Metabolism of Mycobacterium leprae in macrophages

Abstract

The incorporation of "4C-labeled palmitic acid ([U-14C]PA) into the phenolic glycolipid-I (PGL-I) fraction of Mycobacterium keprae was studied in a murine macrophage system in vitro. Peritoneal macrophages from Swiss Webster mice were infected with fresh viable or Formalin-killed M. leprae harvested from infected foo,pads of nulnu mice, and [U-"'C]PA was added to the culture medium. Labeled glycolipid synthesized by live M. leprae was fractionated on a Florisil-silicic acid column and identified as PGL-I by using thin-layer chromatography and localization on a polysulfone membrane with an anti-PGL-I monoclonal antibody. Increased incorporation of (U-"'C]PA into the PGL-I fraction was observed in macrophages infected with only live M. leprae. Treatment of the infected macrophages with rifampin caused a significant reduction in the incorporation of palmitic acid into PGL-I. These preliminary studies suggest that PGL-I synthesis can be used to quantitate the metabolism of M. keprae in macrophages in vitro.The inability to rapidly quantitate the viability of Mycobacterium leprae has vastly impeded progress in leprosy research. To monitor the viability of the noncultivable leprosy bacillus, tedious indirect titrations employing the mouse footpad technique developed by Shepard (21) are required, often taking a year or more to complete (13). Clearly, to more easily screen antileprosy chemotherapeutic regimens or to study host resistance factors in leprosy there is a need to develop alternate and more rapid techniques for determining the viability of M. leprae.M. leprae is a natural intracellular parasite that depends on the internal milieu of the host cell for survival. Within the host cell, M. leprae synthesizes large amounts of a characteristic phenolic glycolipid (PGL-I) which has been shown by Brennan and colleagues to be biochemically unique and immunologically specific for M. leprae (1,9,10). Since mononuclear phagocytes have been shown to serve as important host cells for M. leprae, the present study explores the biosynthesis of PGL-I by the leprosy bacillus in tissue cultures of mouse peritoneal macrophages. The in vitro biosynthesis of PGL-I from 14C-acetate by M. leprae within schwannoma cell lines has been reported by Mukherjee et al. (16). In our studies PGL-I synthesis was monitored in vitro by measuring the incorporation of radiolabeled palmitic acid (PA) into the extractable PGL-I fraction of M. leprae in infected macrophages. The present work describes the methodology developed and reports our preliminary findings on the effects of rifampin on PGL-I synthesis by M. leprae in vitro. MATERIALS AND METHODSMacrophages. Peritoneal cells (PC) were harvested from normal unstimulated Swiss Webster mice by using methods described previously (12). Briefly, PC harvested in heparinized Hanks balanced salt solution containing 10 U of heparin per ml were washed by centrifugation (150 x g for 10 min) and suspended in medium RPMI 1640 (GIBCO Laboratories, Chagrin Falls, Ohio) supplemented with 20% heatinactivated fetal ...

Competency of human-derivedMycobacterium lepraeto use palmitic acid in the synthesis of phenolic glycolipid-I and phthiocerol dimycocerosate and to release C02in axenic culture

“…Utilization of radioactive palmitic acid as a substrate for metabolic actIvItIes of armadillo-or nude mouse-derived M. leprae has been demonstrated in axenic cultures7-1 2 as well as a cell-culture system. 13 In the axenic culture systems, Franzblau7 demonstrated the capacity of nude mouse-derived M. leprae to oxidize palmitic acid and release CO 2 .…”

Mycobacterium leprae

“…Among the mycobacteria, M. leprae has a unique pattern of drug susceptibilities (Shepard, 1971;Shepard et al, 1983;Hastings and Franzblau, 1988). Thus, one could confirm a presumptive identification by measuring drug susceptibility in: 1) inhibition of in vivo growth in the mouse footpad (Shepard, 1971); 2) inhibition of growth in macrophage culture (Mittal et al, 1983;Ramasesh et al, 1987); or 3) effect of the drug on PGL-I synthesis, palmitate oxidation, or ATP generation in bacilli maintained in a synthetic medium (Franzblau, 1988;. Note that the bacilli do not multiply in the systems used by Franzblau and colleagues.…”

“…This test is not entirely specific for M. leprae, since some non-acid-fast bacteria and, more importantly, some mammalian tissues have similar oxidase (phenolase) activities (Prabhakaran, 1967). PGL-I biosynthesis can be followed by measuring the incorporation of 14 Cpalmitate into PGL-I in an M. leprae/macrophage co-culture system (Ramasesh et al, 1987) or in bacilli maintained in a synthetic medium . Since PGL-I is unique to M. leprae, this assay is particularly useful.…”

Axenic incorporation of [U-14C]palmitic acid into the phenolic glycolipid-I of Mycobacterium leprae

“…Mukherjee et al [7] reported on the incorporation of [14C]acetate into PGL-I in M. leprae-infected Schwannoma cells. More recently, Ramasesh et al [8] described the incorporation of [U-14C]palmitic acid ([14C]PA) into the PGL-I of M. leprae residing within mouse peritoneal macrophages. While these systems are undoubtedly valuable for studying host parasite interactions, they preclude determining the effects of physicochemical parameters on PGL-I synthesis due to the intracellular location of the bacillus.…”

“…Addition of unlabelled palmitic acid to viable bacilli had no effect at concentrations ~< 25 /~g/ml but blocked label incorporation by 36% at 50 t~g/ml (data not shown). The identity of the labelled material was confirmed by chromatography on a polysulfone membrane [14], reaction with a specific IgG monoclonal antibody to PGL-I and detection by im- munoperoxidase staining as described by Ramasesh et al [8]. None of the other radiolabelled substrates tested were incorporated into the PGL-I fraction.…”