Enzyme Patterns in the Study of Leptospira1

Abstract

An analysis of intracellular and extracellular leptospiral enzymes was made by use of starch-gel electrophoresis with natural and synthetic substrates. Of 37 serotypes examined for extracellular exterase, all had activity of varying mobility and degree. All extracellular preparations were negative for catalase, phosphatase, and naphthylamidase. Intracellularly, five serotypes were examined, including Leptospira biflexa Patoc I, L. biflexa Waz, L. canicola Moulton, L. icterohaemorrhagiae RGA, and L. pomona S91. Among the enzymes detected by this electrophoretic technique were transaminase and catalase, confirming the results of previous investigators. Further, other enzymes heretofore unreported have been detected. These include esterases, phosphatases, lactic, malic, glutamic, succinic, a-glycerophosphate, and 6-phosphogluconic dehydrogenases, and a naphthylamidase. The presence of these enzymes suggests the existence of tricarboxylic acid, glycolytic, and pentose-related pathways in Leptospira. In addition, enzyme patterns show promise in leptospiral classification. Recent investigations have established the presence of the following enzymes in leptospires: catalase (14, 35), oxidase (13, 20), transaminase (27), and lipase (2, 23, 32). Phosphatase activity was determined by Dymowska and Zakrzewska (9) in culture filtrates of leptospires grown in Korthoff's medium, and results were equivocal. This study was concerned with a broad examination of leptospiral enzymes to (i) gain insight into metabolic sequences, (ii) relate enzyme patterns to classification and taxonomy as has been done for other bacteria (3, 25, 31), and (iii) look for enzymatic differences between avirulent and virulent serotypes of leptospires. MATERIALS AND METHODS Cultures. In this study, 37 strains of leptospires were used (Table 1). All were grown initially in Stuart's (38) medium supplemented with 10% rabbit serum and 0.15% agar. Prior to enzymatic studies, all cultures were grown for two transfers in albumin-Tween 80 liquid medium (10, 11). Before mass cultivation procedures were initiated, the cultures were washed and used as the inocula for extracellular and intracellular enzyme studies. Extracellular enzyme preparation. Cell-free culture 'This is a portion of a dissertation submitted by Stanley S. Green to the faculty of the University of Missouri Graduate School in partial fulfillment of the requirements for the Ph.D. degree in Micro

Enzymatic degradation of H2O2 by Leptospira

“…Media, cultivation, and harvest. Cells were grown in a synthetic medium, SM-7, composed of the following: NaCl, 8 Cells were grown without shaking at 300C in 10-ml volumes in test tubes (20 by 150 mm) with Mortontype caps. Bacteria were harvested when the growth achieved a turbidity of 40 to 50 nepholometer units 672 using a Coleman 9 nephocolorimeter adjusted to 20 nepholometer units with a Coleman 81 nepholos standard, which corresponded to approximately 108 cells/ml.…”

“…Faine reported that a catalase-inactive strain of serovar icterohaemorrhagiae did not produce peroxidase. Green et al (8) found catalase present in the pathogenic canicola Moulton and pomona S-91 strains but absent in the pathogenic icterohaemorrhagiae RGA and L. biflexa strains patoc I and Waz. Baseman and Cox (2) reported a pyrogallol-oxidizing activity in the aquatic isolate L. biflexa B-16.…”

Intermediate Energy Metabolism of Leptospira

“…Such findings might also explain the inability to detect phosphotransacetylase, and lactate and ethanol dehydrogenases, since substrates or products of reactions catalyzed METABOLISM OF LEPTOSPIRA by the latter enzymes might not be further metabolized. Green et al (15) suggested that the acid phosphatase which they found in certain leptospiral serotypes might convert glycerol to glycerol-lphosphate, and that glycerol-i-phosphate would then be metabolized through the glycolytic pathway. No evidence was presented by these authors to support such a role.…”

“…enzyme absent from leptospiral extract; (--->) enzyme not assayed, but presence of enzyme suspected; (--X-->) enzyme not assayed, but presence of enzyme doubted. serotypes examined by Green et al (15) was determined by starch-gel electrophoretic patterns of leptospiral extracts in the presence of lactate and indicator dye. Inability to measure this dehydrogenase in the three strains examined in this study requires further comment.…”

Naphthylamidase Activity of Leptospira1

“…Leptospira serotypes were cultured, and intracellular enzymes were extracted as described previously (5). The method of Goldbarg and Rutenberg (4) was employed to determine naphthylamidase activity for each of the strains used, since this technique is more sensitive than the electrophoretic procedure used in the earlier work.…”