ABSTRACT
A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatography and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death.
Subject(s)
Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Mycobacterium bovis/drug effects , Mycobacterium bovis/metabolism , Proteome/analysis , Proteomics/methods , Bacterial Proteins/analysis , Cell Wall/metabolism , Chromatography, Liquid , Lipid Metabolism , Mass Spectrometry , Proteome/drug effectsABSTRACT
The synthesis and antimycobacterial activity of a series of alpha-methylene-gamma-butyrolactones based on the natural product protolichesterinic acid are described. Compounds 9-12 bearing an allylamide group at the C-4 position showed improved activity with MICs in the range of 6.25-12.5 microg/mL.
Subject(s)
4-Butyrolactone/analogs & derivatives , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Microbial Sensitivity Tests , Mycobacterium bovis/drug effects , Structure-Activity RelationshipABSTRACT
OBJECTIVE: To determine the effect on BCG of n-octanesulphonylacetamide (OSA), a novel compound of the class beta-sulphonylcarboxamides, which has potent in vitro activity against pathogenic mycobacteria. METHODS AND RESULTS: The effect of OSA in BCG was examined using two-dimensional protein electrophoresis. Treatment of BCG with OSA resulted in overexpression of two proteins identified as the b-subunit of ATP synthase (Rv1306) and a 17 kDa heat shock protein (Rv0251c). [35S]Methionine pulse-labelling revealed that overexpression occurred within as little as 3.5 h post-exposure. These results were confirmed by RT-PCR. ATP levels decreased in OSA-treated BCG at 5 min, and 1, 3 and 24 h, with a 64%, 45%, 54% and 73% reduction in ATP, respectively. Only dicyclohexylcarbodiimide (DCCD), a known ATP synthase inhibitor, had a similar effect. No appreciable difference in ATP level was observed in BCG treated with standard antimycobacterial drugs, additional respiratory chain inhibitors or a fatty acid synthase inhibitor at a comparable time-point. Protein synthesis decreased within 5 min of exposure to OSA (56%), DCCD (74%) and thenoyltrifluoroacetone (TTFA) (77%). Ethanol (2.3%) potentiated the activity of OSA. In contrast, no synergic effect was observed with streptomycin and ethanol. Mycolic acid levels decreased 79% with DCCD, 46% with TTFA, a complex II inhibitor, and 43% with OSA compared with untreated controls. CONCLUSIONS: Our results suggest that OSA may interfere directly or indirectly with ATP synthase and possibly other components of the mycobacterial respiratory chain. These effects may hinder energy production, leading to interruption in the synthesis of large macromolecules including proteins and mycolic acids.