Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Abstract

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hylA from the 5' end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E. coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

Molecular Analysis of Cytolysin A (ClyA) in Pathogenic Escherichia coli Strains

“…SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described (21). Western immunoblotting was conducted according to Towbin et al (42) with the modification that the proteins were blotted onto polyvinylidene difluoride membrane using a Tris-glycine transfer buffer (25 mM Tris, 192 mM glycine, (19). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were used to detect the immunoreactive protein bands.…”

Type V Protein Secretion Pathway: the Autotransporter Story

“…Evidence suggests that the MFP (HlyD) and the ABC transporter (HlyB) in E. coli interact before binding of the effector (156). The secretion process begins when a secretion signal located within the C-terminal end of the secreted effector molecule interacts with the ABC transporter protein (224). In general, this signal sequence is specific and is recognized only by the dedicated ABC transporter.…”

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

“…LtxA has been shown to bind calcium as well (41). Finally the C-terminal domain (positions 901-1055) is required for secretion (42).…”