© INSTITUTPASTEUR/ELsEVIEk Paris 1992
Re3. Microbial.
1992, 143, 519-524
Thin-layer chromatography systems for the identification of Myeobacterium tuberculosis, M. bovis BCG, M. kansas/i, M. gastri and M. marinum A. Laszlo ( l } F. Papa (2) and H.L. David ~2~ ¢o Mycohacteriology, Laborat'>ry Centre f o r Disease Control, Ottawa, and :z) Unitd de la tuberculose et des Myc~bactdries, lnstitul Pasteur, 75724 Paris Cedex 15
SUMMARY Knowledge of mycobacterial glyc~lipid antigens and the study of their specificity have resulted in their utilization as species markers. We describe a thin-layer chromatography method which could serve as a useful adjunct for the identification of Mycobacteriurn tuberculosis, M. bovia BCG, M. kansas~i, M. gastri and M. mar/num.
Key-words: Glycol/p/d, Mycghacterium. Tuberculosis; Antigens, Phenolglvcotipids. TLC, $peciation.
IN'[RODUCTION Phenolglycolipids (PGL) have been extensiveiy studied (Randall and Smith, 1953; Smith et aL, 1957, 1960a, |960b ; Gastambide-Odier and Sarda, 1970; Gastambide-Odier and Vill~, 1970; Sarda and Gastambide-Odier, 1967; Viii6 and Gastambide-Odier, 1970). The chemical structures of these myeobacterial substances also known as "mycosides" were identified as phenolphthiocerol dimycocerosates. These structures, differing only by the length and nature of their terminal oligosaccharide chains (table 1), seemed to be confined to a select group of mycobacteria comprising M y c o b a c t e r i u m boris BCG, M . m a r / h u m and M . kansasiL Initial interest in these substances waned and they were all but forgotten in the 1970's.
Submitted November 13, 1991, accepted April 29, 1992.
Interest was rekindled in 1981 following the discovery of a species-specific antigen of M. leprae belonging to the category of the PGL (Hunter and Brennan, 198t). In order to avoid possible confusion with the peptidoglyeolipids of the M. a v i u m complex (mycosides C), the designation of phenolglycolipid (or PGL) was proposed (Hunter and Brennan, 1981).
PGL-BI from M. boris BCG The reported structure included the monosaccharide 2-o-met hyl-ft-D-rhamnopyranosyl (Demarteau-Ginsburg and Lederer, 1963). in a recent reinvestigation, it was established that this glycosyl substituent is rather a 2-o-methyl-a-T rhamnopyranosyl (Daff6 et eL, 19~8; Chatter-
A. L A S Z L O E T AL.
52O
jee et el., 1989). This PGL can be found in all strains of M. bovis BCG and can easily be characterized by thin.layer chromatography (TLC) (Jarnagin et al., 1983). PLG.G! from M, m a r i n u m First discovered in 1965 (Navaikar et al., 1965), M, m a r i n u m ' s PGL was later recognized as a monoglycosylated dimycoeerosyl phenolphthlocerol (Sarda and Gastambide-Odier, 1967), The monosaccharide was identY.fied as a 3-o-methyt-a-L-rhamnopyranosyl (Vill6 and Gastambide-Odier, 19"10). A recent study (Cruaud et aL, 1987) demonstrated that all strains of M . mo.rinum examined, except type strain (ATCC 927), synthesize PGL-G1, the presence of which can easily be demonstrated by TLC.
P G L - K I from M. kansasii and M. gastri
This PGL, originally known as "royceside A " ) was discovered in M . Icansasii (Gastambide-Odier and Sarda, 1970). Its oligosaccharide component is made up of 3 sugars (Gastambide-Odier and Vill6, 1970). New technology and the development of more sophisticated analytical tools has enabled a redefinition of its structure (Fourni6 et at., 1987a, 1987b). It was shown that the oligosaccharide moiety is a tetrasaccharide rather than a trisacearide (table I); this new structure was named PGL-KI. FurtheT investigation has shown that PGL-KI is a species-specific antigen (Papa et al., 1987). -mmunologic study of PGL-KI (Papa et aL, 1987, 1988) has shown that there is a similarity between PGL produced by strains or M. icansasii and that produced by strains of M. gastrL
Subsequently, the chemical identity of these structures has been confirmed (Vercellone et aL. 1988). The abovementioned PC-L's are present in mycobacterlal strains that are frequently found in the clinical setting and which sometimes represent a diagnostic problem for the bacteriologi.qt. The synthesis of PGL is a stable characteristic which constitutes a choice marker for the identification of a species. Therefore, the purpose of this work is to describe a simple method using TLC for the characterization of these substance~ which, in turn, will of be invaluable help in the identification of M, tuberculosis, M . bovis BCG, M . kansasii, M. gastri and M . m a r i n u m .
MATERIALS AND METHODS Bacteria
The following mycobaeterial strains were used as a source of PGL or sutphatide: M. tuberculosis, Canetti strains (CIPT 14001.0059); M. bovis BCG (CIPT 14.004.0001); M. kansasii (ATCC 12478); M. gastri (CIPT 14034 0003); M. marinum (CIPT 14012 0006). PGL-Tbl from hi. tuberculosis The discovery of species-specific glycolipid antigens in some mycohacteria generated the hope of finding similar substances in M. tuberculosis. No species-specific glycolipids were found i- ?4. tuber culosis type strain H37Rv (Hunter et al., 1986). However, a more recent study identified PGL in the M. tuberculosis Canetti strain (Daff6 et al,, 1987). The structure of the major phenolic glycolipid (which constitutes the marker of the Canetti strain), was described as a triglycoi.dimycoserosyl-phenolphthioceroi. The trisaccharide moiety is a l-3dinked tri-o-Me-Fucp-Rhap and 2-o-Me-Rhap. Another PGL with a structure identical to PGL-BI (mycoside B) was also found in small amounts in the Canetti stlain (Daff~ et al., 1988).
l.-J ATCC BV CIPT Fucp t-Iegp
= = = -
A m e r i r a n T y p e Cu]tu~e CoH~czion. bed vglU,-'3e C~i~¢clion Institul Fas~*.ur, TubercuIose. fucopyranosyl. hexanopyranosYl.
Me PGL Rhap SL-I
~ = ~
L o w e n s l e i n - J c n ~ e n (xlledlum). methyl.
phe~olglycolipid, rhamnopyraaosy]. s u l p h a t i d ¢ 1.
T L C S Y S T E M S F O R M Y C O B A C T E R I U M I D E N T I k l C A T,tON
521
"t'able I. Structures o f m a j o r P G L from Mycobacteriunt species.
M. boris (Chatterjce et aL, 1989; Daff6 et aL, 1988) 2-o-Me-,x- L-Rhap- (I ~pher.ol-dimycocerosyL plnhiocc;ol) M. marinum (Viii6 and Gastambide-Odier, 1970) 3-o-Me-~x-L-Rhap-( I--*phenol-dimycoeerosyI phthioceroi) M. leprae (Hunter and Brennan, 198t) 3,6-di-o-Me-~D-Glep-(l --, 4)-2,3-di-o-Me-~-L-Rhap-(I --, 4)-3-o-Me-a-L~Rhap-(l ~ phenol-dimyeocerosyl phlhioeerol) M. kansasii and gastri (Fourni6 et al., 1987a, 1987b) 2,6-dideoxy-4-o-Me-,x-arabino-hexp-(t--*3)-4-o- Ae-2-o-Me-~x-Fucp-2-o- Me-~- Rhap-( 1 ,3)-2,4-di-o-Me-=Rhap-( I--,phenoi-dimycocero~yl phthioeerol) M, tuberculosis (Daff6 et al., t987) 2~3~4-tri-~-M~-~-L-Fuep-( ~ 3 )-~-L-Rhap-( ~~3)~2~-Me-~'-L-Rhap-( ~ p h e n ~ - d i m y c ~ e r ~ s y ~ phthiocerol) M. haemophilum (Bersa et al., 19.'90, 1991) 2,3-di-o-Me-0cL-Rhap-(l ~ 2)-3-o-Me
Re3. Microbial.
1992, 143, 519-524
Thin-layer chromatography systems for the identification of Myeobacterium tuberculosis, M. bovis BCG, M. kansas/i, M. gastri and M. marinum A. Laszlo ( l } F. Papa (2) and H.L. David ~2~ ¢o Mycohacteriology, Laborat'>ry Centre f o r Disease Control, Ottawa, and :z) Unitd de la tuberculose et des Myc~bactdries, lnstitul Pasteur, 75724 Paris Cedex 15
SUMMARY Knowledge of mycobacterial glyc~lipid antigens and the study of their specificity have resulted in their utilization as species markers. We describe a thin-layer chromatography method which could serve as a useful adjunct for the identification of Mycobacteriurn tuberculosis, M. bovia BCG, M. kansas~i, M. gastri and M. mar/num.
Key-words: Glycol/p/d, Mycghacterium. Tuberculosis; Antigens, Phenolglvcotipids. TLC, $peciation.
IN'[RODUCTION Phenolglycolipids (PGL) have been extensiveiy studied (Randall and Smith, 1953; Smith et aL, 1957, 1960a, |960b ; Gastambide-Odier and Sarda, 1970; Gastambide-Odier and Vill~, 1970; Sarda and Gastambide-Odier, 1967; Viii6 and Gastambide-Odier, 1970). The chemical structures of these myeobacterial substances also known as "mycosides" were identified as phenolphthiocerol dimycocerosates. These structures, differing only by the length and nature of their terminal oligosaccharide chains (table 1), seemed to be confined to a select group of mycobacteria comprising M y c o b a c t e r i u m boris BCG, M . m a r / h u m and M . kansasiL Initial interest in these substances waned and they were all but forgotten in the 1970's.
Submitted November 13, 1991, accepted April 29, 1992.
Interest was rekindled in 1981 following the discovery of a species-specific antigen of M. leprae belonging to the category of the PGL (Hunter and Brennan, 198t). In order to avoid possible confusion with the peptidoglyeolipids of the M. a v i u m complex (mycosides C), the designation of phenolglycolipid (or PGL) was proposed (Hunter and Brennan, 1981).
PGL-BI from M. boris BCG The reported structure included the monosaccharide 2-o-met hyl-ft-D-rhamnopyranosyl (Demarteau-Ginsburg and Lederer, 1963). in a recent reinvestigation, it was established that this glycosyl substituent is rather a 2-o-methyl-a-T rhamnopyranosyl (Daff6 et eL, 19~8; Chatter-
A. L A S Z L O E T AL.
52O
jee et el., 1989). This PGL can be found in all strains of M. bovis BCG and can easily be characterized by thin.layer chromatography (TLC) (Jarnagin et al., 1983). PLG.G! from M, m a r i n u m First discovered in 1965 (Navaikar et al., 1965), M, m a r i n u m ' s PGL was later recognized as a monoglycosylated dimycoeerosyl phenolphthlocerol (Sarda and Gastambide-Odier, 1967), The monosaccharide was identY.fied as a 3-o-methyt-a-L-rhamnopyranosyl (Vill6 and Gastambide-Odier, 19"10). A recent study (Cruaud et aL, 1987) demonstrated that all strains of M . mo.rinum examined, except type strain (ATCC 927), synthesize PGL-G1, the presence of which can easily be demonstrated by TLC.
P G L - K I from M. kansasii and M. gastri
This PGL, originally known as "royceside A " ) was discovered in M . Icansasii (Gastambide-Odier and Sarda, 1970). Its oligosaccharide component is made up of 3 sugars (Gastambide-Odier and Vill6, 1970). New technology and the development of more sophisticated analytical tools has enabled a redefinition of its structure (Fourni6 et at., 1987a, 1987b). It was shown that the oligosaccharide moiety is a tetrasaccharide rather than a trisacearide (table I); this new structure was named PGL-KI. FurtheT investigation has shown that PGL-KI is a species-specific antigen (Papa et al., 1987). -mmunologic study of PGL-KI (Papa et aL, 1987, 1988) has shown that there is a similarity between PGL produced by strains or M. icansasii and that produced by strains of M. gastrL
Subsequently, the chemical identity of these structures has been confirmed (Vercellone et aL. 1988). The abovementioned PC-L's are present in mycobacterlal strains that are frequently found in the clinical setting and which sometimes represent a diagnostic problem for the bacteriologi.qt. The synthesis of PGL is a stable characteristic which constitutes a choice marker for the identification of a species. Therefore, the purpose of this work is to describe a simple method using TLC for the characterization of these substance~ which, in turn, will of be invaluable help in the identification of M, tuberculosis, M . bovis BCG, M . kansasii, M. gastri and M . m a r i n u m .
MATERIALS AND METHODS Bacteria
The following mycobaeterial strains were used as a source of PGL or sutphatide: M. tuberculosis, Canetti strains (CIPT 14001.0059); M. bovis BCG (CIPT 14.004.0001); M. kansasii (ATCC 12478); M. gastri (CIPT 14034 0003); M. marinum (CIPT 14012 0006). PGL-Tbl from hi. tuberculosis The discovery of species-specific glycolipid antigens in some mycohacteria generated the hope of finding similar substances in M. tuberculosis. No species-specific glycolipids were found i- ?4. tuber culosis type strain H37Rv (Hunter et al., 1986). However, a more recent study identified PGL in the M. tuberculosis Canetti strain (Daff6 et al,, 1987). The structure of the major phenolic glycolipid (which constitutes the marker of the Canetti strain), was described as a triglycoi.dimycoserosyl-phenolphthioceroi. The trisaccharide moiety is a l-3dinked tri-o-Me-Fucp-Rhap and 2-o-Me-Rhap. Another PGL with a structure identical to PGL-BI (mycoside B) was also found in small amounts in the Canetti stlain (Daff~ et al., 1988).
l.-J ATCC BV CIPT Fucp t-Iegp
= = = -
A m e r i r a n T y p e Cu]tu~e CoH~czion. bed vglU,-'3e C~i~¢clion Institul Fas~*.ur, TubercuIose. fucopyranosyl. hexanopyranosYl.
Me PGL Rhap SL-I
~ = ~
L o w e n s l e i n - J c n ~ e n (xlledlum). methyl.
phe~olglycolipid, rhamnopyraaosy]. s u l p h a t i d ¢ 1.
T L C S Y S T E M S F O R M Y C O B A C T E R I U M I D E N T I k l C A T,tON
521
"t'able I. Structures o f m a j o r P G L from Mycobacteriunt species.
M. boris (Chatterjce et aL, 1989; Daff6 et aL, 1988) 2-o-Me-,x- L-Rhap- (I ~pher.ol-dimycocerosyL plnhiocc;ol) M. marinum (Viii6 and Gastambide-Odier, 1970) 3-o-Me-~x-L-Rhap-( I--*phenol-dimycoeerosyI phthioceroi) M. leprae (Hunter and Brennan, 198t) 3,6-di-o-Me-~D-Glep-(l --, 4)-2,3-di-o-Me-~-L-Rhap-(I --, 4)-3-o-Me-a-L~Rhap-(l ~ phenol-dimyeocerosyl phlhioeerol) M. kansasii and gastri (Fourni6 et al., 1987a, 1987b) 2,6-dideoxy-4-o-Me-,x-arabino-hexp-(t--*3)-4-o- Ae-2-o-Me-~x-Fucp-2-o- Me-~- Rhap-( 1 ,3)-2,4-di-o-Me-=Rhap-( I--,phenoi-dimycocero~yl phthioeerol) M, tuberculosis (Daff6 et al., t987) 2~3~4-tri-~-M~-~-L-Fuep-( ~ 3 )-~-L-Rhap-( ~~3)~2~-Me-~'-L-Rhap-( ~ p h e n ~ - d i m y c ~ e r ~ s y ~ phthiocerol) M. haemophilum (Bersa et al., 19.'90, 1991) 2,3-di-o-Me-0cL-Rhap-(l ~ 2)-3-o-Me
