The intra- and extracellular activities of 2 novel tetramethylpiperidine (TMP)-substituted phenazines, B4121 and B4128 against Mycobacterium tuberculosis H37R (ATCC 27294) were determined and compared with those of clofazimine (B663). Clofazimine, together with B4121 and B4128, were also tested for their activities against drug-resistant strains of M.tuberculosis. Both B4121 and B4128 were significantly more active than clofazimine against M.tuberculosis, including multidrug-resistant clinical strains of this microbial pathogen, demonstrating a lack of cross resistance between the riminophenazines and standard anti-tuberculous drugs. Using M.tuberculosis-infected monocyte-derived macrophages both B4121 and B4128 were found to possess intracellular activity, which was superior to that of both clofazimine and rifampicin. The relationship between anti mycobacterial action of the TMP-subsitituted phenazines and clofazimine and the effects of these agents on microbial PLA2 activity, cation (K+, Ca2+) fluxes and energy metabolism (ATP) was also investigated. PLA2 and cation fluxes were measured by radiometric procedures, while microbial ATP was assayed using a luciferin/luciferase chemiluminescence method. All 3 riminophenazines, particularly B4128 caused dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+-influx and enhancement of uptake of Ca2+. The results of kinetics studies demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapidly-occurring and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA and BAPTA, respectively, individually or in combination, did not prevent the effects of the riminophenazines on mycobacterial PLA2 (enhancement) or K+ transport (inhibition), whereas α-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake. These results demonstrated that the riminophenazine-mediated enhancement of PLA2 is a Ca2+-independent event. The involvement of PLA2 in the antimicrobial activity of the riminophenazines was supported by the observation that added, exogenous Iysophosphotidylcholine (a primary hydrolysis product of PLA2 action on membrane phospholipids) also inhibited K+ transport and growth of mycobacteria. Enhancement of endogenous PLA2 as a mechanism of riminophenazine-mediated disruption of cation transport and antimycobacterial activity was further investigated using the conventional calcium-mobilizing stimuli, calcium ionophore A23187 and thapsigargin. Both agents, but A23187 in particular caused in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Influx of Ca2+ into A23187- and thapsigargin-treated mycobacteria was observed using both radiometric and FURA-2-based spectrofluorimetric procedures. Exposure of the mycobacteria to these agents resulted in an immediate increase in uptake of Ca2+, which implies that enhancement of PLA2 activity in calcium-mobilizing stimuli-treated mycobacteria is Ca2+ dependent. In conclusion, the TMP-substituted phenazines possess anti mycobacterial properties which are superior to those of clofazimine, particularly against intraphagocytic M.tuberculosis. The superior anti mycobacterial properties of these agents is paralleled by their potentiating effects on microbial PLA2 and consequent inhibitory action on uptake of K+, particularly in the case of B4128. Mycobacterial PLA2 and K+ transporters may therefore represent novel targets for antimicrobial chemotherapy. / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2007. / Immunology / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/24462 |
| Date | 04 January 2007 |
| Creators | Matlola, Nthane Martha |
| Contributors | Anderson, Ronald, upetd@up.ac.za |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
| Rights | © 1999 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
Page generated in 0.0026 seconds