Characterization of dominantly negative mutant ClyA cytotoxin proteins in Escherichia coli

Abstract

We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

FIG. 1.

Subcellular localization of ClyA proteins. (A) Immunoblot analysis of ClyA proteins in periplasmic (P), cytoplasmic (C), and membrane (M) fractions from different derivatives of E. coli K-12 expressing wild-type or mutant alleles of the clyA gene. Lanes 1 to 3, strain MC1061/pYMZ80. Lanes 4 to 6, strain MWK11. Lanes 7 to 9, strain YMZ19. Lanes 10 to 12, strain MC1061/pSNW168. Lanes 13 to 15, strain MC1061/pJON63. Lanes 16 to 18, strain MC1061/pMWK16. Lanes 19 to 21, strain BEU616. The relevant characteristics of the strains (ClyA phenotype or mutation and plasmids encoding clyA variants) are shown below the lanes. For immunodetection, a mouse monoclonal antibody was used, as described in Materials and Methods. (B) Immunoblot analysis of the periplasmic and cytoplasmic marker proteins DsbA and H-NS, respectively. Subcellular fractions of strains MC1061/pYMZ80 (lanes 1 to 3), MWK11 (lanes 4 to 6), and MC1061/pJON63 (lanes 7 to 9) were subjected to analysis with anti-DsbA and anti-H-NS rabbit antisera. (C) SDS-PAGE analysis and Coomassie blue staining of proteins in periplasmic (P), inner membrane (IM), outer membrane (OM), and total membrane (M) fractions of strains DH5α/pSNW168 (lanes 2 to 5), BEU616/pJON66 (lanes 6 to 9), and BEU616/pJON70 (lanes 10 to 13). Molecular size markers were included in lane 1, and the sizes are indicated on the left.

FIG. 2.

Single-channel recordings of diphytanoyl phosphatidylcholine-n-decane membranes in the presence of wild-type ClyA (trace A) and the Δ(183-186) (trace B) and Δ(281-303) (trace C) ClyA mutants. The aqueous phase contained 1 M KCl (pH 6) and 10 ng of ClyA wild-type or mutant protein per ml. The applied membrane potential was 20 mV (20°C). Note that the current noise for the single-channel recording of wild-type ClyA is similar to that for the mutants, indicating that the mutation did not induce a major change of the channel structure.

FIG. 3.

Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed from diphytanoyl phosphatidylcholine-n-decane in the presence of 10 ng of wild-type ClyA (A) and the Δ(183-186) (B) and Δ(281-303) (C) mutants per ml. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The aqueous phase contained 1 M KCl. The applied membrane potential was 20 mV (20°C). The average single-channel conductances were 12 nS for 237 single-channel events (wild-type ClyA), 8.5 nS for 95 events [ClyA Δ(183-186) mutant], and 7.5 nS for 181 events [ClyA Δ(281-303) mutant]. Conductance is current divided by voltage.

FIG. 4.

Relative conductance caused by the ClyA Δ(183-186) and ClyA Δ(281-303) mutants in lipid bilayer membranes compared to wild-type ClyA (wt). Membranes were formed from diphytanoyl phosphatidylcholine-n-decane in 1 M KCl, and ClyA proteins were added at a concentration of 100 ng/ml to the aqueous phase. About 30 min after addition of ClyA proteins, the membrane conductance was measured and averaged for three membranes for the individual systems [wild-type ClyA, ClyA Δ(183-186) mutant, and ClyA Δ(281-303) mutant]. The membrane conductance of wild-type ClyA was set to 100%, and the conductances of the two ClyA mutants were calculated relative to that of wild-type ClyA. The membrane activity is given as the mean value ± standard deviation. T = 20°C; V_m = 20 mV.

FIG. 5.

Electron microscope images of formation of ClyA pore assemblies on planar lipid monolayer films. Purified wild-type and mutant ClyA proteins were allowed to assemble by the lipid monolayer crystallization method as described in Materials and Methods. Bars, 25 nm. (A) Wild-type ClyA protein. (B) ClyA Δ(281-303) mutant protein. (C) ClyA Δ(183-186) mutant protein. (D) Wild-type and ClyA Δ(281-303) mutant proteins in a 1:1 ratio. (E) Wild-type and ClyA Δ(183-186) mutant proteins in a 1:1 ratio. (F) Wild-type and ClyA (A183G G184D) mutant proteins in a 1:1 ratio.