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Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease.

ABSTRACT: Microcin E492 (MccE492) is a bactericidal protein secreted by Klebsiella pneumoniae that is active against various species of Enterobacteriaceae. Interaction of MccE492 with target cells leads to the depolarization and permeabilization of their inner membranes. Several MccE492-specific proteins are required for the maturation and secretion of active MccE492. Surprisingly, the expression of only MceA, the polypeptide backbone of MccE492, is shown here to be toxic by itself. We refer to this phenomenon as endogenous MceA bactericidal activity to differentiate it from the action of extracellularly secreted MccE492. The toxicity of endogenous MceA is enhanced by an efficient targeting to the inner membrane. However, a periplasmic intermediate state is not required for MceA toxicity. Indeed, endogenous MceA remains fully active when it is fused to thioredoxin-1, a fast-folding protein that promotes retention of the C terminus of MceA in the cytoplasm. The C-terminal domain of MccE492 is required only for delivery from the extracellular environment to the periplasm, and it is not required for inner membrane damage. A common component is absolutely essential for the bactericidal activity of both endogenous MceA and extracellular MccE492. Indeed, toxicity is strictly dependent on the presence of ManYZ, an inner membrane protein complex involved in mannose uptake. Based on these findings, we propose a new model for cell entry, inner membrane insertion, and toxic activity of MccE492.

SUBMITTER: Bieler S

PROVIDER: S-EPMC1636244 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease.

Bieler Sylvain S Silva Filo F Soto Claudio C Belin Dominique D

Journal of bacteriology 20061001 20

Microcin E492 (MccE492) is a bactericidal protein secreted by Klebsiella pneumoniae that is active against various species of Enterobacteriaceae. Interaction of MccE492 with target cells leads to the depolarization and permeabilization of their inner membranes. Several MccE492-specific proteins are required for the maturation and secretion of active MccE492. Surprisingly, the expression of only MceA, the polypeptide backbone of MccE492, is shown here to be toxic by itself. We refer to this pheno ...[more]

PMID: 17015644

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Project description:Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4'-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

Dataset Information

Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease.

Publications

Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease.

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