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Inhibition of spiralin processing by the lipopeptide antibiotic globomycin
L Béven1, M Le Hénaff, C Fontenelle
1Equipe "Membranes et Osmorégulation," CNRS URA No. 256, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes Cedex, France.
Abstract:
The cyclic lipopeptide globomycin, a specific inhibitor of signal-peptidase II (Lsp A), proved toxic for the mollicute Spiroplasma melliferum with a minimal inhibitory concentration (MIC) in the range 6.25-12.5 microM, about one order of magnitude higher (that is, less efficient) than bee-venom mellitin. SDS-PAGE analysis of cell proteins followed by immunolabeling ("Western blotting") and by crossed immunoelectrophoresis demonstrated that the cleavage of the prespiralin leader peptide was prevented by globomycin. Cell fractionation experiments showed that prespiralin was membrane bound and did not accumulate in the cytoplasm or in the culture medium. Furthermore, the use of the potential-sensitive fluorescent dye 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (diS-C3-[5]) revealed that, in contrast to valinomycin and mellitin, globomycin up to 30 microM has no effect on the electrical transmembrane potential of S. melliferum. This indicates that the toxicity of globomycin for spiroplasma cells is mainly if not exclusively owing to the inhibition of spiralin processing. Added to previously published data, these results suggest that spiralin and probably other lipoproteins of mollicutes are acylated and membrane targeted by a mechanism involving notably the processing of the prelipoprotein precursor by a type II, globomycin-sensitive signal peptidase.
Insights
Globomycin, a signal-peptidase II inhibitor, is toxic to Spiroplasma melliferum by blocking spiralin processing. This suggests lipoproteins in mollicutes require this enzyme for acylation and membrane targeting.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mollicutes are bacteria lacking cell walls, often associated with host organisms.
- Lipoproteins are crucial for bacterial cell membrane structure and function.
- Signal peptidases are essential enzymes for protein processing and secretion.
Purpose of the Study:
- To investigate the toxicity and mechanism of action of globomycin in Spiroplasma melliferum.
- To determine the role of signal-peptidase II (LspA) in mollicute lipoprotein processing.
- To elucidate the pathway of spiralin maturation and membrane targeting.
Main Methods:
- Minimal inhibitory concentration (MIC) determination for globomycin.
- SDS-PAGE, Western blotting, and crossed immunoelectrophoresis for protein analysis.
- Cell fractionation and membrane association studies.
- Measurement of transmembrane potential using a fluorescent dye (diS-C3-[5]).
Main Results:
- Globomycin exhibited toxicity against Spiroplasma melliferum with MICs of 6.25-12.5 microM.
- Globomycin inhibited the cleavage of the prespiralin leader peptide, preventing spiralin processing.
- Prespiralin was found to be membrane-bound and did not accumulate in the cytoplasm or medium.
- Globomycin did not affect the transmembrane potential of S. melliferum cells.
Conclusions:
- Globomycin's toxicity in S. melliferum is primarily due to the inhibition of spiralin processing.
- Signal-peptidase II, targeted by globomycin, is essential for the acylation and membrane localization of spiralin and likely other mollicute lipoproteins.
- This study highlights the critical role of lipoprotein processing in mollicute viability and provides insights into antimicrobial drug targets.