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.

FIG. 1.

(A) Microcin E492 gene cluster from plasmid pJEM15. Open reading frames are represented by arrows. The restriction sites relevant for plasmid constructions are indicated. (B) Amino acid sequence of mature 84-residue MceA. The positions of the stop codons inserted to generate the C-terminally truncated MceA mutants are indicated (-16C, -11C, -6C, and -1C). The posttranslational modification of the C-terminal serine is symbolized by an asterisk. The original ABC signal sequence of preMceA and the signal sequence of PhoA73-MceA are shown underneath. The mutated amino acid in PhoA73-MceA (Q14) is underlined. The signalless construct (Δss-MceA) consists of a methionine residue followed by the mature portion of preMceA.

FIG. 2.

Bactericidal activity of endogenous MceA in the absence of all the other proteins encoded by the MccE492 gene cluster. (A and B) Serial dilutions of overnight cultures of DB503 (rpoA⁺) with pBAD101 (vector), pAB12 (Δss-MceA), or pAB11 (PhoA73-MceA) and of DB512 (rpoA341) with pBAD101 (vector), pAB12 (Δss-MceA), or pAB11 (PhoA73-MceA) were spotted (5 μl) on LB plates without arabinose (A) or with 0.2% arabinose (B) and incubated overnight at 37°C. Results shown here are representative of three independent experiments. (C) Viability assay of cultures of DB503 (rpoA⁺) with pBAD101 (vector), pAB12 (Δss-MceA), or pAB11 (PhoA73-MceA) and of DB512 (rpoA341) with pBAD101 (vector), pAB12 (Δss-MceA), or pAB11 (PhoA73-MceA) after 1 h of arabinose (0.2%) induction. Each value represents the average of three independent cultures. Error bars indicate standard deviations. (D) Synthesis levels of Δss-MceA and PhoA73-MceA. Cultures of AB3 (MccE492-resistant strain; see below) with pBAD101 (vector), pAB12 (Δss-MceA), or pAB11 (PhoA73-MceA) were pulse-labeled as described in Materials and Methods. Proteins were immunoprecipitated with anti-MceA antiserum (lower panel) or with anti-OmpA antiserum (upper panel) and detected by SDS-PAGE and autoradiography.

FIG. 3.

Toxicity of Δss-MceA expression in secY⁺ and prlA4 strains. (A and B) Serial dilutions of overnight cultures of AD85 (secY⁺) with pBAD101 (vector) or pAB12 (Δss-MceA) and of AD413 (prlA4) with pBAD101 (vector) or pAB12 (Δss-MceA) were spotted (5 μl) on LB plates without arabinose (A) or with 0.2% arabinose (B) and incubated overnight at 37°C. Results shown here are representative of three independent experiments. (C) Growth curves of AD85 (secY⁺) with pBAD101 (vector) (□), AD413 (prlA4) with pBAD101 (vector) (▪), AD85 (secY⁺) with pAB12 (Δss-MceA) (▵), and AD413 (prlA4) with pAB12 (Δss-MceA) (▴) in LB supplemented with 0.002% arabinose. Cell growth was followed by recording the A₆₀₀ of the cultures at the indicated times.

FIG. 4.

Bactericidal activity of endogenous MceA-TrxA chimeric proteins. (A and B) Serial dilutions of overnight cultures of DB503 (rpoA⁺) with pBAD101 (vector), pAB12 (Δss-MceA), or pAB16 (Δss-MceA-TrxA) and of DB512 (rpoA341) with pBAD101 (vector), pAB11 (PhoA73-MceA), or pAB15 (PhoA73-MceA-TrxA) were spotted (5 μl) on LB plates without arabinose (A) or with 0.2% arabinose (B) and incubated overnight at 37°C. DB512 (rpoA341) was used instead of DB503 (rpoA⁺) to better monitor a possible change in bactericidal activity between PhoA73-MceA and PhoA73-MceA-TrxA. Results shown here are representative of three independent experiments. (C) Synthesis levels of Δss-MceA and PhoA73-MceA fused or not to TrxA. Cultures of AB3 (MccE492-resistant strain; see below) with pBAD101 (vector), pAB12 (Δss-MceA), pAB11 (PhoA73-MceA), pAB16 (Δss-MceA-TrxA), or pAB15 (PhoA73-MceA-TrxA) were pulse-labeled as described in Materials and Methods. Proteins were immunoprecipitated with anti-MceA antiserum and separated by SDS-PAGE. The lower panel corresponds to the gel portion where Δss-MceA and PhoA73-MceA alone migrate. The upper panel corresponds to the gel portion where Δss-MceA and PhoA73-MceA fused to TrxA migrate.

FIG. 5.

Bactericidal activity of extracellular full-length MccE492 and of C-terminally truncated MceA. (A) Growth inhibition assay of a lawn of MccE492-sensitive cells [BL21(pBR322, pBAD101)] overlaid with 5 μl of overnight culture of AB7 with pBAD101 (vector), pAB10 (ABC-MceA), or pAB10-1C (ABC-MceA-1C) on an LB plate supplemented with 0.2% arabinose. Strain AB7 contains all the genes of the MccE492 system, except mceA, in plasmids pAB4 and pAB7. The plate was incubated overnight at 37°C. Note that both the lawn and the tested cultures are growing on this plate. (B) MceA immunoblot of the media of cultures of AB7 with pBAD101 (vector), pAB10 (ABC-MceA), or pAB10-1C (ABC-MceA-1C). Media were obtained from 24-h cultures in M9 minimal medium supplemented with 0.2% arabinose.

FIG. 6.

Bactericidal activity of endogenous full-length and C-terminally truncated MceA. (A and B) Serial dilutions of overnight cultures of DH5α with pBAD101 (vector), pAB13 (6H-MceA), pAB13-1C (6H-MceA-1C), pAB13-6C (6H-MceA-6C), pAB13-11C (6H-MceA-11C), or pAB13-16C (6H-MceA-16C) were spotted (5 μl) on LB plates without arabinose (A) or with 0.2% arabinose (B) and incubated overnight at 37°C. Results shown here are representative of three independent experiments. (C) Synthesis levels of full-length and C-terminally truncated MceA. Cultures of DH5α with pBAD101 (vector), pAB13 (6H-MceA), pAB13-1C (6H-MceA-1C), pAB13-6C (6H-MceA-6C), pAB13-11C (6H-MceA-11C), or pAB13-16C (6H-MceA-16C) were pulse-labeled as described in Materials and Methods. 6H-tagged proteins were purified and separated by SDS-PAGE. The position of full-length 6H-MceA is indicated by an arrow.

FIG. 7.

Proline uptake in a mannose permease-deficient strain and in a wild-type strain when PhoA73-MceA is produced. Uptake was measured in AB3 (ΔmanXYZ) and in DB503 (wild type) transformed with pAB11 (PhoA73-MceA) either in the absence of arabinose (ara) or after 20 min of arabinose (0.2%) induction. Proline uptake is expressed as the percentage of the slope of uptake measured in uninduced AB3 cultures. Each value represents the average of three independent cultures. Error bars indicate standard deviations.

FIG. 8.

Models for the bactericidal mechanisms of endogenous MceA (A) and extracellular MccE492 (B). MceA and MccE492 are represented as black ellipses with protruding N-terminal signal sequences and C-terminal domains. OM, outer membrane; IM, inner membrane; YEG, SecYEG complex; C*, posttranslationally modified MccE492 C terminus. ±TrxA indicates that the bactericidal activity of endogenous MceA is not affected by the presence of a C-terminal TrxA domain. Instead of Δss-MceA, PhoA73-MceA was chosen here as an example of endogenous MceA. Details are described in the text.