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Variations in the antigenic effects of tunicamycin on Candida albicans
Comp. Immun. Microbiol. infect. Dis. Vol. 16, No. 2, pp. 163-172, 1993 Printed in Great Britain
0147-9571/93$6.00+ 0.00 Pergamon Press Ltd
VARIATIONS IN THE ANTIGENIC EFFECTS OF T U N I C A M Y C I N ON C A N D I D A A L B I C A N S M. N. VESPA,l J. C. LEBECQ,2. N. SIMONETTIt a n d J. M. BASTIDE2 ~Istituto di Microbiologia, Facolt~ di Farmacia, Universitfi degli Studi di Roma, La Sapienza, P. le Aldo Moro 5, 00185 Rome, Italy and 2Facult6 de Pharmacie, Universit6 de Montpellier I, Unit6 de Recherche en Immunologie, 34060 Montpellier, Cedex l, France (Received.for pubfication 14 July 1992) Abstract--We report on the effect of subinhibitory doses of tunicamycin on Candida albicans cells (BP strain high responder NCYC 1466) in a defined medium favourable for expression of the mycelial phase. Tunicamycin inhibited the synthesis of some protein fractions ranging from 40 to 65 kDa, where the immunodominant antigens of C. albicans responsible for the antibody response to systemic mycosis were inhibited. By two-dimensional immunoelectrophoresis, antigen extracts from the cell cultures grown with tunicamycin showed a migration modification and a lower number of precipitation arcs with variation in their height and range. Key words: Candida albicans, tunicamycin, Candida proteins, two-dimensional immunoelectrophoresis. Rrsumr--Est rapport6 l'effet de doses subinhibitrices de tunicamycine sur des cellulesde Candida albicans (souche BP, fort rrpondeur) dans un milieu d~fini favorable h l'expression de la phase mycglienne. La tunicamycine a inhib~ la synth~se de certaines fractions protriques allant de 40 a 65 kDa, dans lesquellesles antigrnes immunodominants de C. albicans responsables de l'induction de la formation d'anticorps dans la mycose systrmique &aient inhibes. Par l'immuno-~lectrophor~sebi-dimensionnelles, dans l'extrait antigenique obtenu ~i partir de cultures s'rtant developpre en pr/~sencede tunicamycine, on a observ6 une modifications dans les caractrristiques de migration, ainsi qu'un nombre moins 61ev6 d'arcs de prrcipitation avec des variations dans leur hauteur et situation. Mots-clefs: Candida albicans, tunicamycine, candida protrines, immuno-61ectrophorrsebi-dimensionelle. INTRODUCTION Candida albicans has two phases: the yeast (Y) phase a n d the mycelial (M) phase, also considered as the invasive form. The mycelial phase begins with a germ tube [1, 2]. This d i m o r p h i s m , the result o f m a n y factors, can also be used as a b r o a d - s p e c t r u m antibiotic, or the t r e a t m e n t of h a e m o p a t h y , l y m p h o m a s , a n d solid t u m o u r s , or o f diseases o f toxic or infectious etiology, particularly in high risk patients a n d in those with a u t o i m m u n e deficiencies ( A I D S ) [3-7]. It is for this reason that the b o d y ' s acquired h u m o r a l i m m u n e mechanisms, i.e. the a n t i c a n d i d a antibodies, have been gaining more importance. These antibodies can vary in relationship to the d i m o r p h i s m of C. albicans a n d its yeast, germ tube, a n d mycelial stages. T u n i c a m y c i n (Mr 840) is a n antibiotic p r o d u c e d by S t r e p t o m y c e s liposuperificans, which belongs to the cyclic peptide family. It c o n t a i n s N - a c e t y l - g l u c o s a m i n e a n d a chain o f fatty *Author for correspondence. 163
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acids that prevent the first reaction of the dolichol pathway leading to N-glycosylation of proteins [8, 9] in eukaryotic cells. The drug's presence in the medium not only inhibits the M-phase in C. albicans, but it also interferes with glycoprotein and mannoprotein biosynthesis [10-12]. The object of our study was to examine the effect of tunicamycin on the antigenic variations found in each of the three phases of C. albicans using twodimensional immunoelectrophoresis. MATERIALS AND METHODS C. albicans BP serotype A high-responder ( N C Y C 1466) was used. The strain was maintained by sub-culturing on slopes of Sabouraud agar. Three different C. albicans antigen extracts were prepared from this strain: (1) from the yeast phase, (2) from the germ tube phase, (3) from the mycelial phase, and an additional fourth sample (4) from the C. albicans treated with 10#g/ml tunicamycin, which was part of the yeast phase (1). Preparation o f the antigenic suspension of C. albicans in Y-phase M S M medium. Yeast Nitrogen Base (Difco) 6.7 g: glucose 10.0 g, asparagine 1.5 g, agar 14.0 g, distilled H~O 1000 ml, p H 6; 250 ml of this medium were put in a 1 litre Roux flask. A preculture was grown on a slant culture of MSM at 26°C for 24h. It was then resuspended in 5ml; of sterile physiological saline solution, inoculated in a l litre Roux flask, and incubated at 26~C for 24 h. The C. albicans cells were then resuspended in 40 ml of sterile physiological saline solution. In each Roux flask, 6 ml of this sample were inoculated and incubated at 2 f f C for 24 h. The C. albicans yeast cells were harvested with a sterile physiological saline solution and sterile glass pellets of 0.5 cm O. They were then centrifuged at + 4 ° C at 2000rpm for 20min, then washed in a sterile physiological saline solution, and rewashed with sterile H 2 0 at + 4 ° C at 2000rpm for 15 rain. Preparation of the antigenic suspension in the germ tube and mycelial phases. An Erlenmeyer flask (250 ml) containing 100 ml of Bacto fluid Sabouraud medium (Difco) was inoculated with a suspension of cells from a 24 h slant culture, and then incubated in a gyratory shaker at 26°C for 18 h at 100 rpm. This was the preculture. The stationary phase cells were centrifuged, washed, and then suspended in 30 ml sterile physiological saline solution; a 2.5 ml aliquot of the cell suspension was transferred to an Erlenmeyer flask (1000 ml) containing 250 ml of M E M (Diagnostic Pasteur, Marnes-la-Coquette, France) medium supplemented with 0.5% N-acetyl-D-glycosamine (Fluka) in a rotary shaker at 37c~C at 100 rpm. The M E M medium was a favourable expression of the transition from the yeast phase to the mycelial phase. The germ tube phase was cultured for 3 h; the mycelial phase was cultured for 7 h. The samples were decanted into sterile glass cylinders to allow the yeast cells to separate from both the germ tube and the mycelium. The samples were harvested by centrifuging at + 4 ° C at 2000 rpm for 20 min. They were washed by
Fig. 1. (Seefacing page.) Two-dimensional immunoelectrophoresisof C. albieans BP strain yeast antigen extract (3 #1) against anti-C, albicans BP strain rabbit antiserum in yeast phase (300,ul). Fig. 2. (Seefacing page.) Two-dimensional immunoelectrophoresis of C. albicans BP strain germ tube antigen extract (3 # 1)against anti-C, albicans BP strain rabbit antiserum in yeast phase (300 ,u1). Fig. 3. (Seefacing page.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain mycelial antigen extract (3/~1) against anti-C, albieans BP strain rabbit antiserum in yeast phase (300/~1). Fig. 4. (Seefacing page.) Two-dimensionalimmunoelectrophoresisof C. albieansBP strain mycelial antigen extract (3 ~1) against anti-C, albicans BP strain rabbit antiserum in mycelialphase (300 ~1).
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Variations in the antigenic effects of tunicamycin on Candida albicans
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centrifugation in a sterile physiological saline solution, then rewashed in sterile H 2 0 at + 4 ° C at 2000 rpm for 15 min. Preparation o f the antigen suspension o f the tunicamycin treated cells. The same preculture and medium were used as for the germ tube and the mycelial phases. Tunicamycin was added in doses of 10 ~tg/ml together with the C. albicans yeast cells. The samples were incubated at 37°C for 7 h in a rotary shaker at 100rpm. They were harvested by centrifugation at + 4 ° C at 2000 rpm for 20 min. They were then washed by centrifugation in sterile physiologic solution, and then rewashed in sterile H 2 0 at +4°C at 2000 rpm for 15 min. Preparation o f the antigen extract o f the different phases. The cells were homogenized in a Braun homogenizer using glass pellets for 15 min. After, they were centrifuged for 20 min at + 4 ° C at 18,000rpm. The supernatant was harvested and sterilized with 0.45 mm Sartorius filters, and then lyophilized. Protein doses. The proteins contained in each antigen were calculated with a micromethod technique [13]. The proteins present in each antigen extract were: (1) antigen yeast extract 0.216 mg/mg (2) antigen germ tube extract 0.215 mg/mg (3) antigen mycelium extract 0.07 mg/mg whereby the yeast cells and tunicamycin were 0.15 mg/mg. Each antigen extract was assayed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in a vertical slab gel apparatus (Pharmacia), using 17% (w/v) acrylamide polymers as running gel and 4.5% acrylamide slab as stacking gel. Electrophoresis migration was performed for 16 h at a constant current of 5 mA in a buffer composed of 6.0 g Tris, 28.8 g glycine, 5.0 g SDS, 1 litre distillated H20, pH = 8.6; Standard used was Mr 14.4-94 kDa (Pharmacia). Each 1 mg/ml antigen extract was solubilized by boiling for 2 min in Tris buffer as above containing SDS 2% (w/v), mercaptoethanol 10% (v/v), glycerol 10% (v/v), bromophenol blue 0.02% (w/v). Two-dimensional immunoelectrophoresis antisera standard. Specific polyvalent anti-C. albicans goat antiserum was obtained from Diagnostic Pasteur (Marnes-la-Coquette, France). Anti-C. albicans serotype A and serotype B antisera were prepared from immunized rabbits with a homogenate of either C. albicans serotype A or serotype B in Freund's complete adjuvant. Antisera were prepared from rabbits weighing 2.5 kg immunized with complete homogenate of yeast cells, and with complete homogenate of mycelium of C. albicans strain BP serotype A for 6 months. The first subcutaneous
Fig. 5. (Seefacingpage.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain germ tube antigen extract (3 # 1)against anti-C, albicans BP strain rabbit antiserum in mycelialphase (300#1). Fig. 6. (See facing page.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain yeast antigen extract (3 #1) against anti-C, albicans BP strain rabbit antiserum in mycelialphase (300 pl). Fig. 7. (See facing page.) Two-dimensional immunoelectrophoresisof C. albicans BP strain yeast antigen extract (3 #1) against anti-C, albicans antiserum standard in yeast phase (PD) (300#1). Fig. 8. (See facing page.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain germ tube antigen extract (3 p l) against anti-C, albicans antiserum standard in yeast phase (PD) (300# l).
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immunization was made with 0.5 ml of total homogenate of yeast plus 0.5 ml of Freund's complete adjuvant. 1 ml of total homogenate of mycelium and 1 ml of Freund's complete adjuvant. After 21 days, intravenous and subcutaneous booster doses were given with complete homogenate of both the yeast and of the mycelium in the same quantities, but without or adjuvant. More booster doses followed weekly until the end of February. From March to June, booster doses were given once a month. Each antigen extract was used at a concentration of 20 mg/ml in a barbital-sodium barbital buffer M 0.02 pH 8.6. Agarose gel A 37,EEO = - 0 . 1 7 , purchased from Industrie Biologique Francaise (Villeneuve-la-Garenne) was used. The gel was prepared at i % w/vol in the barbital-sodium barbital buffer and poured just before use on a Gel Bond " film 85 × 100mm (FMC, Bio Products, Rockland, Maine). 10.0ml agarose were poured at 56°C on Gel Bond ~"' film. Aliquots of 3/~1 of each antigenic preparation were applied to the Gel Bond '~ film. Each antiserum was used at a final concentration of 300/~1 according to the methods described by Lebecq et al. [14]. The final antiserum concentration was at 7.6/~1/cm 2. Veronal-sodium veronal buffer M = 0.02, pH = 8.6 was placed in the electrophoresis chamber: migration I at 50 V for 90 min: migration II at 15 V for 16 h. After, the plates were resuspended in a beaker containing 0.15 M NaCI. The gel was washed by stirring in the salt solution, then it was rinsed with distilled water, pressed, dried, and drained with Coomassie brilliant blue G 250. RESULTS The antigenic extract of C. albicans grown in the presence of tunicamycin showed a lack of high molecular weight proteins above 94 kDa, which were, however, present in the control antigenic extracts. Besides this, a lack of antigen 47 kDa was noted, together with other antigens known as: Mr 44, 52, 48.9 and 59.72 kDa. Antigen Mr 68 kDa was present. Two-dimensional immunoelectrophoresis with homologous and heterologous antisera. The Y-phase antigen extracts showed precipitation arcs of greater intensity and number in the presence of the homologous anti-Y antiserum than in those of the heterologous anti-Y antiserum (DP) (Figs 1-3 and 7--9). The germ tube and mycelial antigen extracts showed greater precipitation arcs towards the cathode both with the homologous anti-Y and anti-M sera, than with the heterologous anti-Y (DP), which was due to the base pH level of the proteins (Figs 2-5, 8 and 9); this was not observed, however, in the yeast antigen extracts (Figs 1, 6 and 7). The antigen extracts of the C. albicans cells grown in the presence of tunicamycin showed only the Y-phase, and thus precipitation arcs of fewer number and lesser intensity than in the same control antigen extracts (heterologous DP antiserum and homologous anti-Y and anti-M antisera) in the rabbits immunized by us (Figs 10-12). Besides this, the
Fig. 9. (See facing page.) Two-dimensional immunoelectrophoresis of C. albicans BP strain mycelial antigen extract (3 ~tl) against anti-C, albicans antiserum standard in yeast phase (PD) (300 #l). Fig. 10. (See facing page.) Two-dimensional immunoelectrophoresis of C. albicans cells BP strain grown in the presence of tunicamycin (10/~ 1/ml); antigen extract (3 # 1) against anti-C, albicans BP strain rabbit antiserum in yeast phase (300#1). Fig. 11. (See facing page.) Two-dimensional immunoelectrophoresis of C. albicans cells BP strain grown in the presence of tunicamycin (101d/ml); antigen extract (3 #l) against anti-C, albicans antiserum standard in yeast phase (PD) (300 #1).
Figs 9-11. (Captions on facing page.) 169
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M
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Fig. 12. Two-dimensional immunoelectrophoresis of C. albicans cells BP strain grown in the presence of tunicamycin (10 gl/ml); antigen extract (3 #1) against anti-C, albicans BP strain rabbit antiserum in mycelial phase (300/~1). Fig. 13. Polyacridamides at 17%. From left to right: antigen extract of C. albicans in yeast phase: antigen extract of C. albicans in mycelial phase; antigen extract of C. albicans in germ tube phase. Standard (Pharmacia); antigen extract of C. albicans cells grown in the presence of tunicamycin ( 10 Ftl/ml).
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precipitation arcs shifted slightly towards the anode; the preciptiation arcs towards the cathode otherwise present in the control sera were lacking. DISCUSSION Tunicamycin is a powerful antibiotic that inhibits the glycoprotein synthesis of fungi, consequently causing a disturbance in the chitine-rich microfibril network. In our study, we used a culture medium MEM favourable expression for filamentation, which was supplemented with 0.5% N-acetyl-D-glucosamine at 37°C [15]. Tunicamycin was observed to inhibit the synthesis of high Mr proteins and other protein fractions ranging from 40 to 65 kDa. Our results are consistent with those of Elorza et al. [8] also as specifically concerns the protein fraction of 30 kDa, which was present in the three antigen control extracts (yeast, germ tube and mycelial), as well as in the C. albicans cell cultures grown in the presence of tunicamycin. These results suggest that tunicamycin not only inhibits the synthesis of the glycoproteins [7-9, 11, 16] but also that of many other proteins, and in particular protein 47kDa. This protein, which is considered an immunodominant antigen, causes the antibody reaction of the host to systemic fungi; it can be found both in the associated cytoplasma of the vacuoles and in the cell walls [6, 17, 18]. The immunodominant antigens described by Strockbine et al. [19] 44 kDa, and by Greenfield et al. [20] 48.9, 59 and 71 kDa, and present in the three antigen control extracts were inhibited by tunicamycin (Fig. 13). This indicates that even in subinhibitory doses, tunicamycin interferes with the metabolism of C. albicans cells and significantly alters their protein synthesis. The antigen extract of C. albicans cells grown in the presence of tunicamycin in the two-dimensional immunoelectrophoresis tests showed both in the cases of the heterologous serum and in the homologous sera a consequently lower number of precipitation arcs, while showing variations in height and range, and migration towards the anode. This indicates that the proteins were inhibited, their charge was slightly modified, and the pH level greatly reduced (Fig. 10). The possibility of having numerous precipitation arcs also with the homologous serum in the M-phase can be explained by the fact that some proteins are common to both the yeast phase and to the M-phase of the C. albicans cells. Manning et al. [5] have reported that the proteins of the yeast and mycetial phases of C. albicans can vary from strain to strain. Besides this, they have demonstrated that the proteins of the mycelial phase appear as a modification of preexisting proteins from the yeast phase rather than as newly synthesized proteins in the yeast phase. Large differences between yeast form and mycelial form cytoplasmic proteins could not be found. The total absence of proteins specific to the mycelial phase can be explained by the fact that in the hyphal phase proteins of the yeast phase are no longer synthesized [5, 15]. This hypothesis is also supported by the work of Sundstrom et al. [21], who have reported that there are slightly modified proteins also in the formation of the germ tube. In our three antigen extracts: yeast, germ tube, and mycelial, the precipitation arcs of the anti-Y sera differed from those of the anti-M serum [20, 22-25]. In particular, the antigen germ tube and mycelial extracts showed a large precipitation arc towards the cathode both with anti-Y and anti-M sera; thus the presence of base pH positively charged proteins could be considered significant for these phases (Figs 2-5, 8 and 9).
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T h e i n h i b i t i o n o f t h e M - p h a s e is c o n s i s t e n t w i t h t h e f a c t t h a t t h e s e c h a r a c t e r i s t i c p r e c i p i t a t i o n a r c s c a n n o t b e f o u n d in t h e a n t i g e n e x t r a c t s o f t h e cells g r o w n in t h e p r e s e n c e of tunicamycin (Figs 10-12). Acknowledgements--We wish to thank Professor Bernard Bizzini, head of the Laboratoire d'Immunochimie des Protein at the lnstitut Pasteur, Paris, for his helpful suggestions in this study. This work was also partially supported by a grant from NATO CNR and CNR Altri Interventi.
REFERENCES 1. Casanova M., Gil M. L., Cardenono L., Martinez J. P. and Sentandreu R. Identification of wall specific antigens synthesized during germ-tube formation by Candida albicans. Infect. Immun. 57, 262-271 (1989). 2. Chattaway F. W., Bishop R., Holmes M. R., Odds F. C. and Barlow A. J. E. Enzyme activities associated with carbohydrate synthesis and breakdown in yeast and mycelial forms of Candida albicans. J. gen. Microbiol. 75, 97-109 (1973). 3. Belsito D. V., Sanchez M. R., Baer R. I., Valentine F. and Thorbecke G. J. Reduce Langerhans' ceils. I antigen and ATPase activity in patients with the Acquired lmmunodeficiency. N. Engl. J. Med. 310, 1279-1293 (1984). 4. Bowen D. U, Lane C. H, and Fauci A. S. Cellular immunity, in AIDS (Edited by Ebbesen P., Biggar R. J. and Melbye M.). Munksgaard, Copenhagen (1984). 5. Manning M. and Mitchell T. C. Morphogenesis of Candida albicans and cytoplasmic proteins associated with differences in morphology, strain, or temperature. J. Bacteriol. 144, 258-273 (1980). 6. Matthews R., Smith D., Midgley J., Burnie J., Clark I. and Conolly M. Candida and AIDS: evidence for protective antibody. Lancet 30, 263-266 (1988). 7. Poulain D., Hopwood V. and Vernes A. Antigenic variability of Candida albicans. Crit. rev. Microbiol. 12, 223-270 (1985). 8. Elorza M. V., Murgui A., Rico H., Miragall F. and Sentandreu R. Formation of a new cell wall by protoplasts of Candida albicans: effect of Papulacandin B, Tunicamycin and Nikkomycin. J. gen. Microbiol. 133, 2315 2325 (1987). 9. Murgui A., Elorza M. V. and Sentandreu R. Effect ofPapulacandin B and calcofluor white on the incorporation of mannoproteins in the wall of Candida albicans blastospores. Biochim. biophys. A cta 881, 215--222 (1985). 10. Bishop C. T., Blank F. and Gardner P. E. The cell wall polysaccarides of Candida albicans: glucan, mannan, and chitin. Can. J. Chem. 38, 869 880 (1960). 11. Elorza M. V., Murgui A. and Sentandreu R. Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls. J. gen. Microbiol. 131, 2209-2216 (1985). 12. Vespa M. N. and Simonetti N. Azione della tunicamicina su Candida albicans. Pol. Med. 95, 1-5 (1988). 13. Sedemak J. and Grossberg S. E. A rapid, sensitive, and versatile assay for protein using Coomassie blue G250. Analyt. Biochem. 79, 544 552 (1977). 14. Lebecq J. C., Salhi S. L. and Bastide J. M. A novel antibody overlay technique for two-dimensional immunoelectrophoresis. J. Immunl. Meth. 66, 219 226 (1984). 15. Chattaway F. W., Holmes M. R. and Barlow A. J. E. Cell wall composition of the mycelial and blastospore forms of Candida albicans. J. gen. Microbiol. 51, 367 376 (1968). 16. Cassone A., Carpinelli G., Angiolella U, Maddaluno G. and Podo F. 31P Nuclear magnetic resonance study of growth and dimorphic transition in Candida albicans. J. gen. Microbiol. 129, 1569-1575 (1983). 17. Matthews R. C., Burnie J. P. and Tabaqchali S. Isolation of immunodominant antigens from sera of patients with systemic candidiasis and characterization of serological response to Candida albicans. J. clin. Microbiol. 25, 230-237 (1987). 18. Matthews R. C., Wells C. and Burnie J. P. Characterisation of the immunodominant 47 kDa antigen of Candida alb&ans. J, med. Microbiol. 27, 227-232 (1988). 19. Strockbine N. A., Largen M. T., Zweibel S. M. and Buckley H. R. Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera. Infect. Immun. 43, 715-721 (1984). 20. Greenfield R. A. and Jones J. M. Purification and characterization of a major cytoplasmic antigen of Candida albicans. Infect. Immun. 34, 469477 (1981). 21. Sundstrom M. R., Nichols J. E. and Kenny G. E. Antigenic difference between mannoprotein of germ tubes and blastospores of Candida albicans. Infect. Immun. 55, 616~520 (1987). 22. Brawner D. L. and Cutler J. E. Variability in expression of cell surface antigens of Candida albicans during morphogenesis. Infect. lmmun. 51, 337-343 (1986). 23. Cailliez J. C. and Poulain D. Analyse cytologique de l'expression d'un 6tiope port6 par les glycoprot6inees excr6te6s par Candida albicans. Ann. Inst. Past. Microbiol. 139, 171-188 (1988). 24. Smail E. H. and Jones J. M. Demonstration and solubilisation of antigens expressed primarily on the surfaces of Candida albicans germ tubes. Infect. lmmun. 45, 74-81 (1984). 25. Vespa M. N. and Simonetti N. Studio sugli antigeni di Candida albicans nella fase lievitiforma e miceliale. Pol. Med. 93, 359-366 (1986).
0147-9571/93$6.00+ 0.00 Pergamon Press Ltd
VARIATIONS IN THE ANTIGENIC EFFECTS OF T U N I C A M Y C I N ON C A N D I D A A L B I C A N S M. N. VESPA,l J. C. LEBECQ,2. N. SIMONETTIt a n d J. M. BASTIDE2 ~Istituto di Microbiologia, Facolt~ di Farmacia, Universitfi degli Studi di Roma, La Sapienza, P. le Aldo Moro 5, 00185 Rome, Italy and 2Facult6 de Pharmacie, Universit6 de Montpellier I, Unit6 de Recherche en Immunologie, 34060 Montpellier, Cedex l, France (Received.for pubfication 14 July 1992) Abstract--We report on the effect of subinhibitory doses of tunicamycin on Candida albicans cells (BP strain high responder NCYC 1466) in a defined medium favourable for expression of the mycelial phase. Tunicamycin inhibited the synthesis of some protein fractions ranging from 40 to 65 kDa, where the immunodominant antigens of C. albicans responsible for the antibody response to systemic mycosis were inhibited. By two-dimensional immunoelectrophoresis, antigen extracts from the cell cultures grown with tunicamycin showed a migration modification and a lower number of precipitation arcs with variation in their height and range. Key words: Candida albicans, tunicamycin, Candida proteins, two-dimensional immunoelectrophoresis. Rrsumr--Est rapport6 l'effet de doses subinhibitrices de tunicamycine sur des cellulesde Candida albicans (souche BP, fort rrpondeur) dans un milieu d~fini favorable h l'expression de la phase mycglienne. La tunicamycine a inhib~ la synth~se de certaines fractions protriques allant de 40 a 65 kDa, dans lesquellesles antigrnes immunodominants de C. albicans responsables de l'induction de la formation d'anticorps dans la mycose systrmique &aient inhibes. Par l'immuno-~lectrophor~sebi-dimensionnelles, dans l'extrait antigenique obtenu ~i partir de cultures s'rtant developpre en pr/~sencede tunicamycine, on a observ6 une modifications dans les caractrristiques de migration, ainsi qu'un nombre moins 61ev6 d'arcs de prrcipitation avec des variations dans leur hauteur et situation. Mots-clefs: Candida albicans, tunicamycine, candida protrines, immuno-61ectrophorrsebi-dimensionelle. INTRODUCTION Candida albicans has two phases: the yeast (Y) phase a n d the mycelial (M) phase, also considered as the invasive form. The mycelial phase begins with a germ tube [1, 2]. This d i m o r p h i s m , the result o f m a n y factors, can also be used as a b r o a d - s p e c t r u m antibiotic, or the t r e a t m e n t of h a e m o p a t h y , l y m p h o m a s , a n d solid t u m o u r s , or o f diseases o f toxic or infectious etiology, particularly in high risk patients a n d in those with a u t o i m m u n e deficiencies ( A I D S ) [3-7]. It is for this reason that the b o d y ' s acquired h u m o r a l i m m u n e mechanisms, i.e. the a n t i c a n d i d a antibodies, have been gaining more importance. These antibodies can vary in relationship to the d i m o r p h i s m of C. albicans a n d its yeast, germ tube, a n d mycelial stages. T u n i c a m y c i n (Mr 840) is a n antibiotic p r o d u c e d by S t r e p t o m y c e s liposuperificans, which belongs to the cyclic peptide family. It c o n t a i n s N - a c e t y l - g l u c o s a m i n e a n d a chain o f fatty *Author for correspondence. 163
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acids that prevent the first reaction of the dolichol pathway leading to N-glycosylation of proteins [8, 9] in eukaryotic cells. The drug's presence in the medium not only inhibits the M-phase in C. albicans, but it also interferes with glycoprotein and mannoprotein biosynthesis [10-12]. The object of our study was to examine the effect of tunicamycin on the antigenic variations found in each of the three phases of C. albicans using twodimensional immunoelectrophoresis. MATERIALS AND METHODS C. albicans BP serotype A high-responder ( N C Y C 1466) was used. The strain was maintained by sub-culturing on slopes of Sabouraud agar. Three different C. albicans antigen extracts were prepared from this strain: (1) from the yeast phase, (2) from the germ tube phase, (3) from the mycelial phase, and an additional fourth sample (4) from the C. albicans treated with 10#g/ml tunicamycin, which was part of the yeast phase (1). Preparation o f the antigenic suspension of C. albicans in Y-phase M S M medium. Yeast Nitrogen Base (Difco) 6.7 g: glucose 10.0 g, asparagine 1.5 g, agar 14.0 g, distilled H~O 1000 ml, p H 6; 250 ml of this medium were put in a 1 litre Roux flask. A preculture was grown on a slant culture of MSM at 26°C for 24h. It was then resuspended in 5ml; of sterile physiological saline solution, inoculated in a l litre Roux flask, and incubated at 26~C for 24 h. The C. albicans cells were then resuspended in 40 ml of sterile physiological saline solution. In each Roux flask, 6 ml of this sample were inoculated and incubated at 2 f f C for 24 h. The C. albicans yeast cells were harvested with a sterile physiological saline solution and sterile glass pellets of 0.5 cm O. They were then centrifuged at + 4 ° C at 2000rpm for 20min, then washed in a sterile physiological saline solution, and rewashed with sterile H 2 0 at + 4 ° C at 2000rpm for 15 rain. Preparation of the antigenic suspension in the germ tube and mycelial phases. An Erlenmeyer flask (250 ml) containing 100 ml of Bacto fluid Sabouraud medium (Difco) was inoculated with a suspension of cells from a 24 h slant culture, and then incubated in a gyratory shaker at 26°C for 18 h at 100 rpm. This was the preculture. The stationary phase cells were centrifuged, washed, and then suspended in 30 ml sterile physiological saline solution; a 2.5 ml aliquot of the cell suspension was transferred to an Erlenmeyer flask (1000 ml) containing 250 ml of M E M (Diagnostic Pasteur, Marnes-la-Coquette, France) medium supplemented with 0.5% N-acetyl-D-glycosamine (Fluka) in a rotary shaker at 37c~C at 100 rpm. The M E M medium was a favourable expression of the transition from the yeast phase to the mycelial phase. The germ tube phase was cultured for 3 h; the mycelial phase was cultured for 7 h. The samples were decanted into sterile glass cylinders to allow the yeast cells to separate from both the germ tube and the mycelium. The samples were harvested by centrifuging at + 4 ° C at 2000 rpm for 20 min. They were washed by
Fig. 1. (Seefacing page.) Two-dimensional immunoelectrophoresisof C. albieans BP strain yeast antigen extract (3 #1) against anti-C, albicans BP strain rabbit antiserum in yeast phase (300,ul). Fig. 2. (Seefacing page.) Two-dimensional immunoelectrophoresis of C. albicans BP strain germ tube antigen extract (3 # 1)against anti-C, albicans BP strain rabbit antiserum in yeast phase (300 ,u1). Fig. 3. (Seefacing page.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain mycelial antigen extract (3/~1) against anti-C, albieans BP strain rabbit antiserum in yeast phase (300/~1). Fig. 4. (Seefacing page.) Two-dimensionalimmunoelectrophoresisof C. albieansBP strain mycelial antigen extract (3 ~1) against anti-C, albicans BP strain rabbit antiserum in mycelialphase (300 ~1).
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Variations in the antigenic effects of tunicamycin on Candida albicans
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centrifugation in a sterile physiological saline solution, then rewashed in sterile H 2 0 at + 4 ° C at 2000 rpm for 15 min. Preparation o f the antigen suspension o f the tunicamycin treated cells. The same preculture and medium were used as for the germ tube and the mycelial phases. Tunicamycin was added in doses of 10 ~tg/ml together with the C. albicans yeast cells. The samples were incubated at 37°C for 7 h in a rotary shaker at 100rpm. They were harvested by centrifugation at + 4 ° C at 2000 rpm for 20 min. They were then washed by centrifugation in sterile physiologic solution, and then rewashed in sterile H 2 0 at +4°C at 2000 rpm for 15 min. Preparation o f the antigen extract o f the different phases. The cells were homogenized in a Braun homogenizer using glass pellets for 15 min. After, they were centrifuged for 20 min at + 4 ° C at 18,000rpm. The supernatant was harvested and sterilized with 0.45 mm Sartorius filters, and then lyophilized. Protein doses. The proteins contained in each antigen were calculated with a micromethod technique [13]. The proteins present in each antigen extract were: (1) antigen yeast extract 0.216 mg/mg (2) antigen germ tube extract 0.215 mg/mg (3) antigen mycelium extract 0.07 mg/mg whereby the yeast cells and tunicamycin were 0.15 mg/mg. Each antigen extract was assayed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in a vertical slab gel apparatus (Pharmacia), using 17% (w/v) acrylamide polymers as running gel and 4.5% acrylamide slab as stacking gel. Electrophoresis migration was performed for 16 h at a constant current of 5 mA in a buffer composed of 6.0 g Tris, 28.8 g glycine, 5.0 g SDS, 1 litre distillated H20, pH = 8.6; Standard used was Mr 14.4-94 kDa (Pharmacia). Each 1 mg/ml antigen extract was solubilized by boiling for 2 min in Tris buffer as above containing SDS 2% (w/v), mercaptoethanol 10% (v/v), glycerol 10% (v/v), bromophenol blue 0.02% (w/v). Two-dimensional immunoelectrophoresis antisera standard. Specific polyvalent anti-C. albicans goat antiserum was obtained from Diagnostic Pasteur (Marnes-la-Coquette, France). Anti-C. albicans serotype A and serotype B antisera were prepared from immunized rabbits with a homogenate of either C. albicans serotype A or serotype B in Freund's complete adjuvant. Antisera were prepared from rabbits weighing 2.5 kg immunized with complete homogenate of yeast cells, and with complete homogenate of mycelium of C. albicans strain BP serotype A for 6 months. The first subcutaneous
Fig. 5. (Seefacingpage.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain germ tube antigen extract (3 # 1)against anti-C, albicans BP strain rabbit antiserum in mycelialphase (300#1). Fig. 6. (See facing page.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain yeast antigen extract (3 #1) against anti-C, albicans BP strain rabbit antiserum in mycelialphase (300 pl). Fig. 7. (See facing page.) Two-dimensional immunoelectrophoresisof C. albicans BP strain yeast antigen extract (3 #1) against anti-C, albicans antiserum standard in yeast phase (PD) (300#1). Fig. 8. (See facing page.) Two-dimensionalimmunoelectrophoresisof C. albicans BP strain germ tube antigen extract (3 p l) against anti-C, albicans antiserum standard in yeast phase (PD) (300# l).
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immunization was made with 0.5 ml of total homogenate of yeast plus 0.5 ml of Freund's complete adjuvant. 1 ml of total homogenate of mycelium and 1 ml of Freund's complete adjuvant. After 21 days, intravenous and subcutaneous booster doses were given with complete homogenate of both the yeast and of the mycelium in the same quantities, but without or adjuvant. More booster doses followed weekly until the end of February. From March to June, booster doses were given once a month. Each antigen extract was used at a concentration of 20 mg/ml in a barbital-sodium barbital buffer M 0.02 pH 8.6. Agarose gel A 37,EEO = - 0 . 1 7 , purchased from Industrie Biologique Francaise (Villeneuve-la-Garenne) was used. The gel was prepared at i % w/vol in the barbital-sodium barbital buffer and poured just before use on a Gel Bond " film 85 × 100mm (FMC, Bio Products, Rockland, Maine). 10.0ml agarose were poured at 56°C on Gel Bond ~"' film. Aliquots of 3/~1 of each antigenic preparation were applied to the Gel Bond '~ film. Each antiserum was used at a final concentration of 300/~1 according to the methods described by Lebecq et al. [14]. The final antiserum concentration was at 7.6/~1/cm 2. Veronal-sodium veronal buffer M = 0.02, pH = 8.6 was placed in the electrophoresis chamber: migration I at 50 V for 90 min: migration II at 15 V for 16 h. After, the plates were resuspended in a beaker containing 0.15 M NaCI. The gel was washed by stirring in the salt solution, then it was rinsed with distilled water, pressed, dried, and drained with Coomassie brilliant blue G 250. RESULTS The antigenic extract of C. albicans grown in the presence of tunicamycin showed a lack of high molecular weight proteins above 94 kDa, which were, however, present in the control antigenic extracts. Besides this, a lack of antigen 47 kDa was noted, together with other antigens known as: Mr 44, 52, 48.9 and 59.72 kDa. Antigen Mr 68 kDa was present. Two-dimensional immunoelectrophoresis with homologous and heterologous antisera. The Y-phase antigen extracts showed precipitation arcs of greater intensity and number in the presence of the homologous anti-Y antiserum than in those of the heterologous anti-Y antiserum (DP) (Figs 1-3 and 7--9). The germ tube and mycelial antigen extracts showed greater precipitation arcs towards the cathode both with the homologous anti-Y and anti-M sera, than with the heterologous anti-Y (DP), which was due to the base pH level of the proteins (Figs 2-5, 8 and 9); this was not observed, however, in the yeast antigen extracts (Figs 1, 6 and 7). The antigen extracts of the C. albicans cells grown in the presence of tunicamycin showed only the Y-phase, and thus precipitation arcs of fewer number and lesser intensity than in the same control antigen extracts (heterologous DP antiserum and homologous anti-Y and anti-M antisera) in the rabbits immunized by us (Figs 10-12). Besides this, the
Fig. 9. (See facing page.) Two-dimensional immunoelectrophoresis of C. albicans BP strain mycelial antigen extract (3 ~tl) against anti-C, albicans antiserum standard in yeast phase (PD) (300 #l). Fig. 10. (See facing page.) Two-dimensional immunoelectrophoresis of C. albicans cells BP strain grown in the presence of tunicamycin (10/~ 1/ml); antigen extract (3 # 1) against anti-C, albicans BP strain rabbit antiserum in yeast phase (300#1). Fig. 11. (See facing page.) Two-dimensional immunoelectrophoresis of C. albicans cells BP strain grown in the presence of tunicamycin (101d/ml); antigen extract (3 #l) against anti-C, albicans antiserum standard in yeast phase (PD) (300 #1).
Figs 9-11. (Captions on facing page.) 169
94 67 43
50 20.1 14.4
Y
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Fig. 12. Two-dimensional immunoelectrophoresis of C. albicans cells BP strain grown in the presence of tunicamycin (10 gl/ml); antigen extract (3 #1) against anti-C, albicans BP strain rabbit antiserum in mycelial phase (300/~1). Fig. 13. Polyacridamides at 17%. From left to right: antigen extract of C. albicans in yeast phase: antigen extract of C. albicans in mycelial phase; antigen extract of C. albicans in germ tube phase. Standard (Pharmacia); antigen extract of C. albicans cells grown in the presence of tunicamycin ( 10 Ftl/ml).
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precipitation arcs shifted slightly towards the anode; the preciptiation arcs towards the cathode otherwise present in the control sera were lacking. DISCUSSION Tunicamycin is a powerful antibiotic that inhibits the glycoprotein synthesis of fungi, consequently causing a disturbance in the chitine-rich microfibril network. In our study, we used a culture medium MEM favourable expression for filamentation, which was supplemented with 0.5% N-acetyl-D-glucosamine at 37°C [15]. Tunicamycin was observed to inhibit the synthesis of high Mr proteins and other protein fractions ranging from 40 to 65 kDa. Our results are consistent with those of Elorza et al. [8] also as specifically concerns the protein fraction of 30 kDa, which was present in the three antigen control extracts (yeast, germ tube and mycelial), as well as in the C. albicans cell cultures grown in the presence of tunicamycin. These results suggest that tunicamycin not only inhibits the synthesis of the glycoproteins [7-9, 11, 16] but also that of many other proteins, and in particular protein 47kDa. This protein, which is considered an immunodominant antigen, causes the antibody reaction of the host to systemic fungi; it can be found both in the associated cytoplasma of the vacuoles and in the cell walls [6, 17, 18]. The immunodominant antigens described by Strockbine et al. [19] 44 kDa, and by Greenfield et al. [20] 48.9, 59 and 71 kDa, and present in the three antigen control extracts were inhibited by tunicamycin (Fig. 13). This indicates that even in subinhibitory doses, tunicamycin interferes with the metabolism of C. albicans cells and significantly alters their protein synthesis. The antigen extract of C. albicans cells grown in the presence of tunicamycin in the two-dimensional immunoelectrophoresis tests showed both in the cases of the heterologous serum and in the homologous sera a consequently lower number of precipitation arcs, while showing variations in height and range, and migration towards the anode. This indicates that the proteins were inhibited, their charge was slightly modified, and the pH level greatly reduced (Fig. 10). The possibility of having numerous precipitation arcs also with the homologous serum in the M-phase can be explained by the fact that some proteins are common to both the yeast phase and to the M-phase of the C. albicans cells. Manning et al. [5] have reported that the proteins of the yeast and mycetial phases of C. albicans can vary from strain to strain. Besides this, they have demonstrated that the proteins of the mycelial phase appear as a modification of preexisting proteins from the yeast phase rather than as newly synthesized proteins in the yeast phase. Large differences between yeast form and mycelial form cytoplasmic proteins could not be found. The total absence of proteins specific to the mycelial phase can be explained by the fact that in the hyphal phase proteins of the yeast phase are no longer synthesized [5, 15]. This hypothesis is also supported by the work of Sundstrom et al. [21], who have reported that there are slightly modified proteins also in the formation of the germ tube. In our three antigen extracts: yeast, germ tube, and mycelial, the precipitation arcs of the anti-Y sera differed from those of the anti-M serum [20, 22-25]. In particular, the antigen germ tube and mycelial extracts showed a large precipitation arc towards the cathode both with anti-Y and anti-M sera; thus the presence of base pH positively charged proteins could be considered significant for these phases (Figs 2-5, 8 and 9).
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T h e i n h i b i t i o n o f t h e M - p h a s e is c o n s i s t e n t w i t h t h e f a c t t h a t t h e s e c h a r a c t e r i s t i c p r e c i p i t a t i o n a r c s c a n n o t b e f o u n d in t h e a n t i g e n e x t r a c t s o f t h e cells g r o w n in t h e p r e s e n c e of tunicamycin (Figs 10-12). Acknowledgements--We wish to thank Professor Bernard Bizzini, head of the Laboratoire d'Immunochimie des Protein at the lnstitut Pasteur, Paris, for his helpful suggestions in this study. This work was also partially supported by a grant from NATO CNR and CNR Altri Interventi.
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