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Human #-defensin 6 self-assembly prevents adhesion and suppresses virulence traits of Candida albicans Phoom Chairatana, I-Ling Chiang, and Elizabeth M. Nolan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01111 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Human α-defensin 6 self-assembly prevents adhesion and suppresses virulence traits of Candida albicans

Phoom Chairatanaa, I-Ling Chianga, and Elizabeth M. Nolana,1

a

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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To whom correspondence may be addressed. Email: [email protected]

Funding Source Statement: This work was supported by The Camille and Henry Dreyfus Foundation, the MIT Research Support Committee (Wade Fund Award), and the MIT Undergraduate Research Opportunities Program (UROP, funding to I.C.). P.C. is a recipient of a Royal Thai Government Fellowship.

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Abbreviations AMA AmpB ATCC BFP CFU DAPI DIC DMEM DMSO F2A HD HD5 HD6 HEPES HIV GFP MOPS MTT PBS RPMI SDM SEM YPD

antimicrobial activity assay amphotericin B American Type Culture Collection blue fluorescent protein colony-forming unit 4’,6’-diamidino-2-phenylindole differential interference contrast Dulbecco’s modified Eagle medium dimethylsulfoxide HD6 variant with Phe2Ala mutation human defensin human α-defensin 5 human α-defensin 6 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid human immunodeficiency virus green fluorescent protein 3-(N-morpholino)propanesulfonic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide phosphate buffered saline Roswell Park Memorial Institute standard deviation of the mean scanning electron microscopy yeast extract-peptone-dextrose


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Abstract HD6 is a host-defense peptide that contributes to intestinal innate immunity and mediates homeostasis at mucosal surfaces by forming non-covalent oligomers that capture bacteria and prevent bacterial invasion into the epithelium. Our present work illustrates a new role of HD6 in defending the host epithelium against pathogenic microorganisms. We report that HD6 blocks Candida albicans adhesion to human intestinal epithelial cells and suppresses two C. albicans virulence traits, namely invasion into human epithelial cells and biofilm formation. Moreover, a comparison of HD6 and a single-point variant F2A that does not form higher-order oligomers demonstrates that the self-assembly properties of HD6 are essential for functional activity against C. albicans. This opportunistic fungal pathogen, which resides in the intestine as a member of the gut microbiota in healthy individuals, can turn virulent and cause a variety of diseases ranging from superficial infections to life-threatening systemic infections. Our results indicate that HD6 may allow C. albicans to persist as a harmless commensal in the gastrointestinal tract. Moreover, HD6 and HD6-inspired molecules may provide a foundation for exploring new antimicrobial strategies that attenuate the virulence traits of C. albicans and other microbial pathogens.


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Fungal pathogens cause a range of infections that can result in serious complications in patient care, especially for immunocompromised individuals and the elderly.1 Candida spp. are the fourth most common cause of hospital-acquired systemic infections in the United States.2,3 C. albicans constitutes a part of the normal flora in healthy individuals and is usually confined to the skin and mucosal surfaces of the oral cavity, gastrointestinal tract, urogenital tract, and vagina.4 Nevertheless, C. albicans can cause superficial and systemic infections in humans. The former include oral, skin, and vaginal candidiasis.5,6 Oral candidiasis is typically non-lethal and common among certain populations of immunocompromised patients; it is a sentinel indicator for HIV disease progression before the appearance of more severe symptoms.7 Systemic candidiasis, in contrast, is associated with a mortality rate of up to 50%, even following treatment with antifungal drugs.8 Both neutropenia and damage of the gastrointestinal mucosa are risk factors for developing a systemic infection.9 Central venous catheters, which allow fungi to directly access the bloodstream, and the use of broad-spectrum antibiotics, which enable fungal overgrowth, can cause systemic candidiasis.10 C. albicans employs virulence factors and fitness traits during infection. The morphological transition between yeast and hyphal forms,11,12 the expression of adhesins13,14 and invasins15 on the fungal cell surface, thigmotropism (a movement in which an organism moves or grows in response to contact stimuli), the formation of biofilms, phenotypic switching, and the secretion of hydrolytic enzymes are considered to be virulence factors.16 The fitness traits include rapid adaptation to fluctuations in environmental pH, metabolic flexibility, robust nutrient acquisition systems, and stress response machineries.17 Targeting specific virulence factors may provide alternative


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strategies to prevent and treat fungal infections, and such approaches are needed because of the emergence of antifungal drug resistance.18 In addition, classical antifungal drugs, such as AmpB and fluconazole, exhibit strong adverse effects in patients.19 The human innate immune system mediates homeostasis and the interplay between the host and microbes at mucosal surfaces, in part by using host-defense peptides.20,21 The intestine continually faces a significant challenge conferred by microbial invaders as well as commensals, and the innate immune system works to protect the intestinal epithelium and maintain homeostatic balance. Paneth cells, secretory cells located at the bases of the crypts of Lieberkühn in the small intestine, contribute to mucosal innate immunity by biosynthesizing and secreting host-defense peptides and proteins.22-24 In humans, Paneth cells express two α-defensins, HD5 and HD6.25-28 Defensins are small (2-5 kDa) cysteine-rich host-defense peptides that generally exhibit broad-spectrum antimicrobial activity in vitro.21,29,30 α-Defensins contain three regiospecific disulfide linkages (CysI—CysVI, CysII—CysIV, CysIII—CysV) in the oxidized form, which stabilize a three-stranded β-sheet fold and confer protease resistance.31-34 The oxidized form of HD6 (Figure 1), in contrast to HD5 and other characterized α-defensins, exhibits negligible in vitro antimicrobial activity.31,34-37 HD6 possesses a unique host-defense mechanism, which involves its remarkable selfassembly properties.34,36 HD6 monomers oligomerize into elongated structures termed “nanonets” that entrap bacteria in the intestinal lumen,36 which prevents the bacteria from invading into host epithelial cells and subsequent dissemination.34,36


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Based on the ability of HD6 to block bacterial invasion,34,36,38 we questioned whether this biological function extends to fungi, which are both commensals and opportunistic pathogens in the gut. We selected to investigate this notion using C. albicans because it is a member of the normal flora and an opportunistic pathogen. We hypothesized that the HD6 oligomers may interact with C. albicans and prevent its surface attachment, which is the first step in both epithelial cell invasion and biofilm formation. In the current work, we investigate whether HD6 provides host defense against C. albicans by examining its effect on fungal adhesion, biofilm formation, and morphology. We report that HD6 prevents C. albicans adhesion to host cells and suppresses its virulence traits. We demonstrate that the self-assembly of HD6 is required for these biological functions. This work expands the host-defense repertoire of HD6 to providing innate immunity against an opportunistic fungal pathogen.

Materials and Methods General Methods. All solvents, reagents, and chemicals were purchased from commercial suppliers and used as received unless noted otherwise. Native HD6 and F2A were prepared and purified as previously described.34 All buffers and aqueous solutions were prepared in Milli-Q water (18.2 MΩcm) and passed through a 0.22-µm filter. All routine optical absorption measurements were performed by using a Beckman Coulter DU 800 UV-visible spectrophotometer maintained at ambient temperature. Extinction coefficients (280 nm) were calculated by using the online tool, ExPASy ProtParam (ε280 = 4845 M-1cm-1 for both HD6 and F2A). Peptide stock solutions


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were routinely prepared in Milli-Q water and concentrations were quantified by using the calculated extinction coefficient. A BioTek Synergy HT plate reader was used to record optical density at 600 nm (OD600) for antimicrobial activity assays and at 550 nm for biofilm formation assays. Solution and buffer pH values were verified by using a Mettler Toledo S20 SevenEasy pH meter. C. albicans Strains and Growth Conditions. The C. albicans strains used in this study are listed in Table S1 (Supporting Information). Fungi from freezer stocks were streaked on YPD (Becton, Dickinson, and Company) agar plates (1% yeast extract, 2% peptone, 2% dextrose, and 2% agar) and incubated at 30 °C for at least 20 h. Single colonies were selected and grown in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) to saturation with shaking (30 °C, 175 rpm, 20 h) prior to each experiment. RPMI medium was prepared by dissolving 10 g RPMI (Life Technologies) in 950 mL of water, and to this solution was added 30.3 g of MOPS (Calbiochem) to achieve a final concentration of 0.145 M. The pH was adjusted to 7.2 by using 4 M NaOH, and the final volume adjusted to 1 L with Milli-Q water. The RPMI medium was then sterile-filtered and used to promote C. albicans hyphal growth in this work. Antimicrobial Activity Assay. For an overnight culture, a colony of C. albicans SC5314 was picked from an agar plate and incubated in 20 mL of YPD in a 250-mL baffled flask with shaking (30 °C, 175 rpm, 18 – 20 h). For a log-phase culture, 400 µL of the overnight culture was added into 20 mL of fresh YPD medium (1:50 dilution) in a 250-mL baffled flask and the diluted culture was incubated at 37 °C with shaking until cells were at mid-log-phase growth (OD600 ≈1.0). A 5-mL portion of the overnight culture at stationary phase (OD600 ≈2.0) or the culture at log-phase was centrifuged (3,600 rpm,


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5 min, 4 °C) to pellet the cells. The supernatant was decanted, and the cells were washed with 5 mL AMA buffer (10 mM sodium phosphate buffer, pH 7.2 supplemented with 1% YPD) and centrifuged (3,600 rpm, 5 min, 4 °C). The cells were resuspended in the AMA buffer to obtain OD600 ≈2.0 for stationary-phase cultures or ≈1.0 for log-phase cultures. Another 1:250 dilution (stationary phase) or 1:40 (log-phase) of these cell suspensions were made with AMA buffer in three (1:10 × 1:5 × 1:5) and two (1:10 × 1:4) steps, respectively, to obtain 106 CFU/mL as the initial inoculum. The assays were conducted in 96-well polystyrene plates (Corning Inc.). To each well was added a 10-µL aliquot of a 10x concentrated aqueous peptide solution (200 µM), AmpB (100 µM in Milli-Q water, Enzo Life Sciences), or sterile Milli-Q water as an untreated control. A 90-µL aliquot of the diluted culture was added to each well, and the plate was incubated for 1 h (37 °C, 150 rpm). The solutions were then serially diluted, and 10-µL drops were spotted on YPD-agar plates. These plates were incubated for 24 h at 37 °C. Colonies were counted and CFU/mL values were calculated. These assays were performed with at least two independently prepared and purified samples of each peptide and in three independent trials. The resulting averages and standard deviations are reported. C. albicans Growth Assay. An overnight culture of C. albicans SC5314 in YPD was prepared as described in the AMA section (vide supra), and the cultures were diluted 1:250 in three steps (1:10 × 1:5 × 1:5) in YPD. To each well of a 96-well plate (polystyrene, flat-bottomed, tissue culture-treated, Corning Inc.) was added 10 µL of a 10x concentrated aqueous peptide solution (200 µM), AmpB (50 µM, Enzo Life Sciences), or sterile Milli-Q water as a no-peptide control. A 90-µL aliquot of the diluted


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fungal culture was added to each well and the plate was incubated with shaking (30 °C, 150 rpm). The optical density at 600 nm (OD600) was recorded at 0, 2, 4, 6, 8, 10, and 24 h using a plate reader. These assays were conducted with at least two independently prepared and purified samples of each peptide and in three independent trials. The resulting averages and standard deviations are reported. C. albicans Adhesion Assay. The experiments were conducted following a literature protocol with modification.15,39 C. albicans adhesion to human T84 intestinal epithelial cells (ATCC CCL-248) was determined by using fluorescence microscopy. A colony of C. albicans SC5314-BFP (Table S1) was grown in 20 mL of YPD in a 250-mL baffled flask with shaking (30 °C, 175 rpm, 20 h). Then, 5 mL of the overnight culture was centrifuged (3,600 rpm, 5 min, 4 °C), washed with 5 mL of RPMI, and resuspended in fresh RPMI. The resulting suspension was then diluted 1:750 in four steps (1:10 × 1:5 × 1:5 × 1:3) with fresh RPMI. A 290-µL aliquot of the diluted culture was immediately added to 10 µL of a 30× concentrated aqueous peptide solution (150, 300, or 600 µM) or sterile Milli-Q water as a no-peptide control and incubated at room temperature for 15 min. T84 cells were routinely cultured in 1:1 (v:v) DMEM and Ham’s F12 medium (Corning Inc.) containing 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and 1.2 g/L sodium bicarbonate, and supplemented with 10% fetal bovine serum (Corning Inc.) and 1% penicillin/streptomycin (Corning Inc.). The detailed protocol for passing T84 cells was previously reported.34 T84 cells were received from ATCC at passage 53 and the cells between passages 59 and 73 were used in these experiments. A 500-µL aliquot of T84 cells (6 × 105 cells/mL) was added to 24-well Costar tissue


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culture plates (Corning Inc.), each well of which contains a sterile 12.7-mm glass cover slip. The cells were incubated on the cover slips at 37 °C and 5% CO2 for 24 h. Next, the T84 cells were washed twice with 500 µL of PBS and incubated with 300 µL of peptide-treated C. albicans in RPMI (the number of inoculum is ≈1 × 105 CFU/mL) at 37 °C and 5% CO2. After 2 or 4 h of incubation, the medium was removed and the T84 cells were washed three times with 500 µL of PBS to remove any non-adhered C. albicans and fixed for 10 min with 500 µL of PBS containing 4% paraformaldehyde and 4% sucrose. The cells were subsequently washed with PBS (2 x 500 µL) and bathed in 500 µL of PBS containing 20 µM SYTO 9 green fluorescent nucleic stain (S-34854, Thermo Fisher) at room temperature for 15 min. The stain solution was then removed. The cells were washed with 500 µL of PBS, bathed in PBS, and mounted onto glass slides. The samples were examined using Zeiss AxioPlan2 upright microscope (W.M. Keck Microscopy Facility, Whitehead Institute, Cambridge, MA). DAPI Zeiss (filter 49) and GFP Chroma filters were used for blue and green channels, respectively. The number of adhered C. albicans cells under each condition was counted from 10 different images, each of which contain ≈200 – 300 T84 cells. The percentage of adhesion represents the ratio of the number of adhered fungi per 100 T84 cells treated with peptide to the number of adhered fungi per 100 T84 cells of the untreated control after 4-h incubation. All adhesion assays were conducted with at least two independently prepared and purified samples of each peptide and in three independent trials and the resulting averages with standard deviations are reported.


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Biofilm Formation Assay. Cultures of C. albicans SC5314 in 5 mL of RPMI were prepared from overnight cultures as described in the adhesion assay (vide supra) except that the overnight culture was diluted 1:250 in three steps (1:10 × 1:5 × 1:5). The biofilm formation assays were conducted following a literature protocol with modification.40,41 A 230-µL aliquot of the C. albicans culture was incubated with 10 µL of a 24× aqueous peptide solution (120, 240, or 480 µM) or sterile Milli-Q water as a nopeptide control and incubated at room temperature for 15 min. The resulting mixture was then added to two wells of a 24-well plate (100 µL per well) and incubated with gentle shaking (37 °C, 50 rpm). After 24 or 48-h incubation, the supernatant was removed by a sterile glass pipette and each well was washed with 100 µL of PBS pH 7.2 three times. An aliquot of MTT (100 µL, 1 mg/mL, pre-filtered, 0.22-µm filter, Alfa Aesar) was added to each well and the plate was incubated for 4 h with gentle shaking (37 °C, 50 rpm). The supernatant was removed and 100 µL of DMSO was added. The plate was incubated for 15 min with shaking (37 °C, 150 rpm). A 90-µL aliquot of the resulting solution was transferred to a new well, and the absorbance at 550 nm was recorded using a plate reader. For the assays where HD6 was removed during the incubation, 400 µL of the C. albicans SC5314 culture in RPMI was treated with 20 µM HD6, and 100 µL of the mixture was added to each of four wells of a 96-well plate. The details of experimental setup are described above. After 24-h incubation, the supernatants from those wells were carefully combined in a sterile 1.7-mL microcentrifuge tube by using a sterile glass pipette. The resulting solution was divided into two tubes (200 µL each) and centrifuged (8,000 rpm, 10 min, 4 °C). The pelleted fungal cells were resuspended in RMPI and


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centrifuged (8,000 rpm, 10 min, 4 °C). Then, the first portion was resuspended in 200 µL of fresh RPMI, whereas the other portion was resuspended in 200 µL of fresh RPMI containing 20 µM HD6. Each suspension was incubated at room temperature for 15 min and 100 µL of each suspension was added to a 96-well plate. The plate was incubated for 24 h with gentle shaking (37 °C, 50 rpm). The MTT assays were then conducted following the procedure described above. All biofilm formation assays were conducted with at least two independently prepared and purified samples of each peptide and in three independent trials and the resulting averages with standard deviations are reported. For the assays in which fungal hyphae were used, the overnight culture of C. albicans SC5314 in YPD was centrifuged (3,600 rpm, 5 min, 4 °C), washed with 5 mL of RPMI, and resuspended in fresh RPMI. The resulting suspension (400 µL) was added into 20 mL of RPMI in a 250-mL baffled flask, and the culture was incubated for 24 h with shaking (37 °C, 150 rpm) to induce hyphae form, which was confirmed by widefield microscopy. Then, the biofilm formation assays were conducted using the overnight culture in RPMI and following the procedure described above. Mature Biofilm Susceptibility Assay. C. albicans biofilms were grown from the overnight culture in RPMI for 24 h with gentle shaking (37 °C, 50 rpm) as described in the biofilm formation assay (vide supra). The supernatant was removed and each well was washed with 100 µL of PBS three times. Then, 100 µL of fresh RPMI without or with 5, 10, 20 µM of HD6, or 20 µM F2A, or 5 µM AmpB was added to wells containing the biofilms. The plate was incubated for another 24 h with gentle shaking (37 °C, 50 rpm). Then, the amount of biofilm was quantified by the MTT assay (vide supra). The


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experiments were conducted with at least two independently prepared and purified samples of each peptide and in three independent trials and the resulting averages with standard deviations are reported. Scanning Electron Microscopy. Cultures of C. albicans SC5314 in 5 mL of RPMI were prepared from overnight cultures as described in the adhesion assay (vide supra) except that the overnight culture was diluted 1:250 in three steps (1:10 × 1:5 × 1:5). A 480-µL aliquot of the diluted fungal culture was combined with 20 µL of a 25x concentrated aqueous peptide solution (500 µM) or sterile Milli-Q water as an untreated control, and incubated at room temperature for 15 min. The resulting mixture was then added to each well of a 24-well plate, which contained a sterile 12.7-mm glass cover slip (Electron Microscopy Sciences), and the plate was and incubated for 24 h with gentle shaking (37 °C, 50 rpm). The supernatant was removed by using a glass pipette, and the samples were fixed in 500 µL of Karnosky fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.06 M Sorensen’s phosphate buffer, pH 7.2) at room temperature for at least 16 h. The samples were then prepared and visualized by SEM as previously described.42 The experiments were conducted with at least two independently prepared and purified samples of each peptide and in three independent trials. Morphological Studies of HD6-treated C. albicans. Cultures of C. albicans HGFP3 (Table S1) in 5 mL of RPMI were prepared from overnight cultures and incubated with peptides in a 24-well plate for 24 h as described in the SEM section (vide supra) except that each well did not contain a glass cover slip. The planktonic fungal cells and the biofilm were collected separately from each well and the samples were


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visualized using Zeiss AxioPlan2 upright microscope (W.M. Keck Microscopy Facility, Whitehead Institute, Cambridge, MA). GFP Chroma filter was used for a green channel. The experiments were conducted with at least two independently prepared and purified samples of each peptide and in three independent trials.

Results and Discussion HD6 Does Not Exert Antifungal Activity against C. albicans. Prior studies of the antimicrobial activity of HD6 have mainly focused on bacteria and demonstrated that HD6 provides neither bacteriostatic nor bactericidal activity against various Gramnegative and positive bacterial strains.34,36,37 It was also reported that both oxidized and reduced (the linear peptide that has six free cysteine thiols) forms of HD6 did not exhibit activity against C. albicans in an agar plate assay where growth inhibition zones were quantified.37 Building upon this prior work, we first conducted antifungal activity assays with native 32-residue HD6 and a single-point variant named F2A, both in the oxidized form with three disulfide linkages. The phenylalanine (F) at position 2 of HD6 contributes to formation of a hydrophobic pocket between four HD6 monomers and hence oligomer formation.31,34 The F2A variant contains an alanine (A) at this position, and we previously reported that this peptide cannot form higher-order oligomers and lacks functional activity against the Gram-positive bacterial pathogen Listeria monocytogenes.34 We examined the antifungal activity of these two peptides against C. albicans SC5314 in liquid culture using either mid-log or stationary phase cultures because the fungi exhibit different metabolic activity depending on the growth phase. We employed AmpB, an antifungal drug used in the clinic,19 as a positive control.


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Neither HD6 nor F2A exhibited antifungal activity against C. albicans in either growth phase (Figure 2A). Moreover, we monitored the growth of C. albicans (30 °C, 150 rpm) in the presence of HD6 and F2A, and these peptides had a negligible effect on the fungal growth rate under these experimental conditions (Figure 2B). In agreement with the prior work,37 our studies indicate that HD6 does not exhibit antifungal activity against C. albicans. Moreover, disruption of the self-assembly does not confer fungicidal activity for HD6, and this observation is reminiscent of our prior study where F2A and other oligomerization-defective HD6 variants displayed no antibacterial activity against Gramnegative and Gram-positive bacterial strains.34 HD6 Prevents C. albicans from Adhering to Human Intestinal Epithelial Cells. Although C. albicans resides in the intestine as a commensal organism, this milieu can be the origin of C. albicans systemic dissemination.43,44 C. albicans infections are initiated by fungal adhesion to the intestinal epithelium followed by invasion into these host cells. Subsequently, C. albicans can enter blood vessels and thereby disseminate to other sites in the host. Because previous studies demonstrated that HD6 confers protection of host cells against invasion by both Gram-positive and Gramnegative bacterial pathogens,34,36 we evaluated whether HD6 also protects host cells from fungi. We performed a series of C. albicans adhesion assays by pre-treating C. albicans expressing BFP in the cytosol with HD6 or F2A, and then adding the mixture to human T84 epithelial cells. Following incubation of the resulting culture, ≈60% of the fungal cells adhered to the T84 cells in the absence of HD6 at 2 h, and this value increased to ≈100% at a 4-h time point (Figures 3, S1, and S2). Pretreatment of the fungi with HD6 resulted in attenuated adhesion (Figures 3, S1, and S2). For instance,


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treatment with 10 µM or 20 µM HD6 resulted in ≈20% or ≈10% adherence, respectively, at the 2-h time point (Figure 3). In contrast, the F2A-treated cultured exhibited comparable fungal adherence to the untreated control (Figure 3), indicating that selfassembly of HD6 is required for inhibiting fungal adhesion to host cells. These results indicate that HD6 attenuates fungal adhesion and subsequent invasion. HD6 Suppresses Biofilm Formation of C. albicans. C. albicans is able to form biofilms on abiotic and biotic surfaces, and biofilm formation is associated with virulence and antifungal drug resistance. Because biofilm formation relies on initial cell adhesion to surfaces45-47 and our data indicate that HD6 reduces fungal adhesion (Figure 3), we examined whether HD6 also prevents biofilm formation by C. albicans. We conducted biofilm formation assays starting with C. albicans in the budding yeast form40,41 and observed that HD6 reduced the amount of biofilm formed in a concentration- and timedependent manner (Figure 4A). For instance, treatment of C. albicans with 20 µM HD6 resulted in only ≈25% and ≈20% of biofilm formed compared to the untreated control after 24 and 48 h, respectively. In contrast, the F2A variant did not suppress biofilm formation of C. albicans. Consistent with the adhesion assays, these data indicate that self-assembly of HD6 is required for this host-defense peptide to prevent the formation of C. albicans biofilms. Because HD6 is not fungicidal against C. albicans, we reasoned that the decrease in biofilm formation would be accompanied by the occurrence of more viable fungal cells in the supernatant. We quantified the number of planktonic cells, which revealed that the supernatant contained ≈106 CFU/mL when C. albicans was pre-treated with HD6. In contrast, no planktonic cells were detected for the untreated or F2A-treated cultures


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(Figure 4B). These results demonstrate that the population of C. albicans shifts from the biofilm to the supernatant when HD6 is present. To investigate whether the effect of HD6 on C. albicans biofilm formation is reversible, we treated C. albicans with HD6 for 24 h, washed the planktonic cells to remove HD6, and then resuspended the cells in fresh medium without the peptide. Once HD6 was removed from the culture, C. albicans formed biofilms to a similar level as the untreated control (Figure 4C). On the other hand, when HD6 was added back to the culture after the wash step, the amount of C. albicans biofilm was reduced to ≈20% relative to the untreated control, which was comparable to the amount of biofilm formed after single treatment with HD6. Thus, the reduction in biofilm formation of C. albicans caused by HD6 is reversible. C. albicans is a polymorphic fungus that can grow either as ovoid-shaped budding yeast, as elongated ellipsoid pseudohyphal cells with constrictions at the septa, or as parallel-walled true hyphae.12,48 Both yeast and hyphal forms of C. albicans can form biofilms; however, this process is typically initiated when the budding yeast form of C. albicans adheres to a surface.47 We therefore questioned whether HD6 is able to inhibit biofilm formation by the hyphal form of C. albicans, and conducted a series of biofilm assays using hyphae instead of budding yeast. First, we confirmed that the fungi completely turned into true hyphae after being cultured in RMPI medium for 24 h prior to the biofilm assays (Figure S3). From the biofilm assay initiated with these hyphae, we observed that HD6 reduced the amount of biofilm formed in a concentration-dependent manner (Figure 4D,E). Moreover, more cells were found in the supernatant and fewer cells in the biofilm when the concentration of HD6 increased (Figure 4A,B,D,E). As


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expected based on our prior assays, F2A did not suppress biofilm formation by the hyphae. We conclude that the ability of HD6 to suppress C. albicans biofilm formation is independent of the form of C. albicans (yeast or hyphae) when the fungi initiate biofilm formation. HD6 Does Not Disrupt C. albicans Biofilms. Next, we performed experiments to determine the effect of HD6 on pre-formed biofilms. We observed that HD6 did not disrupt mature biofilms (Figure 4F), suggesting that HD6 suppresses the biofilm formation only if the peptide is present during the initial step of this process to prevent the fungi from attaching to surfaces. Taken together, our results indicate that HD6 prevents C. albicans invasion and biofilm formation by interfering with the initial fungal adhesion to the epithelial cells or the surfaces (Figures 3, 4, S1, and S2). HD6 Does Not Suppress C. albicans Transition to Filamentous Hyphae. Mucins, the key components of mucus in the intestine, were recently reported to suppress the virulence traits of C. albicans by inhibiting its morphological transition to the virulent hyphal form.49 Moreover, the budding yeast of C. albicans becomes less adhesive by expressing the antiadhesive cell wall protein, Ywp1.50 To determine whether HD6 functions similarly to mucins by suppressing the C. albicans transition to hyphae, we first employed SEM to visualize the morphology of C. albicans planktonic cells and biofilms after treatment with HD6. The resulting images revealed that HD6treated C. albicans looked distinct from the untreated or F2A-treated cells (Figure 5A). The biofilms of the untreated and F2A-treated cultures contained the elongated structure of C. albicans, indicative of hyphae, and the cells were densely packed (Figures 5B and S4). In contrast, the fungal cells agglutinated instead of forming


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biofilms when HD6 was present, resulting in an uneven distribution of fungal cells (Figure 5B). The HD6-treated C. albicans also exhibited the elongated structure (Figure 5B, highest magnification images); however, from visual inspection, these fungi were shorter than the fungi in the biofilms. To confirm that the filamentous structures of C. albicans observed by SEM were true hyphae (Figure 5B), we employed C. albicans HGFP3. This strain has the gene for GFP inserted next to the promoter of HWP1, a gene encoding a hyphal cell wall protein.13 HWP1 is expressed by hyphae, but not by budding yeast. Thus, C. albicans HGFP3 will express GFP only when the cells grow as true hyphae. When C. albicans HFGP3 was treated with HD6 and imaged, we observed no distinct difference in the fungal morphology among untreated, HD6-, and F2A-treated cultures by DIC microscopy (Figure 5C). Moreover, comparable GFP emission was observed for untreated, HD6-, and F2A-treated C. albicans, indicating that the fungi transitioned into true hyphae in both the absence and presence of HD6 (Figures 5C). These data indicate that HD6 does not inhibit the fungal transition to the hyphal form.

Summary and Conclusions In this work, we consider how a self-assembling host-defense peptide deployed by the intestinal innate immune system mediates the virulence of the opportunistic fungal pathogen C. albicans.4 Our current work reveals that HD6, an abundant intestinal αdefensin that is stored in Paneth cells and released into the mucosa, suppresses two virulence traits of C. albicans. It blocks fungal invasion into epithelial cells and biofilm formation. This discovery expands upon two previous reports on HD6 and bacteria,


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which found that HD6 protects host cells from invasion by both Gram-negative36 and Gram-positive bacteria.34 Under the current experimental conditions, HD6 inhibits fungal cell adhesion and biofilm formation at micromolar concentrations (e.g. 10-20 µM). The in vivo concentration of HD6 is unknown, and this value is expected to vary with highest HD6 concentrations found the point of release in the small intestinal crypt. Studies of HD5 suggest that this peptide can reach concentrations up to 450 µg/mL (120 µM)51 in the intestinal crypt, and the reported ratios of HD5 mRNA to HD6 mRNA range from 4:1 to 6:1.52 Provided all mRNAs are translated, these prior analyses indicate that it is possible that HD6 concentrations reach ≈20 µM. Moreover, the current results provide a new example of how endogenous microbe-binding molecules, which include MUC5AC49 and other mucins,53 attenuate microbial virulence and provide barrier function. Indeed, the suppression of virulence traits of C. albicans and other opportunistic pathogens by host-defense peptides, biopolymers, and biopolymer-like molecules may be a general mechanism employed by the host to modulate the behavior of these microbes so that they remain harmless to the host. It is intriguing how the innate immune system employs several types of biomolecules to serve a similar function, and whether unappreciated synergies between these host-defense peptides and proteins exist warrants exploration. In addition, the current work provides a foundation for further mechanistic examination on how HD6 interferes with fungal adhesion. A prior study indicated that HD6 interacts with certain membrane/surface proteins of Salmonella enterica serovar Typhimurium, namely type I fimbriae and flagella.36 We speculate that a generalized model of HD6 interacting with membrane proteins can extend to C. albicans and other


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microbes. We observed that HD6 does not interfere with the C. albicans transition from budding yeast to hyphae; thus, HD6 may interact with certain fungal membrane proteins, such as adhesins, and prevent these proteins from binding to hydrophobic surfaces.15,54 This putative mechanism differs from the anti-virulence model for mucins. Recent work on MUC5AC, a glycoprotein which is expressed in the stomach and in the lungs, revealed that this mucin suppresses several virulence traits of C. albicans via the inhibition of filamentous growth.49 In closing, our current work on HD6 and C. albicans reveals a previously unappreciated function of defensins in suppressing fungal virulence. Fungal invasion into the host epithelium and subsequent dissemination, as well as biofilm formation, pose serious challenges to the host,7 and can complicate the treatment of candidiasis and confer enhanced resistance to antifungal drugs.19 Further studies on how HD6 prevents C. albicans adhesion and biofilm formation may lead to development of new strategies for preventing and treating candidiasis and other microbial infections.

Acknowledgements We thank Professor S. Lindquist (Whitehead Institute, MIT) for providing C. albicans SC5314 and SC5314-BFP, and Professors P. Sundstorm (Dartmouth Medical School) and K. Ribbeck (MIT) for C. albicans strain HGFP3.

Supporting Information. Table of C. albicans strains used in this study (Table S1), additional images of C. albicans adhesion assays with different concentrations of HD6, images of C. albicans morphology in YPD medium, and additional SEM images of C.


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albicans treated with different concentrations of HD6 (Figures S1-S4). This material is available free of charge via the Internet at http://pubs.acs.org.


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[36] Chu, H., Pazgier, M., Jung, G., Nuccio, S.-P., Castillo, P. A., de Jong, M. F., Winter, M. G., Winter, S. E., Wehkamp, J., Shen, B., Salzman, N. H., Underwood, M. A., Tsolis, R. M., Young, G. M., Lu, W., Lehrer, R. I., Bäumler, A. J., and Bevins, C. L. (2012) Human αdefensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets, Science 337, 477-481. [37] Schroeder, B. O., Ehmann, D., Precht, J. C., Castillo, P. A., Küchler, R., Berger, J., Schaller, M., Stange, E. F., and Wehkamp, J. (2015) Paneth cell α-defensin 6 (HD-6) is an antimicrobial peptide., Mucosal Immunol. 8, 661-671. [38] Chairatana, P., Chu, H., Castillo, P. A., Shen, B., Bevins, C. L., and Nolan, E. M. (2016) Proteolysis triggers self-assembly and unmasks innate immune function of a human αdefensin peptide, Chem. Sci. 7, 1738-1752. [39] Wächtler, B., Wilson, D., Haedicke, K., Dalle, F., and Hube, B. (2011) From attachment to damage: defined genes of Candida albicans mediate adhesion, invasion and damage during interaction with oral epithelial cells, PLoS One 6, e17046. [40] Pierce, C. G., Uppuluri, P., Tristan, A. R., Wormley Jr., F. L., Mowat, E., Ramage, G., and Lopez-Ribot, J. L. (2008) A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing, Nat. Protoc. 3, 1494-1500. [41] Liu, R., Chen, X., Falk, S. P., Masters, K. S., Weisblum, B., and Gellman, S. H. (2015) Nylon-3 polymers active against drug-resistant Candida albicans biofilms., J. Am. Chem. Soc. 137, 2183-2186. [42] Chileveru, H. R., Lim, S. A., Chairatana, P., Wommack, A. J., Chiang, I.-L., and Nolan, E. M. (2015) Visualizing attack of Escherichia coli by the antimicrobial peptide human defensin 5, Biochemistry 54, 1767-1777. [43] Gow, N. A. R., van de Veerdonk, F. L., Brown, A. J. P., and Netea, M. G. (2011) Candida albicans morphogenesis and host defence: discriminating invasion from colonization, Nat. Rev. Microbiol. 10, 112-122.


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[44] Nucci, M., and Anaissie, E. (2001) Revisiting the source of candidemia: skin or gut?, Clin. Infect. Dis. 33, 1959-1967. [45] Baillie, G. S., and Douglas, L. J. (1999) Role of dimorphism in the development of Candida albicans biofilms J. Med. Microbiol. 48, 671-679. [46] Chandra, J., Kuhn, D. M., Mukherjee, P. K., Hoyer, L. L., McCormick, T., and Ghannoum, M. A. (2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance, J. Bacteriol. 183, 5385-5394. [47] Finkel, J. S., and Mitchell, A. P. (2011) Genetic control of Candida albicans biofilm development, Nat. Rev. Microbiol. 9, 109-118. [48] Berman, J., and Sudbery, P. E. (2002) Candida albicans: a molecular revolution built on lessons from budding yeast., Nat. Rev. Genet. 3, 918-930. [49] Kavanaugh, N. L., Zhang, A. Q., Nobile, C. J., Johnson, A. D., and Ribbeck, K. (2014) Mucins suppress virulence traits of Candida albicans, mBio 5, e01911-01914. [50] Granger, B. L. (2012) Insight into the antiadhesive effect of yeast wall protein 1 of Candida albicans, Eukaryot. Cell 11, 795-805. [51] Ghosh, D., Porter, E., Shen, B., Lee, S. K., Wilk, D., Drazba, J., Yadav, S. P., Crabb, J. W., Ganz, T., and Bevins, C. L. (2002) Paneth cell trypsin is the processing enzyme for human defensin-5, Nature 3, 583-590. [52] Wehkamp, J., Chu, H., Shen, B., Feathers, R. W., Kays, R. J., Lee, S. K., and Bevins, C. L. (2006) Paneth cell antimicrobial peptides: topographical distribution and quantification in human gastrointestinal tissues., FEBS Lett. 580, 5344-5350. [53] Ogasawara, A., Komaki, N., Akai, H., Hori, K., Watanabe, H., Watanabe, T., Mikami, T., and Matsumoto, T. (2007) Hyphal formation of Candida albicans is inhibited by salivary mucin., Biol. Pharm. Bull. 30, 284-286. [54] Chaffin, W. L. (2008) Candida albicans cell wall proteins, Microbiol. Mol. Biol. Rev. 72, 495544.


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Figure Legends Figure 1. Overview of HD6 structure and function. (A) Structure of oxidized HD6 monomer with disulfide linkages colored in yellow (PDB: 1ZMQ).30 HD6 monomers selfassemble to form large oligomers named “nanonets” that entrap microbes. (B) Primary sequence of 32-residue HD6, secondary structure depiction, regiospecific disulfide bond linkages (solid black lines), and Arg–Glu salt bridge (dashed line).

Figure 2. HD6 is not fungicidal against C. albicans SC5314. (A) Growth of C. albicans in the presence of 20 µM HD6, 20 µM F2A, and 10 µM AmpB against log- and stationary-phase C. albicans SC5314 (t = 1 h, 37 oC). (B) Growth curves of C. albicans SC5314 in the presence of HD6 or F2A in YPD with shaking (mean ± SDM, n = 3). An asterisk indicates that the culture exhibited no colonies on the 10-dilution plate.

Figure 3. HD6 protects human T84 epithelial cells against adhesion of C. albicans SC5314-BFP. The fungi (105 CFU in 300 µL RPMI) were pre-incubated with the indicated peptides for 15 min. (A) Representative images of T84 cells following incubation with varying concentrations of peptides (t = 4 h, T = 37 °C, 5% CO2). The nuclei of T84 cells are colored in green and C. albicans are pseudocolored in red. Scale bar = 20 µm. (B) HD6 reduces the percentage of adhered fungal cells to human T84 colon epithelial cells. The number of adhered fungi was counted from the fluorescent images (e.g. Figures S1 and S2), and the data were normalized to the untreated control after 4-h incubation and plotted (mean ± SDM, n = 3).


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Figure 4. HD6 attenuates C. albicans biofilm formation. (A) The amount of C. albicans biofilm starting from the yeast state in the presence of different concentrations of HD6. (B) The corresponding CFU/mL of C. albicans in the supernatant from (A) after treatment with HD6. An asterisk indicates that no colony was detected. (C) The reduction in biofilm formation of C. albicans by HD6 is reversible. Once HD6 is removed from the supernatant, the fungi form biofilm to a similar level as the untreated cultures. (D) The amount of biofilm formed by hyphal C. albicans SC5314 in the presence of different concentrations of HD6. (E) The corresponding CFU/mL of C. albicans in the supernatant from (D) after treatment with HD6. An asterisk indicates that no colony was detected. (F) The preformed biofilm of C. albicans is resistant to HD6. (A – F) The averages are reported with the error bars representing standard deviations (n ≥ 3).

Figure 5. Morphological studies of C. albicans following treatment with HD6. (A) Macroscopic view of biofilms of C. albicans SC5314 grown in the presence of HD6. The well diameter is 15.6 mm. (B) Representative SEM images of untreated, or F2A-treated, or HD6-treated C. albicans SC5314 (t = 24 h). Scale bar = 100 µm (left and middle columns) and 20 µm (right column). Clumping is observed for HD6-treated cultures in the lower magnification images, and the highest magnification images show the morphologies of individual C. albicans cells. (C) Representative DIC and fluorescence microscopy images of untreated, F2A-treated, or HD6-treated C. albicans HGFP3 after 24 h incubation. These images show regions where individual C. albicans can be discerned to facilitate comparison of GFP emission. Other regions of the HD6-treated cultures showed clumping (not shown). Scale bar = 50 µm.


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Figure 1. (one column)

A self-assembly

"nanonet" microbial entrapment host defense

HD6 monomer

B

β1

coil

β2

turn

β3

AFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL HD6


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Figure 2 (one column)

A

Log-phase Stationary phase

CFU/mL

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* * Untreated

B

HD6 20 µM

F2A 20 µM

AmpB 10 µM


31

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A

DIC

SYTO 9

BFP

Overlay

Untreated

HD6 20 µM

F2A 20 µM

B

Untreated

HD6 5 µM

HD6 10 µM

2h

4h

HD6 20 µM

F2A 20 µM


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Figure 4 (two columns)

A

B

C

Yeast

Absorbance (550 nm)

Absorbance (550 nm)

CFU/mL of Supernatant

Yeast

* HD6 5 µM

HD6 HD6 F2A 10 µM 20 µM 20 µM

D

AmpB 5 µM

tr

HD6 10 µM

E

HD6 20 µM

* *

* *

F2A 20 µM

AmpB 5 µM

U

re nt

ed at

HD6 5 µM

HD6 HD6 F2A 10 µM 20 µM 20 µM

HD6 (20 µM, first 24 h) HD6 (20 µM, second 24 h)

-

+

+

-

+

-

F

Hyphae

CFU/mL of Supernatant

Hyphae

Un

d HD6 te 5 µM ea

Absorbance (550 nm)

U

r nt

d te ea

Absorbance (550 nm)

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tre Un

HD6 ed at 5 µM

HD6 10 µM

HD6 20 µM

F2A 20 µM

U

24 h

re nt

HD6 HD6 HD6 F2A AmpB ed 5 µM 10 µM 20 µM 20 µM 5 µM at

48 h


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A

0h

8h

24 h

48 h

Untreated

HD6 20 µM

F2A 20 µM

B Untreated

HD6 20 µM

F2A 20 µM

C

DIC

GFP

Overlay

Untreated

HD6 20 µM

F2A 20 µM


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TOC Graphic

HD6 oligomers

C. albicans

surface

Mature biofilm


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Human Î±-Defensin 6 Self-Assembly Prevents ... - ACS Publications

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