Properties of Bacillus cereus hemolysin II: A heptameric transmembrane pore (original) (raw)
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FEBS Letters, 1994
Structural changes in staphylococcal cc-hemolysin (aHL) that occur during oligomerization and pore formation on membranes have been examined by using a simple gel-shift assay to determine the rate of modification of key single-cysteme mutants with the hydrophilic sulthydryl reagent, 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonate (IASD). The central glycine-rich loop of aHL lines the lumen of the transmembrane channel. A residue in the loop remains accessible to IASD after assembly, in keeping with the ability of the pore to pass molecules of -1000 Da. By contrast, residues near the N-terminus, which are critical for pore function, become deeply buried during oligomerization, while a residue at the extreme C-terminus increases in reactivity after assembly, consistent with a location in the part of the pore that projects from the surface of the lipid bilayer.
Toxins
Bacillus cereus is the fourth most common cause of foodborne illnesses that produces a variety of pore-forming proteins as the main pathogenic factors. B. cereus hemolysin II (HlyII), belonging to pore-forming β-barrel toxins, has a C-terminal extension of 94 amino acid residues designated as HlyIICTD. An analysis of a panel of monoclonal antibodies to the recombinant HlyIICTD protein revealed the ability of the antibody HlyIIC-20 to inhibit HlyII hemolysis. A conformational epitope recognized by HlyIIC-20 was found. by the method of peptide phage display and found that it is localized in the N-terminal part of HlyIICTD. The HlyIIC-20 interacted with a monomeric form of HlyII, thus suppressing maturation of the HlyII toxin. Protection efficiencies of various B. cereus strains against HlyII were different and depended on the epitope amino acid composition, as well as, insignificantly, on downstream amino acids. Substitution of L324P and P324L in the hemolysins ATCC14579T and B771, re...
Translocation and compartmentalization of Escherichia coli hemolysin (HlyA)
Journal of Bacteriology, 1990
Hemolysin plasmids were constructed with mutations in hlyB, hlyD, or both transport genes. The localization of hemolysin activity and HlyA protein in these mutants was analyzed by biochemical and immunological methods. It was found that mutants defective in hlyB accumulated internal hemolysin, part of which was associated with the inner membrane and was degraded in the late logarithmic growth phase. In an HlyB+ HlyD- mutant, hemolysin was predominantly localized in the membrane compartment. Labeling of these Escherichia coli cells with anti-HlyA antibody indicated that part of HlyA, presumably the C-terminal end but not the pore-forming domains, was already transported to the cellular surface. This finding suggests that HlyB is able to recognize the C-terminal signal of the HlyA protein and to initiate its translocation across the membranes.
E. coli Hemolysin E (HlyE, ClyA, SheA)
Cell, 2000
. Anaerobic expression is controlled by the transcription factor, FNR, such that, upon ingestion and entry into the anaerobic mammalian intestine, HlyE is produced and may then contribute to the colonization of the host (Green and Baldwin, 1997). Unlike HlyA, which is synthesized as a soluble protoxin University of Sheffield, Western Bank Sheffield S10 2TN that requires proteolytic processing and posttranslational acylation to produce the active toxin Stanley et United Kingdom al., 1994), HlyE requires no posttranslational processing (del Castillo et al., 1997; Ludwig et al., 1999). Previous comparisons of the hlyE sequence against the available Summary databases had revealed no similarity to any other known hemolysin or characterized gene product. However, Hemolysin E (HlyE) is a novel pore-forming toxin of Escherichia coli, Salmonella typhi, and Shigella flex-Southern blotting studies had indicated that DNA capable of hybridizing to a hlyE probe is present in all the neri. Here we report the X-ray crystal structure of the water-soluble form of E. coli HlyE at 2.0 Å resolution strains of E. coli tested, including 0157, as well as Shigella flexneri, but not Salmonella D9 (del Castillo et al., and the visualization of the lipid-associated form of the toxin in projection at low resolution by electron 1997). Sequence comparisons reported here confirm that the typhoid fever-causing bacterium Salmonella microscopy. The crystal structure reveals HlyE to be the first member of a new family of toxin structures, typhi and the dysentery-causing organism Shigella flexneri have highly homologous proteins to HlyE encoded consisting of an elaborated helical bundle some 100 Å long. The electron micrographs show how HlyE oligo-in their genomes (Figure 1). These observations suggest that there is a family of HlyE-like hemolysins and that merizes in the presence of lipid to form transmembrane pores. Taken together, the data from these two they are likely to be a significant component of these pathogens' armory of toxins. structural techniques allow us to propose a simple model for the structure of the pore and for membrane Osmotic protection assays and lipid bilayer experiments show that HlyE forms a moderately cation-selec-interaction. tive water-permeable pore of diameter 25-30 Å (Ludwig et al., 1995, 1999; Oscarsson et al., 1999). It is thought Introduction that HlyE pore formation is either part of a mechanism for iron acquisition by the bacterial cell (del Castillo et Recent outbreaks of Escherichia coli-associated food al., 1997) or that it may promote bacterial infection by poisoning have emphasized the importance of identikilling immune cells and causing tissue damage (Ludwig fying and characterizing the various virulence factors et al., 1999). On the basis of hydrophobocity calculaof this organism. This paper reports the structure of a tions, HlyE has been predicted to have one putative recently discovered novel E. coli toxin, variously named transmembrane segment ranging from residues 177 to hemolysin E (HlyE) (Green and Baldwin, 1997; Reingold 203 (Figure 1); in addition, there is a second shorter et al., 1999), cytolysin A (ClyA) (Oscarsson et al., 1996), hydrophobic sequence from residues 89 to 101 (del Casor silent hemolysin A (SheA) (Ludwig et al., 1995). This tillo et al., 1997). structure represents the first member of a new family This paper reports the three-dimensional structure of pore-forming toxins, which are also found in other determination of HlyE from E. coli K12 in its putative pathogenic organisms, including species of Salmonella water-soluble secreted form and the first observations and Shigella (Figure 1). of pore formation in lipid vesicles by electron micros-HlyE is unrelated to the well-characterized pore-formcopy. These investigations reveal how HlyE folds and ing E. coli hemolysins of the RTX family (Coote, 1992), the nature of its association to form pores in the memhemolysin A (HlyA) (O'Brien and Holmes, 1996), and the branes of cells. enterohemolysin encoded by the plasmid borne ehxA gene of E. coli 0157 (Bauer and Welch, 1996). However, Results it is evident that expression of HlyE in the absence of the RTX toxins is sufficient to give a hemolytic phenotype in Structure Solution by X-Ray Crystallography E. coli (Ralph et al., 1998), and hemolytic avian E. coli at 2.0 Å Resolution isolates have been reported that lack the RTX toxins HlyE for crystallization was prepared from a GST-HlyE but possess a close homolog of HlyE (Reingold et al., fusion protein containing a thrombin cleavage site and 1999) (Figure 1), and that HlyE is present in pathogenic overexpressed in E. coli. The sequence of the crystalstrains of E. coli, including E. coli 0157 (del Castillo et lized protein consists of the whole sequence of HlyE al., 1997). It is a protein of 34 kDa that is expressed preceded by a 15-residue linker peptide, a construct that has hemolytic properties in vitro that are indistin-
Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation
Molecular and General Genetics MGG, 1993
Coexpression of pairs of nonhaemolytic HlyA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one HlyA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a nonhaernolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.
Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli
Journal of Bacteriology, 1996
Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also ...
FEMS Microbiology Letters, 1999
Hemolysin II gene from Bacillus cereus VKM-B771 has been sequenced. The deduced primary translation product consists of 412 amino acid residues which corresponds to the protein with an M r of 45.6 kDa. The predicted mature Hly-II protein (residues 32 to 412) is of 42.3 kDa, which is in close agreement with the mini-cell electrophoresis analysis. Hly-II deletion variant lacking 96 C-terminal residues still has hemolytic activity. The protein primary structure analysis revealed no homology with any known Bacillus cytolysins. Significant general homology (31^28% identity) was found between the hemolysin II and Staphylococcus aureus alpha-toxin, gamma-hemolysin (HlgB), and leukocidins (LukF, LukF-R, LukF-PV). The data suggest that hemolysin II belongs to the group of L-channel forming cytolysins.