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Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide
Abstract:
The effects of polymyxin B and polymyxin B nonapeptide (PMBN) on cell envelope integrity in Escherichia coli were compared. Both compounds caused loss of proteins from E. coli K-12 3300(pBR322), although PMBN released less protein than did polymyxin B. The origin of the released protein was determined both by polyacrylamide gel electrophoresis and by using specific enzyme markers (beta-lactamase in periplasm, beta-galactosidase in cytoplasm). The proteins released by both compounds were derived principally from the periplasm, accompanied in the case of polymyxin B by a low level of cytoplasmic proteins. Although polymyxin B and PMBN both caused release of periplasmic proteins, the individual proteins released by the compounds differed. The periplasmic fraction contained six principal polypeptides with molecular weights between 62,000 (polypeptide 1) and 29,000 (polypeptide 6). Polypeptide 6 was identified as the pBR322-encoded beta-lactamase, but the other proteins were not specifically identified. Polymyxin B caused considerable release of polypeptides 1, 2, and 5 with some release of polypeptides 4 and 6. PMBN released polypeptide 1 (trace), 3, 4, and 6 (trace). Scanning electron microscopy showed that polymyxin B and PMBN both caused surface damage in E. coli. However, polymyxin B produced greater morphological changes than PMBN.
Insights
Polymyxin B and polymyxin B nonapeptide (PMBN) both damage Escherichia coli cell envelopes, releasing periplasmic proteins. Polymyxin B caused more significant protein release and cell damage than PMBN.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Polymyxins are critical antibiotics targeting Gram-negative bacteria.
- Understanding their mechanism of action on bacterial cell envelopes is crucial for antibiotic development.
- Polymyxin B nonapeptide (PMBN) is a derivative with potentially altered properties.
Purpose of the Study:
- To compare the effects of polymyxin B and PMBN on the cell envelope integrity of Escherichia coli.
- To identify the specific proteins released from the cell envelope by these compounds.
- To assess the morphological changes induced by polymyxin B and PMBN.
Main Methods:
- Utilized Escherichia coli K-12 3300(pBR322) for experiments.
- Analyzed released proteins using polyacrylamide gel electrophoresis (PAGE).
- Employed enzyme markers (beta-lactamase, beta-galactosidase) to determine protein origin.
- Observed cell surface morphology via scanning electron microscopy (SEM).
Main Results:
- Both polymyxin B and PMBN induced protein release from E. coli, primarily from the periplasm.
- Polymyxin B caused a greater overall protein release, including some cytoplasmic proteins, compared to PMBN.
- Specific periplasmic polypeptides were differentially released by each compound, with PMBN causing less release.
- SEM revealed surface damage, with polymyxin B inducing more pronounced morphological changes.
Conclusions:
- Polymyxin B and PMBN disrupt E. coli cell envelope integrity, leading to protein leakage.
- PMBN appears to be less disruptive to the cell envelope than polymyxin B.
- The differential release of periplasmic proteins suggests distinct interactions or mechanisms of action between polymyxin B and PMBN.