Association of RTX toxins with erythrocytes

Bauer, Margaret E.; Welch, Rodney A.

doi:10.1128/iai.64.11.4665-4672.1996

Cited by 59 publications

(32 citation statements)

References 35 publications

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“…The haemolytic activity of EHEC-Hly resembles that of other members of the RTX toxin family, previously shown to depend strictly on calcium (Bauer and Welch, 1996b). We confirmed these findings for both free and OMVassociated EHEC-Hly.…”

Section: Discussionsupporting

confidence: 88%

“…EHEC-Hly lyses a wide range of cells including human erythrocytes (Schmidt et al, 1995;Bauer and Welch, 1996b;Aldick et al, 2007). Therefore, we compared the kinetics of haemolysis caused by free EHEC-Hly and OMV-associated EHEC-Hly by monitoring haemoglobin release after exposure of red blood cells to each respective toxin preparation.…”

Section: Kinetics Of Ehec-hly-mediated Haemolysismentioning

confidence: 99%

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Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin

Aldick

Bielaszewska

Uhlin

et al. 2009

Molecular Microbiology

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SummaryHaemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by selfaggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHECHly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.

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Section: Discussionsupporting

confidence: 88%

Section: Kinetics Of Ehec-hly-mediated Haemolysismentioning

confidence: 99%

Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin

Aldick

Bielaszewska

Uhlin

et al. 2009

Molecular Microbiology

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“…There are therefore many reasons why a particular cell line might be insensitive to a particular toxin. The toxin may not interact with the membrane, and this may occur for AaltA with human erythrocytes (298) and for LktA with nonbovine erythrocytes (15,42). The toxin may adsorb to the membrane but fail to insert into the lipid bilayer, and this may be the behavior of inactive, nonacylated protoxins.…”

Section: Biological Effects Of Hlya and Related Toxinsmentioning

confidence: 99%

Acylation ofEscherichia coliHemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function

Stanley

Koronakis

Hughes

1998

Microbiol Mol Biol Rev

177

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SUMMARY The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor. This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases. Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups. The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors. While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions. Despite being acylated at two residues, HlyA does not possess a “double-anchor” motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance. The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer. These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation.

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“…Divergent results exist in the literature regarding the effect of Ca 2+ on binding of HlyA to membranes [19–21] and binding experiments were performed with fluorescently labeled hemolysin to address this issue. A potential problem of binding studies relates to the tendency of the unbound toxin to aggregate and be recovered in the membrane fraction after centrifugation.…”

Section: Resultsmentioning

confidence: 99%

Interaction of Escherichia coli hemolysin with biological membranes

Schindel

Zitzer

Schulte

et al. 2001

European Journal of Biochemistry

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Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca 21 to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca 21 , suggesting that Ca 21 is not required for the toxin to enter membranes. However, binding of Ca 21 to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca 21 to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.Keywords: badan; calcium; conformation; Escherichia coli hemolysin, membrane.Escherichia coli hemolysin (HlyA), a 107-kDa protein of 1024 amino acids devoid of cysteine [1], belongs to the family of RTX-toxins [2] that are produced by many Gramnegative organisms. To obtain pore-forming activity, HlyA requires post-translational acylation of two lysine residues, Lys564 and Lys690 [3,4]. The protein harbors a hydrophobic domain between residues 177 and 411, which according to predictions of secondary structure [5] largely assumes helical conformation and might be involved in membrane penetration. The C-terminal half of the molecule contains 12 repeats of the consensus nonapeptide X-Leu-XGly-Gly-X-X-Gly-Asp-Asp-Asp. This repeat sequence spanning residues 739±849 represents a Ca 21 -binding domain and is essential for function [6±9]. In the presence of Ca 21 , HlyA forms pores of < 2 nm diameter in target membranes [10]. Several approaches to study structurefunction analysis of HlyA have been undertaken in the past. CD spectroscopy was used to investigate the effect of Ca 21 on HlyA, but no significant changes in secondary structure could be detected with this method [11]. On the other hand, results obtained from intrinsic fluorescence measurements and trypsin digestion were compatible with a Ca 21 -induced change in conformation [11]. Moayeri and Welch [12] studied the altered accessibility of toxin domains for monoclonal antibodies after the binding of HlyA to membranes. They reported that the far N-terminus and the region between residues 594 and 640 remain accessible in the membrane-bound toxin. No conclusions could be made for the putative transmembrane region and the C-terminal domain, as antibodies for these regions were not available.An important step toward unders...

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Association of RTX toxins with erythrocytes

Cited by 59 publications

References 35 publications

Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin

Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin

Acylation ofEscherichia coliHemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function

Interaction of Escherichia coli hemolysin with biological membranes

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