Identification of acidic, alkaline, and neutral sphingomyelinase activities in Mycobacterium tuberculosis.
BACKGROUND
Sphingomyelinase enzymes are pathogenic factors of several intracellular bacteria species, which have been little studied in Mycobacterium tuberculosis.


METHODS
Cell free extracts from H37Rv and CDC-1551 M. tuberculosis strains were assayed for sphingomyelinase activity by using [N-methyl-14C]-sphingomyelin as substrate. Double-directional thin-layer chromatography was used to separate the substrate and hydrolysis product. Sphingomyelinase activity was analyzed as a function of incubation time, dose, pH and the presence of MgCl2, CaCl2, ZnSO4, HgCl2, MnCl2, CoCl2 and EDTA (1 or 10 mM).


RESULTS
Mycobacterial preparations hydrolyzed [14C]-sphingomyelin, in time- and dose-dependent manners, producing [14C]-phosphorylcholine as a unique product. The activity of H37Rv neutral sphingomyelinase at pH 7.5 was 2.15 times higher than that of CDC-1551. This activity was inhibited 21-82% by Ca2+, Hg2+ or Zn2+ and EDTA, and stimulated 40-117% by Mn2+ and Mg2+. In addition, preparations from both strains showed two peaks of sphingomyelinase, one at pH 5.5 and the other at pH 3.0. However, these activities were 4-22 times lower than that observed at pH 7.5 for strain H37Rv. Preparations from H37Rv, but not those of CDC-1551, hydrolyzed sphingomyelin at pH 8-9, with a specific activity similar to that of the neutral CDC-1551 enzyme.


CONCLUSIONS
Both strains H37Rv and CDC-1551 of M. tuberculosis have cation-dependent acidic and neutral sphingomyelinase-C enzymes, showing the neutral as the major activity. In addition, H37Rv has an alkaline sphingomyelinase-C. The importance of SMases in M. tuberculosis pathogenesis remains to be elucidated.
Vargas-Villarreal
Javier
J
Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, División de Biología Celular y Molecular, Monterey, N.L, Mexico.
Mata-Cárdenas
Benito D
BD
Deslauriers
Manon
M
Quinn
Frederick D
FD
Castro-Garza
Jorge
J
Martínez-Rodrĺguez
Herminia G
HG
Said-Fernández
Salvador
S
D001426:Bacterial Proteins / Q000378:metabolism; D002474:Cell-Free System; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000472:pathogenicity; D009994:Osmolar Concentration; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014774:Virulence
D001426:Bacterial Proteins; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase