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Human Beta-defensins: Differential Activity against Candidal Species and Regulation by Candida albicans

¹ Department of Biological Sciences and Department of Periodontics, ³ Department of Community Dentistry, Case Western Reserve University School of Dental Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4905, USA; and
² Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH, USA;

^* corresponding author, axw47{at}po.cwru.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Oral epithelial cell-derived human beta-defensins-1, -2, and -3 participate in innate immune responses against Candida. We hypothesized that these peptides utilize several mechanisms for protection. Recombinant hBD-1 and -2 were produced with the use of an insect cell/baculovirus expression system, while rhBD-3 was expressed as a fusion protein in E. coli. RhBD-2 and -3 were more effective at killing the candidal species at low micromolar concentrations than was rhBD-1, except for C. glabrata. While this species was relatively resistant to rhBD fungicidal activity, its adherence to oral epithelial cells was strain-specifically inhibited by the rhBDs. C. albicans hyphae were important in regulating hBD2 and -3 mRNA expression in primary human oral epithelial cells. Confocal microscopy of rhBD-2-challenged C. albicans suggests disruption of the fungal membrane. Results support the hypothesis that hBDs control fungal colonization through hyphal induction, direct fungicidal activity, and inhibition of candidal adherence.

KEY WORDS: beta-defensins • Candida • innate immunity • oral epithelial cells

	INTRODUCTION

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The acquired and innate immune systems play important roles in defense against candidiasis, with the latter receiving increased attention in the recent past (Fidel, 2002). Since (1) mucosal epithelial cell-derived peptides, called ‘human ß-defensins’ (hBDs), exhibit antifungal activity (Härder et al., 1997, 2001; Hoover et al., 2003), (2) single-nucleotide polymorphisms in the hBD-1 gene have been associated with low levels of Candida carriage (Jurevic et al., 2003), and (3) hBDs act as chemoattractants toward adaptive immune cells (Yang et al., 1999), one can surmise the importance of these agents in preventing and/or controlling fungal infection at mucosal surfaces.

While the most frequently isolated species in oropharyngeal candidiasis (OPC) is C. albicans, other non-albicans Candida species—including C. parapsilosis, C. krusei, and C. glabrata—have also been implicated (Pankhurst, 2002). The rationale for the present study was to test the hypothesis that endogenous ß-defensins contribute to the innate defense against Candida species by multiple mechanisms which may include antimicrobial action, inhibition of adherence to epithelial cells, and up-regulation in response to hyphal growth.

	MATERIALS AND METHODS

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Candidal Isolates and Growth Conditions
Candida albicans oropharyngeal isolates (OPC76, OPC84, OPC99), a disseminating strain (SC5314) (Gillum et al., 1984), mutant strains HLC54 and HLC84 from SC5314 (Lo et al., 1997), Candida krusei (isolates A91 and MRL-214; both fluconazole-resistant), Candida parapsilosis (ATCC 22019 and isolate A58), and Candida glabrata (isolates 90030 and 2255; both fluconazole-resistant) were used. Clinical isolates were obtained at the Center for Medical Mycology, University Hospitals of Cleveland, and cultured on Sabouraud dextrose agar (SDA) (glucose 40 g, peptone 10 g, agar 20 g/liter) (Difco Laboratories, Detroit, MI, USA). Organisms were identified by the germ tube test (Huppert et al., 1975) and API 20C system (Biomerieux Vitek, Hazelwood, MO, USA). For experimentation, organisms were grown for 16 to 18 hrs in SD broth or yeast nitrogen base (YNB) with 50 mM glucose (Difco Laboratories) at 37°C in a water-bath shaker. After centrifugation (3000 x g, 10 min), yeast cells were re-suspended in phosphate-buffered saline (1x; PBS) without calcium and magnesium (Mediatech Cellgro, Herndon, VA, USA), sonicated (60 Hz; Sonic Dismembrator, Fisher Scientific, Pittsburgh, PA, USA), 4 sec, washed 2x in PBS, and sonicated again. Cells were adjusted to the desired number of yeast cells/mL by hemocytometric counts. Yeast concentrations (CFU per mL) were confirmed by quantitative cultures.

C. albicans Regulation of hBD mRNA in Normal Human Oral Epithelial Cells (NHOECs)
Normal oral tissue overlying impacted third molars was obtained following a protocol approved by the University’s Institutional Review Board, and informed consent was obtained. Cell cultures were prepared from these tissues as described previously (Krisanaprakornkit et al., 1998, 2000). C. albicans SC5314 (parent), HLC54 (suppressed hyphal mutant), and HLC84 (reconstituted hyphal expression) (Table) were incubated with NHOEC monolayers at a multiplicity of infection of 0.1:1 (yeast:cell), for 24 hrs, followed by real-time PCR analysis to quantify hBD mRNA as previously described (Quinones-Mateu et al., 2003).

Killing Assays
Candidal cells were harvested by centrifugation, washed with 10 mM phosphate buffer (PB) (containing 1% TSB), pH 7.4, re-suspended in PB, and adjusted to 2 x 10⁵ cells/mL. Cell aliquotes (25 µL) were incubated with different concentrations (0–10 µM) of rhBD-1, -2, and -3, respectively, to a final volume of 50 µL. Respective reaction mixtures were incubated (37°C, 3 hrs), followed by serial dilution and plating on SDA plates. Colonies were counted 48 hrs later. Results were calculated as a percentage of CFUs relative to untreated controls.

Candida glabrata Adherence to OKF6/Tert Epithelial Cells
We previously showed individual variability between primary epithelial cells from different donors when testing candidal adherence (Ghannoum and Radwan, 1990). Therefore, we conducted these assays with OKF6/Tert cells that were shown previously to behave like primary oral epithelial cells (Feucht et al., 2003). The OKF6/Tert cell line, which was engineered for extended growth through the overexpression of telomerase and the deletion of the p16^INK4a regulatory protein (Dickson et al., 2000), was provided by J. Rheinwald (Harvard Institutes of Medicine, Boston, MA, USA) and maintained according to Dickson et al.(2000). Adherence assays were conducted as described previously (Ghannoum and Radwan, 1990). C. glabrata strain 90030 or 2255, in YNB, was washed, re-suspended in 10 mM PB (containing 1% TSB), and adjusted to 2 x 10⁶ cells/mL. Twenty-five-µL aliquots were mixed with 2.5 µM or 5 µM rhBD-1, -2, and -3, respectively, to a final volume of 50 µL. Cells were incubated at 37°C, 3 hrs, followed by adjustment to 250 cells/mL in Hanks’ Balanced Salt Solution (HBSS) (50 µL vol was added to 9.95 mL HBSS). One mL of this mixture was added to 19 mL of HBSS, giving a final concentration of 250 cells/mL. Tert cells were grown in six-well plates to 95% confluence. After the monolayers were rinsed 2x with HBSS, 1.5 mL candidal cells (from the 250 cells/mL suspension) were added to each well, followed by incubation at 37°C, 30 min. Control wells included candidal cells that were not pre-treated with the rhBDs. Non-adhering candidal cells were removed by aspiration. After being rinsed 3x with HBSS, wells were overlaid with SDA, followed by incubation at 37°C, 24 hrs. Results were calculated as a percentage of CFUs relative to untreated controls.

Confocal Microscopy Analysis
C. albicans OPC 84 or C. glabrata 90030 cells were grown in 10 mL YNB, at 37°C, overnight. After being washed, cells were adjusted to 1 x 10⁷ cells/mL in 10 mM PB. A 10-µM quantity of rhBD-2 was added, bringing the final volume to 100 µL with 10 mM PB. Respective reaction mixtures were incubated at 37°C, for 48 hrs. Alexa Fluor 488 conjugated Concanavalin A (ConA) (50 µg/mL) (Molecular Probes, Eugene, OR, USA) and FUN^® 1 (20 µM) (Molecular Probes) were mixed together in PBS. A 100-µL quantity of the staining solution was added to each tube, mixed, and incubated at 37°C, 35 min. After being washed and centrifuged, cells were re-suspended in PBS and compared with untreated cells by means of a dual scanning confocal microscopy system (LSM 510, Zeiss, Oberkochen, Germany).

Statistical Analysis
Data were expressed as mean ± standard deviation. Two-way analysis of variance and the Student unpaired t test were used with significance set at P < 0.05.

	RESULTS

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C. albicans Hyphal Regulation of ß-defensins
Candidal infection of mucosa is associated with hyphal growth. We therefore tested the role of hyphae in the regulation of hBDs. Using specific hyphal-suppressed mutants of C. albicans SC5314 (Table), we found that yeast cells poorly induced hBD-2 and -3 mRNA when compared with the hyphal-producing parent (Fig. 1). Moreover, hBD mRNA expression levels were directly related to the degree of hyphae produced. Re-introducing hyphal expression in hyphae-suppressed mutants (strain HLC84; Table) caused induction of hBD mRNA expression in NHOECs to levels comparable with those seen in the parent strain (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Real-time PCR analysis of hBD-2 and -3 mRNA induction in NHOECs following challenge with C. albicans SC5314 and respective mutants. C. albicans SC5314 (parent), HLC54 (suppressed hyphal mutant), and HLC84 (reconstituted hyphal expression) were incubated with NHOEC monolayers at a multiplicity of infection of 0.1:1 (Ca:HOEC), 24 hrs, followed by real-time PCR analysis. White bar, hBD2; black bar, hBD3. N = 4. *Significantly different from negative control and ** from SC5314 (p < 0.05; t test).

View larger version (30K): [in this window] [in a new window]   Figure 2. Differential activity of human beta-defensins against candidal species. (A–F) Human beta defensin killing of candidal species. Recombinant hBD-1, -2, and -3 were incubated at increasing concentrations (0–10 µM) with strains of C. albicans (OPC76, OPC84; OPC99; SC5314), C. krusei (A91, MRL-214), C. parapsilosis (A58, ATCC 22019), and C. glabrata (90030, 2255), respectively, 37°C, 3 hrs, followed by serial dilutions, plating on Sabouraud Dextrose agar plates, and counting of colonies 48 hrs later. (A) rhBD-1 killing of C. albicans strains; (B) rhBD-1 killing of Candida krusei, C. parapsilosis, and C. glabrata strains; (C) rhBD-2 killing of C. albicans strains; (D) rhBD-2 killing of Candida krusei, C. parapsilosis, and C. glabrata strains; (E) rhBD-3 killing of C. albicans strains; and (F) rhBD-3 killing of Candida krusei, C. parapsilosis, and C. glabrata strains. Results are calculated as a percentage of colony-forming units when compared with untreated controls. Data are presented as mean ± SD of 3 (n = 3) independent experiments. (G,H) ß-defensin inhibition of Candida glabrata adherence to human oral epithelial cells. C. glabrata strain 90030 (G) or C. glabrata strain 2255 (H) was incubated with either rhBD-1, -2, or -3 (2.5 or 5 µM), at 37°C, for 3 hrs, followed by assay for adherence to confluent monolayers of OKF6/Tert cells. Results are calculated as a percentage of adhering fungi when compared with untreated controls. Data are presented as mean ± SD of 3 (n = 3) independent experiments. Asterisks show a significant difference from control values at *P < 0.05 and **P < 0.005 by Student’s unpaired t test.

Confocal Microscopy Analysis of C. albicans and C. glabrata after Incubation with rhBD-2
To examine the effects of hBDs on candidal structure, we incubated C. albicans and C. glabrata with ConA and FUN 1 fluorescent dyes, which stain the cell wall and indicate metabolic activity, respectively. Untreated C. albicans OPC 84 yeast cells showed uniform cell wall thickness and intact outer envelopes (Fig. 3A, green staining). Moreover, fungal cells had intense red staining by FUN 1 within each cell (Fig. 3A). When compared with the control, rhBD-2-treated cells showed dramatic changes in C. albicans, with evidence of thinning and dissolution of the cell walls (Fig. 3B, arrows) and a distinct lack of FUN 1 staining within the cells (Figs. 3B, 3C). Fig. 3C also shows cytoplasmic debris (arrow), which could have resulted from cell lysis. In contrast, C. glabrata 90030 did not show differences in either envelope thickness, metabolic activity, or cytoplasmic debris between untreated (Fig. 3D) and rhBD-2 treated cells (Fig. 3E).

View larger version (47K): [in this window] [in a new window]   Figure 3. Confocal analysis of C. albicans OPC 84 and C. glabrata 90030 after incubation with rhBD-2. C. albicans OPC 84 or C. glabrata 90030 yeast cells were respectively incubated with rhBD-2, at 37°C, for 48 hrs. Cells were then incubated with a staining solution containing Alexa Fluor 488 conjugated Concanavalin A (ConA) (50 µg/mL) (Molecular Probes, Eugene, OR, USA) and FUN® 1 (20 µM) (Molecular Probes) in PBS, 37°C, 30 min. Stained cells were observed with the use of a dual scanning confocal microscopy system (LSM 510, Zeiss, Oberkochen, Germany). (A) Normal C. albicans cells showing uniform membrane thickness (green boundaries) and normal metabolic activity (red inside yeast cells); (B,C) C. albicans after incubation with rhBD-2, showing a lack of FUN 1 staining within the cells, indicating severe metabolic deficiencies, and evidence of thinning of the cell walls (B, arrows); (C) shows leakage of cytoplasmic material (arrow); (D) C. glabrata 90030 showing uniform membrane thickness (green boundaries) and normal metabolic activity (red inside yeast cells); (E) C. glabrata after incubation with rhBD-2 does not show appreciable differences in either envelope thickness, metabolic activity, or cytoplasmic debris when compared with untreated cells (D).

	DISCUSSION

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Strain-selected activity of our rhBDs against candidal species was not as pronounced as that reported by Joly et al.(2004). This may be due to differences in sources of the hBDs—i.e., commercial vs. our recombinant forms, the use of radial diffusion vs. our direct killing assays in solution, and the need for additional scrutiny of the strains tested.

C. glabrata strain-specific resistance to hBD-mediated inhibition of adherence (Figs. 2G, 2H) may be due to the interaction of hBDs with the C. glabrata EPA1 adhesin that mediates adherence to human epithelial cells (Cormack et al., 1999), and could imply differences in virulence between strains. Moreover, since we used immortalized cells, the in vivo biological significance of these results needs to be addressed. Finally, defensin interference with microbial adherence appears to be site-specific—i.e., PMN-derived -defensins enhance bacterial adherence to respiratory epithelial cells (Gorter et al., 2003), while in Crohn’s disease, increased bacterial adherence appears to be attributed to impaired defensin expression (Folwaczny et al., 2003).

The inability of the rhBDs to kill C. glabrata, an emerging cause of oropharyngeal candidiasis (Redding et al., 2004), suggests a specific inhibitory mechanism of this species against these peptides. Since hBDs interact electrostatically with microbial membranes, leading to membrane perturbation and cell death (Weinberg et al., 1998), the key may lie in fewer negatively charged membrane phospholipids between C. glabrata and the susceptible species. However, since hBD-3, which expresses a higher net cationic charge than the other two hBDs (Schibli et al., 2002), was not significantly more effective in killing susceptible fungi than was hBD-2, as determined by a two-way analysis of variance, electrostatic interactions alone cannot explain the fungicidal mechanism(s) of the hBDs. A possible key in understanding hBD antifungal activity may come from observations showing that the alpha defensin, human neutrophil defensin 1, shares features very similar to those of human histatin 5 (Hst 5) in killing C. albicans (Edgerton et al., 2000). Both peptides appear to bind the recently identified 70-kDa yeast-membrane-associated heat-shock protein Ssa1/2p (Li et al., 2003), resulting in ATP-mediated cytotoxicity (Edgerton et al., 2000). Conducting fungicidal assays with Ssa mutants in normal and under heat-shock conditions (Li et al., 2003) could determine if hBDs act similarly. Moreover, investigating the expression of Ssa proteins in C. glabrata could also shed light on why hBDs demonstrate low fungicidal activity against this species, since Hst5 has also been shown to be less effective against C. glabrata when compared with other Candida species (Nikawa et al., 2001). Finally, a recent report identified fungal glucosylceramides as antifungal targets for plant and insect defensins (Thevissen et al., 2004).

C. albicans hyphae, but not yeast cells, induced expression of hBD2 and -3 in NHOECs. Since hyphae are structures that establish an invasive candidal infection, hBDs may be eliciting protection by acting as chemokines in recruiting human neutrophils (Niyonsaba et al., 2004), and immature dendritic cells and T-cells (Yang et al., 1999). Additionally, our in vitro experiments demonstrated fungicidal and adherence inhibition activity of rhBD-2 well within the upper limit of normal in vivo expression levels (Sawaki et al., 2002), and were conducted in low salt and serum conditions, in keeping with the actual environment at the oral mucosal interface (Mandel, 1972). These results support our hypothesis that endogenous beta defensins contribute to the innate defense against Candida species by controlling fungal colonization at the oral mucosal barrier.

	ACKNOWLEDGMENTS

 
We thank Dr. Tom McCormick from the Department of Dermatology, Case Western Reserve University and University Hospitals, Cleveland, OH, for timely suggestions and valuable advice. This study was supported by NIH/NIDCR grants 1RO-1 DE13992 (AW), 1RO-1 DE12589 (AW), and RO-1 DE13932 (MG). Confocal studies were supported by the Core Facility of the Comprehensive Cancer Center of CWRU and University Hospitals of Cleveland (P30 CA43703).

Received October 13, 2003; Last revision January 21, 2005; Accepted January 28, 2005

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