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Prostaglandin production during growth of Candida albicans biofilms

Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK

Correspondence L. Julia Douglas J.Douglas{at}bio.gla.ac.uk

Received May 25, 2005
Accepted July 12, 2005

Both biofilms and planktonic (suspended) cells of Candida albicans synthesized extracellular prostaglandin(s) during growth at 37 °C, but biofilm cells secreted significantly more prostaglandin(s) when production was determined on the basis of cell dry weight. Prostaglandin synthesis by both cell types was sensitive to the cyclooxygenase inhibitors aspirin, diclofenac and etodolac. A morphological mutant blocked in two signalling pathways (cph1/cph1 efg1/efg1) produced prostaglandin levels similar to those of the parent strain, but formed yeast-only biofilms. These results suggest that prostaglandin production could be a significant virulence factor in biofilm-associated infections, although its role in C. albicans morphogenesis remains unclear.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Prostaglandins are lipid molecules with a range of biological activities, including the regulation of immune responses. In mammalian systems they are synthesized from arachidonic acid, which is formed from phospholipids via the action of two cyclooxygenase (COX) isoenzymes, COX-1 and COX-2 (Dannhardt & Kiefer, 2001; Harris et al., 2002). Prostaglandins are now known to be produced by various eukaryotic micro-organisms as well as by mammalian cells, and they have recently been identified in pathogenic fungi (Noverr et al., 2001, 2002, 2003). Culture supernatants from both Candida albicans and Cryptococcus neoformans contained prostaglandins, and treatment of either yeast with the COX-inhibitor indomethacin significantly reduced cell viability (Noverr et al., 2001). Purified fungal prostaglandin, partially characterized as a PGE (prostaglandin E) series lipid, was shown to enhance germ tube formation by C. albicans, and affected chemokine production by mammalian cells (Noverr et al., 2001).

Little is known about the role of prostaglandins in fungal biology. They may function as regulators of gene expression, possibly in relation to dimorphism in C. albicans. Considering their known effects on mammalian cell biology, prostaglandins secreted by pathogenic fungi could also promote colonization and chronic infection by these organisms. Colonization frequently involves the formation of a biofilm on host surfaces (Douglas, 2003). Recently, we investigated the effects of various COX inhibitors on C. albicans biofilms (Alem & Douglas, 2004). Etodolac, diclofenac, and most notably aspirin, dramatically decreased biofilm formation by up to 95 %. Aspirin was active against growing and fully mature biofilms; its effect was dose-related, and it produced significant inhibition at pharmacological concentrations. However, it had relatively little effect on dimorphism in Candida biofilms. Treatment with different COX inhibitors such as etodolac, on the other hand, resulted in biofilms that consisted almost entirely of yeast cells. Overall, these results suggested that COX-dependent synthesis of fungal prostaglandin(s) might act as a regulator for biofilm development in C. albicans. In the present study, we have compared prostaglandin synthesis and its sensitivity to COX inhibitors during growth of both biofilms and planktonic (suspended) cells of C. albicans. We have also determined prostaglandin production by biofilms and planktonic cells of a morphological mutant of this organism.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Organisms.

Three strains of C. albicans were used in this study. C. albicans GDH 2346 was isolated at Glasgow Dental Hospital from a patient with denture stomatitis. C. albicans SC5314 and its morphological mutant, C. albicans HLC54 (genotype ura3 : : 1 imm434/ura3 : : 1 imm434 cph1 : : hisG/cph1 : : hisG efg1 : : hisG/efg1 : : hisG-URA3-hisG) (Lo et al., 1997), were kindly provided by N. A. R. Gow, University of Aberdeen, Scotland, with permission from G. R. Fink. All strains were maintained on slopes of Sabouraud dextrose agar.

Medium and culture conditions.

Organisms were grown in yeast nitrogen base medium containing 50 mM glucose, as previously described (Alem & Douglas, 2004). For biofilm studies, washed cell suspensions from 24 h cultures were adjusted to an optical density of 0.8 at 520 nm. Biofilms were formed on small disks (surface area 0.5 cm²) cut from polyvinyl chloride Faucher tubes (French gauge 36; Vygon), as reported previously (Baillie & Douglas, 1999a). Briefly, the disks were placed in the wells of 24-well Nunclon tissue culture plates, and a standardized cell suspension (80 µl) was applied to the surface of each one. Initially, incubation lasted for 1 h at 37 °C (adhesion period). Nonadherent organisms were removed by washing, and the disks were then incubated for further periods (up to 48 h) while submerged in 1 ml growth medium (biofilm formation). For planktonic (suspended) cultures, standardized cell suspension (100 µl) was used to inoculate yeast nitrogen base medium (50 ml, in 250 ml Erlenmeyer flasks) giving a cell density of 4 x 10⁴ cells ml^–1. Cultures were incubated at 37 °C in an orbital shaker at 60 r.p.m. for time periods of up to 48 h.

COX inhibitors.

Stock solutions (5 mM) of the COX inhibitors diclofenac and etodolac (Sigma) were prepared in DMSO. Stock solutions (5 mM) of aspirin (acetylsalicylic acid; Sigma) were prepared in ethanol. For experiments with both planktonic cells and biofilms, COX inhibitors were used at a final concentration of 50 µM. Inhibitors were added at the time of inoculation for planktonic cultures. For biofilm cultures, inhibitors were added following the 1 h adhesion period, at time zero of the subsequent incubation period. Controls included disks with no cells, and disks with solvent but no COX inhibitor.

Scanning electron microscopy.

Scanning electron microscopy of biofilms was carried out after fixation with glutaraldehyde and treatment with osmium tetroxide, uranyl acetate and ethanol, as described previously (Hawser & Douglas, 1994).

Determination of prostaglandin concentration by ELISA.

Prostaglandin production was determined in culture supernatants of planktonic cells and biofilms after concentrating the supernatant 10-fold by freeze-drying. At the end of the incubation period, planktonic cells were centrifuged at 3000 r.p.m. for 3 min. The supernatants were filtered through non-pyrogenic filters (pore size 0.2 µm) and portions (5 ml) were freeze-dried. For biofilms, after removal of disks with their adherent cells, the remaining medium from 12 wells was similarly filtered through non-pyrogenic filters (pore size 0.2 µm) and portions (5 ml) were freeze-dried. All freeze-dried samples were reconstituted by the addition of 0.5 ml distilled water and analysed for prostaglandins using a prostaglandin-screening enzyme immunoassay kit (Cayman Chemicals), according to the manufacturer's instructions. This ELISA detects PGE₂, PGE₁, PGF₂ and PGF₁, and to a lesser extent PGF₃, PGD₂, PGE₃ and thromboxane B₂. It does not detect the PGA class, PGB₁, 15-keto PGE₂, 13,14-dihydro-15-keto PGF₂ or misoprostol. For standard curves, PGE₂ was diluted in a 10-fold concentrate of yeast nitrogen base medium. To check for possible interference by COX inhibitors, controls were included that contained a 10-fold concentrate of medium plus inhibitor.

Determination of cell dry weight.

Growth of planktonic cells and biofilms was monitored by the determination of cell dry weight. At the end of the incubation period, portions of planktonic cell cultures (3 ml) were collected on preweighed cellulose nitrate filters (0.45 µm pore size; 25 mm diameter) and given three washes with water (5 ml). The filters were dried to constant weight at 37 °C, and the dry weights of cells per filter were calculated. Cell dry weights were determined in triplicate. After biofilm formation, 12 disks with their adherent cells were transferred to 12 ml 0.15 M PBS, pH 7.2, and vortexed vigorously. Portions (3 ml) of the resulting cell suspensions were then collected on preweighed cellulose nitrate filters, and processed as described above.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Prostaglandin production by biofilms and planktonic cells of C. albicans GDH 2346

Prostaglandin production was determined at intervals over a 48 h period for both biofilms and planktonic cells growing in yeast nitrogen base medium containing 50 mM glucose. Preliminary experiments showed that one or more components of Sabouraud dextrose medium interfered with the assay for prostaglandins using the prostaglandin-screening enzyme immunoassay kit. Arachidonic acid, which has been used previously to promote prostaglandin secretion by C. albicans (Noverr et al., 2001), also appeared to interfere with this assay. There was no interference when the organisms were grown in yeast nitrogen base medium, but the levels of prostaglandin detected were rather low. Routinely, therefore, culture supernatants from either biofilms or planktonic cells were concentrated 10-fold by freeze-drying before the prostaglandin assay. Biofilms were normally grown on small PVC discs, and the supernatants from 12 disks pooled for use in prostaglandin determinations.

Prostaglandin synthesis was barely detectable after incubation for 10 h, at concentrations of 1.4 ± 0.8 pg ml^–1 (mean ± SEM) and 0.5 ± 0.5 pg ml^–1 for planktonic and biofilm cells, respectively (Fig. 1a, b). After 10 h, however, prostaglandin production increased rapidly to a maximum of 61.9 ± 2.7 pg ml^–1 for planktonic cells and 49.1 ± 2.7 pg ml^–1 for biofilm cells after 48 h. Throughout the entire growth period there was little correlation between prostaglandin concentration and cell density, suggesting that prostaglandin does not function as a quorum-sensing molecule. During the process of quorum sensing, signal molecules accumulate in cultures and at a threshold population density (quorum) interact with cellular receptors that control the expression of specific target genes. In C. albicans two signal molecules have been identified: farnesol, which acts as a negative signal and inhibits the formation of hyphae (Hornby et al., 2001), and tyrosol, which acts as a positive signal and promotes hyphal formation (Chen et al., 2004). Like prostaglandins, both of these molecules are relatively hydrophobic and of low molecular mass.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Relationship between prostaglandin production (open squares) and cell dry weight (solid squares) of (a) planktonic cells and (b) biofilms of C. albicans GDH 2346 during growth over 48 h. The results are the means ± SEM of three independent experiments carried out in duplicate.

When prostaglandin production was calculated as a function of cell dry weight, it became apparent that biofilm cells produce significantly more prostaglandin than do planktonic cells (Fig. 2). In vivo, such a localized concentration of prostaglandin could significantly enhance fungal colonization and pathogenesis. Experiments with mammalian cells have already shown that purified C. albicans prostaglandin down-regulates chemokine production, tumour necrosis factor alpha production and splenocyte proliferation, but up-regulates interleukin 10 production (Noverr et al., 2001). Pathogen–host interactions of this type could contribute to the persistence of Candida infections like chronic vaginitis, which is now thought to be biofilm-associated (Domingue et al., 1991; Costerton et al., 2003).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Prostaglandin production expressed as a function of cell dry weight for planktonic cells (open circles) and biofilms (solid circles) of C. albicans GDH 2346. The results are the means ± SEM of three independent experiments carried out in duplicate.

Effects of COX inhibitors on prostaglandin production by biofilms and planktonic cells of C. albicans GDH 2346

Previous work showed that aspirin, diclofenac and etodolac had the greatest inhibitory effects on the growth of Candida biofilms (Alem & Douglas, 2004). In the present investigation, these inhibitors were used at a relatively low concentration of 50 µM. Aspirin at this concentration was shown by our earlier study to decrease biofilm formation by about 20 % after 48 h (Alem & Douglas, 2004). Here, aspirin significantly reduced prostaglandin synthesis by both biofilms and planktonic cells (Table 1). For biofilms, prostaglandin production was only 48.6 % of that of control cells after 24 h (P < 0.001), but had recovered to 77.4 % after 48 h (P < 0.01). Both diclofenac and etodolac also significantly decreased prostaglandin synthesis by biofilms after 48 h (P < 0.05). Taken together with our earlier findings (Alem & Douglas, 2004), these results demonstrate a strong correlation between decreased prostaglandin levels and decreased biofilm formation following exposure to COX inhibitors, thus supporting the notion that COX-dependent synthesis of prostaglandins may play a role in regulating biofilm development.

Prostaglandin production by C. albicans SC5314 and its morphological mutant HLC54 (cph1/cph1 efg1/efg1)

To investigate further a possible role for Candida prostaglandin in hyphal formation during biofilm development, prostaglandin production was determined in a mutant with defined defects in two filamentation pathways (cph1/cph1 efg1/efg1) and its wild-type strain C. albicans SC5314. The mutant is a URA3/ura3 heterozygote constructed in strain CAI4, which is derived from the clinical isolate SC5314, and contains deletions of both chromosomal copies of URA3 (Lo et al., 1997). In the mutant, the mitogen-activated protein (MAP) kinase and Ras-cAMP pathways, involving transcription factors Cph1 and Efg1, respectively, are blocked. The mutant is capable of growth in the yeast form only, whereas strains SC5314 and CAI4 can grow in both yeast and hyphal forms.

Prostaglandin levels were measured for biofilm and planktonic cells of both strains after 24 and 48 h. As noted already with strain GDH 2346, biofilm cells of both the wild-type and mutant strains produced significantly more prostaglandin than did planktonic cells (P < 0.001 to P < 0.05; Fig. 3). For example, biofilm cells of strain SC5314 secreted 234.2 ± 0.6 pg (mg dry wt)^–1 (mean ± SEM) after 48 h, whereas planktonic cells of the same strain produced only 53.8 ± 3.9 pg (mg dry wt)^–1. However, there was no significant difference between prostaglandin production by biofilm cells of the mutant and parent strains after 48 h (Fig. 3). Scanning electron microscopy demonstrated that biofilms of the parent strain possessed a morphology typical of C. albicans biofilms on PVC catheter disks, i.e. a basal region of densely packed yeast cells with an overlying, mostly hyphal layer (Fig. 4a; Baillie & Douglas, 1999b). The mutant also produced substantial biofilms, but in this case the structures consisted of yeast cells only (Fig. 4b). This finding is in marked contrast to previous reports that such mutants fail to produce any biofilms (Lewis et al., 2002; Ramage et al., 2002), although in these other studies the growth medium used was RPMI 1640, not yeast nitrogen base.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Prostaglandin production after 24 h (white bars) and 48 h (black bars) by planktonic cells and biofilms of C. albicans SC5314 and its morphological mutant HLC54 (cph1/cph1 efg1/efg1). The results are the means ± SEM of two independent experiments carried out in duplicate.

View larger version (108K): [in this window] [in a new window]   Fig. 4. Scanning electron microscopy images of 48 h biofilms of C. albicans SC5314 (a) and its morphological mutant HLC54 (cph1/cph1 efg1/efg1) (b). Bars, 10 µm.

Overall, our experiments with the morphological mutant demonstrate that the genetic defects of this strain did not affect its ability to secrete prostaglandin. Moreover, biofilms of this strain, consisting entirely of yeast cells, produced significantly more prostaglandin than did planktonic cells. Thus, although prostaglandin from either fungal or mammalian sources can promote germ tube formation in C. albicans (Kalo-Klein & Witkin, 1990; Noverr et al., 2001), its exact role in fungal morphogenesis and biofilm development is obviously complex and remains unclear.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Mohammed Alem is the recipient of a research studentship from the Ministry of Health, Saudi Arabia. We are indebted to Margaret Mullin for expert assistance with electron microscopy.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES