Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 25, Num. 3, 2007, pp. 241-244

Indian Journal of Medical Microbiology, Vol. 25, No. 3, July-September, 2007, pp. 241-244

Original Article

Phenotypic switching and its influence on expression of virulence factors by Candida albicans causing candidiasis in human immunodeficiency virus-infected patients

Antony G, Saralaya V, Bhat GK, Shivananda PG

Dept. of Microbiology, Kasturba Medical College, Mangalore - 575 001, Karnataka
Correspondence Address: Dept. of Microbiology, Kasturba Medical College, Mangalore - 575 001, Karnataka, gkbhat61@yahoo.co.in

Date of Submission: 06-Dec-2006
Date of Acceptance: 15-Apr-2007

Code Number: mb07067

Abstract

Purpose : The purpose of the present study was to determine the degree of expression of virulence factors such as adherence, cell surface hydrophobicity (CSH) and production of proteinase by different morphological forms of Candida albicans causing oral candidiasis in human immunodeficiency virus (HIV)-infected individuals.
Methods : C. albicans 3153A and two strains isolated from oral thrush in HIV infected individuals were induced to undergo phenotypic switching by exposure to UV light and the degree of expression of virulence factors by the different morphological forms was studied.
Results : Three different morphological forms of C. albicans were obtained namely, star (S), wrinkled (W) and ring (R) types from the original smooth (O) variety. It was found that proteinase production was greatest with the W type followed by the R type and O type. The S type produced the least proteinase. Expression of cell surface hydrophobicity and adherence was greatest in the O type followed by the R and then the W type and finally the S type.
Conclusions : The differential expression of virulence factors occurs with different phenotypic forms of C. albicans and this may provide a particular morphological type with a distinct advantage over other types in causing candidiasis.

Keywords: C. albicans, phenotypic switching, virulence, candidiasis, human immunodeficiency virus

The transition of Candida spp . from a harmless commensal to an unrelenting pathogen is a fine line and one that is attributable to an extensive repertoire of virulence determinants selectively expressed under suitable predisposing conditions. ^[1] Candida spp . colonizing the oral cavities of human immunodeficiency virus (HIV) infected individuals are subjected to selective pressure that may lead to the emergence of strains with altered genotypic/phenotypic characteristics and enhanced expression of virulence factors. Most strains of C. albicans are known to be capable of switching spontaneously, reversibly and at high frequencies between a number of general phenotypes distinguishable by colony morphology. ^[2],[3] Switching has been demonstrated to regulate a number of phenotypic characteristics involved in pathogenesis such as adhesion, expression of cell surface hydrophobicity (CSH) and secretion of proteinases. ^[4],[5]

The aim of the present study was to investigate the link between high frequency phenotypic switching and pathogenicity of C. albicans . We compared the degree of expression of virulence factors such as secretion of proteinases, expression of CSH and the ability to adhere to human buccal epithelial cell (BEC) by various morphological forms of C. albicans isolated from oral thrush in HIV- infected individuals.

Materials and Methods

Organisms
Three strains of C. albicans namely C. albicans 3153A, CA-O28 and CA-O68 were used in the present study. C. albicans 3153A was procured from the National Collection of Pathogenic Fungi, Mycology Reference Laboratory, London, U.K, and used as the control. C. albicans 3153A produces virulence factors such as proteinase, phospholipase, adherence, cell surface hydrophobicity (CSH) and exhibit phenotypic switching. ^[6],[7],[8] C. albicans CA-O28 and CA-O68 were isolated from HIV patients with oral thrush using standard method. ^[9]

Induction of phenotype switching
C. albicans was grown in liquid Lee′s medium ^[10] supplemented with 70 mg arginine ml ^-1 and 0.1 mM ZnSO ₄ at 25˚C for 24h. The cells were harvested by centrifugation and suspended in sterile distilled water and counted using a haemocytometer. About 10 ⁶ cells were taken and suspended in 100 mL sterile water in a petri dish and exposed to UV light (15W with a emission wave length of 254 nm, total energy output 31 J m ^-2 ) for 5 sec. An aliquot of 1 ml of irradiated cell suspension was taken and diluted in sterile water to obtain a final concentration of 1 x 10 ³ cells ml ^-1 . 100 ml containing about 100 cells was spread on plates containing Lee′s medium with 2% agar and incubated at 25˚C for 7-14 days and observed for different colony morphological forms. ^[11]

A single colony from each of the different phenotypes of C. albicans obtained was used for the estimation of proteinase production, adherence to BEC and for the expression of CSH. After each assay, the colony phenotype was verified by plating 10 µl of the cell suspension on to a Lee′s medium agar plates and incubating the plates at 25 ^˚ C for seven days.

Growth conditions
For estimation of proteinase production, cells from each of the different phenotype colonies were taken and suspended in 1 mL of sterile distilled water and counted with a hemocytometer. Erlenmeyer′s flasks containing 10 mL Macdonald and Odds medium were inoculated with 10 ⁶ cells/ml and incubated at 25^˚ C for 7 days. ^[12] The broth culture was centrifuged and the supernatant was used for the estimation of extracellular proteinase.

Assay of proteinase
The sample of culture supernatant (0.2 mL) was mixed with 0.8 ml substrate (1% bovine serum albumin (BSA) in 0.025M sodium citrate buffer, pH 3.2) and incubated at 37^˚ C for three hours. The reaction was halted by the addition of 2.0 mL of 5% trichloroacetic acid (TCA) resulting in precipitation of BSA. The tubes were kept at 4^˚ C overnight and centrifuged at 2000 rpm for 20 minutes. Proteolysis was determined by measuring the absorbance of the soluble peptides at 280 nm. For control, substrate was added to the culture supernatant and immediately treated with TCA. The absorbance of controls was subtracted from test samples to obtain values for enzyme activity. ^[12] The experiments were repeated four times and mean (± SD) of the readings was determined.

Adherence assay
The adherence assay described by Kimura and Pearsall was used with minor modification. ^[13] Buccal epithelial cells (BEC) were obtained from the buccal mucosa of a single healthy donor, on the day of the assay. BEC were washed thrice in PBS (pH 7.2) and finally suspended in PBS. Standardized suspensions of human BEC (1x10 ⁵ cells per ml of PBS, 0.5 mL) and yeast cells (1x10 ⁷ yeast cells per mL in PBS, 0.5 mL) were mixed and incubated at 37^˚ C with gentle shaking for 45 min. Epithelial cells were then washed with PBS to remove unattached yeasts, collected by filtration, fixed by methanol on to a microscopic slide and stained by Gram stain. The number of adherent yeast cells on each of 100 epithelial cells was counted for each preparation. The experiments were repeated four times and mean (± SD) of the readings was determined.

Polystyrene microsphere assay for CSH
The CSH assay described by Hazen and Hazen was used with some minor modification. ^[14] Blue dyed polystyrene microspheres (Sigma) having a diameter of 0.8 ±0.1 µM, were used in the study. A working solution containing approx. 9x10 ⁸ microspheres/ml in ice cold PBS was prepared from a stock of colloidal suspension of microspheres (10% solids). Equal volumes (200 ml) of microsphere suspensions and yeast cells (5x10 ⁶ cells/ml of PBS) were mixed, rapidly equilibrated to room temperature and vortexed for 30 sec. Cell surface hydrophobicity was determined as the percentage of yeast cells (from at least 100) with three or more attached microspheres, when viewed by bright field microscopy at 400x. The experiments were repeated four times and mean (± SD) of the readings was determined.

Statistical analysis
The expression of virulence factors by the different morphological forms of C. albicans were compared using the Kruskal Wallis Anova test.

Results

Induction of phenotype switching
Upon UV light treatment, the smooth (O) form of C. albicans demonstrated three variant phenotypes namely ring (R), wrinkled (W) and star type (S) [Figure - 1].

Proteinase activity of each phenotype, expressed as OD ₂₈₀ varied dramatically among colony phenotypes [Table - 1]. While there was not much difference in the activities of all three strains tested, (2.242 by CA-O28 to 1.915 by C. albicans 3153A for smooth (O) phenotype, statistically not significant) there was significant difference (P< 0.01) in proteinase activity between switch phenotypes of the same strain. Proteinase activity was highest with the ′W′ type (2.847) of C. albicans CA-O28, followed closely by the ′R′ type (2.645) and then ′O′ type (2.242). The ′S′ type was observed to produce least amounts of proteinase when compared to the parent ′O′ type of C. albicans . This pattern of proteinase production was similar to all three strains of C. albicans tested.

Effect on adherence
The adhesion of ′O′ type cells to buccal epithelium was significantly greater than adhesion of ′R′ type, ′W′ type or ′S′ type [Table - 2], but not significantly greater than the adhesion of ′R′ type [Table - 2]. The adherence of smooth type (′O′ type) of C. albicans strain CA - O28 to human buccal epithelial cells is shown in [Figure - 2].

Effect on CSH
The Cell surface hydrophobicity of ′O′ type cells was significantly greater than CSH of ′R′ type, ′W′ type or ′S′ type, but not significantly greater than the CSH of "R" type [Table - 3]. The adherence of polystyrene microspheres to smooth type of C. albicans CA - O68 is shown in [Figure - 3].

Discussion

The present study indicates that the degree of expression of virulence factors such as CSH, adherence and proteinase does vary significantly among the different phenotypes of C. albicans . It was observed that proteinase production was greatest with the wrinkled (W) phenotype, followed by the ring (R) and smooth (O) types. The star (S) type was observed to produce the least proteinase. A previous study has shown that switching of laboratory strains of C. albicans plays an important role in regulating the transcription of certain secreted aspartyl proteinase genes SAP1 and SAP3. ^[15] Therefore, the variation in secretion of proteinases by the different morphological types observed might be due to the differential regulation of proteinase genes.

Variation was also observed in the ability of the different phenotypes of C. albicans to adhere to BEC and in their expression of CSH. The generalized hierarchy of adherence to BEC was as follows: smooth (O) > ring (R) > wrinkled (W) > star (S). The expression of CSH by the different phenotypes also exhibited a similar pattern. Changes in the adhesion and CSH due to phenotypic switching has been reported in earlier studies. ^[4]

C. albicans is known to exhibit both yeast form and hyphal form in culture during phenotypic switching. It has been demonstrated that the percentage of yeast cells in a particular population has a significant effect on the adherence of C. albicans . Such phenomenon occurs more frequently with the smooth (O) type, which has been shown to adhere better to mucosal epithelia, probably due to formation of germ tubes rather than a population of C. albicans which has a greater percentage of hyphal forms (star type). ^[8]

High frequency phenotypic switching has been shown to occur at an elevated level in C. albicans causing infection in HIV infected individuals. ^[16] This might not only result in the emergence of variant phenotypes expressing different levels of virulence determinants, but also presumably, exhibiting very different combinations of virulence traits. Thus, a high level of spontaneous variability in such populations would provide them with the advantage of rapid adaptation and this might provide a particular morphological type with a distinct advantage over other types in causing candidiasis.

References

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