Sabouraudia (1977). 15, 297-303
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CELL W A L L A N A L Y S I S OF A N A D E N I N E - R E Q U I R I N G M U T A N T OF T H E Y E A S T - L I K E F O R M OF PARACOCCIDIOIDES BRASILIENSIS S T R A I N I V I C Pb9
GIOCONDA SAN-BLAs, F. SAN-BLAs, ELIZABETE ORMAECHEAAND L. E. SERRANO
Centro de Microbiolog'ga y Biolog~a Celular Instituto Venezolano de Investigaciones Cientificas (IVIC) Apartado 1827, Caracas, Venezuela An adenine-requiring mutant of Paracoccidioidesbrasiliensisstrain IVIC Pb9 was isolated after treatment of the yeast-like (Y) form with nitrosoguanidine. Cell wall analysis of this mutant (strain IVIC Pbl41) showed an increase in the amount of a-l,3-glucan and a virtual disappearance of the antigenic galactomannan. At the same time, a higher degree of virulence was observed for the mutant. These results agree with the hypothesis presented before (16) about a relationship between cell wall polysaccharides and pathogenicityin P. brasiliensis.
Paracoccidioides brasitiensis, an inhperfect fungus pathogenic for humans, undergoes thermal dimorphism (9) developing a yeast-like (Y) form when grown at 37°C and a mycelial (M) form at room temperature. Kanetsuna & Carbonell (6) and Kanetsuna, Carbonell, A z u m a & Yamamura (7), indicated that glucans in the Y form of P. brasiliensis strain I V I C Pb9 were structured mainly in the a-l,3 configuration (95 ~) with fl-glucans in a very low proportion (5 ~ ) . Recent investigations on this strain (16) have shown that the degree of virulence of the Y-form of different strains of P. brasiliensis seems to be directly related to the amount of a-l,3-glucan in their cell wails. Continuous subculturing in vitro for several years of strain I V I C Pb9 reduced the amount of its e-l,3-glucan rendering the strain avirulent for different species of mice. However, when isolated from experimentally infected hamsters (strain I V I C Pb9H), the amount of e-l,3-glucan increased up to 10 times the amount in the parental strain, while being able to infect and kill mice (16). Also when the Y-like form of Pb9 was grown in vitro in the presence of fetal calf serum (17) or human serum (San-Bias, unpublished results), the amount of this glucan and its virulence resembled strain Pb9H. San-Bias, San-Blas & Cova (15), described the isolation of a morphological mutant of the Y-form of strain Pb9. This mutant ( I V I C Pbl40) replaced the fibrillar e-l,3-glucan by an amorphous 1,3-mannan and was unable to produce disease or to be recovered from experimentally infected animals. With this data, it has been suggested (16) that the presence of c~-1,3-glucan in the cell wall of the Y-form of this fungus may play a role in the pathogenicity of this microorganism. In order to obtain more information on this matter, mutants of strain Pb9 with higher content of a-1,3-glucan were tested for virulence. This paper describes cell wall analysis on a morphological and auxotrophic mutant, strain I V I C Pbl41, more virulent than the parental strain Pb9 and with a higher content of a- 1,3-glucan in its cell wall. 297
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Organism The origin of P. brasiliensis strain I V I C Pb9, and the method of obtaining exponentially growing populations of the Y-like form have been described elsewhere (14). Incubation was carried out at 37°C. Growth conditions These have been described previously (15). G G Y medium (10) consisted of: glucose, 20 g; glycine, I0 g; yeast extract (Difco), 2 g; per liter of distilled water, and was used as the standard solid medium. Soft and hard agar media were prepared by the addition of 6 or 12 g per liter of bacto agar (Difco) respectively. Minimal medium (4) consisted of; ( N H 4 ) 2 S O 4 , 5 g; K H 2 P O 4 , 1 g; M g S O 4 - 7 H 2 0 , 0"5 g; CaC12.2H20 , 0"1 g; NaC1, 0"1 g per liter of distilled water. Sterilized glucose was added to a final concentration of 5 g per liter. This medium was supplemented with methionine (250/2g/ml) (12) and thiamine (20 #g/ml) (15). Brain heart infusion broth (BHI) (BBL, Division ofBioQuest, Cockeysville, Md.) was used as the standard broth for growing the Y-form of this fungus. All media were adjusted to p H 7"4 with 1N K O H before autoclaved at 120°C for 15 min. Isolation andpurification of mutants These have been described elsewhere (13). Fifty ml of an exponentially growing population of the Y-like form of P. brasiliensis I V I C Pb9 in B H I broth containing 2 x 106 colony-forming-units per ml (c.f.u./ml), were washed and suspended in 100 ml of 0"1 M citrate buffer p H 5 containing 50 #g/ml N-methyl-N'-nitro-N'nitrosoguanidine (NTG) (Sigma). The cell suspension was incubated at 37°C for 30 min and washed several times with buffer to remove the mutagen. The cell residue was suspended in 100 ml of fresh B H I broth and further incubated in a 500 ml Erlenmeyer flask on a gyrotary shaker at 37°C for 72 hours to allow segregation of the mutant phenotypes. The cell suspension was then diluted and plated out on GGY agar plates to give between 30 and 300 colonies per plate. Single isolated colonies were picked out at r a n d o m and patched with toothpicks in a geometrical pattern onto agar plates. These were incubated until they showed confluent growth and were then replica-plated on minimal agar plates; patches which failed to grow on minimal and grew normally on complete medium were selected for further studies. Samples of the presumptive auxotroph patches were suspended in 5 ml of B H I broth in test tubes and incubated at 37°C, on a reciprocating shaker. Samples from the cultures were diluted in saline, and plated out on G G Y agar plates to give single well separated colonies. These were replica-plated on minimal agar to confirm the auxotrophic phenotype. Characterization of the nutritional requirements were determined by growing single colonies of the mutants on minimal agar plates supplemented with single or different combinations of amino acids, vitamins and bases. Animal experimentation This has been described elsewhere (16). Adult outbred male hamsters weighing an average of 90 g and male white mice (strains N M R Y / I V I C and BALB/c), weighing 20 to 25 g, were inoculated intraperitoneally with 1 ml of a Y-like suspension of the fungal strain containing 2 x 106 c.f.u. A total of 12 animals of each species was used in testing each culture. T h e y were sacrificed every month up to 6 months, and examined for paracoccidioidomycotic nodules on the mesentery, peritoneal surface and viscera. Portions, or whole viscera were minced, inoculated on mycosel
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agar (BBL, Division of BioQuest, Cockeysville, Md., USA) slants, and incubated at 37°C. Y-like colonies that developed on mycosel were subcultured and kept on Sabouraud agar.
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Preparation andfractionation of cell walls They were carried out as before (15). By such method, three cell wall fractions were obtained: an alkali-insoluble fraction 1, and alkali-soluble acid precipitable fraction 2 and an alkali-soluble non-precipitable fraction 3. Analytical procedures Lipid, sugar, amino acid and amino sugar determinations, periodate oxidations of polysaccharides, and infrared spectra were carried out as before (15). Amino acids were automatically analyzed after hydrolysis of the samples (15) by means of a Beckman model Multichrom B amino acid analyzer. Absolute values for each amino acid were calculated by comparison with a standard mixture (Beckman Instruments, Co.) of the 17 most common amino acids. RESULTS
P. brasiliensis strain I V I C Pbl41 was one of eight adenine-requiring mutants isolated by the treatment described (15). T h e colony morphology of this mutant was similar to that of the parental strain I V I C Pb9 when grown on complex agar media at 37°C. However, in contrast witt~ the parental strain, the mutant shows a dark brown colour, shared by some other adenine-requiring strains obtained by the same procedure. No detectable differences from the parental strain I V I C Pb9 were shown by the Y-like cells of the mutant when observed in the light microscope or in the morphology of the colonies when transformed to the M form. Autopsy performed on all animals, sacrificed in the first and second months, revealed paracoccidioidomycotic nodules of various sizes scattered through the mesentery. Nodules from mice inoculated with Pb9, treated with vital stain (14), revealed only dead cells, most of them without protoplasm. No viable cells were recovered from these nodules. These paracoccidioidomycotic nodules were not present in the rest of the mice sacrificed afterward. Also no other sign of disease was found in mice and none of them died during the experiment. O n the contrary, mice inoculated with strain Pbl41 were found to contain evidences of infection in the viscera and diaphragm. Viable cells were recovered from minced viscera and mesenteric nodules since the first month of infection. Autopsy performed in mice which died during the experiment revealed gross evidence of the disease. Hamsters which have been demonstrated to be sensitive to strain I V I C Pb9 (16) were also sensitive to strain I V I C Pbl41. Table 1 summarizes the differences in chemical composition of P. brasiliensis I V I C Pbl41 cell wall and its fractions compared to the parental strain I V I C Pb9 and the mutant I V I C Pbl40, both reported before (15). The composition of fraction 1 was very m u c h the same as the parental strain I V I C Pb9 analyzed before (15). The hydrolysate contained a high amount of amino sugars (50 ~ of the total fraction) which when analyzed by thin-layer chromatography produced a spot of glucosamine. Infrared absorption of this fraction produced a pattern identical to that reported before for chitin (15). Automatic analysis of neutral sugars showed the presence of only glucose. Although no detailed analyses were done
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on this glucan, the presence of an absorption b a n d at 890 c m - I in its infrared spectrum suggested a/3-configuration for this glucan. A m i n o acids were also present in higher amounts t h a n the parental strain. I t was not determined, though, whether they were present as isolated proteins or b o u n d u n d e r the form ofglycoproteins. Fraction 2 increased its a m o u n t from 3.2 °/o ( I V I C Pb9) to 24.0 ~ ( I V I C Pbl41). I n both cases, hexoses built up virtually the whole of the fraction, glucose being the only sugar component. Periodate oxidation gave no reaction, suggesting the possible existence of 1,3-1inkages. Infrared analysis p r o d u c e d a spectrum identical to those reported before for a-l,3-glucans (15). I n this way, fraction 2 of P. brasiliensis I V I C Pbl41 was identified as an a-l,3-glucan virtually identical to that found in the parental strain.
TABLE I.--CHEMICAL COMPOSITION OF STRAINS OF P. brasiliensis
WALLS
FROM VARIOUS
(Y-FoRM)
IVIC Pb9
IVIC Pbl41
41 "5 16.9 60"5 16"2
44-0 12'0 53"0 27-0
3'2 I00.0 0.0 0'0
24.0 90-0 0.8 5.0
55'0
32-0
20-4 0'0 19.2 60-0
3' 7 1-0 0.7 80'0
Fraction 1
Yield* Hexoses (glucose) Amino sugars Amino acids Fraction 2
Yield* Hexoses (gIucose) Amino sugars Amino acids Fraction 3
Yield* Hexoses (mannose and galactose) Amino sugars Amino acids Lipids
*The amount of each class of compound is expressed as a percentage of the fraction. The yield represents percentage of the weight of the wall.
T h e alkali-soluble, non-precipitable fraction 3 of the m u t a n t P b l 4 1 contained mostly lipids, contrary to the parental strain which showed some sugars as galactom a n n a n and proteins (20 ~ each) (16). A m i n o acid analysis of whole cell walls (Table 2) indicated that no significant differences were observed a m o n g the amounts of the various amino acid components of the proteins. Slight variations were observed in the amounts of lysine, arginine, alanine, methionine, isoleucine, leucine and tyrosine present in the parental and the m u t a n t strains. T h e table also includes values reported before for P . brasiliensis I V I C Pb9 (8).
AN ADENINE-REQUIRING MUTANT OF P. BRASILIENSIS TABLE 2.--AMINO ACID ANALYSES OF WHOLE CELL WALLS OF
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P.
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brasiliensis (REsIDUES/MGCELLWALL)
Lysine Itistidine Arginine Asparticacid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleueine Leucine Tyrosine t'henylalanine
Strain IVIC Pb9*
Strain IVIC Pb9t
Strain IVIC Pbl41t
4.38 5.08 1.06 4.16 3.78 3"58 4"60 3.00 5.51 3-92 1-64 0.97 1.25 2"22 -1.64
4.82 2"43 3.09 5-68 3.96 4.22 6.64 3-78 5.54 5.53 3.73 0'91 3-05 5'54 0'43 2-41
5-61 2' 13 2-32 5"27 4-06 4.21 6-39 4.84 5.56 4-89 3.24 0-43 2"61 6"29 0-72 2.27
*As reported by Kanetsuna et al. (1969). "~Asfound during the course of this work.
DISCUSSION Experiments on several pathogenic fungi have suggested that a variety of factors seem to be related to pathogenicity. Di Salvo and Denton (3) studied the possible relationship between lipid content and virulence in several strains of Blastomyces dermatitidis and found that the amount of lipids increased from 6"8 ~ in the Y-phase of non-virulent strains to 12"3 ~ in virulent strains. The authors, however, did not explain the fact that the saprophytic M phases contained more than twice the lipids than the corresponding Y-phases. Instead, Nielsen (11) working with six strains of Histoplasma capsulatum could not correlate, lipids to virulence. Cox and Best (2) observed a higher amount of a-glucan in the cell walls of several strains of B. dermatitidis extracted from patients when compared to strains isolated from soil; however, they emphasized the high amount of phosphorous, probably as phospholipids, found in the more virulent strains as responsible for pathogenicity. In Cryptococcus neoformans, the capsule has been postulated as the structure responsible for pathogenicity in this fungus (5). In the mutant strain ofP. brasiliensis described in this study, slight differences were observed on the amino-acid composition of the proteins from the cell walls of both the parental and mutant strains of P. brasiliensis. Also some differences in the amount oflipids in each case were observed. Nothing, though, could be concluded about the significance of these variations since they could not be related to degrees of virulence in other strains of P. brasiliensis (15, 16). Instead, more importance was attached to the differences observed in the cell wall glucans. Previous reports (6, 7) indicated that glucans in the Y-like form of P. brasiliensis were structured mainly in the e-l,3configuration. It was also reported (16) that during continuous subculturing in vitro of this strain, its pathogenicity decreased and at the same time its cell wall corn-
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position varied r e m a r k a b l y , m a i n l y with regards to the a m o u n t s of e - l , 3 - g l u c a n a n d g a l a c t o m a n n a n . I t was suggested t h e n that the g l u c a n was only r e q u i r e d by the fungus w h e n the pathogenic Y-like form infected the host, b e i n g reduced w h e n continuously s u b c u l t u r e d in vitro. T h e corresponding increase in g a l a c t o m a n n a n with the decrease of the e - l , 3 - g l u c a n could also play a role in the virulence of a p a r t i c u l a r strain. A z u m a , K a n e t s u n a , T a n a k a , Y a m a m u r a & Carbonell, (1) showed that the g a l a c t o m a n n a n s usually present in fraction 3 of various d i m o r p h i c fungi, i n c l u d i n g P. brasiliensis, played a n i m p o r t a n t role as a n t i g e n i c d e t e r m i n a n t s , the cross-reactions found a m o n g p r e p a r a t i o n s from these fungi b e i n g actually due to the fact that all of these g a l a c t o m a n n a n s h a d c o m m o n structures. T h e cell wall analysis of the Y-like form of the m u t a n t strain I V I C P b l 4 1 revealed a n increase of the a - l , 3 - g l u c a n (fraction 2) while the g a l a c t o m a n n a n (fraction 3) virtually disappeared w h e n c o m p a r e d with the p a r e n t a l strain. This altered cell wall composition m a d e the m u t a n t a likely c a n d i d a t e for higher v i r u l e n c e i n view of the hypothesis stated above (16) where strains of P. brasiliensis with high a m o u n t s of ~ - l , 3 - g l u e a n a n d low quantities of the i m m u n o g e n i c fraction ( g a l a c t o m a n n a n ) must be highly virulent. T h e results o b t a i n e d i n the e x p e r i m e n t a l infection of l a b o r a t o r y animals support this hypothesis, suggesting some relationship b e t w e e n cell wall polysaccharides a n d pathogenicity i n P. brasiliensis.
R.ESUMEN Se aisl6 un mutante de Paraccccidioides brasiliensis cepa IVIC Pb9 auxotrofo para adenina luego de tratar la fase levaduriforme del hongo con nitrosoguanidina. Los an~tlisisde pared celular del mutante (cepa IVIC Pbl41) mostraron un incremento en la cantidad de ~-l,3-gluc~n y una casi total desaparicidn de la fraccidn antig~nica de galactoman~n. A1 mismo tiempo, se observ6 una mayor virulencia en el mutante. Estos resultados estfin de acuerdo con la hipdtesis planteada anteriormente (San-Bias et al., 1977) acerca de una relaci6n entre los polisaefiridos de la pared celular y la patogenicidad en P. brasiliensis. REFERENCES 1. AZUMA,I., KANETSUNA,F., TANAKA,Y., YAMAMURA,Y. & CARBONELL,L. M. (1974). Chemical and immunological properties of galactomannans obtained from Histoplasma duboisii, Histoplasma capsulatum, Paracoccidioides brasiliensis and Blastomyces dermatitidis. Mycopathologia & Mycologia Applicata, 54, 111-125. 2. Cox, R. A. & B~ST, G. K. (1972). Cell wall composition of two strains of Blastomyces dermatitidis exhibiting differences in virulence of mice. Infection and Immunity, 5, 449M~53. 3. DI SALVO, A. & D~NTON,J. F. (1963). Lipid content of four strains of Blastom~ces dermatitidis of different mouse virulence. Journal of Bacteriology, 85, 927-931. 4. GILA~Db G. L. & LAPPER, N. C. (1962). Nutritional studies on the yeast phase of Blastomvces dermatitidis. Journal of Bacteriology, 83, 219-227. 5. I s ~ , C. M., BULM~R,G. S. & FELTON,F. G. (1968). An evaluation of various environmental factors affecting the propagation of Cryptococeus neoformans. Mycopathologia et Mycologia Applicata, 35, 81-90. 6. K.~ETSONA,F. & CARBONELL,L. M. (1970). Cell wall glueans of the yeast and mycelial forms of Paracoccidioides brasitiensis. Journal of Bacteriology, 101, 675-680. 7. KANETSUNA,F., CARBONELL,L. M., AZUMA,I. & YAMAMORA,Y. (1972). Biochemical studies on the thermal dimnrphism of Paracoccidioides brasiliemis. Journal of Bacteriology, 110, 208-218. 8. KANETSUNA,F., CARBON~LL,L. M., MORENO,R. E. & RODRIGOEZ,J. (1969). Cell wall composition of the yeast and mycelial forms of Paracoccidioides bra~iliensis. Journal of Bacteriology, 97, 1036-1041. 9. NICV,~RSON,W.J. (1948). Enzymatic control of cell division in microorganisms. Nature, London, 162, 241-245. 10. NICKE~SON,W.J. & EDWARDS,G. A. (1949). Studies on the physiological basis of morphogenesis in fungi. I. The respiratory metabolism of dimorphic pathogenic fungi. Journal of General Physiology, 33, 41-55.
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11. NIELSEN, H. S.JR. (1966). Variations in lipid content of strains ofHistoplasma capsulatum exhibiting different virulence properties for mice. Journal qfBacteriology, 91 273-277. 12. RAMIREz-MARTiNEZ,J. R. & RODRfGUEZ,J. (1972). Nutritional studies on a methionine-requlring strain of Paracoecidioides brasiliensis. Mycopathologia & Mycologia Applicata, ,t6, 341-350. 13. SAN-BLAs, F. & CENTRNO, SONrA (1977). Isolation and preliminary characterization of auxotrophic and morphological mutants of the yeast-like form of Paracoccidioidesbrasiliensis. Journal of Bacteriology, 129, 138-144. 14. SAr~-BLAS, F. & COVA, L . J . (1975). Growth curves of the yeast-like form of Paracoeeidioides brasiliensis. Sabouraudia, 13, 22-29. 15. SAN-BLAs, F., SAN-BLaS, GIOCONDA & COVA, L. J. (1976). A morphological mutant of Paracoccidioides brasiliensis strain IVIC Pb9. Isolation and wall characterization. Journal of General Microbiology, 93, 209-218. 16. SAN-BLAS, GIOCONDA, SAN-BLA$, F. • SERRANO, L. E. (1977). Host-parasite relationships in the yeast-like form of Paracoccidioides brasiliensis strain IVIC Pb9. Infection and Immunity, 15, 343-346. 17. SAN-BLAs, GIOCONDA & VERNET, DOLOP.~S (1977). Induction of the synthesis of cell wall a-l,3-glucan in the yeast-like form of Paracoccidioides brasiliensis strain IVIC Pb9 by fetal calf serum. Infection and Immunity, 15, 897-902.
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CELL W A L L A N A L Y S I S OF A N A D E N I N E - R E Q U I R I N G M U T A N T OF T H E Y E A S T - L I K E F O R M OF PARACOCCIDIOIDES BRASILIENSIS S T R A I N I V I C Pb9
GIOCONDA SAN-BLAs, F. SAN-BLAs, ELIZABETE ORMAECHEAAND L. E. SERRANO
Centro de Microbiolog'ga y Biolog~a Celular Instituto Venezolano de Investigaciones Cientificas (IVIC) Apartado 1827, Caracas, Venezuela An adenine-requiring mutant of Paracoccidioidesbrasiliensisstrain IVIC Pb9 was isolated after treatment of the yeast-like (Y) form with nitrosoguanidine. Cell wall analysis of this mutant (strain IVIC Pbl41) showed an increase in the amount of a-l,3-glucan and a virtual disappearance of the antigenic galactomannan. At the same time, a higher degree of virulence was observed for the mutant. These results agree with the hypothesis presented before (16) about a relationship between cell wall polysaccharides and pathogenicityin P. brasiliensis.
Paracoccidioides brasitiensis, an inhperfect fungus pathogenic for humans, undergoes thermal dimorphism (9) developing a yeast-like (Y) form when grown at 37°C and a mycelial (M) form at room temperature. Kanetsuna & Carbonell (6) and Kanetsuna, Carbonell, A z u m a & Yamamura (7), indicated that glucans in the Y form of P. brasiliensis strain I V I C Pb9 were structured mainly in the a-l,3 configuration (95 ~) with fl-glucans in a very low proportion (5 ~ ) . Recent investigations on this strain (16) have shown that the degree of virulence of the Y-form of different strains of P. brasiliensis seems to be directly related to the amount of a-l,3-glucan in their cell wails. Continuous subculturing in vitro for several years of strain I V I C Pb9 reduced the amount of its e-l,3-glucan rendering the strain avirulent for different species of mice. However, when isolated from experimentally infected hamsters (strain I V I C Pb9H), the amount of e-l,3-glucan increased up to 10 times the amount in the parental strain, while being able to infect and kill mice (16). Also when the Y-like form of Pb9 was grown in vitro in the presence of fetal calf serum (17) or human serum (San-Bias, unpublished results), the amount of this glucan and its virulence resembled strain Pb9H. San-Bias, San-Blas & Cova (15), described the isolation of a morphological mutant of the Y-form of strain Pb9. This mutant ( I V I C Pbl40) replaced the fibrillar e-l,3-glucan by an amorphous 1,3-mannan and was unable to produce disease or to be recovered from experimentally infected animals. With this data, it has been suggested (16) that the presence of c~-1,3-glucan in the cell wall of the Y-form of this fungus may play a role in the pathogenicity of this microorganism. In order to obtain more information on this matter, mutants of strain Pb9 with higher content of a-1,3-glucan were tested for virulence. This paper describes cell wall analysis on a morphological and auxotrophic mutant, strain I V I C Pbl41, more virulent than the parental strain Pb9 and with a higher content of a- 1,3-glucan in its cell wall. 297
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Organism The origin of P. brasiliensis strain I V I C Pb9, and the method of obtaining exponentially growing populations of the Y-like form have been described elsewhere (14). Incubation was carried out at 37°C. Growth conditions These have been described previously (15). G G Y medium (10) consisted of: glucose, 20 g; glycine, I0 g; yeast extract (Difco), 2 g; per liter of distilled water, and was used as the standard solid medium. Soft and hard agar media were prepared by the addition of 6 or 12 g per liter of bacto agar (Difco) respectively. Minimal medium (4) consisted of; ( N H 4 ) 2 S O 4 , 5 g; K H 2 P O 4 , 1 g; M g S O 4 - 7 H 2 0 , 0"5 g; CaC12.2H20 , 0"1 g; NaC1, 0"1 g per liter of distilled water. Sterilized glucose was added to a final concentration of 5 g per liter. This medium was supplemented with methionine (250/2g/ml) (12) and thiamine (20 #g/ml) (15). Brain heart infusion broth (BHI) (BBL, Division ofBioQuest, Cockeysville, Md.) was used as the standard broth for growing the Y-form of this fungus. All media were adjusted to p H 7"4 with 1N K O H before autoclaved at 120°C for 15 min. Isolation andpurification of mutants These have been described elsewhere (13). Fifty ml of an exponentially growing population of the Y-like form of P. brasiliensis I V I C Pb9 in B H I broth containing 2 x 106 colony-forming-units per ml (c.f.u./ml), were washed and suspended in 100 ml of 0"1 M citrate buffer p H 5 containing 50 #g/ml N-methyl-N'-nitro-N'nitrosoguanidine (NTG) (Sigma). The cell suspension was incubated at 37°C for 30 min and washed several times with buffer to remove the mutagen. The cell residue was suspended in 100 ml of fresh B H I broth and further incubated in a 500 ml Erlenmeyer flask on a gyrotary shaker at 37°C for 72 hours to allow segregation of the mutant phenotypes. The cell suspension was then diluted and plated out on GGY agar plates to give between 30 and 300 colonies per plate. Single isolated colonies were picked out at r a n d o m and patched with toothpicks in a geometrical pattern onto agar plates. These were incubated until they showed confluent growth and were then replica-plated on minimal agar plates; patches which failed to grow on minimal and grew normally on complete medium were selected for further studies. Samples of the presumptive auxotroph patches were suspended in 5 ml of B H I broth in test tubes and incubated at 37°C, on a reciprocating shaker. Samples from the cultures were diluted in saline, and plated out on G G Y agar plates to give single well separated colonies. These were replica-plated on minimal agar to confirm the auxotrophic phenotype. Characterization of the nutritional requirements were determined by growing single colonies of the mutants on minimal agar plates supplemented with single or different combinations of amino acids, vitamins and bases. Animal experimentation This has been described elsewhere (16). Adult outbred male hamsters weighing an average of 90 g and male white mice (strains N M R Y / I V I C and BALB/c), weighing 20 to 25 g, were inoculated intraperitoneally with 1 ml of a Y-like suspension of the fungal strain containing 2 x 106 c.f.u. A total of 12 animals of each species was used in testing each culture. T h e y were sacrificed every month up to 6 months, and examined for paracoccidioidomycotic nodules on the mesentery, peritoneal surface and viscera. Portions, or whole viscera were minced, inoculated on mycosel
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agar (BBL, Division of BioQuest, Cockeysville, Md., USA) slants, and incubated at 37°C. Y-like colonies that developed on mycosel were subcultured and kept on Sabouraud agar.
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Preparation andfractionation of cell walls They were carried out as before (15). By such method, three cell wall fractions were obtained: an alkali-insoluble fraction 1, and alkali-soluble acid precipitable fraction 2 and an alkali-soluble non-precipitable fraction 3. Analytical procedures Lipid, sugar, amino acid and amino sugar determinations, periodate oxidations of polysaccharides, and infrared spectra were carried out as before (15). Amino acids were automatically analyzed after hydrolysis of the samples (15) by means of a Beckman model Multichrom B amino acid analyzer. Absolute values for each amino acid were calculated by comparison with a standard mixture (Beckman Instruments, Co.) of the 17 most common amino acids. RESULTS
P. brasiliensis strain I V I C Pbl41 was one of eight adenine-requiring mutants isolated by the treatment described (15). T h e colony morphology of this mutant was similar to that of the parental strain I V I C Pb9 when grown on complex agar media at 37°C. However, in contrast witt~ the parental strain, the mutant shows a dark brown colour, shared by some other adenine-requiring strains obtained by the same procedure. No detectable differences from the parental strain I V I C Pb9 were shown by the Y-like cells of the mutant when observed in the light microscope or in the morphology of the colonies when transformed to the M form. Autopsy performed on all animals, sacrificed in the first and second months, revealed paracoccidioidomycotic nodules of various sizes scattered through the mesentery. Nodules from mice inoculated with Pb9, treated with vital stain (14), revealed only dead cells, most of them without protoplasm. No viable cells were recovered from these nodules. These paracoccidioidomycotic nodules were not present in the rest of the mice sacrificed afterward. Also no other sign of disease was found in mice and none of them died during the experiment. O n the contrary, mice inoculated with strain Pbl41 were found to contain evidences of infection in the viscera and diaphragm. Viable cells were recovered from minced viscera and mesenteric nodules since the first month of infection. Autopsy performed in mice which died during the experiment revealed gross evidence of the disease. Hamsters which have been demonstrated to be sensitive to strain I V I C Pb9 (16) were also sensitive to strain I V I C Pbl41. Table 1 summarizes the differences in chemical composition of P. brasiliensis I V I C Pbl41 cell wall and its fractions compared to the parental strain I V I C Pb9 and the mutant I V I C Pbl40, both reported before (15). The composition of fraction 1 was very m u c h the same as the parental strain I V I C Pb9 analyzed before (15). The hydrolysate contained a high amount of amino sugars (50 ~ of the total fraction) which when analyzed by thin-layer chromatography produced a spot of glucosamine. Infrared absorption of this fraction produced a pattern identical to that reported before for chitin (15). Automatic analysis of neutral sugars showed the presence of only glucose. Although no detailed analyses were done
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on this glucan, the presence of an absorption b a n d at 890 c m - I in its infrared spectrum suggested a/3-configuration for this glucan. A m i n o acids were also present in higher amounts t h a n the parental strain. I t was not determined, though, whether they were present as isolated proteins or b o u n d u n d e r the form ofglycoproteins. Fraction 2 increased its a m o u n t from 3.2 °/o ( I V I C Pb9) to 24.0 ~ ( I V I C Pbl41). I n both cases, hexoses built up virtually the whole of the fraction, glucose being the only sugar component. Periodate oxidation gave no reaction, suggesting the possible existence of 1,3-1inkages. Infrared analysis p r o d u c e d a spectrum identical to those reported before for a-l,3-glucans (15). I n this way, fraction 2 of P. brasiliensis I V I C Pbl41 was identified as an a-l,3-glucan virtually identical to that found in the parental strain.
TABLE I.--CHEMICAL COMPOSITION OF STRAINS OF P. brasiliensis
WALLS
FROM VARIOUS
(Y-FoRM)
IVIC Pb9
IVIC Pbl41
41 "5 16.9 60"5 16"2
44-0 12'0 53"0 27-0
3'2 I00.0 0.0 0'0
24.0 90-0 0.8 5.0
55'0
32-0
20-4 0'0 19.2 60-0
3' 7 1-0 0.7 80'0
Fraction 1
Yield* Hexoses (glucose) Amino sugars Amino acids Fraction 2
Yield* Hexoses (gIucose) Amino sugars Amino acids Fraction 3
Yield* Hexoses (mannose and galactose) Amino sugars Amino acids Lipids
*The amount of each class of compound is expressed as a percentage of the fraction. The yield represents percentage of the weight of the wall.
T h e alkali-soluble, non-precipitable fraction 3 of the m u t a n t P b l 4 1 contained mostly lipids, contrary to the parental strain which showed some sugars as galactom a n n a n and proteins (20 ~ each) (16). A m i n o acid analysis of whole cell walls (Table 2) indicated that no significant differences were observed a m o n g the amounts of the various amino acid components of the proteins. Slight variations were observed in the amounts of lysine, arginine, alanine, methionine, isoleucine, leucine and tyrosine present in the parental and the m u t a n t strains. T h e table also includes values reported before for P . brasiliensis I V I C Pb9 (8).
AN ADENINE-REQUIRING MUTANT OF P. BRASILIENSIS TABLE 2.--AMINO ACID ANALYSES OF WHOLE CELL WALLS OF
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brasiliensis (REsIDUES/MGCELLWALL)
Lysine Itistidine Arginine Asparticacid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleueine Leucine Tyrosine t'henylalanine
Strain IVIC Pb9*
Strain IVIC Pb9t
Strain IVIC Pbl41t
4.38 5.08 1.06 4.16 3.78 3"58 4"60 3.00 5.51 3-92 1-64 0.97 1.25 2"22 -1.64
4.82 2"43 3.09 5-68 3.96 4.22 6.64 3-78 5.54 5.53 3.73 0'91 3-05 5'54 0'43 2-41
5-61 2' 13 2-32 5"27 4-06 4.21 6-39 4.84 5.56 4-89 3.24 0-43 2"61 6"29 0-72 2.27
*As reported by Kanetsuna et al. (1969). "~Asfound during the course of this work.
DISCUSSION Experiments on several pathogenic fungi have suggested that a variety of factors seem to be related to pathogenicity. Di Salvo and Denton (3) studied the possible relationship between lipid content and virulence in several strains of Blastomyces dermatitidis and found that the amount of lipids increased from 6"8 ~ in the Y-phase of non-virulent strains to 12"3 ~ in virulent strains. The authors, however, did not explain the fact that the saprophytic M phases contained more than twice the lipids than the corresponding Y-phases. Instead, Nielsen (11) working with six strains of Histoplasma capsulatum could not correlate, lipids to virulence. Cox and Best (2) observed a higher amount of a-glucan in the cell walls of several strains of B. dermatitidis extracted from patients when compared to strains isolated from soil; however, they emphasized the high amount of phosphorous, probably as phospholipids, found in the more virulent strains as responsible for pathogenicity. In Cryptococcus neoformans, the capsule has been postulated as the structure responsible for pathogenicity in this fungus (5). In the mutant strain ofP. brasiliensis described in this study, slight differences were observed on the amino-acid composition of the proteins from the cell walls of both the parental and mutant strains of P. brasiliensis. Also some differences in the amount oflipids in each case were observed. Nothing, though, could be concluded about the significance of these variations since they could not be related to degrees of virulence in other strains of P. brasiliensis (15, 16). Instead, more importance was attached to the differences observed in the cell wall glucans. Previous reports (6, 7) indicated that glucans in the Y-like form of P. brasiliensis were structured mainly in the e-l,3configuration. It was also reported (16) that during continuous subculturing in vitro of this strain, its pathogenicity decreased and at the same time its cell wall corn-
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position varied r e m a r k a b l y , m a i n l y with regards to the a m o u n t s of e - l , 3 - g l u c a n a n d g a l a c t o m a n n a n . I t was suggested t h e n that the g l u c a n was only r e q u i r e d by the fungus w h e n the pathogenic Y-like form infected the host, b e i n g reduced w h e n continuously s u b c u l t u r e d in vitro. T h e corresponding increase in g a l a c t o m a n n a n with the decrease of the e - l , 3 - g l u c a n could also play a role in the virulence of a p a r t i c u l a r strain. A z u m a , K a n e t s u n a , T a n a k a , Y a m a m u r a & Carbonell, (1) showed that the g a l a c t o m a n n a n s usually present in fraction 3 of various d i m o r p h i c fungi, i n c l u d i n g P. brasiliensis, played a n i m p o r t a n t role as a n t i g e n i c d e t e r m i n a n t s , the cross-reactions found a m o n g p r e p a r a t i o n s from these fungi b e i n g actually due to the fact that all of these g a l a c t o m a n n a n s h a d c o m m o n structures. T h e cell wall analysis of the Y-like form of the m u t a n t strain I V I C P b l 4 1 revealed a n increase of the a - l , 3 - g l u c a n (fraction 2) while the g a l a c t o m a n n a n (fraction 3) virtually disappeared w h e n c o m p a r e d with the p a r e n t a l strain. This altered cell wall composition m a d e the m u t a n t a likely c a n d i d a t e for higher v i r u l e n c e i n view of the hypothesis stated above (16) where strains of P. brasiliensis with high a m o u n t s of ~ - l , 3 - g l u e a n a n d low quantities of the i m m u n o g e n i c fraction ( g a l a c t o m a n n a n ) must be highly virulent. T h e results o b t a i n e d i n the e x p e r i m e n t a l infection of l a b o r a t o r y animals support this hypothesis, suggesting some relationship b e t w e e n cell wall polysaccharides a n d pathogenicity i n P. brasiliensis.
R.ESUMEN Se aisl6 un mutante de Paraccccidioides brasiliensis cepa IVIC Pb9 auxotrofo para adenina luego de tratar la fase levaduriforme del hongo con nitrosoguanidina. Los an~tlisisde pared celular del mutante (cepa IVIC Pbl41) mostraron un incremento en la cantidad de ~-l,3-gluc~n y una casi total desaparicidn de la fraccidn antig~nica de galactoman~n. A1 mismo tiempo, se observ6 una mayor virulencia en el mutante. Estos resultados estfin de acuerdo con la hipdtesis planteada anteriormente (San-Bias et al., 1977) acerca de una relaci6n entre los polisaefiridos de la pared celular y la patogenicidad en P. brasiliensis. REFERENCES 1. AZUMA,I., KANETSUNA,F., TANAKA,Y., YAMAMURA,Y. & CARBONELL,L. M. (1974). Chemical and immunological properties of galactomannans obtained from Histoplasma duboisii, Histoplasma capsulatum, Paracoccidioides brasiliensis and Blastomyces dermatitidis. Mycopathologia & Mycologia Applicata, 54, 111-125. 2. Cox, R. A. & B~ST, G. K. (1972). Cell wall composition of two strains of Blastomyces dermatitidis exhibiting differences in virulence of mice. Infection and Immunity, 5, 449M~53. 3. DI SALVO, A. & D~NTON,J. F. (1963). Lipid content of four strains of Blastom~ces dermatitidis of different mouse virulence. Journal of Bacteriology, 85, 927-931. 4. GILA~Db G. L. & LAPPER, N. C. (1962). Nutritional studies on the yeast phase of Blastomvces dermatitidis. Journal of Bacteriology, 83, 219-227. 5. I s ~ , C. M., BULM~R,G. S. & FELTON,F. G. (1968). An evaluation of various environmental factors affecting the propagation of Cryptococeus neoformans. Mycopathologia et Mycologia Applicata, 35, 81-90. 6. K.~ETSONA,F. & CARBONELL,L. M. (1970). Cell wall glueans of the yeast and mycelial forms of Paracoccidioides brasitiensis. Journal of Bacteriology, 101, 675-680. 7. KANETSUNA,F., CARBONELL,L. M., AZUMA,I. & YAMAMORA,Y. (1972). Biochemical studies on the thermal dimnrphism of Paracoccidioides brasiliemis. Journal of Bacteriology, 110, 208-218. 8. KANETSUNA,F., CARBON~LL,L. M., MORENO,R. E. & RODRIGOEZ,J. (1969). Cell wall composition of the yeast and mycelial forms of Paracoccidioides bra~iliensis. Journal of Bacteriology, 97, 1036-1041. 9. NICV,~RSON,W.J. (1948). Enzymatic control of cell division in microorganisms. Nature, London, 162, 241-245. 10. NICKE~SON,W.J. & EDWARDS,G. A. (1949). Studies on the physiological basis of morphogenesis in fungi. I. The respiratory metabolism of dimorphic pathogenic fungi. Journal of General Physiology, 33, 41-55.
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11. NIELSEN, H. S.JR. (1966). Variations in lipid content of strains ofHistoplasma capsulatum exhibiting different virulence properties for mice. Journal qfBacteriology, 91 273-277. 12. RAMIREz-MARTiNEZ,J. R. & RODRfGUEZ,J. (1972). Nutritional studies on a methionine-requlring strain of Paracoecidioides brasiliensis. Mycopathologia & Mycologia Applicata, ,t6, 341-350. 13. SAN-BLAs, F. & CENTRNO, SONrA (1977). Isolation and preliminary characterization of auxotrophic and morphological mutants of the yeast-like form of Paracoccidioidesbrasiliensis. Journal of Bacteriology, 129, 138-144. 14. SAr~-BLAS, F. & COVA, L . J . (1975). Growth curves of the yeast-like form of Paracoeeidioides brasiliensis. Sabouraudia, 13, 22-29. 15. SAN-BLAs, F., SAN-BLaS, GIOCONDA & COVA, L. J. (1976). A morphological mutant of Paracoccidioides brasiliensis strain IVIC Pb9. Isolation and wall characterization. Journal of General Microbiology, 93, 209-218. 16. SAN-BLAS, GIOCONDA, SAN-BLA$, F. • SERRANO, L. E. (1977). Host-parasite relationships in the yeast-like form of Paracoccidioides brasiliensis strain IVIC Pb9. Infection and Immunity, 15, 343-346. 17. SAN-BLAs, GIOCONDA & VERNET, DOLOP.~S (1977). Induction of the synthesis of cell wall a-l,3-glucan in the yeast-like form of Paracoccidioides brasiliensis strain IVIC Pb9 by fetal calf serum. Infection and Immunity, 15, 897-902.
