INTERNABONAL JOURNAL. OF LEPROSY^ Volume 50, Number 2Printed in the U.S.A.

Stimulation of Growth of Mycobacteriumlepraemurium in Cell-Free Liquid

Medium by DL-Aspartic Acid'

Masahiro Nakamura'

In order to improve NC ( '), NC-5 ( 5), andND-5 (") media, soluble starch ($. 15), andliposome ('") were added to them. The ef-fects of these ingredients on the prolongedgrowth of Mycobacterium lepraemuriumwere tested because NC, NC-5, and ND-5media are still useful to obtain primary mul-tiplication of M. hyruenturium from an in-fected mouse. In addition, the effects ofamino acids and vitamins (") on possiblecontinuous growth of M. /epraenutriumhave been tested in the Nakamura system.During these investigations, it was recentlyfound that DL-aspartic acid markedly stim-ulated the growth of the bacilli in cell-freeliquid medium. This interesting result hasbeen reported in preliminary form (u).

The purpose of the present paper is todescribe in detail the factors affecting theactivity of aspartic acid on the growth of afastidious microorganism, M. lepraemu-rium.

MATERIALS AND METHODSM. lepraenturium. M. lepraemurium

Hawaiian strain (M-71) were obtained froma subcutaneous leproma experimentallyproduced in a C„H mouse. The leproma washomogenized with a glass homogenizer inM/30 Sorensen buffer (pH 7.0) containing0.1% v/v calf set -urn. Before use the bacil-lary suspension was appropriately dilutedwith Sorensen buffer containing calf serum.

Culture media tested. The compositionsof the culture media were as follows:1) ND base.

Dubos powder"^0.5 g

' Received for publication on 14 December 1981;accepted for publication in revised form on I March1982.

2 1M. Nakamura. M.D., Professor of Microbiology.Department of Microbiology, Kurume UniversitySchool of Medicine, Kurume, 830, Japan.

Dubos powder ( Liken Co., Japan) is composed of

adenosine^ 1.0 mgthymidine^ 2.0 mgthioglycollate^1.0 mgsuccinamide^42.0 mgwater^ 38.0 ml

The pH was adjusted to the values indicat-ed in the tables of the results, then auto-claved.

2) ND. To sterilized ND base medium (38ml), 5 ml calf serum was added aseptically.3) ND-5. The following supplements (3.0 ml)which were previously sterilized by Milli-pore filtration were added to 40 ml of sterileND medium. In this case, the ingredientsof ND base medium were dissolved in 35ml of water.

Supplements:5% a-ketoglutarate^0.5 ml0.3% 1-cysteine HCI

^0.5 ml

0.02% heroin^ 1.0 ml0.5% cytochrome c^0.5 ml0.03% NADH^

0.5 ml

Hemin was dissolved in 0.01 N NaOH.

4) NDDLI). Dextran (MW: 100,000-200,000), 82.0 mg (") and liposome, 0.6 ml,were added to 38 ml of ND base medium,autoclaved, then 5 ml of sterile calf serumwas added (ND containing dextran and li-posome).5) NDDLP-5. Dextran, 82 mg, and lipo-some, 0.6 ml, were added to 35 ml of NDbase medium, autoclaved, and then 5 mlsterile calf serum and 3 ml of supplementswere added (ND-5 containing dextran andliposome).

K FLPO., 1.3 g, NaMPO., • 12F1,0 2.2 g, asparagine 2.0 g,peptone 5.2 g, MgS0., 0.1 g, ferric ammonium citrate0.01 g, ZnSO, 0.0001 g, CuSO., 0.0001 g, CaC1, 0.0005g, malachite green 0.002 g and Tween 80 0.5 g (total11.3127 g).

193

194^ International Journal of Leprosy^ 1982

TABLE I. Effect of DL-aspartic acid on the ,f,, rowth of Mycobacterium lepraemuriumin the presence or absence of dextral' and liposome.

Base mediumDextran

andliposome

Asparticacid

pll ofmedium

Inoculumsize

x 1W/tube

Foldincrease"

ND 0.02% 6.0 3.0 14.6ND + 0.02% 204.2ND-5 0.02% 6.8 3.1 22.3ND-5 (1.02% 188.5

" After 6 weeks of incubation at 30°C.

6) NDLA. DL-aspartic acid was added toNDDLP medium.

7) NDLA -5. DL-aspartic acid was added toNDDLP-5 medium.

Preparation of medium. Dextrin, lipo-some, DL-aspartic acid and dimethyl sulf-oxide (DMSO) were added to ND mediumbefore sterilization, the pH was adjustedwith 20% NaOH or I N HCI, then the me-dium was autoclaved. Liposomes were pre-pared as follows: lecithin (Wako Co., Ja-pan), 80 mg and cholesterol, 10 mg (WakoCo., Japan) were dissolved in 2 ml of CHC1 ; :then the chloroform was evaporated withnitrogen gas. After evaporation, 10 ml ofdistilled water was added to the lecithin-cholesterol mixture, which was then ho-mogenized by ultrasonication, 19.5 KR,, 3A(Kaijo Electric Co., Japan, T-A-4280 type)for 5 min. The homogenate was kept in thecold until use.

Menadione (Sigma Chemical Co., St.Louis, Missouri, U.S.A.) was used for pre-paring vitamin K„ solution: 8 mg of mena-dione was dissolved in 10 ml of distilledwater, diluted 1000 fold with distilled water,then sterilized by filtration through a Mil-lipore filter (pore size, 0.22 p.). Para-ami-nobenzoic acid (MBA) (1 mg/ml of H 2 O)also was sterilized by filtration.

Cultivation and assessment of the growthof M. lepraemurium. Two-tenths of a milli-liter of M. hp/wenn/•em suspension wasinoculated into 43 ml of culture medium,which was distributed in 5 ml or 7 ml quan-tities into test tubes (10.5 x 1.3 cm) fittedwith sterile rubber stoppers. Cultivation wascarried out at 30°C. Growth of M. leprae-murium was estimated six weeks after in-oculation by Hanks' pinhead method (").

Growth rates were calculated by the ratiobetween the number of bacilli at "0 - timeand that after six weeks' cultivation. Growth

TABLE 2. Effect of close of DL -aspartic acid on the grmt•th of Mycobacterium leprae-murium.

AsparticBase medium acidpH of

medium

Inoculumsize

x 10"/tube

Foldincrease"

N DDLP • K„ 6.0 2.3 43.80.01% 216.I0.02% 188.0

NDDLP • DMSO-5K„ 7.3 8.7 22.9(1.01% 61.00.02% 64.00.1% 48.90.2% 36.9

NDDLP• DMSO-5PABA 6.8 3.5 24.20.02% 175.9

" After 6 weeks of incubation at 30`C.

50, 2^Nakamura: Aspartic Acid Growth of M. lm.^195

TABLE 3. Effect of pH ou the growth ofMycobacterium lepraemurium in culturemedium containing DL-aspartic acid.

Culturemedium

pH ofmedium

Volumeof

culturemedium

Inoculum

x10"/tube

Foldincrease"

NDLA-5 6.0 7 ml 3.2 3.66.3 88.96.6 219.37.0 75.07.3 47.3

NDLA 6.0 7 ml 4.8 100.06.5 58.57.0 13.27.3 3.2

NDLA 6.0 5 ml 4.8 138.26.5 13.17.0 6.67.3 3.1

" After 6 weeks of incubation at 30°C.

patterns were morphologically observed bythe sediment smear method (SSM) ( 7).

RESULTSEffect of DL-aspartic acid in the presence

or absence of dextran and liposome. DL -aspartic acid was added to ND and ND-5 me-dium and to the same media containing dex-tran and liposome, and the effect of asparticacid on the growth of M. lepraenuirium wastested. The media were distributed in 7 mlquantities into test tubes. The results ob-tained are summarized in Table I. Thegrowth of M. lepraenutrium was markedlystimulated by the addition of DL-asparticacid at a final concentration of 0.02% to themedia containing dextran and liposome;whereas no effects were observed whenaspartic acid was added to plain ND andND-5 medium. After this experiment, DL-aspartic acid was compared with D- and L-aspartic acid. The results suggest that DL-aspartic acid was slightly more effectivethan the others, but the differences werenot significant.

Effect of concentration of DL -aspartic acid.In order to determine the optimal concen-tration of aspartic acid, it was added toNDDL-K„, NDDLP-DMS0-5K„, andNDDLP-DMSO-SPABA media at final con-centrations from 0.01% to 0.2%. The mediawere distributed in 7 ml quantities into test

FIG. I. Early growth pattern of M. lepraemuritim

grown in NDLA medium, 2 weeks' cultivation at 30'C(high magnification x 1000).

tubes. The results obtained demonstrate, asshown in Table 2, that 0.01% and 0.02%were optimal. Higher concentrations re-sulted in a decreased effect.

Effect of pH of the culture medium con-taining DL-aspartic acid. Table 2, row 2shows that in NDDLP• DMS0-5K„ medi-um, aspartic acid had no significant effect.Therefore, it was thought that the effect ofpH was much more important for asparticacid in medium containing dextran and li-posome than in plain culture medium. Asshown in Table 3, the effect of pH rangingfrom 6.0 to 7.3 was tested. In the case ofNDLA-5 medium, the optimal pH was 6.6.On the other hand, pH 6.0 was the optimumfor NDLA medium, with either 7 ml or 5ml per tube. These results strongly indicatethat selection of the optimal pH is the mostimportant factor for the growth of M. le-praentrium in media containing dextran,liposome and aspartic acid.

Morphological features of M. lepraemu-riunz grown in NDLA and NDLA-5 media.

196^

International Journal of Leprosy^ 1982

FIG. 2. Morphological features of M. /co -act/whim grown in IN1LA-5 medium. pH 6.6, 7 ml/tube (left: lowmagnification x40: right: high magnification x

The early growth pattern of M. lepraemu-rium cultivated in NDLA medium showsfeatures typical of those usually observedin M. tuberculosis (Fig. I). After six weeksof cultivation at 30°C, however, a majorityof the bacilli grown in NDLA at pH 6.0, 7ml per tube, and in NDLA-5 medium, werenonsolid, beaded, and elongated, and thebacilli were loosely clustered, as shown inFigure 2. On the other hand, bacilli inNDLA, at pH 6.0, 5 ml per tube, were quitesolid, and the clumps of bacilli appearedtight (Fig. 3) Therefore, from the point ofview of morphological features, it could beconsidered that the best condition wasNDLA medium, pH 6.0, 5 ml per tube.

DISCUSSIONSince 1972 it has been reported that M.

/epractifurium quantitatively multiplies inthe synthetic liquid culture media NC, NC-5, and ND-5 ( 1 • 5 ."). These results were def-initely confirmed by Dhople and Hanks ( I • 2 ).However, it was noted by Dhople andHanks as well as by the author that growth

stopped after six or eight weeks of culti-vation at 30°C. Therefore every effort wasmade to obtain prolonged and continuousgrowth. Finally Dhople and Hanks (Lep-rosy Scientific Memoranda 1978, Memo L-938) attained continuous growth of M. le-praemurium by substitutions of malic acid,dithiothreitol, and 6-aminolevulinic acid fora-ketoglutarate, cysteine and hemin, re-spectively. On the other hand, the authorhad been searching for a growth factor nec-essary for prolonged growth. During inves-tigations of the effects of amino acids andvitamins, it was found that DL-aspartic acidhas a very strong growth stimulating effect( 1 2) .

As shown in Table 1, the effect of aspar-tic acid seems to be limited, because thestimulating effect was observed only whenaspartic acid was added to medium con-taining dextran and liposome. It is not yetclear which is essential for aspartic acid,dextran, or liposome, or both.

Besides this problem, there is a most in-teresting and important problem which

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50, 2^Nakamura: Aspartic A cid Growth of M. Int.^197

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Fin. 3. Morphological features of M. lepractuttrium grown in NDLA medium, pH 6.0, 5 ml/tube (left: lowmagnification x40; right: high magnification x 1000).

should be solved in the future; namely, whyasparagine has no such effect, because as-paragine is closely related to aspartic acid,and asparagine is frequently used as a ni-trogen source for the growth of cultivablemycobacteria. In some cases, asparagine ismore effective than aspartic acid. In addi-tion, Dubos powder, which is used in NDbase medium, contains asparagine at a finalconcentration of 0.2% in the complete me-dium. Nevertheless, no marked stimulatingeffect like that of aspartic acid was ob-served in ND medium containing dextranand liposome (Table 2, row 1). However,there is one possible explanation for thisfact—that the concentration of aspara-gine in Dubos medium is too high, 0.2%was slightly toxic even in the case of as-partic acid. In the future, lower concentra-tions of asparagine should be tested in theNC-5 system ( 5 ) in which Kirchner mediumminus asparagine is employed as the base.According to Kato's report (Leprosy Sci-entific Memoranda, 1980, Memo L-1117/1),DMSO was tried but it is still not decided

whether DMSO is essential or not for thegrowth of M. lepraemu•ium.

As far as pH is concerned, the authorindicated previously that pH 6.8 is optimalin the NC-5 system. In the present study itwas also noted that pH 6.6 was optimal inNDLA-5 medium. In contrast, it wasstrongly indicated that pH 6.0 was optimalin NDLA medium. This value correspondswith those reported by Ogawa and Hiraki(") and Portaels and Pattyn ("). The differ-ence in optimal pH between NDLA andNDLA-5 media might be caused by a-ke-toglutarate, because the optimal pH of allmedia containing a-ketoglutarate, for in-stance, NC-5 and ND-5 medium, is 6.6 to6.8. This evidence clearly suggests that theremight be different metabolic pathways be-tween a-ketoglutarate and aspartic acid. Inthe future, the effect of aspartic acid shouldbe compared with that of oxaloacetate, be-cause oxaloacetate is much more closelyrelated to aspartic acid than (Y - ketogluta -

rate.Beyond these problems, the data pre-

198^ biternatiomil Journal qf Leprosy^ 1982

sented here strongly indicate, as recentlypointed out by PottleIs and Pattyn '), thatthe most important and essential factor forthe growth of M. leprocomrium is optimalpH; the growth is strictly influenced by thepH of liquid medium just as has been dem-onstrated for solid media, but the optimalpH varies with the composition of the cul-ture medium.

Therefore, three essential factors in-volved in the Nakamura system, i.e., a-ke-toglutarate, 33% air space, and cultivationat 30'C, which were determined by the an-alytical studies of Dimple and Hanks ('),should be changed when a new culture sys-tem is established, because, in general, es-sential factors for cultivation are character-ized by different substrates.

Finally, morphological features are knownto be important parameters to detect theactivity of mycobacteria. The results ob-tained here demonstrate that the solid cellstructure of M. lepraenueriton was ob-served when NDLA medium at pH 6.0, 5ml per tube, was used. An attempt is beingmade to subculture the bacilli under theconditions described above.

SUMMARYThe growth of M. /epraemurium in cell-

free liquid medium was strongly stimulatedby addition of DL-aspartic acid at a finalconcentration of 0.0IY to 0.02Y to ND andND-5 media containing dextran and lipo-some. No effect of DL-aspartic acid wasobserved when it was added to ND and ND-5 media without dextran and liposome. Op-timal pH of the culture medium is criticalfor the stimulating effect of DL-asparticacid, and it varies with the composition ofthe medium; the optimal pH was 6.0 in NDmedium containing dextran and liposome(NDLA), and was 6.6 in NDLA mediumsupplemented with a-ketoglutarate. 1-cys-teine HCI, hemin, and cytochrome c(NDLA-5). A possible mechanism of the ef-fect of aspartic acid is discussed.

RESUMENLa incorporaciOn de acido DL-aspartico en una con-

centrackin final del 0.01 al en los medios ND yND-5, libres de celulas, conteniendo dextrana y lipo-somas, estimulO marcadamente el crecimiento del M.lepraemurium. Cuando el ticido DL-aspartico se adi-

cionti a los medios NI) y ND-5 en ausencia de dex-trana y liposomas, no se observe) ningiin efecto esti-mulante. Un pl I tiptimo del cultivo es critic() part elelect° estimulante del acid() DI.-aspartico y varia conla composicitin del medio. LI pl l tiptimo fue de 6.0 enel medio ND con dextrana y liposomas (NI/I,A) y de6.6 en el meth() NDLA suplementado con (gamma)-cetoglutarato, 1-cisteina HCI, hemina y citocronto CINDLA-5). Se discute un posible mecanismo del efectodel acido aspartico.

RESUMELa croissanec de M. lepotemurium dans tin milieu

liquids sans celltdes a etc fortement stimulee parl'addition (Tackle DL-aspartique a tine concentrationfinale de 0,01 it 0,02Ci, aux milieux ND et ND-5 con-tenant du dextran et des liposomes. II n'a etc noteaucun diet de l'acide DI.-aspartique it la suite de sonaddition it des milieux ND et ND-5 sans dextran niliposomes. Le choix d'un pH optimal pour le milieude culture est dune importance critique pour Ferretstimulant de l'acide DL-aspartique; cc pH vane avecla composition du milieu. l,e pH optimal etait de 6,0dans le milieu ND contenant du dextran et des lipo-somes INDI,A), et de 6,6 dans le milieu NDA enrichien (gamma)-cetoglutarate. de la 1-cysteine HCI, del'hemine. et du cytochrome-c INDLA-5). On discutedu mecanisme possible (Faction de l'acide aspartique.

Acknowledgments. This work was partially support-ed by grants from the U.S.-Japan Cooperative MedicalScience Program and the Sasakawa Memorial HealthFoundation. The author is also indebted to Miss To-moko Takeda for her skillful technical assistance.

REFERENCESI. Dinio.t., A. NI. and HANKS, J. IL Factors that

influence the growth of M. lepracuntrium in theNakamura's system. Int. J. Lepr. 44(1976) 18-26.

2. Darin.(), A. NI. and HANKS, J. H. In vitro growthof .1/. teprattutrium. an obligate intracellular mi-crobe. Science 22 (1977) 379-381.

3. HANKS, J. H. Microscopic counts of mycobac-teria. Conversion factors for pinhead methods. Int.J. Lepr. 36 (1968) 76-77.

4. NAKANtuRA, NI. Multiplication of Mycobacte-rium lepraemurium in cell-free medium containinga-ketoglutaric acid and cytochrome c. J. Gen.Microbiol. 73 (1972) 193-195.

5. NAKAN1URA, NI. Quantitative multiplication ofMycobacterium lepraemurium in a cell-free liquidmedium INC-5). J. Gen. Nlicrobiol. 82 (1974) 385—391.

6. NAKANIURA, NI. A new cell-free liquid mediumfor rapidly culturing Mycobacterium lepracaut-rium (ND-5 medium). Proc. Jap. Acad. 51 (1975)707-711.

7. NAKAN1URA, NI. Multiplication of Mycobacte-rium lepraemurium in cell-free liquid medium:

50, 2^Nakamura: A vortic Acid Growth of M. Int.^199

Growth evaluation: Sediment smear method

(SSW. La Lepro 46 (1977) 98-103.

8. NAKANIURA, M. Stimulation effect of soluble

starch on the growth of Mycobacterium leprae-

muritim in cell-free liquid medium. Preliminary re-

port. Kurume Med. J. 26 (1979) 397-400.

9. NAKANIURA, M. Growth stimulating effect of

dextral) on M. tepraemurium in vitro in cell-free

liquid medium. Jap. J. Lepr. (1982) (in press).10. NAkxxtuRi, M. Growth stimulation of Myco-

bacterium lepraemurium by liposome in cell-free

liquid medium. Jap. J. Lepr. (1981) (in press).I I. NAKAMURA, M. Growth stimulation of Myco-

bacterium lepracmurium in cell-free liquid medi-

um by vitamin K„ and II,- Jap. J. Lepr. 50 (1981)

135-138.12. NAKAMURA, M. Enhanced growth of Alyrobm-

terium lepraenutritan in cell-free liquid mediumwith DL-aspartic acid. Kurume Med. J. (1981) (inpress).

13. OGAwx, T. and HIRAKI, M. Studies on murineleprosy bacillus V. Growth in relation to concen-

trations of monopotassium phosphate, sodium

glutamate and glycerol in the Iii egg yolk medium

and relation to the pH of the medium. KitasataoArch. Exp. Med. 45 (1972) 25-31.

14. Poizimi.s. F. and Pxyrl . N. S. R. Parameters in-fluencing the in vitro growth of Mycobacteriumlepractnurium. Int. J. Leprosy 49 ( 1981) 194-197.

15. SENGUPTA, U. and NAKANIURA, M. Growth ofMycobacterium lepraemurium in cell-free liquidmedium containing soluble starch: Evaluation by

generation time and morphological observation.Lepr. India 53 (1981) 23-28.