INFECTION AND IMMUNITY, Mar. 1977, p. 897-902Copyright © 1977 American Society for Microbiology

Vol. 15, No. 3Printed in U.S.A.

Induction of the Synthesis of Cell Wall a-1,3-Glucan in theYeastlike Form of Paracoccidioides brasiliensis Strain IVIC

Pb9 by Fetal Calf SerumGIOCONDA SAN-BLAS* AND DOLORES VERNET

Centro de Microbiologia y Biologia Celular, Instituto Venezolano de Investigaciones Cientificas, Apartado1827, Caracas, Venezuela

Received for publication 19 October 1976

In vitro subculturing of the yeastlike form of Paracoccidioides brasiliensisstrain IVIC Pb9 leads to the disappearance of a-1,3-glucan as a main componentof its cell wall. However, the addition of fetal calf serum to the growth mediuminduces the synthesis ofthis polysaccharide. It is suggested that the synthesis ofa-1,3-glucan in the cell wall of the yeastlike form ofP. brasiliensis is induced byexternal factors.

Experiments in our laboratory (17) suggestthat the synthesis of a-1,3-glucan in the cellwall of the yeast (Y)-like form of Paracoccidi-oides brasiliensis is halted when the strain issubcultured in vitro for many years and that itcan be induced by inoculating the fungus intoanimals. At the same time, these experimentssuggest that the virulence of the strain is di-rectly correlated to the presence of a-1,3-glucanin its cell wall.

Since fetal calf serum (FCS) has been re-ported to have biological activities of variouskinds on mammalian and avian cells in culture(18) and on Trypanosoma cruzi (14), similarexperiments with P. brasiliensis were de-signed. In this way, we hoped to observe aninduction of a-1,3-glucan synthesis in vitrocomparable to that seen in vivo. This papersummarizes the results of these experiments.(A portion of this work was submitted by D.Vernet in partial fulfillment of the require-ments for the Licenciado en Biologia degree atthe Universidad Central de Venezuela.)

MATERIALS AND METHODSOrganism and growth conditions. P. brasiliensis

strain IVIC Pb9 was used for this study. The Y-likeform was cultured at 370C on a gyratory shaker for 6days in 500-ml Erlenmeyer flasks containing 100 mlof brain heart infusion agar (BBL, Division of Bio-Quest, Cockeysville, Md.) to which increasingamounts of FCS (Grand Island Biological Co.,Grand Island, N.Y.) (0, 2, 5, 10, and 15% [vol/vol])were added. When FCS fractions (obtained as de-scribed below) were added, amounts equivalent to aconcentration of 5% (vol/vol) FCS were used.

Preparation, fractionation, and analysis of cellwalls. These were done as described before (16). Cellwalls of the Y-like form of P. brasiliensis were iso-lated from whole cells by passing the material

through a Ribi cell fractionator model RF-1 (IvanSorvall Inc., Norwalk, Conn.) with pressures of40,000 to 50,000 lb/in2. Cell walls and debris wererecovered by centrifugation at 8,000 x g for 15 min.Resuspension of the pellet in 60% sucrose and cen-trifugation at 2,000 x g for 10 min separated a pelletcontaining the cell walls from a milky suspensioncontaining the cytoplasmic material. Cell wallswere carefully washed with water and dialyzed toliberate any residual sucrose. After lyophilization,cell walls were ready for fractionation as follows.Yeast cell walls were suspended in 1 N NaOH (10mg/ml) and gently stirred at room temperature for 1h. After centrifugation at 8,000 x g for 15 min, thesupernatant was collected, and the procedure wasrepeated three times, combining all the superna-tants. The alkali-insoluble precipitate was thenwashed with water until it reached pH 7 and thenwith ethanol, acetone, and ether, in that order. Thewhite powder was called fraction 1. The supernatantwas neutralized by the addition of glacial acetic acidand left overnight at 4°C. The suspension was thencentrifuged as before. The supernatant and the pre-cipitate were collected and dialyzed separatelyagainst distilled water. After lyophilization, an al-kali-soluble fraction that was precipitable with acid(fraction 2) and an alkali-soluble fraction that wasnonprecipitable with acid (fraction 3) were obtained.The yield of each fraction varied according to theexperiment.

Sugar characterization was performed by meansof a JEOL 3BC amino acid analyzer (Japan Electricand Optical Laboratories, Tokyo, Japan) that hadbeen modified for sugar analysis. Noncorrosive so-dium bicinchoninate (13) was used as the reagent.The conditions of chromatographic elutions were

as follows: stationary phase, JEOL LC-R3 (JapanElectric and Optical Laboratories); eluent, 0.35 MH3BO3, pH 9.6; column size, 0.8 by 15 cm; flow rate ofreagent and eluent, 0.46 ml/min; reaction bath tem-perature, 80°C; column temperature, 40°C; absorb-ance, 570 nm; chart speed, 30 mm/h; and sample, 50to 200 ,g of hydrolyzed polysaccharide.

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898 SAN-BLAS AND VERNET

To determine the structure of the polysaccharidecomponent of fraction 2, a-1,3-glucan from Aspergil-lus niger (9) was used as a standard in the analyticalprocedures.The total organic phosphorus was estimated by

the combined methods of Ames (1) and Chen et al.(4).FCS fractionation. FCS (GIBCO) was precipi-

tated at 4VC under constant agitation (14) by addingsaturated, neutralized (NH4)2S04 (ultrapure;Schwarz/Mann) dropwise to a 50% final concentra-tion. After 2 h, the precipitate was collected bycentrifugation at 15,000 rpm for 30 min at 4VC. Thesupernatant was then exhaustively dialyzed againstwater for 72 h. The precipitate was washed threetimes with 50% saturated (NH4)2S04. The final pel-let (2 g) was dissolved in 0.01 M phosphate bufferand dialyzed against the same buffer for 48 h at 40C.The protein precipitate was subsequently chro-

matographed in a DE22 cellulose column (6 by 70cm; Whatman, Chromedia) equilibrated with 0.01 Mphosphate buffer at pH 7.5. Fractions were elutedstepwise with increasing NaCl concentrations (Fig.1). Fractions were pooled as indicated, dialyzedagainst water for 48 h at 4VC, lyophilized, and keptfrozen until use.

Preparations of samples for electron microscopy.Y-like cells were harvested from culture media withand without 5% FCS at 0 and 5 days of growth andprepared for electron microscopy according to themethod of Freeman and Spurlock (8). Ultrathin sec-tions were obtained as described by Carbonell (3).

RESULTSTable 1 summarizes the differences in the

amount and chemical composition of the cellwall of P. brasiliensis IVIC Pb9 cultured invitro in the absence and presence of increasingamounts of FCS. The total amount of cell wallsin whole cells increased from 15 to 60% as a

0.0 MNoCI 1005MNaCI0IMNaCl

function of the concentration of FCS in media.In all cases fraction 1 remained more or less thesame, whereas the amounts of fraction 2 in-creased from 3% in medium without FCS up to23% in FCS-supplemented media. At the sametime, fraction 3 also varied, its concentrationbeing inversely proportional to the amount offraction 2.To study which component of FCS was re-

sponsible for these changes in the cell wallcontent and composition, a partial fractionationof serum was carried out. First, precipitationwith 50% ammonium sulfate was done. Boththe precipitate and the supernatant weretested, and the precipitate was found to be re-sponsible for the induction ofchanges in the cellwall composition (Table 2). Ion-exchange chro-matography of this precipitate fraction (Fig. 1)was carried out as described in Materials andMethods, and each fraction was individuallyadded to the culture media in which P. brasi-liensis was growing.The results of this experiment are summa-

rized in Table 2; the distribution of cell wallfractions according to the serum fraction pres-ent in the medium is shown. Although all FCSfractions seemed to induce the production offraction 2 in the cell wall, FCS fractions D3a andD3b were the most effective. Cell wall fraction 1remained almost invariable and was not fur-ther analyzed.

Detailed analyses of fraction 2 from each cellwall preparation were carried out to determinetheir actual composition (Table 3). In all cases,this fraction produced 96 to 98% hexoses andtraces of proteins and phosphorus. Sugar analy-ses indicated the presence of glucose as the sole

1015 M NoCI 0.20 M NaCI I0 30 MNaCI1 040 MNaCI

120 180 240 300 360 420 480 540 600 660 720 780 840

Fraction Ng

FIG. 1. Ion-exchange chromatography ofthe 50% ammonium sulfate FCS precipitate. Conditions are givenin the text.

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INDUCTION OF a-1,3-GLUCAN IN P. BRASILIENSIS 899

TABLE 1. Variations in the distribution ofP.brasiliensis cell walls and their fractions withincreasing amounts ofFCS in the medium

% Cell% FCS walls in % Frac- % Frac- % Frac-

whole tion 1 tion 2 tion 3cells

0 15.2 41.5 3.2 55.32 22.2 47.3 17.0 36.05 35.0 52.0 18.0 29.310 53.5 52.3 23.0 24.715 60.0 56.4 22.0 20.3

TABLE 2. Cell wall composition ofP. brasiliensisIVIC Pb9 (Y form) cultured in vitro with and without

FCS or its fractions

Cell wall fractions (%)bFCS fraction in BHIa F

ControlSupernatant, 50%

(NH4)2SO4Precipitate, 50%

(NH4)2SO4Serum fractions,

ex-DEAE-celluloseDID2D3aD3bD4aD4bD5D6D7

'raction Fraction1 2

41.5 3.238.8 6.1

Fraction3

55.354.9

57.6 18.9 23.4

55.454.854.849.841.852.436.635.740.1

12.322.528.428.522.020.820.315.314.3

32.322.716.721.736.240.843.149.045.6

a BHI, Brain heart infusion agar.b Figures are the mean value from three experi-

ments.

sugar component (Fig. 2B). Periodate oxidationgave no reaction, suggesting that glucose unitswere joined through 1,3-linkages. Further-more, infrared spectra (Fig. 3) were identical tothose of previously reported a-1,3-glucans (15)having a peak at 840 cm-1, which is characteris-tic of a-glucans. Also a peak at 1,525 cm-',which corresponded to proteins (15), was pro-duced. For these reasons, an a-1,3-glucan struc-ture was postulated for this polysaccharide.Preparations of fraction 3 were also analyzed

in some detail. The results suggest that theamount of hexoses varied from 17.3 to 30.0% inthe various materials analyzed, whereas theamino acid content remained between 20 and25%; the rest consisted of lipids. In all cases,sugar analyses of fraction 3 produced glucose(11%), galactose (35%), and mannose (54%)(Fig. 2C) in a molar ratio of 1:3.2:4.9. Thisrelationship was almost the same for all frac-tions 3 analyzed.

Electron microscopic observations of wholecells showed that the thickness of the cell wallincreased from 60 to 80 nm for cells grown inFCS-free medium (Fig. 4a) to 130 to 160 nm forcells grown in medium supplemented with 5%FCS (Fig. 4b), which correlated with the in-creased percentage of cell walls in each case(Table 1).

TABLE 3. Analysis offraction 2 ofP. brasiliensisIVIC Pb9 cell walls (Y form)

Composition of fraction 2

FCS fraction in me- Odium Hexoses Amino Organic

(%) acids M phs M

Supernatant, 50% 100 0.0 0(NH4)2SO4

Precipitate, 50% 97.00 1.90 0.39(NH4)2SO4

Serum fraction,ex-DEAE-cellulose

DI 98.80 0.08 0.00D2 100.70 0.00 0.03D3a 98.20 0.09 0.50Dab 97.20 0.16 0.02D4a 96.80 0.00 0.03D~jb 93.50 1.69 0.00D5 101.80 0.00 0.04D6 98.10 1.71 0.00D7 94.00 2.36 0.01

A c

g.5S

(i)i

S

c

FIG. 2. Automatic sugar analysis of. (A) stan-dard mixture; (B) alkali-soluble, acid-precipitablecell wall fraction 2 of P. brasiliensis; (C) alkali-soluble, unprecipitable cell wall fraction 3 ofP. bras-iliensis. Conditions are given in the text.

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900 SAN-BLAS AND VERNET

Wavelength (,)5 3.5 4 5 6 7 8 9 102.6

4000 3600 3200 2800 2400 2000 1800 1600 1400 200 1000 500 600

Wave number (cm-1)

FIG. 3. Infrared spectra of: (1) commercial chitin(Calbiochem, Los Angeles, Calif.); (2) ,¢1,3-glucanfrom P. brasiliensis; (3) a-1,3-glucan from A. niger;(4) a-1,3-glucan from P. brasiliensis.

DISCUSSIONPrevious results in our laboratory (17)

showed that the synthesis of a-1,3-glucan in thecell wall of P. brasiliensis could be induced bypassage of the fungus through animals. Thescope of this paper was to determine whethersome factors present in the serum could inducethe same effect and also whether there wereany other chemical changes in the cell wallstructure.FCS and FCS fractions obtained by ion-ex-

change chromatography of the 50% ammoniumsulfate precipitation induced in various degreesthe synthesis of fraction 2 of the P. brasiliensiscell wall; FCS fractions D:ia and Dlb were themost active (Tables 1 and 2). At the same time,an increase in fraction 2 corresponded to a de-crease in the proportion of fraction 3 in the cellwall.To determine whether fractions 2 and 3 of

this fungal cell wall corresponded to a-1,3-glu-can and galactomannan, respectively (11), ad-ditional analyses were performed on these ma-terials. Fraction 2 was identical to the a-1,3-glucan that had been previously reported for P.brasiliensis and A. niger (11); some protein(less than 2%) was apparently completed to the

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polysaccharide. Cox and Best (6) suggested thatthe presence of phosphorus in the alkali-solublefraction of Blastomyces dermatitidis (i.e., a-1,3-glucan) was related to the differences in thedegree of virulence observed among variousstrains of this fungus. Our studies do not seemto give any particular emphasis to the role ofphosphorus.With regard to fraction 3, it can be said that

previous reports (10) indicated that the polysac-charide component of this fraction was mainlya galactomannan with very little glucose in it(molar ratio, glucose-galactose-mannose, 1:12.3:16.5). However, our results suggest ahigher proportion of glucose (molar ratio, glu-cose-galactose-mannose, 1:3.2:4.9) as an inte-gral part of the galactomannan. Recently,Azuma et al. (2) reported that these polysaccha-rides, present in various pathogenic dimorphicfungi, were serologically active and that thecross-reactions observed among these fungiwere due to common antigenic structures intheir galactomannans. Whether the introduc-tion of glucose moieties in as much as 10% ofthe whole polysaccharide inP. brasiliensis cul-tured in the presence of FCS may, in turn,produce some variations in the antigenic re-sponse remains to be studied.

Electron microscopic observations of yeastcells grown in FCS-free and FCS-containingmedia showed that the variations in the per-centage of the contribution of the cell wall tothe whole cell (Table 1) correlated with changesin the thickness of the cell wall. In fact, the cellwall of P. brasiliensis (Y form) grown in vitrofor several years was only 60 to 80 nm thick(Fig. 4a) compared to 200 to 600 nm reportedbefore (4). The growth of this strain in FCS-supplemented medium doubled the thickness ofthe fungal cell wall to 130 to 160 nm (Fig. 4b).

It is possible that the increase of a-1,3-glucanin P. brasiliensis grown in animals (17) or inmedia supplemented with FCS may have someeffect on the biochemical mechanisms that con-trol the pathogenicity of this fungus. One ofthese mechanisms refers to those factors, theso-called aggresins (19), not necessarily toxic,which promote microbial growth in vivo by in-hibiting host defense mechanisms by means ofinhibition in the action of phagocytes or byinhibition of ingestion. Among the latter aresome surface and capsular products resistant toany attack by phagocytes, such as the capsularpolysaccharides of Streptococcus pneumoniae(7), the Vi antigen (poly-N-acetyl-D-galactosa-minouronic acid) ofSalmonella typhi (5), or thepoly-D-glutamic acid ofBacillus anthracis (12).We suggest that the a-1,3-glucan present in P.

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INDUCTION OF a-1,3-GLUCAN IN P. BRASILIENSIS 901

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FIG. 4. Electron micrographs of. (a) yeastlike cells ofP. brasiliensis grown in brain heart infusion agar;(b) same cells grown in brain heart infusion agar supplemented with FCS. x15,000. The bar represents 500nm.

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902 SAN-BLAS AND VERNET

brasiliensis may well fall into this category. Infact, preliminary results in our laboratory sug-

gest that cells of P. brasiliensis IVIC Pb9 are

more virulent if they are cultured in FCS-sup-plemented medium prior to inoculation in mice(strain BALB/cj).The effect of serum upon the in vitro growth

of eukaryotic cells has been reported only twicebefore (14, 18). Furthermore, O'Daly (14) madethe first account dealing with the identificationof serum factors that stimulated the division ofT. cruzi in vitro. In that case, those factorswere identified with five proteins from the su-pernatant of 50% ammonium sulfate. In thecase reported in this paper, the factor(s) induc-ing the synthesis of a-1,3-glucan in P. brasi-liensis seems to be protein(s) from the precipi-tate. In both cases, it is noteworthy that FCSfactors induce effects similar to those inducedby the host. Experiments to determine the ex-act nature of the inducing factor(s) are cur-rently in progress.

ACKNOWLEDGMENTS

We are deeply indebted to F. San-Blas and J. A. O'Dalyfor fruitful discussions, F. Kanetsuna and Angela Restrepofor critical reading of this manuscript, and Domitila Ordazand E. J. Merino for skilled technical assistance.

LITERATURE CITED

1. Ames, B. N. 1966. Assay for inorganic phosphate, totalphosphate and phosphatases, p. 115-118. In S. P.Colowick and N. 0. Kaplan (ed.), Methods in enzy-mology, vol. 8. Academic Press Inc., New York.

2. Azuma, I., F. Kanetsuna, Y. Tanaka, Y. Yamamura,and L. M. Carbonell. 1974. Chemical and immunolog-ical properties of galactomannans obtained from His-toplasma duboisii, Histoplasma capsulatum, Paracoc-cidioides brasiliensis and Blastomyces dermatitidis.Mycopathol. Mycol. Appl. 54:111-125.

3. Carbonell, L. M. 1967. Cell wall changes during thebudding process of Paracoccidioides brasiliensis andBlastomyces dermatitidis. J. Bacteriol. 94:213-223.

4. Chen, P. S., Jr., T. Y. Toribara, and H. Warner. 1956.Microdetermination of phosphorus. Anal. Chem.28:1756-1758.

5. Clark, W. R., J. McLaughlin, and M. E. Webster. 1958.An aminohexuronic acid as the principal hydrolytic

component of the Vi antigen. J. Biol. Chem. 230:81-89.

6. Cox, R. A., and G. K. Best. 1972. Cell wall compositionof two strains ofBlastomyces dermatitidis exhibitingdifferences in virulence for mice. Infect. Immun.5:449-453.

7. Dubos, R. J., and J. G. Hirsch. 1965. Bacterial andmycotic infections of man, 4th ed. J. B. LippincottCo., Philadelphia.

8. Freeman, J. M., and B. 0. Spurlock. 1962. A new epoxyembedment for electron microscopy. J. Cell Biol.13:437-443.

9. Johnston, I. R. 1965. The partial acid hydrolysis of ahighly dextrorotatory fragment of the cell wall ofAspergillus niger. Isolation of the a-1,3-linked dex-trin series. Biochem. J. 96:659-664.

10. Kanetsuna, F., L. M. Carbonell, I. Azuma, and Y.Yamamura. 1972. Biochemical studies on the thermaldimorphism of Paracoccidioides brasiliensis. J. Bac-teriol. 110:208-218.

11. Kanetsuna, F., L. M. Carbonell, R. E. Moreno, and J.Rodriguez. 1969. Cell wall composition of the yeastand mycelial forms of Paracoccidioides brasiliensis.J. Bacteriol. 97:1036-1041.

12. Keppie, J., H. Smith, and P. W. Harris-Smith. 1955.The chemical basis of the virulence of Bacillus an-thracis. III. The role of the terminal bacteraemia indeath of guinea pigs from anthrax. Br. J. Exp. Pa-thol. 36:315-322.

13. Mopper, K., and E. M. Grindler. 1973. A new noncorro-sive dye reagent for automatic sugar chromatogra-phy. Anal. Biochem. 56:440442.

14. O'Daly, J. A. 1975. Serum proteins promoting [3H] thy-midine uptake by Trypanosoma (Schizotrypanum)cruzi (Chagas) in vitro. J. Protozool. 24:550-555.

15. San-Blas, F., G. San-Blas, and L. J. Cova. 1975. Amorphological mutant of Paracoccidioides brasilien-sis strain IVIC Pb9. I. Isolation and cell wall charac-terization. J. Gen. Microbiol. 93:209-218.

16. San-Blas, G., and L. M. Carbonell. 1974. Chemical andultrastructural studies on the cell wall of the yeast-like and mycelial forms of Histoplasma farcimi-nosum. J. Bacteriol. 119:602-611.

17. San-Blas, G., F. San-Bias, and L. E. Serrano. 1977.Host-parasite relationships in the yeastlike form ofParacoccidioides brasiliensis strain IVIC Pb9. Infect.Immun. 15:343-346.

18. Temin, H. M., R. W. Pierson, Jr., and N. C. Dulak.1972. The role of serum in the control of multiplica-tion of avian and mammalian cells in the culture, p.49-81. In G. H. Rothblat and V. J. Cristofalo (ed.),Growth, nutrition, and metabolism of cells in culture.Academic Press Inc., New York.

19. Wilson, G. S., and A. A. Miles. 1964. Topley and Wil-son's principles of bacteriology and immunity. Ed-ward Arnold Ltd., London, England.

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