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. 267-271

Indian Journal of Medical Microbiology, Vol. 25, No. 3, July-September, 2007, pp. 267-271

Brief Communication

The use of mtt [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2h- tetrazolium bromide]-reduction as an indicator of the effects of strain-specific, polyclonal rabbit antisera on Candida albicans and C. krusei

Arseculeratne SN, Atapattu DN, Kumarasiri R, Perera D, Ekanayake D, Rajapakse J

Department of Microbiology, Faculty of Veterinary Sciences, University of Peradeniya
Correspondence Address: Department of Microbiology, Faculty of Veterinary Sciences, University of Peradeniya
chubby@sltnet.lk

Date of Submission: 06-Oct-2006
Date of Acceptance: 06-Mar-2007

Code Number: mb07074

Abstract

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro , although previously published work has shown that specific antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing. The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti- Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.

Keywords: Strain-specific antibody, complement, Candida albicans, Candida krusei, viability, in vitro

Candida albicans and C. krusei are of increasing importance as opportunistic pathogens on account of the increasing prevalence of states of immune compromisation. This investigation was done to provide a greater understanding of humoral defences against infections by Candida species .

As far as we are aware, there are only two reports on the effects of specific antibody on Candida cells in vitro . ^[1],[2] It is generally assumed that antibody and complement alone (i.e., without cells) cannot kill Candida ^[3] and that, in general, the role of antibody immunity in fungal infections is a controversial subject. ^[4] Although the role of B-cells and antibody has been studied, understanding of its relative importance has not ensued. ^[5]

The MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide]-reduction test has been used earlier on Candida albicans after treatment with heat, formaldehyde, hypochlorous acid or amphotericin B ^[6] and to determine the sensitivity of Candida albicans and Aspergillus spp. to therapeutic anti-fungal agents. ^[7] MTT-reduction was demonstrated to correlate with the viability of the antifungal agent-treated Candida cells on culture. ^[6] Moragues et al ^[2] also used the MTT-reduction to investigate the effects of anti- Candida monoclonal antibodies in vitro; that appears to be the only report based on the use of MTT for this purpose.

The investigations described here with C. albicans and C. krusei and their strain-specific antibodies, were done to investigate the effect of sera containing strain-specific anti- Candida antibodies on the viability of C. albicans and C. krusei, in vitro . The tests were based on the capacity of viable cells to reduce MTT to formazan which was assayed by spectrophotometric quantitation of optical density (OD) after its extraction with acid-propanol, with the OD taken as a measure of the metabolic status and the total, viable mass of the Candida cells.

Materials and Methods

C. albicans and C. krusei were isolates from clinical material and identified by the API system. Stock cultures on Sabouraud agar were sub-cultured on brain heart infusion (BHI) broth for -three to six days at 37 ^o C.

Preparation of anti-Candida sera
Strain-specific antibodies were used in view of the heterogeneity of strains of C. albicans and because the protective efficacy of antibodies is dependent on epitope-specificity.

Antigens were extracted from Candida cells of the two test strains with 1% Triton-x100 in PBS pH 7.6 at 4 ^o C. After quantitation of protein content (Bichinconinic acid method, Sigma, Missouri, USA), rabbits were immunized beginning with antigen (100 µg protein per dose) in Freund′s complete adjuvant, followed after one week by antigen in Freund′s incomplete adjuvant, weekly for four weeks followed by two booster doses with an interval of one week.

Pre-immunisation and post-immunization sera were assayed for antibody with their corresponding Triton-extracted antigen by the Immuno dot-blot method, with monoclonal anti-rabbit total Ig-peroxidase conjugate (Sigma, MO, USA) as tracer and 3-amino-9-ethyl carbazole (Sigma, MO, USA) as substrate-chromogen. The antisera against C. albicans and C. krusei had titres of anti- Candida antibody of over 800.

The initial dilutions of antisera (1/100) in PBS were heated at 56 ^o C for 20 min to inactivate residual complement, before addition to the Candida cell deposits. Each batch of guinea pig serum was assayed for complement activity by the conventional anti-sheep red cell haemolysin method.

The pre-immunization sera had no detectable anti- Candida antibody at this initial dilution (1/100).

Reagents
0.2M phosphate buffered saline (PBS) at pH 7.4 was used to dilute sera and dissolve MTT. Anti- Candida antibodies were titrated by Immuno-dot-blot assay. ^[8]

Aliquots of freshly obtained guinea pig (gp) sera without added preservative were stored at -20 ^o C. Aliquots of guinea pig sera preserved with Richardson′s complement preservative, were also stored at -20 ^o C. The guinea pig serum that was used as a source of complement had no detectable anti- Candida antibody.

Solutions containing 0.5 mg/mL of MTT (Sigma, MO, USA) in PBS were used for viability assays on the deposits of the Candida cells.

Tests with antisera
Centrifuged (400g x 10 minutes) deposits from 25 µL of the Candida cells in triplicate or quadruplicate were treated with 25 µL volumes of the following reagents in a typical experiment:

1. heated guinea pig serum (1/20, basal control).
2. unheated guinea pig serum (1/20, complement)
3. pre-immunization (rabbit) serum (1/100)
4. pre-immunization (rabbit) serum (1/100) with added (1/20) guinea pig serum
5. post-immunization (rabbit) serum (1/100)
6. post-immunization (rabbit) serum (1/100) with added (1/20) guinea pig serum
   The diluted rabbit sera in (c), (d), (e) and (f) were heated, after dilution, at 56 ^o C for 20 min to inactivate residual complement. Tests were done on each Candida species with complement with and without complement preservative.
7. Cells with 10% formalin (40% formaldehyde) in PBS incubated at room temperature for 18h were used as a positive-control for inhibition of MTT-reduction. ^[9]

Twenty-five µL of PBS were added when necessary to equalize total volumes. Cells with heated guinea pig serum rather than cells in PBS alone, were used as a basal control for comparison with cells that were treated with pre- or post-immunisation sera and/or complement because the former suspensions showed significantly higher cell-content probably due to their protein content and were therefore comparable with the serum-containing samples.

The tubes, after side-tapping to mix the cell-deposit with the reagents, were incubated statically at room temperature (RT, 28 ^o C ambient) for 18h.

Estimation of total cell mass and cell-viability
Total cell-counts in blood-cell counting chambers, as indicators of lysis, were not done as the Candida cells were often clumped. The MTT-reduction test, on the other hand, provided a measure of both the total viable cell mass and its metabolic capacity, irrespective of clumping of the yeast cells and hence only this test was used as a measure of total cells and their viability in these experiments.

The MTT test
The centrifuged deposits of control or treated cells were washed twice with PBS and were then treated with approximately 50 µL of the MTT solution (0.5 mg/mL PBS); the tubes, after mixing of the contents by side-tapping, were incubated at 37 ^o C for three hours. The tubes containing MTT-cell mixtures were centrifuged (400 x g for 10 minutes) to deposit the cells, the supernatant MTT solution was pipetted out and then acid-isopropanol (95 mL isopropanol with 5 mL 3N HCl) was added to the coloured cell deposit. After stirring of the acid-alcohol-treated deposit with a glass rod, the mixture was allowed to react for five minutes, followed by centrifugation at 400 g for 10 min. One hundred microlitre of the purple-blue coloured supernatant that contained the solubilized formazan in each sample was added to a well in a 96-well microtitre plate for spectrophotometry at 570 nm in an ELISA reader. Acid-isopropanol was used as a blank in the readings.

Tests were repeated with each Candida strain, with or without complement, at least twice.

Statistical comparisons
Statistical analysis was done using Student′s "t" test for the comparison of mean values (SPSS, version 10.01) of the ODs of the supernatants from tube-1 as basal cell control versus tubes 2-7, for each species of Candida .

Results

C. krusei
[Table - 1] shows the results from a typical experiment in which C. krusei and preserved guinea pig serum as a source of complement were used. Ten percent formalin, as a positive control biocide, gave a highly significant decrease of MTT-reduction.

With unheated gp serum in sample 2, the OD was higher than (not significant) with heated gp serum (control sample 1) probably indicating a higher growth-promoting effect of the unheated serum. The absence of a reduction of OD indicated that complement alone had no inhibitory effect and that there were no thermo labile agents with anti- Candida effects in normal serum.

Pre-immunization serum alone (sample 3) also did not reduce the OD; on the contrary the OD was significantly higher than that with the control sample 1 containing heated gp serum (and with sample 2 that contained unheated gp serum), an effect that could conceivably be due to higher growth promoting capacity of rabbit over guinea pig serum.

Pre-immunization serum with complement (sample 4) showed a minor, non-significant reduction of OD. With strain-specific antibody, without complement (sample 5) and with complement (sample 6), no reduction of OD was found. The absence of a reduction of OD with samples containing antiserum, with or without complement, indicated that specific antibody did not cause the inactivation or lysis of C. krusei .

C. albicans
Similar results [Table - 2] were obtained with C. albicans . Ten percent formaldehyde as a potent control biocide gave a highly significant reduction of OD (sample 7). With unheated gp serum (sample 2) the OD value was (non-significantly) higher than in heated serum (control sample 1) probably because the unheated serum was more growth-promoting than the heated serum. The absence of a reduction of OD indicated that complement alone had no inhibitory effect and that there were no thermolabile agents with anti-Candida effects in normal serum.

With pre-immunization (rabbit) serum (sample 3) the OD was significantly higher suggesting, that rabbit serum was more growth-promoting than gp serum. In sample 4 that contained both rabbit and gp sera the OD was significantly higher probably due to increase of growth resulting from the increased volume of serum (50 µL) than in samples 1, 2 or 3 (25 µL). Specific antibody without complement (sample 5) or with complement (sample 6) did not cause a reduction of OD indicating that there was no antibody-mediated lysis or inactivation of C. albicans . The significantly higher OD values in samples 4, 5 and 6 would, again, probably be due to a greater growth-promoting capacity of rabbit over guinea pig serum, for C. albicans more than for C. krusei . The increase rather than a decrease of OD of the formazan in some samples could indicate a higher cell mass due to transformation of the yeast phase to the mycelial phase in the presence of the respective sera; our experiments, however, were concerned primarily with an inhibition of the yeast and therefore a reduction of formazan-OD. The yeast-mycelial transformation would be of relevance to the protective role of antibody in vivo.

Replicate tests with both Candida species gave similar results.

Discussion

In our experiments, counts of yeast cells in blood-cell counting chambers and viable counts in culture as additional tests of cell viability, after their exposure to the antisera and complement, were abandoned on account of gross clumping of the cells that caused wide variations in the counts in replicate samples. The pitfall of viable counts of Candida cells was referred to by Chilgren et al ., ^[1] who quoted the findings and conclusions of Louria and Brayton (1964) who used viable counts of Candida albicans on culture plates. They interpreted the reduction of counts of the yeast after exposure to normal human sera found by the latter authors as having indicated a lethal action of the sera on the yeast, whereas Chilgren et al ., ^[1] explained the reduction of counts as rather having been due to clumping and not a lethal action. Moragues et al ., ^[2] used monoclonal anti- Candida antibodies in vitro with the MTT- reduction test as an indicator of cell viability and demonstrated three anti- Candida activities including a fungicidal effect. However, whether the antibodies they used were strain specific, was not stated.

Several non-specific antifungal agents have been described, including collectins, defensins and microbicidal peptides and metal chelators or iron-binding proteins. ^[10],[11] An inhibitory factor that was active on Cryptococcus neoformans only in high concentrations of serum, that caused disruption and fragmentation of the yeast cells and which was not complement or immunoglobulin, has been described. ^[12]

On the other hand, Chilgren et al ., ^[1] used high concentratons of sera, 0.1 mL yeast cell-suspension to 1 mL of serum, but found that normal sera neither affected the growth of, nor were lethal to C. albicans . Lehrer and Cline ^[13] also reported that normal human serum did not kill C. albicans . Greenfield, reviewing the literature, stated that human serum is not fungicidal against Candida . ^[5] Using the MTT-reduction method, we too found no evidence of inhibition of either Candida species by antibody-free rabbit or guinea pig sera in the higher dilutions that we tested. Murphy ^[10] considered fungal cells to be protected by chitin with carbohydrate polymers; endospores of R. seeberi that possess chitin in their walls as in Candida , is also resistant to damage by specific antibody, in vitro . ^[14]

Kozel described the role of Mannan-binding protein (MBP), in normal sera that binds, lectin-wise, to carbohydrate structures in yeasts leading to activation of the antibody-C1q-independent classical pathway, but stated that "direct killing via formation of a membrane-attack-complex has not been convincingly demonstrated with pathogenic fungi". ^[15] Fungi are resistant to lysis by the terminal components of the complement system even in the presence of specific antibody. ^[16] C. albicans activates complement on incubation with normal serum but does not undergo complement-mediated lysis or killing by serum. ^[17] Our results also indicate that complement alone had no inhibitory or lytic effect on either Candida species in vitro . However, it was demonstrated experimentally in vivo, by Han et al ., ^[18] that complement is essential for protection against haematogenously disseminated candidiasis by specific antibody.

Protective effects of antibody in vivo could include inhibition of yeast-mycelial transformation, ^[4] prevention of adherence to host cell surfaces, ^[3] toxin neutralization, antibody-dependent cell-mediated cytotoxicity, ^[11] neutralization of immuno-modulating fungal products, neutralization of extracellular proteases and promotion of opsonic phagocytosis. Anti-Candida antibody with complement has been claimed to be protective against disseminated candidiasis, although Murphy ^[10] earlier stated that the protective nature of anti- Candida antibodies in both mucosal and systemic forms of the disease is another unsettled issue.

The controversies regarding the anti-fungal effects of antibodies appear to be explainable on the basis of the existence of multiple antibodies of varying epitope-specificity, their concentrations and ability to fix complement. Our demonstration of the absence of an in vitro anti- Candida effect of polyclonal serum needs re-investigation with monoclonal, strain-specific antibodies, in view of the comment that "the uncertainty regarding the importance of antibody in immunity against fungi despite several decades of study suggests that experiments with polyclonal sera may not be conclusive". ^[4] It should, however, be noted that even among monoclonal antibodies, protective and non-protective antibodies exist. ^[4] Moreover, investigations with monoclonal antibodies in vitro are needed because the identification of the protective, non-protective or deleterious effects of these antibodies has thus far been made through in vivo rather than in vitro experimentation.

Our results indicate that complement alone or strain-specific antibody with or without complement, caused neither cell lysis nor metabolic inhibition of either C. krusei or C. albicans as indicated by the MTT-reduction assay. Similar conclusions were derived from experiments with complement, specific antibody and endospores of Rhinosporidium seeberi . ^[14] It is noteworthy that rhinosporidial endospores and Candida cell walls are thick and possess chitin.

Our results with Candida spp. are in contrast to the lysis of gram negative bacteria, This contrast is probably attributable to differences in the structure of the cell walls of Candida and R. seeberi on the one hand and bacteria on the other.

While the protective role of specific anti- Candida antibody remains controversial, a consensus exists that specific cell-mediated immunity rather than humoral immunity is the major mechanism of adaptive immunity against fungal infections and in candidiasis.

Acknowledgement

References

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