Purification and characterisation of isocitrate dehydrogenase and malate dehydrogenase from Mycobacterium tuberculosis and evaluation of their potential as suitable antigens for the serodiagnosis of tuberculosis

Tubercleand Lung Disease (1996)77, 454-461 © 1996 PearsonProfessionalLtd

Purification and characterisation of isocitrate dehydrogenase and malate dehydrogenase from Mycobacterium tuberculosis and evaluation of their potential as suitable antigens for the serodiagnosis of tuberculosis R. 13hman, M. Ridell Department of Medical Microbiology and Immunology, Giiteborg University, G6teborg, Sweden S U M M A R Y. Setting: Enzymes from Mycobacterium tuberculosis are potent antigens and might thus be of interest in the serodiagnosis of tuberculosis. Objective: The purpose of the study was to purify and characterize the two enzymes isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) from, M. tuberculosis and to evaluate their potential in the serodiagnosis of tuberculosis. Design: The two enzymes were analysed for specificity by electrophoresis and then purified by means of affinity chromatography using reactive dyes and ion exchange chromatography. The two isolated enzyme fractions were analysed by ELISA, using antisera against related organisms. They were then tested as antigens in ELISA together with sera from tuberculous patients and controls. Results: The electrophoretical analyses showed that the two enzymes each differed markedly from the corresponding enzymes of other mycobacteria. The serological analyses, however, could not distinguish between either IDH or MDH from other mycobacteria, but organisms of other genera, such as Nocardia, gave much weaker responses. When IDH and MDH were tested with sera from tuberculous patients and controls the former gave clearly higher optical density values than the latter. Conclusion: The enzymes/antigens IDH and MDH may be of value in developing a serological test for tuberculosis. The latter fraction seemed particularly capable of discriminating patients from controls. R E S U M E . Cadre: Les enzymes provenant de Mycobacterium tuberculosis sont de puissants antig~nes et pourraient d~s lors ~tre int~ressants pour le s~rodiagnostic de la tuberculose. Objectif: Cette ~tude vise h purifier et ~ caract~riser les deux enzymes isocitrate d~hydrog~nase (IDH) et malate d~hydrog~nase (MDH) de M. tuberculosis et h ~valuer leurs potentialit~s dans le s~rodiagnostic de la tuberculose. Schema: Les deux enzymes ont ~t~ analys~s par ~lectrophor~se en ce qui concerne leur specificitY. Ils ont ~t~ purifi~s ensuite par la chromatographie d'affinit~ en utilisant des colorants r~actifs et la chromatographie par ~change d'ions. Les deux fractions enzymatiques isol~es ont ~t~ analys~es par ELISA en utilisant des antisera contre des organismes apparent~s. Elles ont par ailleurs ~t~ test~es comme antig~nes dans une r~action ELISA faisant appel h des sera de patients atteints de tuberculose et des sera de sujets-contr61e. R~sultats: Les analyses ~lectrophor~tiques ont montr~ que chacun des deux enzymes de M. tuberculosis est nettement different des enzymes correspondants provenant d'autres mycobact~ries. Toutefois, les analyses s~rologiques sont incapables de distinguer I'IDH ou la MDH provenant d'autres mycobact~ries alors que des organismes d'autres genres, par exemple les Nocardia, donnent des r~ponses beaucoup plus faibles. Lorsque r I D H et la MDH sont test~es avec des sera de patients atteints de tuberculose et de sujets-contr61e, les premiers donnent des valeurs de densit~ optique nettement sup~rieures aux seconds. Conclusion: Les enzymes/antig~nes IDH et MDH peuvent ~tre int~ressants pour d~velopper un test s~rologique pour la tuberculose. C'est particuli~rement la deuxi~me fraction (MDH) qui semble bien apte h discriminer les patients d'avec les sujets-contr61e.

Correspondenceto: Dr R. Ohman, Departmentof Medical Microbiologyand Immunology,G6teborgUniversity,Guldhedsgatan 10, S-41346G/Steborg,Sweden.Tel:+46 31 604723:Fax: + 46 31 820160. Paper received 26 April 1995. Finalversion accepted8 May 1996. 454

Purification of two dehydrogenases from Mycobacterium tuberculosis 455 R E S U M E N. Marco de referencia: L a s e n z i m a s p r o v e n i e n t e s de Mycobacterium tuberculosis son a n t i g e n o s p o t e n t e s , r a z 6 n p o r la c u a l p u e d e n s e t de inter6s en el s e r o d i a g n 6 s t i c o de l a tuberculosis. Objetivo: E1 p r o p 6 s i t o de este e s t u d i o fue d e p u r i f i c a r y c a r a c t e r i z a r dos e n z i m a s p r o v e n i e n t e s de M. tuberculosis, i s o c i t r a t o d e s h i d r o g e n a s a ( I D H ) y m a l a t o d e s h i d r o g e n a s a ( M D H ) , p a r a e v a l u a r su u t i l i d a d en el s e r o d i a g n 6 s t i c o de la t u b e r c u l o s i s . M~todo: L a e s p e c i f i c i d a d de las dos e n z i m a s rue a n a l i z a d a p o r electroforesis. L a p u r i f i c a c i 6 n se realiz6 p o r m e d i o d e la c r o m a t o g r a f i a de a f i n i d a d , u t i l i z a n d o tinci6n r e a c t i v a y c r o m a t o g r a f i a de i n t e r c a m b i o de iones. L a s dos f r a c c i o n e s d e e n z i m a s a i s l a d a s f u e r o n a n a l i z a d a s con la t6cnica E L I S A u t i l i z a n d o a n t i s u e r o s c o n t r a m i c r o o r g a n i s m o s e m p a r e n t a d o s . Ademfis fu6ron s o m e t i d a s a test E L I S A , c o m o antigenos, con s u e r o p r o v e n i e n t e d e p a c i e n t e s con t u b e r c u l o s i s y de c o n t r o l e s sanos. Resultados: L o s anfilisis electrofor6ticos m o s t r a r o n que c a d a u n a de las dos e n z y m a s se d i f e r e n c i a b a n o t a b l e m e n t e de las e n z i m a s c o r r e s p o n d i e n t e s d e o t r a s m i c o b a c t e r i a s . Sin e m b a r g o , los exfimenes serol6gicos no p u d i e r o n h a c e r la distinci6n con la I D H o la M D H p r o v e n i e n t e s de o t r a s m i c o b a c t e r i a s , p e r o o r g a n i s m o s de o t r o s g6neros c o m o Nocardia d i e r o n r e s p u e s t a s m u c h o mils d6biles. C u a n d o la I D H y la M D H f u e r o n sometid a s a test con s u e r o p r o v e n i e n t e de de p a c i e n t e s con t u b e r c u l o s i s y controles, los p r i m e r o s d i e r o n c l a r a m e n t e v a l o r e s d e d e n s i d a d 6 p t i c a (OD) mils altos que los fiitimos. Conclusi6n: L a s e n z i m a s / a n t i g e n o s I D H y M D H p u e d e n t e n e t u n v a l o r en el d e s a r r o l l o de u n test serol6gico p a r a la t u b e r c u l o s i s . E n especial la filtima f r a c c i 6 n p a r e c i 6 c a p a z de d i s c r i m i n a r e n t r e p a c i e n t e s y controles.

INTRODUCTION Tuberculosis is a disease of major importance and its incidence is increasing in many parts of the world. 1 A better vaccine, a better tuberculin and more rapid and sensitive diagnostic methods than the present ones are therefore imperative in order to control the spread of the disease. A prerequisite for development in these fields is increased knowledge concerning the antigenic repertoire of Mycobacterium tuberculosis and related organisms. Many Mi tuberculosis antigens have been revealed, but only a small number of them have been purified for individual characterization. 2 Enzymes are antigens and in all likelihood of crucial importance in this context, and studies using purified M. tuberculosis enzymes are therefore essential° We have earlier shown that several mycobacterial enzymes are strong antigens. 34 In the present study two such enzymes, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH), were analysed for species specificity and subsequently purified from M. tuberculosis by means of affinity chromatography, using reactive dyes and ion exchange chromatography. The potential of the purified enzyme fractions in the serodiagnosis of tuberculosis was evaluated.

MATERIALS AND METHODS Organisms A strain of M. tuberculosis isolated from a Swedish patient in 1991 and designated 4610/91 was used. The strain showed all the traditional properties of M. tuberculosis, such as typical colony form, cord formation, niacin positivity and full sensitivity to five first-line antimycobacterial chemotherapeutic agents. In addition four strains of the M. tuberculosis complex, i.e.M, tuberculosis' and M. bovis bacille Calmette-Gurrin (BCG) and nine strains representing seven nontuberculous

mycobacterial species (see Table) were included. The strains were grown as surface culture on liquid or solid Sauton m e d i u m for 2 - 6 weeks and the cells were harvested, washed and kept at - 7 0 ° C until use.

Crude protein preparations The bacterial cells were sonicated for 3 min at full effect in a Heat systems sonicator at 375W. The suspensions were centrifuged at 30 000 g for 15 rain and the supernatants were sterilized by filtration through a 0.22 g m filter and kept at - 7 0 ° C until use.

Enzyme electrophoresis The electrophoretic migration of the enzymes 6 was analysed as earlier described, 7,s using specific staining for IDH and MDH. 4,9.~°Dark blue dots indicated enzyme activity in both IDH and MDH.

Table Etectrophoretic migration of IDH and MDH in t4 mycobacterial strains representing nine species 34. avium ATCC 25291 T M. avium CCUG 29221 M. avium CCUG 29233 M. bovis BCG Danish substrain M. bovis BCG Moscow substrain M. fortuitum NCTC 10394 T M. intracellulare TMC 1403 M. kansasii GA468 M. phlei NCTC 8151 T M senegalense NCTC 10956 T M. simiae ATCC 25275 T M. tuberculasis Swedish patient strain 9829/87 M. tuberculosis Swedish patient strain 4610/91 34. tuberculosis Swedish patient strain 141/89

IDH

MDH

M M F VF VF VS F S VS VS VF VF VF VF

M M M F F M M S VF S F F F

T = Type strain. IDH = isocitrate dehydrogenase; MDH = malate dehydrogenase. VS = very slow; S = slow; M = medium; F = fast; VF = very fast.

456 Tubercleand Lung Disease

Purification of MDH and IDH The purification of IDH and MDH from M. tuberculosis 4610/91 was performed by affinity chromatography, using reactive dye as ligands: Reactive Blue 160 was used for MDH and Reactive Violet 5 for IDH. 11,12The dyes were mixed with Sepharose CL-6B (Pharmacia, Uppsala, Sweden), the proportion of dye being 1% of the Sepharose. Subsequently 1 M NazCO 3 was added to raise the pH to 10.5-11. The mixture was incubated at 56°C for 2h. The coupled dye-Sepharose was then poured into columns and washed extensively with distilled water to remove unbound dye. To the columns was added starting buffer made up of 30 mM K2HPO4, 30 mM NaC1, and 2 mM MgC1, pH being adjusted to 7.0 by adding 0.5 M succinic acid in 2 M acetic acid. The crude protein preparation from M. tuberculosis 4610/91 was added to the columns, which were then washed extensively with starting buffer to remove unbound material. Purification of lDH with Reactive Violet 5 1. After adding the crude protein preparation to the column with dye-Sepharose and subsequent washing, the column was again washed with 1 M NaC1 in starting buffer, which released some protein bands but not IDH Then an amount of Veronalbuffer pH 8.6 (electrophoresis buffer) 13 corresponding to five times the column volume was passed through the column and the eluted material was collected for further purification and called IDH-F1. 2. An ion exchange column was set up using DEAESepharose-CL6B (Pharmacia) as medium. IDH-F1 was dialysed against 25 mM K2HPO4 buffer adjusted to pH 7.4 with 1 M HCI added to the DEAE-column. Extensive washing was then performed. Elution was carried out by setting up a gradient from 10 mM to 60 mM NaCI in 0.5 M K2HPO4, pH 7.4. IDH was released from the column when the buffer reached about 0.15 M NaC1. Fractions with high IDH-activity were pooled and designated IDH-F2. Purification of MDH with Reactive Blue 160 1. After adding the crude protein preparation to the column with dye-Sepharose and subsequent washing, elution was performed by passing five column volumes of starting buffer with 0.4 mM NAD, 10 mM malic acid adjusted to pH 7.0.14 The eluted material was collected and fractions with high MDH activity were pooled and designated MDH-F1. 2. Ion exchange chromatography was performed in the same way as for IDH. MDH came off the column when the buffer reached about 0.25 M NaC1. The fractions with high MDH activity were collected and designated MDH-F2.

Enzyme detection The fractions were tested concerning enzyme activity by applying the samples dropwise to DEAE-papers

(Whatman DE-81, Maidstone, UK) and incubating the papers in staining solution specific to each enzyme. In addition to IDH and MDH, activity was tested for the following seven enzymes: alcohol dehydrogenase (ADH), diaphorase (DIA), glucose phosphate isomerase (GPI), hydroxybutyrate dehydrogenase (HBDH), lactate dehydrogenase (LDH), mannose phosphate isomerase (MPI), 6 phosphogluconate dehydrogenase (6PG). 4,8q°

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) The standard technique based on the Laemmli buffer system was used. Ready-made gradient gels 4-15% were purchased from Bio-Rad (Richmond, CA, USA) Staining was done with Silver Stain Plus from BioRad.

Two-dimensional immunoelectrophoresis (2D-IE) The 2D-IE technique by Samuelson 15was used, in which glass plates, 50 mm x 70 ram, and 170 gl antiserum are employed. General protein staining was performed with Crosein scarlet red.

Sera Hyperirnmune sera against cell extracts from the 24 organisms were prepared in rabbits, as earlier described? Sera from 30 patients with tuberculosis and from 25 healthy blood donors were tested. Among the 30 patients, 19 had pulmonary tuberculosis, whereas the remaining 11 patients had other forms, such as lymphadenitis, miliary tuberculosis, etc. All cases of tuberculosis were bacteriologically confirmed and 11 of them were smearpositive. The sera were kept at -20°C.

Enzyme-linked immunosorbent assay (ELISA) analyses The sera were analysed by ELISA technique as follows. The antigens were diluted in phosphate buffered saline (PBS): IDH-f2 to a concentration of 100ng/ml and MDH-F2 to a concentration of 500 ng/ml. The plates were charged with 100 ml antigen solution per well of NUNC (Roskilde, Denmark) Immuno Maxisorb microtiter plates and incubated overnight. All plates were subsequently washed three times with PBS and blocked with 0.1% bovine serum albumin (BSA) in PBS and incubated at 37°C for 30 min, followed by washing once in PBS. The sera were diluted 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200, and 1/6400 in PBS containing 0.05% Tween and 0.1% BSA. 100 ml of serum-dilutions was added to each well and incubated for 90 min. Washing three times as above with PBS-Tween was followed by the addition of peroxidase conjugated anti antisera. When rabbit sera were tested, anti-rabbit IgG (H+L) peroxidase conjugate Jackson 111-035-045 diluted

Purification of two dehydrogenases from Mycobacterium tuberculosis 457

1/4000 was used. When human sera were tested, antihuman IgA+IgG+IgM (H+L) peroxidase conjugate Jackson (Baltimore, PA, USA) 309-305-064 diluted 1/2200 was used. The plates were incubated for 90 rain. After washing as above reactions were developed by ortophenylene diamine (OPD) - 10 mg of OPD dissolved in 10 ml of 0.1 M sodium citrate buffer pH 4.5 to which was added 4 ml of 30% H202 immediately before use. The optical density (OD) of each well was read at 450 nm after 15 rain and after 25 rain. The OD values were calculated in relation to standard sera. All reactions, except for the albumin blocking, were performed at room temperature.

RESULTS The results of the electrophoretic analyses of IDH and MDH from the mycobacterial strains are given in the Table and Figure 1. The MDH of the M. boris BCG strains and the MDH of the M. tuberculosis strains were similar in electrophoretic mobility, but differed markedly from the MDH of the other mycobacterial species tested. Likewise, the IDH of M. bovis BCG was similar to that of M. tuberculosis, but differed clearly from the IDH of the other mycobacterial strains included. However, the IDH of M. simiae had the same migration as the IDH of M. boris BCG and M. tuberculosis (Fig. 1).

The fractions obtained after the purification process were analysed by SDS-PAGE. The protein band patterns revealed are shown in Figure 2. The number of bands in IDH-F2 was five. Two of them were wide and diffuse, with molecular weights of about 35 and 50 kD. The remaining three bands were more distinct with moleculax weight of 64, 95 and 115 kD, respectively, the 95 kD band being particularly strong. In MDH-F2 two wide bands were revealed as having about 35 and 50 kD. When the samples were analysed by 2D-IE, the number of precipitates for IDH-F2 was seven and for MDH-F2 two, compared to crude antigen having about 50 antigens, and IDH-FI about 15 and MDH-F1 about eight precipitinogens. The IDH-F2 fraction was shown to be rich in IDH-activity but devoid of MDH-activity as well as of the seven other enzymes tested. The MDH-F2 fraction was tested in the same way and found to be rich in MDH-activity, but none of the other eight tested enzymes (including 1DH) could be detected. Figure 3 is a compilation of the results obtained by ELISA analyses, using IDH-F2 and MDH-F2 as antigens and sera against extracts from disintegrated whole cells of organisms of Mycobacterium and related genera. The sera represented 24 different species, divided into three groups, and the mean values of OD are given for each group. Group I consisted of the five M. tuberculosis complex strains, Group II of 13 nontuberculous mycobacteria, and Group III of six organisms referred to genera related to Mycobacterium. No significant difference between the sera of the two mycobacterial groups (I and II) was obtained either for IDH-F2 or for MDHF2. The reactions of the sera against the nontuberculous mycobacteria (Group II) were in several cases greater than the reactions against the sera against M. boris BCG and M. tuberculosis (Group I). The results thus show that

kD

kD

97 66

~--~97 t , , ~ 66

45 31 ~,,,~

~,,4:45

21 ~ - , ~ 14 ~ , , ~

,,-.4:21 14

1

Fig. l~Electrophoretic analyses of mycobacterial isocitrate dehydrogenase (1DH).

2

3

4

5

6

7

1. Protein weight markers 2. IDH-F2, Reactive Violet 5 + DEAE sample 3. I D H - F | , Reactive Violet 5 sample 4. Crude antigen Tb 4610/91 5. MDH-F1, Reactive Blue 160 sample 6. MDH-F2, Reactive Blue 160 + DEAE sample 7. Protein weight markers Fig. ~-SDS-PAGE analyses of the fractions from M. tuberculosis

458 Tubercle and Lung Disease OD 2

IDH-F2

1

MDH-F2

T

0,5

o Group

~

H

iH

i

II

III

Fig. 3---Crossreactivity of fractions IDH-F2 and MDH-F2 from M. tuberculosis analysed by ELISA. The 24 sara used were produced in rabbits against 5 strains of the M. tuberculosis complex (Group I), 13 strains of nontuberculous mycobacteria (Group II) and 6 strains of various genera related to Mycobacterium (Group III). The bars represent the mean value _+SD of the optical density (OD) for each group. Using IDH-F2 as antigen: Group I sera 1.01 _+0.23; Group II sera 1.10 _ 0.36; Group III sera 0.31 _+0.11. Using MDH-F2 as antigen: Group I sera 0.91 _+0.28; Group II sera 1.07 _+0.29, group III sera 0.34 +_0.10. The figures are given for a serum concentration of 1/100 and reaction time of 15 min. S t r a i n s o f each g r o u p

Group I (open bars): M. tuberculosis Swedish patient strain 9829/87, M. tuberculosis Swedish patient strain 4610/91, M. tuberculosis Swedish patient strain 141/89, M. bovis BCG Danish substraiu, M. bovis BCG Swedish substrain, Group II (hatched bars): M. avium ATCC 25291 T, M. chelonae ss abscessus ATCC 19977 T, M. farcinogenes NCTC 10955 T, M. fortuitum NCTC 10394 T, M. intracellulare TMC 1403, M. kansasii ATCC 12478 T, M. marinum ATCC 927 T, M. peregrinum GA 739, M. phlei NCTC 8151 T, M. scrofulaceum Schaefer P29, M. simiae ATCC 25275 T, M. smegmatis ATCC 19420 T, M. vaccue ATCC 15483 T Group III (filled bars): Nocardia otitidiscaviarurn NCTC 14629 T, Nocardiopsis dassonvillei GB468, Streptomyces griseus ISP 523, Str. flavopersicus GB 476, Str. netropsis GB475, Tsukamurella paurometabolurn (Gordona aurantiaca) NCTC 10741 T T = type strain.

the IDH from different mycobacterial species, although separable at electrophoresis, were serologically unseparable and the same results were obtained for MDH. However, the sera against the related organisms, i.e. Nocardia, Nocardiopsis, Streptomyces and Tsukamurella

(Group IIl) gave much weaker responses than did the sera against the mycobacteria (Groups I and II). Similar results were obtained for both antigen fractions. IDH-F2 and M D H - F 2 were also used as antigens in E L I S A analyses of human sera. Figures 4 A and B demonstrate the results obtained when sera from 30 patients with tuberculosis were analysed together with 25 healthy blood donors, most of w h o m were B C G vaccinated. The sera from the patients had higher OD values than did the controls. W h e n choosing a cut-off of 1.000 for IDH-F2 sensitivity would be 60% and specificity 92%. Likewise a cut-off of 0.500 for M D H - F 2 would give a sensitivity of 90% and specificity 88%. Using M D H - F 2 as antigen gave more discriminating values between the patients and the healthy controls than using IDH-F2. In general, however, similar results were obtained for both antigens, i.e. those with high OD values in one test mostly had high ones in the other, and vice versa. The patients with high OD values i.e. the 18 patients with >1.000 using

IDH-F2 and the 13 patients with >0.750 using M D H - F 2 , included both smear-positive and smear-negative patients and also several patients with extra-pulmonary tuberculosis. One serum sample from a healthy control reacted very strongly with both antigens (>1.250); it has not been possible, however, to further analyse the status of this person.

DISCUSSION

Enzyme electrophoretic analyses of the mycobacterial strains show that IDH from the tested M. tuberculosis and M. bovis B C G strains migrates similarly and that the M D H of these organisms is also similar (Table and Fig. 1). The M D H and IDH, respectively, of the other species tested differed clearly in their electrophoretic migration, except for the IDH of M. simiae, which migrated in the same way as that of M. tuberculosis and M. boris BCG. Previous analyses in which 16 strains of M. bovis, M bovis B C G and M. tuberculosis were analysed together with strains representing M. intracellulare, M. m a r i n u m and M. phlei gave the same results for M D H J The analyses of IDH also showed marked differ-

Purification of two dehydrogenasesfromMycobacterium tuberculosis 459

OD

OD

IDH-F2

MDH-F2

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2.000

[] 1.750

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1.500

1.500

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-

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---

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00 0 0 0

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Dir neg patients

O

O O O

0.250

I

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0 g

-

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Dir pos patients

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Dir neg patients

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Healthy controls

ELISA analyses of sera from patients with tuberculosis and healthy controls, using the fractions IDH-F2 (Fig. 4A) and MDH-F2

(Fig. 4B) fromM. tuberculosis as antigens. The optical density values are givenfor a serumconcentrationof 1/100 and a reaction time of 15 rain. ences between the 16 M. tuberculosis complex organisms and the other mycobacteria. However, some variation was also registered among the former 16 organisms in relation to IDH, probably due to the higher resolving capacity of the technique used. The combined result of the present and the previous studies 5 indicate that both the IDH and MDH enzymes are mutually similar among the M. bovis, M. bovis BCG and M. tuberculosis strains, but differ clearly from the corresponding enzymes of many other mycobacterial species. The specificity of the two enzymes IDH and MDH was an important reason for their purification. Only a small number of mycobacterial proteins have so far been isolated for individual studies? There is therefore a need to find methods suitable for the purification of such proteins, particularly those from M. tuberculosis. Most protein purification methods are based on hydrophilic systems in which mycobacterial lipids often interfere. Affinity chromatography with reactive dyes has been used advantageously for purification of many different substances, particularly a large number of different enzymes, 1,16-19and this technique does not seem to be affected by the remaining lipids to the same extent as

many other methods. Another advantage of reactive dyes in purification is that the proteins are retained in their native form, which is of importance for studies of different antigens, immunogens and enzymes. The choice of dyes was based on data from previous studies. 11,~2Reactive Violet 5 was selected as the most suitable dye for isolating IDH, due to simple and efficient elution with Veronalbuffer. IDH also bound to Reactive Green 19 but the yield was lower. Reactive Blue 160 was considered the most suitable dye for MDH. Reactive Red 120 was also tested and gave a higher yield, but also more contaminating bands, which were difficult to remove by other purification methods) 1 In the present study it was not possible to isolate the two enzymes to homogeneity with preserved enzyme activity. Several methods were tested for further purification, e.g. hydrophobic interaction chromatography, gel filtration, and electroelution, but these methods did not increase the extent of purification or they inactivated enzyme activity, thus preventing the possibility to identify the pertinent enzymes. It has been shown that IDH from other bacterial species falls into two different categories. The IDH of

460

Tubercle and Lung Disease

some organisms have a monomer form with a molecular weight ranging between 80 and 100 kD while IDH of others have a dimer form in which the subunits are in the range around 45 kD. 2°-23For example, IDH of Bacillus stearothermophilus is a dimer in which the subunits have a molecular weight of 45 kD and the native enzyme 92 kD. 23 The present results could therefore not clarify whether or not the IDH of M. tuberculosis is a monomer or a dimer. The MDH from several different organisms has been analysed and seems also to fall into two categories. All the eucaryotic cells tested and several bacteria have a dimer form with a molecular weight in the range of between 40-70 kD. The MDH of all the Bacillus tested species and some other grampositive bacteria tested is a tetramer with a molecular weight ranging between 120-150kD. 24The same molecular weight for MDH was also found in archeabacteria, which represent organisms very distantly related to eubacteriaY In both groups the subunit molecular weight is around 32-36 kO. 26 These two types of enzymes did not cross react immunologically, but within each group a wide cross reactivity was recorded. 26 Judging from these data, it is not possible to conclude whether or not the MDH of M. tuberculosis has a tetramer or a dimer form. The fractions were purified from a recently isolated strain of M. tuberculosis and not from the type strain H37Rv or any other well-known strains of M. tuberculosis. The reason for choosing a recently isolated strain, instead of any of those which are well analysed at many laboratories, is that the latter may have lost or changed certain characteristics during many years of cultivation in vitro. While the enzyme electrophoresis analyses demonstrated specificity of the two enzymes, the ELISA analyses could not differentiate between either MDH-F2 or IDH-F2 from M. tuberculosis complex and the nontuberculous mycobacterial species. The reactions of the polyclonal rabbit sera against the nontuberculous mycobacteria were actually greater than the reactions of the sera against M bovis BCG and M. tuberculosis. The explanation may be that several of the nontuberculous mycobacteria, growing far more rapidly than the M. bovis BCG and M. tuberculosis are better immunogens. However, when sera against organisms from other, although related, genera were included, much weaker responses were obtained than for the mycobacterial sera. The mycobacterial IDH thus differed serologically from IDH of the other genera, such as Nocardia and Streptomyces. Likewise mycobacterial MDH differed from MDH of the other genera included. Diagnosis of tuberculosis is presently mainly based on microscopy and/or culture. The former technique has low sensitivity and the latter is time consuming. There is, therefore, a need for a more rapid diagnostic method with high sensitivity, which is suitable in developing countries. Attempts to develop a serodiagnostic test for tuberculosis have thus been made for many yearsY -29 Many different antigens have been used, both lipids 3°-31

and proteins, 32-34but serodiagnostic tests are not generally used at routine diagnostic laboratories. Previous studies have demonstrated that the enzymes IDH and MDH of M. tuberculosis and other mycobacteria are potent antigens and thus of potential interest in the serodiagnosis of tuberculosisY In the present study it was shown that sera from patients with tuberculosis react more strongly than sera from healthy blood donors with the antigens IDH-F2 and MDH-F2. The latter antigenic fraction seems particularly efficient in discriminating between patients and controls. Most serodiagnostic tests developed so far have demonstrated wide variations in response among both tuberculosis patients and controls. One explanation might be that different stages of the disease can give different antibody responses. It is also possible that some antigens, particularly glycolipids, are not common to all M. tuberculosis strains. In all likelihood, no single antigen is alone suitable for a reliable serodiagnostic test for tuberculosis. However, a combination of several antigens, both proteins and lipids, may be of greater value in this respect. It is likely that the role of protein-antigens in such a combined test is mainly to give sufficient sensitivity, and glycolipid-antigens sufficient specificity. Further studies are needed, testing more antigens and more sera; the latter should include sera from patients with diseases other than tuberculosis. IDH and MDH are enzymes of the citric acid cycle and most likely located intracellularly. It is probable that intracellular proteins are released during an infection. Consequently, the presence of antibodies against these antigens is a clear indication of ongoing tuberculosis. The importance of these antigens in protection is more dubious. It has been postulated that only secreted proteins are of value for protection. 35 Since MDH and IDH are potent immunogens the possibility cannot be excluded that these antigens/enzymes have an impact also on protection against the development of disease. Studies investigating their potential in this respect should therefore be performed. It would also be of interest to study the capacity of these antigens as skin-test reagents, i.e. as tuberculin.

Acknowledgements This study was supported by the King Oscar II Jubilee Fund of the Swedish Heart Lung Fotmdation, the Gtteborg Medical Society, the Swedish Society of Medicine, and the Oscar and Hanna Bj/Srkbom Foundation. The authors are grateful to Ms Gun Wallerstrtm's skill with the ELISA analyses.

References 1. Crofton J. Tuberculosis: A global review. IUATLD Newsletter 1994; 12: 2-7. 2. Young D B, Kanfmann S H E, Hermans P W M, Thole J E R Mycobacterial protein antigens: a compilation. Mol Microbiol 1992; 6 (2): 33-145. 3. ()hman R, Ridell M. Selective enzyme staining procedure for the characterization of mycobacterial immunoprecipitates. Int Arch Allergy appl Immun 1986; 79: 145-148. 4. Ridell M, Ohman R, Wallerstrtm G. Characterization of

Purification of two dehydrogenases from Mycobacterium tuberculosis

5.

6.

7.

8. 9. 10. 11. 12. 13.

14.

15.

16. 17.

18. 19.

20.

21.

22.

mycobacterial immuno-precipitates by selective staining of enzymes. J Gen Microbiol 1987; 133: 1983-1986. (~hman R, Ridell M. Enzymatic and antigenic analyses of strains of Mycobacterium bovis, M. bovis BCG and M. tuberculosis. Current Microbiol 1995; 30: 161-165. Selander R K, Caugant D A, Octmaan H, Musser J M, Gilmour M N, Wittman T S. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol 1986; 51: 873-884. Mclellan T, Ramshaw J A M. Serial electrophoretical transfers: A technique for the identification of numerous enzymes from single polyacrylamid gels. Biochem Genetics 1981; 9: 647-654. Ridell M. Enzyme electrophoresis in taxonomy of mycobacteria. Ann Soc Belg Med Trop 1993; 73:35-39 Harris H, Hopkinson D A. Handbook of enzyme electrophoresis in human genetics. North Holland, Amsterdam, 1976. Rothe G M. Electrophoresis of enzymes. Springer Verlag, Berlin Heidelberg: 1994. Boyer P M, Hsu J T. Protein purification by Dye-Ligand Chromatography. Adv Biochem Engineering 1993; 49: 1-44. Atldnson T, Hammond P, Hartwell R et al. Triazine-dye affinity chromatography. Biochem Soc Trans 1981; 9: 290-239. Svendsen P J, Weeke B, Johansson B G. Chemical solutions, equipment and general procedures. Scand J Irnmunol 1983; 17 (suppl 10): 3-20. Smith K, Sundarem T K, Kernick M, Wilkinson A E. Purification of bacterial malate dehydrogenase by selective elution from triazinyl dye affinity column. Biochem Biophys Acta 1982; 708: 17-25. Samuelson L. A thin gel layer technique for improved resolution in two-dimensional immunoelectrophoresis. Int Arch Allergy Appl Immunol 1986; 79: 140-144. Lowe C R, Pearson J C. Affinity chromatography on immobilized dyes. Methods in Enzymology 1984; 104: 97-113. Lowe C R, Burton S J, Pearson J C, Clonis Y, Stead V. Design and application of bit-mimetic dyes in biotechnology. J Chromatogr 1986; 376: 121-130. Scopes R K. Strategies for enzyme isolation using dyeligand and related adsorbents. J Chromatogr 1986; 376: 131-140. Clonis Y D. The application of reactive dyes in enzyme and protein downstream processing. Crit Rev Biotechnol 1988; 7: 4; 263-279. Muro-Pastor M I, Florencio F J. Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechcystic sp. PCC 6803. Eur J Biochem 1992; 203: 99-105. Ishii A, Suzuki M, Sahara T, Takada Y, Sasaki S, Fununaga N. Genes encoding two isocitrate dehydrogenase isoenzymes of a psychrophilic bacterium Vibrio sp. strain ABE-I. J Bacteriol 1993; 175 (21): 6873-6880. Leyland M L, Kelly D J. Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme

23.

24.

25.

26.

27.

28. 29.

30.

31.

32.

33.

34.

35.

461

specificity from the photocynthetic bacterium Rhodomicrobium vannielii. Eur J Biochem 1991; 202: 85-93. Howard R L, Becker R R. Isolation and some properties of the Triphosphopyridine nucleotide isocitrate dehydrogenase from Bacillus stearothermophilus. J Biol Chem 1970; 245(1): 3186-3194. Murphey W H, Kitto G B, Everse J, Kaplan N O. Malate dehydrogenase a survey of molecular size measured by gel filtration. Biochemistry 1967; 6(2): 603-609. Grossebtitter W, Hartl T, Gtrish H, Stezowski J J. Purification and properties of malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum. Biol Chem 1986; 367: 457-463. Sundaram T K, Wright I P, Wilkinsson A E. Malate dehydrogenase from thermophilic and mesophilic bacteria. Molecular size, subunit structure, amino acid composition, immunochemical homology and catalytic activity. Biochemistry 1980; 19:2017-2022 Daniel T M. Antibody and antigen detection for the immunodiagnosis of tuberculosis: Why not? What more is needed? Where do we stand? J Infect Dis 1988; 158: 678-680. Ridell M. Serodiagnosis of tuberculosis. Eur J Respir Dis 1988; 1: 587-588. Grange J M, Laszlo A. Serodiagnostic tests for tuberculosis: a need for assessment of their operational predictive accuracy and acceptability. Bull World Health Organ 1990; 68: 571-576. Martin Casabona N, Gonzales Fuente T, Arcalis Acre L, Otal Entraigas J, Vidal Pla R. Evaluation of phenolglycolipid antigen (PGL-Tbl) from M. tuberculosis in the serodiagnosis of tuberculosis. Acta Leprologica 1989; 7(suppll): 89-93. Ridell M, Wallerstr6m G, Minnikin D E, Bolton R C, Magnusson M. A comparative serological study of antigenic glycolipids from Mycobacterium tuberculosis. Tubercle Lung Dis 1992; 73: 101-105. Jackett P S, Bothamley G H, Batra H V, Mistra A, Young D, Ivanyi J. Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis. J Clin Microbiol 1988; 26(11): 2313-2318. Verbon A, Weverling G J, Kuijper S, Speelman P, Jansen H M, Kolk A H J. Evaluation of different tests for the diagnosis of tuberculosis and the use of likelihood ratios in serology. Am Rev Respir Dis 1993; 148: 378-384. Zheng Y J, Wang R H, Lin Y Z, Daniel T M. Clinical evaluation of the diagnostic value of measuring IgG antibody to 3 mycobacterial antigen preparations in the capillary blood of children with tuberculosis and control subjects. Tubercle Lung Dis 1994; 75: 366-370. Andersen P, Askaard D, Ljungquist L, Bennedsen J, Heron I Proteins released from Mycobacterium tuberculosis during growth. Infect Immun 1991; 59(6): 1905-1910.