Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC (original) (raw)
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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 ...
Journal of Biological Chemistry, 2000
␣-Hemolysin (HlyA) is a secreted protein virulence factor observed in certain uropathogenic strains of Escherichia coli. The active, mature form of HlyA is produced by posttranslational modification of the protoxin that is mediated by acyl carrier protein and an acyltransferase, HlyC. We have now shown using mass spectrometry that these modifications, when observed in protein isolated in vivo, consist of acylation at the ⑀-amino groups of two internal lysine residues, at positions 564 and 690, with saturated 14-(68%), 15-(26%), and 17-(6%) carbon amide-linked side chains. Thus, HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures, and the substrate specificity of the HlyC acyltransferase appears to differ from that of the closely related CyaC acyltransferase expressed by Bordetella pertussis.
Journal of Biological Chemistry, 2000
␣-Hemolysin (HlyA) is a secreted protein virulence factor observed in certain uropathogenic strains of Escherichia coli. The active, mature form of HlyA is produced by posttranslational modification of the protoxin that is mediated by acyl carrier protein and an acyltransferase, HlyC. We have now shown using mass spectrometry that these modifications, when observed in protein isolated in vivo, consist of acylation at the ⑀-amino groups of two internal lysine residues, at positions 564 and 690, with saturated 14-(68%), 15-(26%), and 17-(6%) carbon amide-linked side chains. Thus, HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures, and the substrate specificity of the HlyC acyltransferase appears to differ from that of the closely related CyaC acyltransferase expressed by Bordetella pertussis.
Molecular and General Genetics, 1988
A sequence (hlyR) of about 600 bp which enhances the expression of hemolysin (HlyA) more than 50-fold was identified in the plasmid pHly152-specific hemolysin (hly) determinant. Deletion of this entire hlyR sequence led to the same low level of hemolysin synthesis and excretion as that expressed by the recombinant plasmid pANN202-312. HlyR was active in cis but its activity was orientation-dependent. The enhancing sequence, hlyR, is separated from the promoter phlyI transcribing hlyC, hlyA and possibly hlyB by more than 1.5 kb including an IS2 element. Stepwise removal of the hlyR sequence from its 5′ end by exonuclease III (ExoIII) digestion yielded several types of deletion mutants which expressed decreasing amounts of hemolysin. A similar observation was made when hlyR was shortened by ExoIII from its 3′ end, which suggests that more than one functional region may be present in the hlyR sequence. A deletion of 717 bp within the adjacent IS2 element reduced the activity of hlyR only slightly, indicating that IS2 is not directly involved in the enhancement mechanism but that it may support an optimal positioning in hlyR relative to the hly promoter. The nucleotide sequence of hlyR is rich in A+T and does not contain an extended open reading frame, but exhibits several sequence motives that may represent sites for protein binding and DNA bending.
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.
Molecular and General Genetics MGG, 1997
The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two dierent mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3¢-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These eects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed speci®c interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3¢-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm.
Microbiology-sgm, 2010
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 b-lactamase when the latter was fused to a Cterminal 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 b-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.
Molecular Microbiology, 1996
The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated. Each domain required 15-30 amino acids for basal recognition and 50-80 amino acids for wild-type acylation. The two domains (FA1 and FAII) competed with each other in cis and in trans for HlyC. The affinity of FA1 for HlyC is approximately four times greater than that of FAll resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess CaZ1 prevented acylation of both lysines. The lack of primary sequence identity between FA1 and FAll domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.
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-
Role of lipopolysaccharide on the structure and function of a-hemolysin from Escherichia coli
Chem Phys Lipids, 2005
␣-Hemolysin (HlyA) is a protein toxin (107 kDa) secreted by some pathogenic strains of E. coli. Several studies suggested the relationship between HlyA and lipopolysaccharide (LPS). We have studied experimentally the role of LPS on the stability and function of this toxin. The HlyA conformation in both, LPS-free and LPS-bound forms was investigated by tryptophan fluorescence. Studies about HlyA thermal and chemical denaturation indicated that its stability increased in the presence of LPS. On the other hand, the presence of negative and polar residues on the LPS reduced the tendency of HlyA to self-aggregation, and they may be the reservoir of calcium, cation essential for the lytic action of this toxin on red blood cells. These results suggest that HlyA and LPS are combined mainly via hydrophobic force to form an active toxin which stability is favored by the LPS.