (2, 13). However, caspofungin targets both chitins

and, in specific, β-1,3-glucans. In both caspofungin-treated and sequentially-treated biofilms, the

upregulation of chitin synthase genes alludes to the cell wall stress mechanisms which the cells

use to compensate for the lack of chitin in their cell walls from caspofungin. Previous studies have

highlighted that fluconazole, upon treatment, embeds itself inside the extracellular matrix structure

as a form of resistance (4, 17). When caspofungin is only used as a treatment for biofilms, it

immediately begins activity to target glucans on the cell walls and extracellular matrix, so biofilms

experience slight upregulation of their chitin and glucan synthase genes. However, in sequentially-

treated biofilms, as fluconazole remains on the surface, more expression of chitin synthase and

glucan synthase genes is needed to have the same effect of maintaining cell wall integrity against

caspofungin.

Conclusions and Future Work:

This study identifies the mechanisms by which Candida albicans biofilms gain resistance

towards echinocandins and azoles and in specific, towards caspofungin and fluconazole. Results

provide further reason to believe that biofilm resistance is a multifactorial process that entails the

participation of components from the extracellular matrix, cell wall, cell membrane, as well as

other factors that contribute to surface remodeling. The upregulation of genes modulating chitin

synthesis and β-1,3-glucans during caspofungin and sequential treatment highlights the cell stress

that biofilms undergo. The biofilms, therefore, in an attempt to maintain cell wall integrity,

strengthen and reinforce defense mechanisms such as cell wall thickness to counter the invasive

potency of the drug.

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This study is unique in that it not only identifies the response mechanisms of certain drugs

in biofilm conditions, but it also further delves into the mechanisms of such interactions. The

conclusions align with current literature on C. albicans biofilm resistance. The data portrays a

systematic and conclusive report to further elucidate the complexity of interactions relating to the

phenomenon of resistance to caspofungin and fluconazole. This research seems to be on track and

logical in terms of its approach. Future aims include analysis of cell fluidity and phospholipid

content in order to determine if resistance causes cell walls to become more compact or fluid. This

relationship would explain why caspofungin is not able to find its target due to its inability to

penetrate the wall. There will additionally be a screening of cell wall mutants of C. albicans

including mutants of the GPI anchor proteins, ergosterol, and chitin to see if certain mechanisms

are imperative in mediating resistance. A major possibility is also two-dimensional electrophoresis

to measure protein content differences in treated and untreated biofilms, especially under

sequential conditions. Future experiments will hopefully provide more insight into reasons why

antifungal drugs act unusually when used against biofilms.

Illustrations and Legends:

Figure 1. C. albicans biofilms both in vitro (left) and in vivo (right). Images portray similarities in structure between clinical isolates and in vitro grown biofilms. Image reference Finkel, et al (8).

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Figure 2. Metabolic activity quantified through XTT of C. albicans biofilms in vitro. Sequential treatment (far right) with fluconazole and caspofungin shows resistance to drugs. Concentrations of 1 mg/ml and 0.25 µg/ml of fluconazole and caspofungin, respectively, were used. Caspofungin shows the least amount of metabolic activity, 30% that of untreated biofilms.

Figure 3. Transmission electron microscopy of C. albicans biofilm cell walls. Cell walls when exposed to fluconazole treatment are about 30% thicker than those of untreated C. albicans biofilms. Addition of fluconazole causes the cell wall to look increasingly robust.

Figure 4. Quantification of ergosterol levels in biofilms. Levels of ergosterol are altered due to treatment of antifungal drugs on C. albicans biofilms. Absorbance was taken at 260 nm. Fluconazole-treated biofilms show a 50% decrease in ergosterol. Caspofungin-treated biofilms increase almost twofold with relation to ergosterol levels.

Figure 5. Venn diagram of an elevated gene expression pattern. Upregulated genes of treated biofilms of C. albicans were compared to untreated cells

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Figure 6. Venn diagram of a repressed gene expression pattern. Downregulated genes of treated biofilms of C. albicans were compared to untreated cells.

Figure 7. Metabolic activity quantified through XTT. Metabolic levels of ergosterol added exogenously were analyzed through a microtiter plate reader. With the addition of ergosterol externally, biofilms gain increased resistance to caspofungin (far right).

Table 1. Differentially regulated genes at each treatment condition for microarray analyses.

Table 2. Real time PCR data of differentially expressed genes. C. albicans biofilms show increased expression of ergosterol in caspofungin (ERG4, ERG5, ERG6, ERG7, ERG9, ERG11). Sequentially treated biofilms also portray overexpression of ergosterol. In sequential conditions, chitin genes are overexpressed, and GSC1 (glucan gene) increases almost 5 fold.

Genes FLC vs Untreated CAS vs Untreated SEQ vs Untreated ERG3 +3.7 No change +7.5

ERG4 No change +1.8 +1.8

ERG5 +2.5 +2.2 +4

ERG6 +6.8 +2.5 +20

ERG7 No change +1.7 No change

ERG9 +1.4 +1.8 +2.4

ERG10 No change No change +2.3

ERG11 +4 +4 +7

CHS2 No change No change +1.9

CHS3 No change No change +1.9

CHS8 No change +2.8 +2.4

GSC1 No change +1.5 +5.3

00.10.20.30.40.50.60.7

Control Caspofungin Caspofungin withErgosterol

Abs

orba

nce

at 4

90 n

m

20

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