Pharmacokinetic of ALA and h-ALA induced porphyrins in the models Mycobacterium phlei and Mycobacterium smegmatis.

Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins. It is also important to know the distribution of the photosensitizer in bacteria at the microscopic level. The present results examine the accumulation of photosensitizers by Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. The kinetics of porphyrin synthesis after treatment with the precursors ALA and h-ALA were studied. The goal was to describe the biosynthesis and the pharmacokinetics of sensitizers in both bacterial strains using fluorescence microscopy and spectroscopy. We could show that both Mycobacterium strains enrich porphyrins after ALA and h-ALA administration detected by fluorescence peaks at about 620nm. By HPLC analyses the major porphyrin could be identified as coproporphyrin. In the future we will apply the new knowledge in in vitro and in vivo experiments to strains of M. tuberculosis, M. leprae and M. bovis and examine cell destruction by PDI.

Restoration of active transport of solutes and oxidative phosphorylation by naphthoquinones in irradiated membrane vesicles from Mycobacterium phlei.

Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK(9)(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K(1), menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K(1). Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca(2+). These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation.

Ultraviolet susceptibility of BCG and virulent tubercle bacilli.

To test the effectiveness of irradiating the upper air of a room with ultraviolet light at reducing the concentration of airborne tubercle bacilli, the susceptibility to the germicidal effects of ultraviolet light, Z, was determined for various mycobacteria. Virulent tubercle bacilli and bacille Calmette-Guérin (BCG) were equally susceptible to ultraviolet radiation, whereas Mycobacterium phlei had 10 times their resistance (Z, approximately one-tenth that for M. tuberculosis). The effectiveness against BCG of upper air ultraviolet irradiation in a room was tested directly by nebulizing BCG into the air of the room and monitoring its rate of disappearance. With one 17-watt fixture operating, the rate of disappearance increased 6-fold; with 2 fixtures operating (46 watts total), the rate of disappearance increased 9-fold. This implies that under steady-state conditions, the concentrations of airborne organisms with ultraviolet light(s) on would have been one-sixth and one-ninth, respectively. The increase in rate of decay of the airborne organisms using 1 fixture was equivalent to 10 air changes per hour, whereas that using 2 fixtures was approximately 25 air changes per hour (range: 18 to 33 air changes per hour). These increments are less than those reported previously for Serratia marcescens, because the Z value for BCG is approximately one-seventh that for serratia. These findings with BCG are believed to be directly applicable to virulent tubercle bacilli.

Different mechanisms of energy coupling for transport of various amino acids in cells of Mycobacterium phlei.

Whole cells of Mycobacterium phlei were shown to actively accumulate proline, leucine, lysine, tryptophan, histidine, glutamine, and glutamic acid to different steady state levels. The transport of proline, in contrast to that of other amino acids, was found to be insensitive to various respiratory inhibitors, e.g. cyanide, arsenate, azide, and sulfhydryl reagents. However, oxygen was an obligatory requirement for the uptake of proline, as well as for the other amino acids. The results indicate that the energy requirements for proline uptake are different from those of other amino acids. In contrast to the system from Escherichia coli, the mode of energy transduction for the uptake of proline, glutamine, and glutamic acid is different even though these amino acids are shock resistant in the M. phlei system.