Shiga toxins, their intracellular transport and involvement in human disease
The Shiga toxins comprise a family of related protein toxins secreted by certain types of bacteria. Shiga toxin (Stx) is produced by Shigella dysenteriae, whereas the Shiga-like toxins, Stx1 and Stx2, with a few known isoforms, are secreted by specific strains of Escherichia coli named Shiga-toxin-producing E. coli (STEC). Stx1 is virtually identical to Stx, differing in only one amino acid residue, whereas the Stx2 isoforms share less sequence similarity with Stx (∼60%) and are immunologically distinct. In spite of the differences in their amino acid sequence, all the Stx isoforms share the same overall toxin structure and mechanism of action, and unless otherwise specified, for the remainder of this review the singular term ‘Stx’ will refer to the family of Shiga toxins in general (for recent reviews on Stx see Sandvig et al., 2009; Johannes and Römer, 2010).
Gastrointestinal infection with STEC serotypes might be followed by the life-threatening complication haemolytic uremic syndrome (HUS), which is defined by haemolytic anaemia, thrombocytopenia and acute renal failure (Palermo et al., 2009). HUS predominantly affects young children, and although the mechanism is not entirely resolved, the disease involves Stx-induced damage to kidney cells. The STEC serotypes associated with HUS produce isoforms of Stx1 or Stx2, or a combination of these, and the most severe disease outcome is associated with Stx2 (Boerlin et al., 1999). It should be noted that, unlike STEC, S. dysenteriae bacteria invade host cells, and only S. dysenteriae serotype 1 expresses Stx (for a recent review about diseases caused by S. dysenteriae see Schroeder and Hilbi, 2008).
The Shiga toxins are composed of an enzymatically active A moiety that is non-covalently attached to a pentameric binding moiety (Fig. 1A–C). The whole natural toxin is commonly referred to as the ‘holotoxin’. The B moiety binds specifically to the sugar domain of the glycosphingolipid globotriaosylceramide (Gb3) (Fig. 1D) in the plasma membrane of target cells and mediates uptake and intracellular transport of the toxin (Sandvig et al., 2009; Johannes and Römer, 2010). More than one endocytic pathway seems to be involved in Stx entry, as both clathrin-dependent and clathrin-independent toxin uptake has been identified in different cell types (Sandvig et al., 1989; Khine and Lingwood, 1994; Schapiro et al., 1998; Nichols et al., 2001; Lauvrak et al., 2004; Saint-Pol et al., 2004; Torgersen et al., 2005; Römer et al., 2007). The exact contribution of each pathway to toxin uptake is difficult to determine, as inhibition of one pathway might lead to upregulation of another (Damke et al., 1995). Notably, Stx seems to have the ability to stimulate its own uptake (Torgersen et al., 2005; Lauvrak et al., 2006), and recently, the toxin was reported to induce tubule formation (Römer et al., 2007). To which extent tubule-induction contributes to toxin uptake is unresolved and has been suggested to be minor (Hansen et al., 2009).
The sorting of Stx into the retrograde transport pathway from early endosomes to the Golgi apparatus is a highly regulated process (for recent reviews, see Johannes and Popoff, 2008; Sandvig et al., 2009; Torgersen et al., 2010). An overview of components reported to be involved in this process is shown in Fig. 2. These include, for instance, SNARE proteins, Rabs, kinases, sorting nexins, scaffold proteins and cytoskeletal elements. During intracellular transport, primarily in the endosomes, the Stx A moiety is cleaved by furin into the A1 and A2 fragments (Fig. 1B) (Garred et al., 1995). The enzymatically active A1 fragment remains bound to the A2 fragment via a disulfide bond during the transport from the Golgi apparatus to the endoplasmic reticulum (ER), and is released upon exposure to the reducing conditions in the ER. The A1 fragment is then translocated into the cytosol, possibly via the Sec61 complex involved in ER-associated degradation of misfolded proteins (Yu and Haslam, 2005). The A1 fragment is able to escape ubiquitination and cytosolic degradation, due to the virtual absence of lysine residues, and its N-glycosidase activity irreversibly modifies the ribosomal 28S RNA, leading to inhibition of protein synthesis. Although this may by itself lead to cell death, Stx has also been shown to induce apoptosis via induction of ribotoxic- and ER stress signals (Smith et al., 2003; Lee et al., 2008), or even via signal transduction induced by Gb3-ligation (Mangeney et al., 1993; Taga et al., 1997; Tetaud et al., 2003; Kovbasnjuk et al., 2005). The mechanism(s) whereby Stx kills cells still needs further clarification and seems at least in some cases to depend on the cell type (Tesh, 2010). However, in most cell types, retrograde transport of the toxin to the ER is a prerequisite for Stx toxicity.
The Shiga toxin receptor, Gb3
The receptor for Shiga toxin in human cells is Gb3. The glycosphingolipids (GSLs) are a subtype of glycolipids that are synthesized by the addition of sugar molecules to a ceramide backbone. The metabolic pathways of GSLs branch at the point of lactosylceramide (Gal-β1→4Glc-β1→Cer) into the lacto-, ganglio-, and globo-series (Hakomori, 2008). The globoseries of GSLs are unique in having an α1→4Gal structure at the internal core, resulting in an unusual conformational structure distinct from that of the other series. Gb3 is the first product in the globoseries of GSLs, and is synthesized by the addition of galactose to lactosylceramide in a reaction catalysed by Gb3 synthase (α-1,4-galactosyltransferase). For the molecular structure of Gb3 see Fig. 1D.
Binding of Shiga toxin to Gb3
Binding of Shiga toxin to Gb3 is complex (Peter and Lingwood, 2000; Pina et al., 2007; Lingwood et al., 2010b), and although much has been learned, the Stx–Gb3 interaction is far from being completely understood. Crystallographic studies have indicated that in the context of the StxB pentamer, each of the five B-chains has three potential Gb3 binding sites (Ling et al., 1998), so that at least in theory, one Stx molecule can simultaneously bind up to 15 Gb3s. Mutational analysis of the Gb3 binding sites in StxB indicates that at least two of the sites are required for StxB to be able to bind to Gb3, whereas optimal binding involves all three binding sites (Soltyk et al., 2002). Furthermore, other studies have indicated that optimal interaction between Stx and Gb3 requires a mixture of Gb3 species with different fatty acid chain lengths in their ceramide backbone moieties (Pellizzari et al., 1992) combined with an optimal organization of Gb3 species (Nyholm et al., 1996), as well as a favourable surrounding lipid environment in the plasma membrane itself (Arab and Lingwood, 1996). As one example of the possible implication of differences in such factors in vivo, a recent study reported that due to differential membrane Gb3 organization in paediatric versus adult renal glomeruli, Stx binds stronger to the former (Khan et al., 2009). These findings may at least in part explain why STEC-induced HUS is mainly a paediatric disease.
Differences in the fatty acid chain lengths in the ceramide backbone of Gb3 may alter not only the binding characteristics of Gb3 to Stx, but also the intracellular routing of the Gb3/Stx complex. Whereas the sphingosine part of the ceramide backbone in general appears with a constant number of 18 carbon atoms, the number of carbon atoms in the fatty acid part varies, normally appearing within a range of 16–24 carbon atoms (C16-C24). There are large cell type-dependent differences in the species composition of Gb3 (Raa et al., 2009). In general, however, the most abundant Gb3 species contains C24, whereas the second most abundant species contains C22, C18 or C16. Gb3 species with short fatty acid chain lengths (C16 or C18) have been associated with enhanced retrograde transport of Stx (Arab and Lingwood, 1998; Raa et al., 2009). So far there is little information available regarding the species composition of Gb3 in different tissues.
Methods to detect the Stx receptor Gb3 in cells and tissues
To evaluate the possibilities of using Stx for imaging or therapy, one needs to investigate the distribution of Gb3, and the ability of Gb3 to bind Stx in human cells and tissues. Different methods have been used for this purpose, and we therefore provide a brief overview of those that are most commonly used. Advantages and disadvantages of these methods are summarized in Table 1.
|Mass spectrometry (Müthing and Distler, 2010; Raa et al., 2009)||Identification and quantification of all||Expensive equipment.|
|Gb3 species.||Special knowledge needed.|
|Very sensitive.||Sample must be homogenized and extracted before analysis.|
|TLC with orcinol or overlay assays (Lingwood et al., 2010a; Müthing and Distler, 2010)||Rapid visualization of several samples.|
Do not need expensive equipment.
|Relationship between signal intensity and amount of Gb3 present may be complicated to interpret when using overlay assays.|
|Precise species composition not obtained.|
|Sample must be homogenized and extracted before analysis.|
|Fluorescence microscopy/Immunohistochemistry (Salhia et al., 2002; Falguières et al., 2008)||Direct visualization of cells.||Not possible to obtain reliable quantitative data of total Gb3 content or the species composition.|
|Discriminate between tumour cells and surrounding tissue.|
|Flow cytometry (LaCasse et al., 1999; Tetaud et al., 2003)||Measure distribution of cellular Gb3 expression in a sample.|
Possible to estimate the surface level of Gb3 of non-permeabilized cells.
|Not possible to obtain reliable quantitative data of total Gb3 content or the species composition.|
Mass spectrometry. Due to recent development within the field of mass spectrometry (MS), the total amount of Gb3 and the relative content of different Gb3 species in tissue or cell extracts can now be routinely analysed by MS. Using high-resolution MS, the analysis may be performed with direct injection of the extracts into the MS (so-called ‘shot-gun analysis’), i.e. without any chromatographic separation of the samples. The MS analysis also offers the possibility to obtain information about the total cellular/tissue lipidome, including the content of other GSLs, which may give additional important information about the samples (Raa et al., 2009). We anticipate that direct MS will play an increasing role in analysis of Gb3 in the future, but so far most studies have used the more traditional methods described below.
TLC with orcinol staining or overlay assays. Analysis of lipid extracts from cells or tissues may be performed by various chromatographic methods combined with different types of detection. In practice, chromatographic analysis of Gb3 is today mainly performed by thin layer chromatography (TLC) (for reviews see Lingwood et al., 2010a; Müthing and Distler, 2010). Usually high-performance TLC (HPTLC) plates are used. Glycolipids can be visualized by staining carbohydrates with a mixture of orcinol and sulfuric acid. Information about the identity and approximate quantity of the bands can be obtained by comparing the mobility of the band of interest with a reference, or a set of known glycolipid standards. Alternatively, to obtain information about the amount of binding of Stx or anti-Gb3 antibodies to the Gb3 species on the TLC plate, the plate can be overlaid with Stx, a Stx derivative or an anti-Gb3 antibody, followed by overlay with a secondary antibody conjugated to a detection moiety for visualization of the bound molecules. Thus, the TLC overlay methods are used to indicate the total cellular levels of Gb3 in the cells/tissue sample. Based on the differential motility of Gb3 species with, e.g. long or short fatty acid chain lengths in their ceramide backbone, one can also obtain indications on the relative expression of different Gb3 species, which can be further identified by MS. The MS identification is important, since a single band on a HPTLC plate is likely to contain different species of Gb3 (Arab and Lingwood, 1998; Lingwood et al., 2010b).
Fluorescence microscopy/immunohistochemistry of cryosections. These methods are based on visualization of cellular Gb3 by use of Stx/StxB or anti-Gb3 antibodies followed by secondary antibodies conjugated to fluorescent molecules or enzymes, to allow for detection. Alternatively, Stx/StxB or the anti-Gb3 antibodies may be directly coupled to fluorescent/enzymatic moieties. These methods give an indication of the in situ total cellular level of Gb3 if the cells are permeabilized before staining (which is normally the case), or in situ surface levels of Gb3 if staining is performed at 4°C on non-permeabilized preparations. Importantly, in the case of a tumour sample, one can obtain information on the origin of the Gb3-expressing cells, i.e. if they are the cancer cells and/or cells from the surrounding tissue. This is in contrast to MS and chromatographic methods, which only aim at measuring the total levels of Gb3 in the sample.
Flow cytometry. By use of flow cytometry one can obtain information on single-cell expression levels of Gb3 in a large number of cells (normally ≥ 10 000 cells are analysed), and thereby obtain information on the distribution of cellular Gb3 expression levels in the sample. Similarly to the method mentioned above (fluorescence microscopy/immunohistochemistry of cryosections), this method is based on staining of cellular Gb3 by use of Stx/StxB or anti-Gb3 antibodies followed by fluorescently labelled secondary antibodies, or alternatively the use of directly coupled Stx/StxB or anti-Gb3 antibodies. Surface levels of Gb3 can be determined if staining is performed at 4°C on non-permeabilized cell preparations. The origin of the Gb3-expressing cells can be assessed by immunofluorescence-based counterstaining with antibodies towards known cell markers.
Function and expression of Gb3 in humans
Apart from its suggested involvement in negative selection of tonsillar B cells during affinity maturation (Taga et al., 1997), the normal function of Gb3 in humans is unknown. Although not fully characterized, Gb3 appears to show a relatively restricted expression in normal human tissues, being mostly found in kidney epithelium and endothelium (Lingwood, 1994; Khan et al., 2009), in microvascular endothelial cells in intestinal lamina propria (Miyamoto et al., 2006; Schuller et al., 2007), in platelets (Cooling et al., 1998), and in subsets of germinal centre B lymphocytes (Murray et al., 1985; Gregory et al., 1988; Mangeney et al., 1991). A low level of Gb3 expression has been reported in monocytes (van Setten et al., 1996), and in monocyte-derived macrophages and dendritic cells (Falguières et al., 2001). Recent reports indicate that Gb3 is also expressed by intestinal pericryptal myofibroblasts (Schuller et al., 2007), neurons (Obata et al., 2008) and endothelial cells in the central nervous system (Johansson et al., 2006; Obata et al., 2008), and the possible implications of this needs to be further clarified. For example, since primary cultures of human cerebral capillary and microvascular endothelial cells are found to be largely resistant to Stx (Arab et al., 1998; Ramegowda et al., 1999; Hughes et al., 2002), it remains to be determined to what extent Gb3 is expressed at the cell surface of human brain endothelial cells, and whether Stx is toxic to these cells in vivo. It should be kept in mind that with regard to the sensitivity of cells to the toxic action of Stx, cell surface expression of Gb3 is not always sufficient (Sandvig et al., 1992; Jacewicz et al., 1994), since in general the retrograde transport step to the Golgi and ER is required for Stx toxicity. Moreover, as pointed out above, the expression of different species of Gb3 may alter the binding and intracellular routing of Stx.