Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin.
Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of beta-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the beta-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.
Jumpertz
Thorsten
T
Institute of Biochemistry, Heinrich Heine University, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.
Chervaux
Christian
C
Danone Research Center, Daniel Carasso, Paliseau, Cedex 91767, France.
Racher
Kathleen
K
Water Resources, Indian and Northern Affairs (INAC-AINC), Canada.
Zouhair
Maria
M
Institut de Génetique et Microbiologie, URA 1354, Université Paris Sud, Bâtiment 409, 91405 Orsay Cedex, France.
Blight
Mark A
MA
Institut de Génetique et Microbiologie, URA 1354, Université Paris Sud, Bâtiment 409, 91405 Orsay Cedex, France.
Holland
I Barry
IB
Institut de Génetique et Microbiologie, URA 1354, Université Paris Sud, Bâtiment 409, 91405 Orsay Cedex, France.
Schmitt
Lutz
L
Institute of Biochemistry, Heinrich Heine University, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D006461:Hemolysis; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D001618:beta-Lactamases / Q000378:metabolism
D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D001618:beta-Lactamases