title: Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin.
abstract: Haemolysin 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 self-aggregation. 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 EHEC-Hly 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.
authors.affiliation: Institute of Hygiene, University of Münster, 48149 Münster, Germany. thomas.aldick@ukmuenster.de
authors.forename: Thomas
authors.initials: T
authors.lastname: Aldick
authors.forename: Martina
authors.initials: M
authors.lastname: Bielaszewska
authors.forename: Bernt Eric
authors.initials: BE
authors.lastname: Uhlin
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Humpf
authors.forename: Sun Nyunt
authors.initials: SN
authors.lastname: Wai
authors.forename: Helge
authors.initials: H
authors.lastname: Karch
mesh_terms: D001427:Bacterial Toxins / Q000378:metabolism*; D054324:Enterohemorrhagic Escherichia coli / Q000378:metabolism*; D004912:Erythrocytes / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D055550:Protein Stability; D022161:Transport Vesicles / Q000378:metabolism* / Q000382:microbiology
chemical_list: D001427:Bacterial Toxins; C097321:EHEC-hlyA protein, E coli; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins