Accepted Manuscript Title: Hemolysin of enterohemorrhagic Escherichia coli: Structure, transport, biological activity and putative role in virulence Mini-Review Author: Martina Bielaszewska Thomas Aldick Andreas Bauwens Helge Karch PII: DOI: Reference:

S1438-4221(14)00056-3 http://dx.doi.org/doi:10.1016/j.ijmm.2014.05.005 IJMM 50828

To appear in: Received date: Revised date: Accepted date:

27-3-2014 9-5-2014 11-5-2014

Please cite this article as: Bielaszewska, M., Aldick, T., Bauwens, A., Karch, H.,Hemolysin of enterohemorrhagic Escherichia coli: Structure, transport, biological activity and putative role in virulence Mini-Review, International Journal of Medical Microbiology (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Manuscript

1 Hemolysin of enterohemorrhagic Escherichia coli: Structure, transport,

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biological activity and putative role in virulence

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Mini-Review

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Martina Bielaszewska1*, Thomas Aldick1, Andreas Bauwens, Helge Karch

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Institute of Hygiene, University of Münster, Robert-Koch-Str. 41, 48149 Münster, Germany

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These authors contributed equally

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Running title: EHEC hemolysin as EHEC virulence factor

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Mailing address: Institut für Hygiene, Universität Münster, Robert-Koch-Str. 41, 48149 Münster,

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Germany

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Phone: +49-251/83 59965

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Fax: +49-251/83 55341

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E-mail: [email protected]

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Corresponding author: Dr. Martina Bielaszewska

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Summary Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea, bloody diarrhea and

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hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy affecting the renal glomeruli,

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the intestine, and the brain. The pathogenesis of EHEC-mediated diseases is incompletely

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understood. In addition to Shiga toxins, the major virulence factors of EHEC, the contribution of

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EHEC hemolysin (EHEC-Hly), also designated EHEC toxin (Ehx), which is a member of the

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RTX (repeats-in-toxin) family, is increasingly recognized. The toxin and its activation and

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secretion machinery are encoded by the EHEC-hlyCABD operon, in which EHEC-hlyA is the

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structural gene for EHEC-Hly and the EHEC-hlyC product mediates post-translational activation

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of EHEC-Hly; the EHEC-hlyB- and EHEC-hlyD-encoded proteins form, together with

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genetically unlinked TolC, the type I secretion system that transports EHEC-Hly out of the

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bacterial cell. EHEC-Hly exists in two biologically active forms: as a free EHEC-Hly, and an

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EHEC-Hly associated with outer membrane vesicles (OMVs) that are released by EHEC during

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growth. The OMV-associated form results from a rapid binding of free EHEC-Hly to OMVs

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upon its extracellular secretion. The OMV association stabilizes EHEC-Hly and thus

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substantially prolongs its hemolytic activity compared to the free toxin. The two EHEC-Hly

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forms differ by their mechanism of toxicity toward human intestinal epithelial and microvascular

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endothelial cells, which are the major targets during EHEC infection. The free EHEC-Hly lyses

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human microvascular endothelial cells, presumably by pore formation in the cell membrane. In

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contrast, the OMV-associated EHEC-Hly does not lyse any of these cell types, but after its

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cellular internalization via OMVs it targets mitochondria and triggers caspase-9-mediated

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apoptosis. The proinflammatory potential of EHEC-Hly, in particular its ability to elicit secretion

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of interleukin-1β from human monocytes/macrophages, might be an additional mechanism of its

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putative contribution to the pathogenesis of EHEC-mediated diseases. Increasing understanding

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3 of molecular mechanisms underlying interaction of EHEC-Hly with target cells as well as the

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host cell responses to the toxin supports the involvement of EHEC-Hly in the pathogenesis of

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EHEC-mediated diseases and forms a basis for prevention of the EHEC-Hly-mediated injury

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during human infection.

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Key words: EHEC hemolysin (EHEC-Hly); EHEC toxin (Ehx); RTX toxin; enterohemolysin;

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enterohemolytic phenotype; cell lysis; apoptosis; mitochondria; outer membrane vesicles.

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Introduction Enterohemorrhagic Escherichia coli (EHEC) O157:H7/H- (non-motile) and a large

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number of non-O157:H7 serotypes (the most common of which are O26:H11/H-, O103:H2,

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O111:H8/H-, and O145:H28/H-) cause diarrhea, hemorrhagic colitis, and the hemolytic-uremic

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syndrome (HUS) (Karch et al., 2005; Karmali, 2004; Mellmann et al., 2008; Tarr et al., 2005).

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HUS, the most severe clinical outcome of an EHEC infection, is characterized by hemolytic

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anemia, thrombocytopenia, and acute renal insufficiency (Tarr et al., 2005). The precise

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mechanisms that underly these hematologic and renal impairments are unknown. However,

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thrombotic microangiopathy resulting from microvascular endothelial damage and affecting the

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renal glomeruli, the intestine, and the brain (Habib, 1992; Richardson et al., 1988) is the primary

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event in the pathogenesis of HUS (Karpman et al., 2010; Tarr et al., 2005; Zoja et al., 2010).

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Shiga toxins (Stxs) are the key virulence factors of EHEC involved in the endothelial injury

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(Bauwens et al., 2011; Bielaszewska and Karch, 2005; Karpman et al., 2010; Obrig et al., 2010;

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Zoja et al., 2010) but also other toxins produced by EHEC strains can trigger or contribute to this

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process (Bielaszewska and Karch, 2005; Karch et al., 2006). Several possible candidates include

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cytolethal distending toxin (Bielaszewska et al., 2005; Friedrich et al., 2006), subtilase cytotoxin

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(Paton et al., 2004), EHEC vacuolating toxin (Bielaszewska et al., 2009) and a pore-forming

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cytotoxin designated EHEC hemolysin (EHEC-Hly; encoded by EHEC-hlyA) (Schmidt et al.,

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1995) or EHEC toxin (Ehx; encoded by ehxA) (Bauer and Welch, 1996a). EHEC-Hly belongs to

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the repeats-in-toxin (RTX) family, a large group of bacterial toxins that share several common

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features (Frey and Kuhnert, 2002; Linhartová et al., 2010; Welch et al., 1995; Welch, 2001;

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Wiles and Mulvey, 2013) including: i) the presence of glycine-rich nonapeptide repeats located in

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the carboxy (C)-terminal region of the proteins; ii) possession of a hydrophobic domain in the

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amino (N)-terminal region, which is involved in pore formation in the target membranes; iii)

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5 requirement for post-translational activation of pro-toxins (prior to their extracellular secretion)

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via fatty acylation of particular internal lysine residues; iv) secretion from the bacterial cells via

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the type I secretion system; v) post-secretion activation by binding calcium ions (Ca2+) within the

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nonapeptide repeats region, which leads to a toxin conformational change and facilitates its

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interaction with the host cell membrane. In this review we summarize current knowledge on

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structure-function relationships of EHEC-Hly, regulation of its expression, its extracellular

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transport, biologically active forms and a putative role in the pathogenesis of EHEC-mediated

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diseases.

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Structure-function relationships of EHEC hemolysin

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EHEC-Hly was characterized by Schmidt et al. (Schmidt et al., 1994, 1995, 1996a) as a

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novel E. coli hemolysin encoded on a large plasmid of EHEC O157:H7 (pO157), and a new

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member of the RTX family. By cloning and sequencing a fragment of pO157 responsible for a

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typical enterohemolytic phenotype (a narrow incomplete hemolysis on enterohemolysin agar

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distinct from a complete hemolysis caused by α-hemolysin) (Beutin et al., 1989), the authors

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identified an operon of four genes (EHEC-hlyC, EHEC-hlyA, EHEC-hlyB, EHEC-hlyD). These

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genes demonstrated 61% to 69% nucleotide sequence homologies to genes of the hlyCABD

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operon that encodes E. coli α-hemolysin (Welch and Pellet, 1988), the prototype member of the

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RTX family (Welch, 2001; Wiles and Mulvey, 2013). In this operon, EHEC-hlyA is the structural

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gene encoding EHEC-Hly, a 107-kDa protein, which mediates the typical enterohemolytic

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phenotype (Schmidt et al., 1995). Analogous to α-hemolysin (Goebel and Hedgpeth, 1982;

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Holland et al., 2005; Issartel et al., 1991; Wagner et al., 1983), EHEC-hlyB- and EHEC-hlyD-

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encoded proteins have been proposed to mediate transport of EHEC-Hly out of the bacterial cell,

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and EHEC-hlyC product to be required for post-translational activation of EHEC-Hly (Bauer and

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6 Welch, 1996a; Schmidt et al., 1995, 1996a). EHEC-Hly resembles α-hemolysin not only in the

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operon organization, but also in the protein domain structure (Schmidt et al., 1995). It consists of:

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i) a N-terminal stretch of about 200 mainly hydrophobic amino acids that is similar to a stretch in

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α-hemolysin that mediates insertion into the target cell membrane and is thus essential for pore

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formation (Schindel et al., 2001; Welch, 2001; Wiles and Mulvey, 2013); ii) a tandem array of 13

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glycine-rich nonapeptide repeats located between amino acids 706 and 832, which is responsible

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for Ca2+ binding in α-hemolysin (Ludwig et al., 1988); and iii) a C-terminal secretion signal

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recognized by the type I secretion system (Linhartová et al., 2010; Wiles and Mulvey, 2013). The

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presence of additional key structures in EHEC-Hly was proposed based on sequence alignment

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with related RTX toxins (Cortajarena et al., 2002, 2003; Stanley et al., 1998). These include two

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potential acylation sites at positions K550 and K675 (Stanley et al., 1998), which correspond to K564

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and K690 in α-hemolysin (Stanley et al., 1994), and which are, similar to those in α-hemolysin

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(Stanley et al., 1994), obligatory for hemolytic activity of EHEC-Hly (Aldick et al., 2009). This

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demonstrates that like α-hemolysin (Stanley et al., 1994), EHEC-Hly requires fatty acylation of

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the two internal lysine residues for its biological activity (Aldick et al., 2009). Moreover, in

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analogy to α-hemolysin, the amino acids H841 and D845 have been proposed to be required for

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calcium-dependent conformation of the toxin (Cortajarena et al., 2002, 2003).

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Regulation of expression of EHEC hemolysin

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Transcription of the EHEC-hlyA gene in EHEC O157 strains is under the control of the

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GrlR-GrlA regulatory system, which is encoded within the locus of enterocyte effacement (LEE)

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pathogenicity island (Saitoh et al., 2008). Deletion in grlR, encoding the negative regulator GrlR,

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and overexpression of the positive regulator GrlA significantly induced the enterohemolytic

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phenotype, indicating that GrlA positively regulates EHEC-hlyA transcription and EHEC-Hly

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7 production (Saitoh et al., 2008). In addition, transcriptional regulators Ler and LrhA, which both

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positively regulate expression of genes located within the LEE (Barba et al., 2005), also act,

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independently of each other and of GrlA, as activators for EHEC-Hly expression (Iyoda et al.,

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2011). In contrast, the heat-stable nucleoid-structuring (H-NS) protein, a global negative

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transcription regulator (Dorman 2004), represses transcription of EHEC-hlyA and EHEC-Hly

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production in EHEC O157 (Li et al., 2008) and non-O157 (Rogers et al., 2009; Scott et al., 2003).

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This process is temperature-dependent, being significantly more efficient at 30°C than at 37°C

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(Li et al., 2008), similar to that reported for α-hemolysin (Madrid et al., 2002). Analysis of the

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mechanism of the H-NS-mediated repression of the EHEC-hly operon in EHEC O91 strain B2F1

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demonstrated that H-NS binds to a 88 bp region of DNA upstream of the EHEC-hlyC start codon,

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which contains the putative promoter of the operon (Rogers et al., 2009). Overexpression of the

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small RNA DsrA, which inhibits H-NS synthesis (Sladjeski and Gottesman, 1995) increased

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EHEC-hlyA transcription at 30°C in the wild-type EHEC O157:H7 strain, but not in its hns

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deletion mutant; at 37°C, DsrA overexpression increased EHEC-hlyA transcription independent

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of hns genotype (Li et al., 2008). These data indicate that DsrA influences transcription of the

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EHEC-hly operon by two different ways, one of which (at lower temperature) is H-NS-dependent

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and the other (at higher temperature) is H-NS-independent (Li et al., 2008). Moreover, mutation

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of the rpoS gene encoding the RpoS σ factor, which mediates the general stress response in E.

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coli (Hengge-Aronis, 2002) completely abolished EHEC-hlyA transcription, regardless of

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temperature or DsrA overexpression, demonstrating RpoS σ factor to be an essential and primary

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regulator of EHEC-Hly expression (Li et al., 2008). Taken together, regulation of EHEC-Hly

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expression is a complex process which is likely to be influenced, in addition to temperature (Li et

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al., 2008), also by a variety of other environmental factors including those encountered by EHEC

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during their passage through the human gastrointestinal tract (Brockmeyer et al., 2011; Rashid et

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al., 2006). Such in-host regulations might further modulate virulence of EHEC strains in a host-

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specific manner.

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Pore formation by EHEC hemolysin and its target cell spectrum

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EHEC-Hly is a pore-forming toxin that is cytolytic towards erythrocytes and other

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eukaryotic cells (Aldick et al., 2007; Bauer and Welch, 1996a; Schmidt et al., 1995, 1996b). As

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in other RTX toxins, the cytolytic activity of EHEC-Hly depends strictly on calcium and is heat

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labile (Bauer and Welch, 1996a). A detailed analysis of its pore-forming characteristics using

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artificial lipid bilayer membranes demonstrated that EHEC-Hly forms transient (a few seconds

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lifetime), ion-permeable and cation-selective pores with an average diameter of ~2.6 nm

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(Schmidt et al., 1996b). This pore size was confirmed by osmotic protection experiments, in

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which sucrose and raffinose (molecular diameter 0.9 and 1.2 - 1.4 nm, respectively) did not

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inhibit EHEC-Hly-mediated hemolysis, but no hemolysis occurred in the presence of dextran 4

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(molecular diameter ~ 3 nm) (Schmidt et al., 1996b). The size of pores formed by EHEC-Hly is

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similar to that of pores caused by α-hemolysin in erythrocyte membranes (Bhakdi et al., 1986).

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Like EHEC-Hly-mediated pores, the pores caused by α-hemolysin were only transient as

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suggested by the failure to visualize them using electron microscopy (Bhakdi et al., 1986). In

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contrast to α-hemolysin, where oligomerization of the toxin monomers within the target

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membrane is required for pore formation (Ludwig et al., 1993; Herlax et al., 2009), it remains yet

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unknown whether EHEC-Hly acts in a monomeric form or as an oligomer. Furthermore, the steps

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leading to the pore formation, which involve for α-hemolysin the initial membrane anchoring via

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acyl groups linked to the lysine residues, followed by insertion of the hydrophobic domain into

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the membrane, which is promoted by electrostatic forces or by interaction with a cell surface

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receptor (β2 integrin CD11a/CD18) (Lally et al., 1997, 1999; Linhartová et al., 2010; Welch

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2001; Wiles and Mulvey, 2013) remain to be elucidated for EHEC-Hly. Analysis of the spectrum of cells that are lysed by EHEC-Hly revealed that it is distinct

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from that of α-hemolysin. In an early study (Bauer and Welch, 1996a), α-hemolysin was shown

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to lyse with a high efficiency sheep and human erythrocytes, bovine lymphoma cells (BL-3 line),

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and two different human lymphoma cell lines (Raji, Daudi). In contrast, EHEC-Hly lysed

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erythrocytes from both species, but only bovine and not human lymphoma cells (Bauer and

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Welch, 1996a). Moreover, α-hemolysin and EHEC-Hly differ in their specific abilities to bind to

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and lyse erythrocytes in vitro. In particular, EHEC-Hly binds to these cells at least 100-fold less

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efficiently and has 40-fold lower specific activity than α-hemolysin (Bauer and Welch, 1996a,

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1996b). The EHEC-Hly lytic activity against BL-3 cells was also substantially lower than that of

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α-hemolysin (Bauer and Welch, 1996a). The structural basis underlying these differences remains

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unknown. Despite the initial observation that the host cell lytic spectrum of EHEC-Hly is

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narrower than that of α-hemolysin (Bauer and Welch, 1996a), a recent demonstration of lytic

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activity of EHEC-Hly against human microvascular endothelial cells (Aldick et al., 2007)

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suggests that it may be broader than originally thought.

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A typical phenotype of EHEC-Hly, which facilitates identification of EHEC-Hly-

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producing E. coli strains in patients´ stool samples, is a narrow turbid zone of hemolysis, which

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appears after overnight incubation on blood agar containing washed erythrocytes and Ca2+

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(enterohemolysin agar), but not on a standard blood agar (Beutin et al., 1989; Schmidt et al.,

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1995). This phenotype is clearly distinct from that caused by α-hemolysin, which produces a

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complete, wide hemolytic zone on enterohemolysin agar already after 3-4 h of incubation, and a

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complete hemolysis on standard blood agar overnight (Beutin et al., 1989). The level of

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expression of the enterohemolytic phenotype varies among EHEC strains. While most of them

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10 cause the typical enterohemolytic phenotype with a narrow turbid lysis zone (non-sorbitol-

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fermenting EHEC O157:H7/H-, O26:H11/H-, O111:H8/H-, O145:H28/H-), some strains express a

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strong enterohemolytic phenotype, which resembles that of α-hemolysin (EHEC O103:H2), or a

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very weak or no hemolysis (sorbitol-fermenting EHEC O157:H-, some EHEC O111:H-), despite

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the presence of a functional EHEC-Hly operon (Bielaszewska et al., 2008; Brunder et al., 2006;

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Eklund et al., 2006; Schmidt and Karch, 1996; Schmidt et al., 1995, 1999). Mechanisms

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responsible for these differences might be at the level of regulation of transcription, expression

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and/or secretion, or involve interactions of EHEC-Hly with other virulence factors of EHEC

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(Brockmeyer et al., 2011).

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The unique hemolytic phenotype displayed by EHEC-Hly-producing EHEC (and also

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some enteropathogenic E. coli) strains prompted the original designation of the hemolytic

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determinant as “enterohemolysin” (i.e., hemolysin associated with E. coli causing enteric disease)

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(Beutin et al., 1988, 1989) to distinguish it from α-hemolysin produced by extraintestinal

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pathogenic E. coli. However, further studies demonstrated that two phage-encoded proteins

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(Ehly1 and Ehly2) proposed to represent different types of enterohemolysin based on their

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abilities to confer the hemolytic phenotype on E. coli K-12 laboratory strains (Beutin et al., 1990,

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1993; Stroeher et al., 1993), do not directly cause hemolysis. Instead, Ehly1 and Ehly2 lyse the

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recipient E. coli K-12 strains, which then release cytolysin A (ClyA) (Oscarsson et al., 2002).

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ClyA, a pore-forming protein with hemolytic activity (del Castillo et al., 1997) is, in fact, the

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erythrocyte membrane-disrupting effector (Oscarsson et al., 2002). Because of these findings, and

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in consideration of structural differences between EHEC-Hly (Ehx) and the Ehly1 and Ehly2

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proteins as evidenced by published sequences (Beutin et al., 1993; Stroeher et al., 1993), we

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believe that it is inappropriate to retain the term “enterohemolysin” as a synonym for EHEC-Hly

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(Ehx) as it is used in the literature at present.

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Biologically active forms of EHEC hemolysin By investigating the status of EHEC-Hly in supernatants of bacterial cultures, two

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biologically active forms of the toxin have been identified: a free, soluble EHEC-Hly, and an

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EHEC-Hly associated with outer membrane vesicles (OMVs) (Aldick et al., 2009), blebs of the

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bacterial outer membrane, which are released by EHEC and other Gram-negative bacteria during

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growth (Aldick et al., 2009; Beveridge, 1999; Ellis and Kuehn, 2010; Kulp and Kuehn, 2010).

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The OMV-associated form of EHEC-Hly results from a rapid binding of free EHEC-Hly to

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OMVs due to its high affinity to these structures (Aldick et al., 2009; Bielaszewska et al., 2013).

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Analysis of the kinetics of EHEC-Hly secretion, OMV production and EHEC-Hly-OMV

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association in overnight broth cultures of EHEC-Hly-producing strains demonstrated that the

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percentage of OMV-associated EHEC-Hly progressively increased during time, reaching >70%

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in logarithmic phase and >80% in stationary phase of growth. In contrast, the level of free EHEC

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remained low throughout the whole culture period indicating that the majority of EHEC-Hly

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secreted by growing bacteria rapidly binds to OMVs (Bielaszewska et al., 2013). Similar to

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OMVs from other Gram-negative bacteria (Balsalobre et al., 2006; Beveridge, 1999; Ellis and

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Kuehn, 2010; Kulp and Kuehn, 2010), the EHEC-Hly-carrying OMVs are spherical structures

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between 50 and 150 nm, surrounded with a membrane bilayer and composed of the outer

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membrane proteins, lipopolysaccharide, periplasmic proteins and DNA (Aldick et al., 2009;

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Bielaszewska et al., 2013). EHEC-Hly is localized on the exterior of OMVs and is tightly

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associated with the OMV membrane (Aldick et al., 2009; Bielaszewska et al., 2013). Based on

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these data, the following pathway for the secretion of EHEC-Hly has been proposed (Fig. 1):

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After translation and post-translational modification by EHEC-HlyC, EHEC-Hly is secreted out

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of the bacterial cell via the type I secretion system involving EHEC-HlyB, EHEC-HlyD and the

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product of the genetically unlinked tolC gene. Analogous to the well-studied type I secretion

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12 system for α-hemolysin (Holland et al., 2005), EHEC-HlyB, EHEC-Hly D and TolC plausibly

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function as an ABC transporter with a cytoplasmic ATP domain that provides energy, a

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membrane fusion protein and an outer membrane protein, respectively. After recognizing EHEC-

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Hly via its C-terminal secretion signal, these three proteins form a channel spanning the entire

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bacterial envelope that allows translocation of EHEC-Hly from the bacterial cytoplasm directly

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into the extracellular space, without a periplasmic intermediate. Because of its high affinity to

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OMVs, this free EHEC-Hly rapidly binds to vesicles released by EHEC (Fig. 1). Moreover,

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because EHEC-Hly was also observed in association with the outer membrane of bacterial cells

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(Bielaszewska et al., 2013), it could, alternatively (though in a lesser extent), become a part of

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OMVs directly during their shedding from the bacterial cells, similar as has been proposed for

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OMV-associated α-hemolysin (Balsalobre et al., 2006). Both EHEC-Hly (Aldick et al., 2009) and

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α-hemolysin (Balsalobre et al., 2006) associate with OMVs independently of their acylation

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status.

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In vitro studies of hemolytic activity of the free and OMV-associated EHEC-Hly

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demonstrated that similar to the free toxin, the OMV-associated EHEC-Hly binds to human

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erythrocytes and causes hemolysis (Aldick et al., 2009). The hemolytic activity of both free and

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OMV-associated EHEC-Hly is strictly dependent on acylation of the toxin at positions K550 and

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K675 and on the presence of Ca2+ (Aldick et al., 2009). The OMV-associated EHEC-Hly has dual

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roles in its action on erythrocytes: it mediates the OMV binding to these cells, as well as

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hemolysis (Aldick et al., 2009). The interaction of OMV-associated EHEC-Hly with erythrocytes

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is substantially delayed (hemolysis after 15 h) compared to that of free EHEC-Hly (hemolysis

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after 30 min) (Aldick et al., 2009); this is plausibly due to the necessity of a structural

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rearrangement of the OMV-bound toxin before it can bind and lyse erythrocytes (Aldick et al.,

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2009). The association with OMVs increases the stability of EHEC-Hly and thus substantially

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13 (~80 times) prolongs its hemolytic activity compared to the free form (Aldick et al., 2009)

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suggesting a biologically favourable assembly of the toxin within OMVs. However, despite their

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hemolytic activities in vitro, an involvement of any form of EHEC-Hly in hemolytic anemia, one

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of the defining clinical characteristics of HUS, remains obscure. The hemolysis during HUS has

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been assumed to result from mechanical breakdown of erythrocytes during their passing through

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thrombi-occluded small vessels (Karpman et al., 2010; Tarr et al., 2005).

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Putative involvement of EHEC hemolysin in the pathogenesis of EHEC diseases

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Distinct modes of cytotoxicity of free and OMV-associated EHEC-Hly towards human intestinal

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epithelial and microvascular endothelial cells

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Studies that investigated a possible contribution of EHEC-Hly to the pathogenesis of

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EHEC-mediated diseases focused on the toxin´s effects on the intestinal epithelial cells, the first

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barrier encountered by EHEC during human infection, and microvascular endothelial cells, the

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major targets during HUS. Aldick and colleagues (Aldick et al., 2007) reported that Stx-negative,

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EHEC-Hly-producing E. coli O26:H11/H- strains that were isolated as the only etiological agents

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from stools of several patients with HUS were cytotoxic towards human brain microvascular

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endothelial cells (HBMEC). The cytotoxicity was mediated by EHEC-Hly as demonstrated by the

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loss of cytotoxicity in one strain that spontaneously lost EHEC-hlyA and EHEC-Hly production,

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and by the ability of purified free EHEC-Hly to cause a time- and dose-lysis of HBMEC (Aldick

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et al., 2007). These data strongly indicate that free EHEC-Hly possesses an endothelium-

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damaging capacity and could thus contribute to the microvascular injury during HUS.

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The role of OMV-associated EHEC-Hly in the pathogenesis of EHEC-mediated diseases

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was investigated in a recent study (Bielaszewska et al., 2013). Using Caco-2 and HBMEC as

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models for intestinal epithelial and microvascular endothelial cells, respectively, this study

Page 13 of 31

14 demonstrated that in contrast to free EHEC-Hly (Aldick et al., 2007), the OMV-associated

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EHEC-Hly did not lyse these cells but triggered their apoptosis (Bielaszewska et al., 2013). A

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detailed analysis of this process revealed that unlike free EHEC-Hly, which remained restricted

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to the cell membrane, the OMV-associated toxin was internalized by HBMEC and Caco-2 cells

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and trafficked into endo-lysosomes. Upon endosomal acidification, EHEC-Hly separated from

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OMVs, escaped from the lysosomes, most likely via pore-formation in lysosomal membrane, and

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translocated to mitochondria, plausibly using its mitochondrial targeting signal, which was

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recently identified in its N-terminal region (Kisiela et al., 2010). The presence of EHEC-Hly in

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mitochondria resulted in cytosolic cytochrome c release and apoptotic cell death via activation of

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caspase-9 (Bielaszewska et al., 2013). The proposed model of intracellular trafficking and action

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of OMV-associated EHEC-Hly is presented in Figure 2. The apoptotic potential of OMV-

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associated EHEC-Hly towards human intestinal epithelial and microvascular endothelial cells

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indicated a novel, non-lytic mechanism of EHEC-Hly involvement in the pathogenesis of EHEC-

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mediated diseases.

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The different effects of free EHEC-Hly (lysis) and OMV-associated EHEC-Hly

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(apoptosis) may be due to the fact that the amount of EHEC-Hly associated with OMVs is

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approximately 10-fold less than that required for an efficient endothelial lysis (Aldick et al.,

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2007; Bielaszewska et al., 2013). A dose-dependent mechanism of cytotoxicity was also shown

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for other RTX toxins, which in high doses cause cell lysis via their pore-forming activity,

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whereas in low, sublytic doses they trigger a plethora of other biological effects including

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apoptosis (Atapattu and Czuprynski, 2005; Bhakdi et al., 1989, 1990; Czuprynski and Welch,

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1995; Korostoff et al., 1998; Laestadius et al., 2002; Lally et al., 1999; Suttorp et al., 1990; Wiles

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and Mulvey, 2013). Since the amount of EHEC-Hly produced during infection and reaching the

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target cells is unknown, it is impossibble to speculate which of the above effects of the toxin

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Page 14 of 31

15 might take place in vivo. However, the observations that free EHEC-Hly rapidly and irreversibly

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loses its biological activity in vitro at 37°C (Aldick, 2008) and that its OMV association

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significantly prolongs its activity (Aldick et al., 2009) suggest that the OMV-associated EHEC-

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Hly, rather than free EHEC-Hly, might be the pathophysiologically relevant form of the toxin

325

(Aldick et al., 2009; Bielaszewska et al., 2013).

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Interaction of EHEC-Hly with EspP

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In a study of interaction of EHEC-Hly with other EHEC virulence factors it was shown

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that EspPα, a highly proteolytical subtype of EHEC serine protease EspP (Brockmeyer et al.,

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2007) cleaves both free and OMV-associated EHEC-Hly (Brockmeyer et al., 2011). The cleavage

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of free EHEC-Hly abolished its hemolytic activity and its cytolytic potential towards

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microvascular endothelial cells (Brockmeyer et al., 2011). Notably, the OMV-associated EHEC-

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Hly was cleaved when EHEC-Hly-containing OMVs and EspPα were co-incubated in a cell-free

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system (Brockmeyer et al., 2011). However, the presence of intestinal epithelial cells, by which

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EHEC-Hly-containing OMVs are rapidly internalized (Bielaszewska et al., 2013), protected the

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OMV-associated EHEC-Hly from EspPα-mediated cleavage and inactivation, as indicated by the

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toxin´s ability to retain its apoptotic potential (M. Bielaszewska, unpublished data). The

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observation that EspPα was not able to inactivate OMV-associated EHEC-Hly under conditions

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mimicking those in the human gut during infection makes the relevance of the interaction

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between EHEC-Hly and EspPα in the pathogenesis of EHEC diseases questionable. Moreover,

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this observation further supports the benefit of EHEC-Hly association with OMVs for the toxin´s

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potential to subvert host cell functions.

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16 345

Proinflammatory potential of EHEC-Hly In addition to its cytotoxicity towards microvascular endothelial and intestinal epithelial

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cells, the proinflammatory potential of EHEC-Hly might play a role in the pathogenesis of EHEC

348

infections. Two independent studies demonstrated that EHEC-Hly elicited release of the

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proinflammatory cytokine interleukin-1β from the human monocyte/macrophage line THP-1

350

(Taneike et al., 2002; Zhang et al., 2012). This could contribute to the inflammatory response

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which occurs during HUS, as indicated by increased levels of circulating interleukins,

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chemokines, soluble adhesion molecules, and growth factors in HUS patients (Karpman et al.,

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2010; Zoja et al., 2010). Monocytes and macrophages are the major sources of the inflammatory

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mediators (Zoja et al., 2010). Moreover, interleukin-1β is one of the cytokines that upregulate the

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Stx receptor globotriaosylceramide (Gb3) on human endothelial cells (Ramegowda et al., 1999;

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van de Kar et al., 1992), the process resulting in an increased Stx cellular binding and toxicity

357

(van de Kar et al., 1992). Induction of interleukin-1β by EHEC-Hly may thus contribute to the

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pathogenesis of HUS by augmenting the effect of Stx on the endothelium.

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Other evidence supporting involvement of EHEC-Hly in the pathogenesis of EHEC diseases Several indirect pieces of evidence support the involvement of EHEC-Hly in the

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pathogenesis of human diseases. First, the toxin is expressed during infection as indicated by the

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development of serum antibodies against EHEC-Hly in most patients with HUS (Schmidt et al.,

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1995; Xu et al., 2002) and by increased EHEC-hlyA transcription levels in stools of patients with

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EHEC infection (Rashid et al., 2006). Second, EHEC-hlyA transcription is significantly

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upregulated upon contact of EHEC-Hly-producing bacteria with human intestinal epithelial cells,

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compared to the transcription during culture in LB broth (Brockmeyer et al., 2011). Third, the

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transcription level of the EHEC-hlyA gene was found to be significantly higher in highly

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Page 16 of 31

17 pathogenic compared to less pathogenic EHEC O157:H7 strains (Abu-Ali et al., 2010). Fourth,

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epidemiological studies demonstrate that EHEC-Hly is produced by the vast majority of EHEC

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serogroups most frequently associated with HUS (Mellmann et al., 2008), including both LEE-

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positive (O157, O26, O103, O111, O145) and LEE-negative (O91, O113) strains (Beutin et al.,

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2004; Bielaszewska et al., 2007, 2009; Elliot et al., 2001; Newton et al., 2009; Paton et al., 1999;

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Schmidt and Karch, 1996; Sonntag et al., 2004; Zhang et al., 2007). The presence of EHEC-hlyA

375

together with stx2 and eae (encoding the adhesin intimin) in an EHEC strain was found to be a

376

risk factor for a severe clinical outcome of the infection including HUS development (Boerlin et

377

al., 1999).

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Distribution of EHEC hemolysin among E. coli strains of different pathotypes

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In addition to its common presence in EHEC associated with HUS, the EHEC-hlyA gene

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also occurs (and if so studied, it is expressed) in a subset of other Stx-producing E. coli, mostly

382

LEE-negative, which cause diarrhea in humans (Beutin et al., 2004; Friedrich et al., 2003; Paton

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et al., 2001, 2004), as well as in strains isolated from feces of domestic animals (mainly cattle and

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sheep) and foods of animal origin (Cobbold et al., 2008; Cookson et al., 2007; dos Santos et al.,

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2010; Fremaux et al., 2006; Guth et al., 2003; Hornitzky et al., 2001; Lorenz et al., 2013; Slanec

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et al., 2009; Zweifel et al., 2005). Similar to LEE-positive EHEC (Brunder et al., 2006; Burland

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et al., 1998; Fratamico et al., 2011; Schmidt et al., 1995; Yan et al., 2012), the EHEC-hly operon

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is also located on a large plasmid in LEE-negative EHEC strains (Leyton et al., 2003; Newton et

389

al., 2009; Paton et al., 2001, 2004). Allelic profiling of EHEC-hlyA genes from LEE-positive and

390

LEE-negative EHEC strains demonstrated that despite overall nucleotide sequence identity of

391

>95%, the EHEC-hlyA sequences from LEE-positive and LEE-negative EHEC segregated into

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two distinct clades (Lorenz et al., 2013; Newton et al., 2009); this suggests that the large plasmids

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Page 17 of 31

18 that harbor the EHEC-hlyCABD operons in LEE-positive and LEE-negative EHEC belong to

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different evolutionary lineages (Boerlin et al., 1998; Newton et al., 2009). Distinct allelic profiles

395

and phylogeny of EHEC-hlyA were also reported in LEE-positive and LEE-negative Stx-

396

producing E. coli isolated from animals and foods (Cookson et al., 2007; Lorenz et al., 2013).

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EHEC-hlyA is also present and expressed in a subset of stx-negative, eae-positive E. coli

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strains classified as atypical enteropathogenic E. coli (aEPEC) (Afset et al., 2006; Bielaszewska

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et al., 2008; Cookson et al., 2007; Müller et al., 2007; Scaletsky et al. 2009; Trabulsi et al., 2002).

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The EHEC-hlyA-positive strains within this pathotype consist of two subgroups (Bielaszewska et

401

al., 2008). One group are stx-negative, eae-positive strains that belong to serotypes frequently

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found among EHEC causing HUS and bloody diarrhea (O26, O103, O145, O157), are closely

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phylogenetically related to the corresponding EHEC, and share with them typical non-stx

404

virulence profiles (Bielaszewska et al., 2007, 2008; Mellmann et al., 2005, 2009). Such strains

405

plausibly derived from EHEC by stx loss during infection (Bielaszewska et al., 2007, 2008;

406

Mellmann et al., 2005, 2009) and have been therefore designated EHEC-LST (EHEC that lost

407

Stx) (Bielaszewska et al., 2008). The other group of EHEC-hlyA-positive aEPEC are strains

408

belonging to a broad spectrum of other, non-EHEC serotypes, which have been associated with

409

uncomplicated diarrhea (Afset et al., 2006; Bielaszewska et al., 2008; Scaletsky et al., 2009);

410

these strains presumably represent “genuine” aEPEC because they are phylogenetically distant

411

from EHEC and EHEC-LST (Bielaszewska et al., 2008; Tennant et al., 2009). In some studies,

412

the presence of EHEC-hlyA in such strains was associated with the ability to cause diarrhea

413

(Afset et al., 2006) suggesting that the ability of EHEC-Hly to damage human intestinal epithelial

414

cells (Bielaszewska et al., 2013) might also contribute to the pathogenesis of EPEC infections.

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415

In E. coli strains of other pathotypes including typical EPEC, enteroaggregative E. coli,

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enterotoxigenic E. coli, enteroinvasive E. coli, and extraintestinal pathogenic E. coli EHEC-hlyA

Page 18 of 31

19 was not found (Müller et al., 2007). It is also absent from non-pathogenic, commensal E. coli

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isolates (Müller et al., 2007). Screening for enterohemolytic phenotype on enterohemolysin agar

419

thus facilitates identification of EHEC-Hly-producing EHEC, other Stx-producing E. coli and

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aEPEC strains in patients´ stool samples, as well as in samples from food and the environment.

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421 Conclusions

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EHEC-Hly is an EHEC virulence factor of increasingly recognized importance. The

424

OMV-associated EHEC-Hly might be the pathophysiologically relevant form of the toxin.

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Further studies are necessary to elucidate the molecular basis of EHEC-Hly association with

426

OMVs, mechanisms of EHEC-Hly interactions with lysosomes and mitochondria, and to

427

determine a complex spectrum of biological effects elicited by EHEC-Hly (and its different

428

forms) in pathogenetically relevant human cells. Moreover, the in-host factors that influence the

429

EHEC-Hly expression need to be identified and the significance of interaction(s) of EHEC-Hly

430

with other EHEC virulence factors in the context of human infection needs to be clarified. The

431

involvement of EHEC-Hly in virulence of EHEC, as suggested by in vitro studies, should be

432

confirmed and further explored using suitable animal models. Understanding of complex

433

pathophysiological processes elicited by EHEC-Hly in host cells and their underlying

434

mechanisms forms a basis for prevention of the EHEC-Hly-mediated injury during human

435

disease. Finally, because the human host is probably the last chain-link in a number of different

436

environments where EHEC occur (animals, water, soil, food), deciphering the EHEC-Hly role in

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EHEC environmental survival and adaptation is needed to unravel a complete biological potential

438

of this toxin.

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Acknowledgements The research on EHEC hemolysin in the laboratory of Prof. Dr. Helge Karch has been

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supported by the Deutsche Forschungsgemeinschaft (DFG) grant KA 717/5-1 and the DFG

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Sonderforschungsbereich (SFB: Collaborative Research Consortium) 1009, project B04. We

445

thank Nicola Holden (The James Hutton Institute, Dundee, UK) for critical reading of the

446

manuscript and Phillip I. Tarr (Washington University School of Medicine, St. Louis, Mo., USA)

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for discussions during revision of the manuscript.

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21 References

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29 Figure 1. Proposed pathway for secretion and OMV association of EHEC-Hly.

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EHEC-Hly is secreted via a type I secretion system formed by HlyB, HlyD and TolC from the

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bacterial cytoplasm into the extracellular space. Due to its high affinity to outer membrane

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vesicles (OMVs) released from bacterial cells, the majority of the free secreted toxin gets rapidly

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OMV-associated. OM, bacterial outer membrane; CM, cytoplasmic membrane.

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Figure 2. Model of intracellular trafficking and action of OMV-associated EHEC-Hly.

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1. After its secretion from EHEC bacteria and association with OMVs, the OMV-associated

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EHEC-Hly is endocytosed by target cells and trafficked into endosomes. 2. During endosomal

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acidification via the H+ ATPase the neutral pH drops to acidic, which induces separation of

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EHEC-Hly from OMVs. 3. The separated toxin plausibly damages the endosomal/lysosomal

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membrane by its pore-forming activity in order to release from lysosomes. As a consequence of

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the membrane damage, the lysosomal pH increases. 4. EHEC-Hly released from lysosomes

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translocates into mitochondria. 5. This results in cytosolic cytochrome c release, which leads to

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activation of caspase-9 and apoptotic cell death. 6. Presence of the proton ATPase inhibitor

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bafilomycin A1 (BafA1) inhibits endosomal acidification and hereby prevents EHEC-Hly to be

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separated from OMVs and translocated to mitochondria. (The figure was reproduced from

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Bielaszewska et al., 2013, PLoS Pathog 9, e1003797).

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Hemolysin of enterohemorrhagic Escherichia coli: structure, transport, biological activity and putative role in virulence.

Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea, bloody diarrhea and hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy affecting ...
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