Ribosome-inactivating Proteins (Ricin and Related Proteins) || Shiga Toxins

Ribosome-inactivating Proteins: Ricin and Related Proteins, First Edition. Edited by Fiorenzo Stirpe and Douglas A. Lappi.

© 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.

97

7 Shiga Toxins: The Ribosome-inactivating Proteins from Pathogenic BacteriaMaurizio Brigotti

Dipartimento di Medicina Specialistica, Università di Bologna, Italy

Introduction

Adhesiveness, invasiveness, and delivery of toxins are considered to be the main weapons of the bacteria responsible for diseases in humans and animals. The former virulence factors allow pathogenic bacteria to interact with eukaryotic cells, to multiply and to penetrate host tissues, whereas the production of toxins induces specific effects on target cells (exotoxins) and broad or systemic effects in the host (endotoxins). The interplay between these virulence factors is of prime importance in the development of infectious diseases. Stxs (Shiga toxins) are produced by various bacteria including Shigella dysente-riae type 1, which is responsible for bacillary dysentery in humans, and by a restricted subset of Escherichia coli strains, hence called STEC (Shiga toxin-producing E. coli), which have a causative role in the pathogenesis of hemorrhagic colitis and HUS (hemolytic uremic syndrome) in children.1

Interestingly, these bacterial toxins and the RIPs (ribosome-inactivating proteins) from plants were found to share the same enzymatic mechanism of action on ribosomes (see below) and hence Stxs are considered the bacterial branch of this large family of enzymes. Recently, another bacterial RIP from the soil bacterium Streptomyces coelicolor has been described.2 This protein, however, differs from Stxs since it is not toxic to intact cells and is apparently not related to human diseases.

Purification of Shiga Toxins

The involvement of Stxs in the pathogenic processes of the above-mentioned human diseases was established after the purification to homogeneity of the cytotoxin produced by Shigella. This was accomplished by Olsnes and colleagues,3, 4 starting from bacterial culture media or bacterial lysates, through a laborious purification procedure based on repeated chromatographic steps, sucrose gradient centrifugation, and non-denaturating polyacrylamide gel electrophoresis. The addition of carrier proteins (rabbit hemoglobin, bovine serum albumin) and radioiodination of the partially purified toxin were exploited to avoid loss of toxin activity and to facilitate detection. Since then, many different methods have been devised enabling isolation of higher amounts of purified Stxs and preserving their cytotoxic and enzymatic activities. To date, the most simple and useful methods are

98 RIBOSOME-INACTIVATING PROTEINS

those based on the capture of the toxins present in crude bacterial lysates or in culture media with the immobilized specific receptor. Typical examples are the affinity chromatographies with globot-riose-fractogel,5 P1 glycoprotein,6, 7 and Synsorb pK.8 These methods are not time-consuming and induce only minimal perturbation of the toxic sample. This preserves the cytotoxic activity (binding specificity for target cells and intracellular enzymatic activity) and also the known ability of Stxs to be recognized by PMN (polymorphonuclear leukocytes) in the blood stream of the infected hosts. The latter interaction is probably a crucial step in the pathogenesis of HUS and/or in the innate response of the host, as recently reviewed.9 Proper folding is required for Stxs to be recognized by PMN and it has been suggested that single-step purification procedures would yield Stxs with the proper conformation, whereas more complicated multi-step methods may induce partial unfolding of Stxs and loss of binding to PMN.9, 10 It is worth noting that toxicity is preserved in partially unfolded Stxs, that only lose the binding activity for PMN.10

Structure and Mechanism of Action of Shiga Toxins

The AB5 Structure

Suitable and functional molecular models have often been re-proposed during evolution, as in the structure of some bipartite bacterial exotoxins. The classical AB5 model comprises an enzymatic A chain non-covalently bound to a pentamer of B chains which bind cell receptors. Cholera toxin, E. coli heat-labile toxin, and all the Stxs variants share this structure, although the binding speci-ficity as well as the type of enzymatic activity greatly differ among them. The Stxs family comprises several toxic molecules: Stx (Shiga toxin) is the single prototype produced by Shigella, whereas numerous cytotoxins are elaborated by STEC, even though two variants prevail: Stx1 (Shiga toxin 1) and Stx2 (Shiga toxin 2).1 The former is immunologically indistinguishable from Stx since it differs by a single amino acid residue located in the A chain (Thr45 in Stx, Ser45 in Stx1).11 Conversely, Stx2 is not neutralized by antibodies to Stx since it has less than 60% similarity with the prototype toxin, both at protein and at gene levels.12 Stx1 and Stx2 are phage-encoded and require induction for full expression,13, 14 whereas Stx is encoded by genes located on the Shigella chromosome.11

Crystallographic studies have detailed the structure of Stxs variants and the reciprocal interac-tions between A (32 kDa) and B chains (7.7 KDa; Figure 7.1).15 The B chains, each folded in a single α-helix, are arranged in a pentamer which form a ring delimiting an opening. In this space, a single α-helix belonging to the C-terminus moiety of the A chain deeply interacts with the five B subunit α-helices forming a non-covalent bridge between the two different chains of the holotoxin. The structure is reinforced with antiparallel β-sheets formed by strands belonging to each B subunit that, on the outside of the B pentamer, encircle the A/B contact regions (Figure 7.1). It is of note that the arrange-ment and the fold of the B-chain pentamer of Stxs are similar to those of the B subunits of the E. coli heat-labile toxin. Stx A chain harbors the active site in a cleft closely resembling the one in the A subunit of ricin, the well-known plant RIP from the seeds of Ricinus communis.15, 16 The folding of ricin A chain and of a portion of Stx A chain (called A1 fragment, see below) are very similar, since they possess about 150 structurally equivalent amino acid residues and more than 20% of them are identical.15, 16 More importantly, there are several invariant amino acid residues present in the active sites of the two toxins (Tyr-77, Val-78, Ser-112, Tyr-114, Glu-167, Ala-168, Arg-170, and Trp-203; numbers as in Stx) and this represents a further evolutionary convergence between molecules belonging to quite different biological kingdoms.15, 16 A comprehensive review of the structural behaviors of RIPs is given in Chapter 8.

SHIGA TOXINS: THE RIBOSOME-INACTIVATING PROTEINS FROM PATHOGENIC BACTERIA 99

Intracellular Activation

Both Stx1 and Stx2 share the requirement of intracellular activation to fully express the toxic activity. The reduction of a single disulfide bond connecting A and B subunits in ricin allows the A chain to reach the cellular targets.17 In the case of Stxs the A/B chain interactions are non-covalent and the disulfide bond in fact connects two fragments of the A subunit arising from the intracellular proteolytic activity (Figure 7.2). In eukaryotic cells, during endocytosis, the membrane-anchored protease furin cleaves the A chain of Stxs18 forming two different fragments: A1 (~28 kDa) endowed with the enzymatic activity and A2 (~4 kDa) interacting with the B chains (Figure 7.2). The reduction of the disulfide bond is mandatory for the disengagement of the active site-containing A1 moiety and, in the case of Stx1, also for boosting the enzymatic activity. In fact, analysis of the kinetic constants of the enzymatic reaction catalyzed on ribosomes (see below) revealed similar substrate affinities for holotoxin, isolated A chain, and A1 fragment (K

m ~ 1 μM), whereas the K

cat of the A1

fragment was 100 and 1000-fold higher than those of the A chain and holotoxin, respectively.19

Ribosomes and DNA as Intracellular Targets

Stxs have been classified as RNA N-glycosidases acting on eukaryotic ribosomes, even though the term glycosylases was proposed to indicate their action on nuclear DNA (Figure 7.3). Both the enzymatic activities involve the cleavage of a glycosidic bond connecting the base adenine to the sugar of the corresponding nucleic acid. Stxs express their activity on 28S rRNA in the large

A chain

B chains

Figure 7.1 Detailed structure of Stx1 depicted as ribbon diagram. Reproduced with permission15 with modifications. The

fracture between A1 and A2 is due to proteolysis.


ribosomal subunit, specifically removing a single adenine residue20 located in a conserved stem-loop region21 recognized by elongation factors during protein synthesis.22 The ribosomal functional impairment is thus related to the inhibition of the binding of elongation factors to ribosomes and the consequence is the irreversible arrest of translation in cells. The specific injury and the particular functional impairment induced by ricin and Stxs acting on ribosomes are indistinguishable, as the plant and bacterial toxins share the same mechanism of action. A more detailed description of this topic is presented in Chapter 2.

Shortly after the discovery of the mechanism of action on ribosomes, a method for the detection of the adenine released by the enzymatic activity of these toxins was devised. The base can be readily detected by HPLC (high pressure liquid chromatography) after conversion into its fluorescent etheno-derivative.23 By this method, the glycosylase activity of plant ribosome-inactivating pro-teins24, 25 and Stxs26 on isolated DNA in vitro was demonstrated. Although these results provided an important contribution to the field, the effect on nuclear DNA in whole cells and the relationship with the ribotoxic activity were only pinpointed several years later. The time-course of the enzy-matic activities of Stx1, Stx2, and ricin acting on ribosomes and DNA was measured in human endothelial cells.27, 28 The two bacterial toxin variants showed overlapping time-courses of ribosome inactivation, while the ribotoxic activity of ricin was shown to occur later, probably reflecting dif-ferent modes of internalization. The glycosylase activity on DNA was expressed at the same time (ricin) or 3 hours after (Stx1, Stx2) the inhibition of translation, even though the damage to nuclear DNA was not secondary to the inhibition of protein synthesis, nor to apoptosis.27, 28 In fact, nuclear

A2 A1

B

B

B B

B

S–S

Figure 7.2 Schematic structure of the A/B interactions in Stxs. (This figure also appears in the color plate section.)

AdenineStxs

StxsAdenineAdenine

DNA

28S rRNA

X

XXX

X

Adenine

Adenine

Figure 7.3 Enzymatic activity of Stxs on ribosomal RNA and nuclear DNA. (This figure also appears in the color plate

section.)


DNA damage peaked several hours before the onset of the earliest markers of apoptosis in intoxi-cated cells,27, 28 thus indicating that the ordered apoptotic DNA digestion with formation of discrete fragments is clearly distinguishable from the primary and direct DNA damage induced by the toxins. It should be noted that nuclear DNA damage was quantified by a sensitive and simple method, the “halo assay,” performed in alkaline conditions, which allows the conversion of DNA apurinic sites in single strand DNA breaks to be detected by microscopic observation.29, 30

The glycosylase activity of Stxs appears rather broad in specificity as these enzymes are capable of removing multiple adenines from DNA in vitro26, 31 and in whole cells.27 Their activity is very similar to that of DNA repair enzymes that preserve DNA homeostasis in the nucleus by removing inappropriate, mismatched, or damaged bases. There are few significant examples of DNA mis-match repair enzymes, either in bacteria (MutY) or in man (MYH), which specifically recognize the normal base adenine as well as Stxs do. By comparing the kinetics of Stx1 acting on DNA with those of known DNA glycosylases acting on adenine in mismatch, it is noteworthy that the speci-ficity constant (K

cat/K

m) of Stx1 (4.2 μM-1/min-1)32 fits within the range of values (0.5–39 μM-1/min-1)

obtained with MutY and MYH,33, 34 showing that the bacterial toxin is as efficient in removing adenine from DNA as other known DNA glycosylases. However, the consequences of these enzy-matic actions are quite dissimilar, since Stxs would induce toxic, mutagenic, and cancerogenic effects rather than DNA repair, as previously demonstrated in the case of plant RIP.35 The situation is complicated by the ability of Stxs to inhibit the repair of oxidative and alkylative DNA lesions via a mechanism involving impairment of BER (base excision repair).36 It has been shown that some plant RIPs35 remove adenines from poly(ADP-ribose), a polymer produced by the enzymatic activity of PARP (poly(ADP-ribose) polymerase) and involved in signaling DNA damage and in orches-trating DNA repair.37, 38 As observed in vitro,35 an extensive depurination might promote early deg-radation of poly(ADP-ribose) polymer and, if this happens in the nucleus of intoxicated cells, the PARP-dependent nuclear DNA repair processes could be affected. These authors also demonstrated the transforming activity of plant RIP for 3 T3 fibroblasts.35 Thus, the genotoxicity of Stxs on mam-malian cells would be the result of direct (DNA damaging activity) and indirect effects (DNA repair inhibition), being also influenced by the presence of other DNA targeting species.

Binding Properties of Shiga Toxins

The binding properties of the two main Stxs variants are rather specific and involve a multivalent interaction of their B chains to neutral glycosphingolipids, mainly globotriaosylceramide (Gb3Cer) and to a lesser extent to globotetraosylceramide (Gb4Cer). Gb3Cer is expressed on the membrane of few human cells, that is endothelial cells lining the microvasculature of the kidney, brain, and intestine; mesangial cells; glomerular epithelial cells; and tubular epithelial cells in the kidney.39–42 The receptor distribution explains the involvement of the gut in hemorrhagic colitis and of kidney and brain in HUS, as consequences of human infections by the widely diffused STEC. Stxs bind to the membrane of sensitive cells and are then endocytosed by different pathways involving lipid rafts, the latter defined as membrane microdomains in which cholesterol, glycosphingolipids, sphingomyelin, dipalmitoylphosphatidylcholine, and several proteins are assembled.43 To reach their intracellular targets, Stxs present in endosomes must be retrogradely transported through the Golgi apparatus to the endoplasmic reticulum where the active A1 fragment is translocated to the cytosol and to the nucleus.44–48 Although Gb3cer is required as the Stxs receptor, glycosphingolipids in general are important for the transport of Stxs from endosomes to Golgi once the toxin has bound the specific receptor.49 Moreover, in vertebrates, glycosphingolipids are composed of a sphingosine


core linked to a fatty acid which differs in chain length (mostly C16 to C24) and degree of desatura-tion.43 This heterogeneity in GB3Cer lipoforms might condition the intracellular transport and des-tination of Stxs, since different fatty chains would mediate different interactions with other membrane components in lipid drafts.50, 51 Chapter 2 of this book compares and contrasts ricin and Stxs, giving the molecular details of trafficking in eukaryotic cells.

Role of Shiga Toxins in the Pathogenesis of HUS

HUS is the life-threatening sequela of intestinal infections caused by STEC and, in the Western world, is the leading cause of acute renal failure in early childhood.52–54 Typical STEC strains, such as O157:H7, live in the gut of ruminants, particularly cattle, without provoking any symptoms in animals.55 Therefore, hemorrhagic colitis and HUS should be considered zoonoses, with vegetables or undercooked bovine meat contaminated with fecal bovine specimens being the main vehicles of the diseases.52–54

Pathogenesis of HUS

The hallmark of HUS is the presence of thrombotic microvascular lesions confined to few organs and in particular to the renal glomeruli, the gastrointestinal tract, and the brain. Microvascular endo-thelial injury is considered the most important pathogenic event in HUS, and the targeted endothe-lial cell subtypes are known to express Gb3Cer receptors for Stxs.1 It is recognized that the toxins have a causative role in HUS and this is consistent with data obtained in patients and in vitro at both cellular and molecular levels.1, 53

The histopathology of HUS in humans has been investigated in detail, and the main features observed in renal glomeruli include changes in capillary wall thickness, swelling and detachment of endothelial cells from the basement membrane, and deposition of fibrin and platelets with congested rather than ischemic glomeruli.56, 57 This explains the acute renal failure, the first com-ponent of the triad characterizing HUS, closely related to the presence of microthrombi, which compromise blood supply by narrowing or occluding the capillary lumen.58, 59 In HUS, platelets are found activated and massively engaged in microthrombi, as well as aggregated in blood and thus removed by the reticuloendothelial system. This large consumption of platelets is the cause of the severe thrombocytopenia seen in HUS patients, the second component of the triad.58, 59 HUS patients complete the triad by developing hemolytic anemia, whose main features are the formation of fragmented erythrocytes, often with shapes resembling helmets, that are removed from the blood by the reticuloendothelial system. The lesions to red blood cells are mechanically induced, since these cells are forced to pass through the partially occluded capillaries in renal glomeruli.58, 59 In keeping with the notion that Stxs are the main actors in the development of HUS, many investigations have been carried out to shed more light on the mechanisms under-lying the pathophysiology of the syndrome on the basis of the toxin actions on targeted organs and cells.

After the consumption of food contaminated by STEC and a short incubation time (about 3 days) patients develop watery diarrhea with cramping abdominal pain (Figure 7.4).1, 53 Interestingly, this non-specific symptom is not related to the action of Stxs, but rather to the particular and intimate mode of adhesion of these E. coli strains to the epithelial cells of the gut. STEC interacting with human intestinal mucosa are capable of translocating various bacterial


effectors into enterocytes through an injection device called the Type III secretion system.60 This leads to intimate adhesion to the gut, and the deriving, attaching and effacing lesions cause pro-found modifications and perturbations in epithelial cells that lose brush borders and accumulate actin-derived pedestals beneath the site of membrane contact.1, 59 The derangement of the intestinal lining alters the mechanisms of absorption of the bowel, thus explaining the watery diarrhea. In a short time (1–2 days), a large proportion of infected patients may develop bloody diarrhea that is the common prodromal symptom in most HUS cases (Figure 7.4).1, 53 In this case the pathogenetic process involves Stxs: these are produced by the non-invasive bacteria and translocate through the gut epithelium reaching the lamina propria, although human enterocytes apparently do not possess specific Stxs receptors.61 Passage of the toxins through epithelial tight junctions loosened by the adherence of STEC and/or by the opposite passage of PMN in the inflamed bowel have been observed.62–64 Recently, a new Gb3Cer-independent mechanism explaining transcellular transcytosis and centered on the actin-driven macropynocitotic activity of human enterocytes has been described.65 Interestingly, Stxs uptake by these cells also stimu-lates apical secretion and depletion of galectin-3, impairing the functions of transporters and structural brush border proteins, thus contributing to the onset of diarrhea.66 Once in the gut lamina propria and absorbed in the circulation, Stxs find Gb3Cer on intestinal endothelial cells, which in turn become intoxicated.61, 62 Mucosal and submucosal edema, hemorrhage with focal areas of necrosis, and thrombotic microangiopathy are the histopathological changes observed in the bowel of patients with hemorrhagic colitis and are related to the action of toxins on gut endothelia.56, 67 After about a week from the onset of bloody diarrhea, HUS develops in a low proportion (~15%) of patients (Figure 7.4).

Linking Shiga Toxins to the Pathogenesis of HUS

During the journey from the gut to the kidney and the brain, Stxs may encounter several blood components. These interactions are believed to be important in the pathogenesis of HUS for two different reasons. On the one hand, Stxs may exploit binding to blood cells to be shuttled into the

Days Stxs involvement

+ main role

+ definite role

– no role

10

5

3

0

Bloody diarrhea

Watery diarrhea

abdominal pain

STEC ingestion

HUS

Figure 7.4 Time-course of STEC infections in humans and role of Stxs in the related diseases. HUS (hemolytic uremic

syndrome); STEC (Shiga toxin-producing E. coli).


ultimate target cells. On the other hand, the challenge to circulating cells may induce responses related to the subsequent pathogenetic steps, such as the intoxication of endothelial cells or the prothrombotic state observed in HUS. Indeed, Stxs have been found to bind in vitro to red cells,68 monocytes,69 platelets,70–72 and PMN.73–75 While the interaction with the former cells is related to the presence on their membrane of Gb3Cer lipoforms similar to those on target endothelial cells, PMN interact with Stxs through a low affinity unknown receptor.73 The transfer of Stxs from PMN to the high affinity Gb3Cer receptors of human endothelial cells has been demonstrated in vitro.73, 76 Moreover, PMN-bound Stxs have been detected in patients with HUS,77, 78 hence the role of these cells in transporting the toxins in blood has been envisaged, although is as yet unproven, in HUS patients. Strikingly, the A chain of ricin and two different single-chain RIPs interact with the same PMN receptor, indicating a specific role for these cells in the recognition of foreign toxins.79

Monocytes triggered by Stxs showed up-regulation and secretion of IL-1 and TNF-α,69 two potent inflammatory mediators that, in turn, enhance Gb3Cer expression on endothelial cells.80, 81 This would render renal endothelial lining more susceptible to toxin action, and so HUS is probably fomenting in the kidney during the prodromal gastrointestinal phase.

Platelet activation and aggregation induced by Stxs have been observed.72 as well as binding of Stxs to platelets activated by other stimuli.71 In both cases, these interactions appear to be involved in the development of thrombocytopenia (massive engagement in damaged body sites or removal of platelet aggregates by the reticuloendothelial system) and in microthrombi formation. Recently, however, Stxs have also been found on blood cell complexes.82, 83

This is particularly intriguing, since two STEC virulence factors added to human blood, such as LPS from the widely diffused O157:H7 STEC strain and Stx2, induce the formation of platelet–monocyte or platelet–neutrophil aggregates containing activated thrombocytes and leu-kocytes.83 Moreover, platelet- or monocyte-derived microparticles are generated upon these treatments and, interestingly, the combined (Stx2, O157LPS) challenge leads to higher amounts of microparticles expressing tissue factor, a key activator involved in fibrin generation and thus related to thrombogenesis.83 Indeed, HUS patients show higher platelet–leukocyte aggregates in blood and increased levels of plasmatic and aggregate-borne tissue factor than do healthy chil-dren.83 Stx2 has been shown to be present on these circulating aggregates and this further high-lights the direct involvement of these bacterial toxins in the prothrombotic states observed during the natural course of HUS.83

Stxs also have a clear-cut role in directly activating the complement system via the alternative pathway and in delaying the protecting action of factor H for host cells.84 This was confirmed in HUS patients who have circulating platelets, leukocytes, and microparticles bearing activated complement components on their surfaces.82 The same study also confirmed the involvement of Stxs and O157LPS in this phenomenon.82 This is in keeping with old and recent unexplained evi-dence on the activation of the complement system in HUS patients.85, 86 Complement is a key weapon of innate immunity, deeply related to the inflammation response, thus activation of complement by Stxs may well induce direct destruction of kidney tissues and indirect inflammatory injuries.

It should be noted that a small number of sporadic or familial HUS cases are caused by known genetically-driven defects (atypical HUS) of key complement regulators (loss of function muta-tions) or of C3 convertase components (gain of function mutations).87 The former mutations reduce the defenses of endothelial cells against complement attacks, whereas the second mutations result in hyperactive C3 convertase, the step at which the alternative or classical complement pathways converge.87 A novel therapy for atypical HUS was developed by the administration of a monoclonal


antibody to C5 (eculizumab) preventing the generation of the pro-inflammatory peptide C5a and the formation of the membrane-attack complex.88, 89 Since the involvement of complement might represent a common thread between atypical HUS and typical STEC-induced HUS, treatment with eculizumab was proposed in a recent German outbreak (see below).90 Nevertheless, the fact that HUS develops in a relatively low proportion of STEC-infected children suggests that genetic or acquired factors concur with STEC virulence factors in triggering HUS. One can hypothesize that particular polymorphisms of normal genes regulating complement rather than genetic defects are involved in typical STEC-induced HUS. However, it should be borne in mind that the pathogenesis of typical HUS is centered on the interaction between Stxs and endothelial cells endowed with the specific toxin receptor. Therefore, explanations of what happens during typical HUS on the basis of non-endothelial cell responses to Stxs or LPS also need to take account of the actions of the toxin on the endothelial lining of target organs.

Stxs act on sensitive human endothelial cells triggering an array of responses: intoxicated cells might undergo apoptosis, produce pro-inflammatory cytokines, or up-regulate their adhesion molecules. Microvascular endothelial cells treated with Stxs express P selectin, an adhesion molecule implicated in platelet deposition, also being a specific high affinity ligand for C3.91 Moreover, Stxs promote C3 activation that results in C3a generation and the related pro-inflamma-tory response.91 Other findings have revealed a more complicated inflammatory scenario, since endothelial cells challenged with Stxs up-regulate the expression of several pro-inflammatory cytokine messengers and of the corresponding proteins. This has been demonstrated in different laboratories as recently reviewed9 and, in particular, in a straightforward study with human endothelial cells in which microarray analysis of thousands of genes showed the up-regulation of about 25 pro-inflammatory messengers after treatment with both Stx1 and Stx2.92 The up- regulating events were also demonstrated at the protein level, and this is not obvious since Stxs are strong inhibitors of translation.92

How can the toxic effect induced by Stxs be related to these up-regulating effects? Endothelial cells after intoxication even by small concentrations of Stxs suffer from two different stresses: ribo-toxic stress and ER (endoplasmic reticulum) stress. Ribotoxic stress is a direct consequence of the depurinating activity of 28S RNA by Stxs, as well as by other RIPs such as ricin.93 On the other hand, ER-stress is probably initiated by the production of misfolded or incomplete proteins after toxin action on the protein synthesis machinery.94 The former stress leads to the activation of MAPK (mitogen-activated protein kinases) cascades culminating in the transcription of the above men-tioned pro-inflammatory cytokine messengers through activation of proper transcription factors (NF-kB and AP-1).9, 94 The same signaling pathway might also be important in regulating apoptosis of intoxicated cells as does a prolonged ER-stress.94 Indeed, endothelial cells might choose to par-ticipate in their own demise when multiple signaling pathways, such as ribotoxic stress, ER-stress, DNA damage, interaction with Gb3Cer (B chain), or with Bcl-2 (Stx1 A chain),28, 48, 94–96 converge in the induction of apoptosis.

Thus, Stxs contribute to the development of HUS by eliciting a plethora of effects on endothelial and non-endothelial cells that can be categorized as: (i) pro-apoptotic effects, (ii) pro-thrombotic effects, and (iii) pro-inflammatory effects. Stxs seem to be a multi-faceted agonist stimulating eukaryotic cells to devise multiple concurrent and interacting responses involved in the pathogenesis of this syndrome. Estimating the relative importance of the different Stxs-induced pathways, their relationships, and the precise hierarchy would help further understanding of the pathogenesis of HUS and, in particular, the obscure mechanisms underlying the transition between hemorrhagic colitis and HUS. The relevance of Stxs in human pathology was exemplified dramatically during the 2011 German outbreak discussed below.


The 2011 German STEC Outbreak

In May–July 2011, a public health issue arose from the sudden appearance of a large STEC outbreak that caused a remarkable number of deaths among adults in Germany and other European countries. The unusual STEC strain responsible for the outbreak (E. coli O104:H4) was improperly re-named by the media as a killer bacterium, leading to widespread alarm in the population. Although STEC has been studied for many decades by scientists, this strain displayed an unexpectedly high level of virulence, likely related to an unusual combination of virulence determinants. The German strain produced Stx2 and exhibited a particular pattern of adherence to human intestinal epithelial cells, called enteroaggregative adhesion.97, 98 This pattern differed considerably from that caused by the interaction of the known STEC strains with human intestine. A similar combination was found in the 1990s in a different E. coli strain (O111:H2) that caused a more confined HUS outbreak in French children.99, 100

In the third week of May 2011, the Robert Koch Institute in Germany noted an abnormally high proportion of patients with HUS and bloody diarrhea caused by STEC.98 Since then, and during the fully evolved outbreak, many attempts have been undertaken by the German Public Health Authorities to identify the source and vehicle of the infection. Initially, cucumbers were incrimi-nated and were consequently banned in several European countries with considerable economic consequences. The unusual combination of virulence factors in the etiologic agent of the outbreak98 was identified in Denmark by Flemming Scheutz (World Health Organization, Collaborating Centre for Reference and Research on Escherichia and Klebsiella, Copenhagen) at the end of May 2011 in a traveling German patient.97 A few days later, the European Union Reference Laboratory for E. coli, directed by Alfredo Caprioli (Istituto Superiore di Sanità, Rome, Italy), developed a novel real-time PCR procedure for the detection of the German strain in food.97 The new test was shared with the National Reference Laboratories of the different European Countries for food testing. The actions of the European Public Health Authorities appeared to be rapid, efficient, interactive, and integrated. By virtue of this synergism and of more carefully conducted analysis, a correlation between the consumption of fenugreek sprouts and this food-borne STEC outbreak emerged, hence exonerating cucumbers.101 Overall the German outbreak caused about 4000 STEC-induced cases of diarrhea and 22% of these patients developed HUS, with an unusually high incidence, as well as an unexpectedly high mortality (about 50 cases).102 Moreover, close to 50% of HUS patients developed neurologic complications and needed dialysis103 and, most importantly, 88% of the patients were adult (median 42 years) and 68% of them were female.104 Since HUS is the main cause of acute renal failure in early childhood, the epidemic profile of the German outbreak appears idiosyncratic.104 Horizontal genetic exchange with acquisition of the prophage encoding Stx2 by an enteroaggregative E. coli O104:H4 strain, otherwise able to cause only diarrhea, was found to be the mechanism allowing the emergence of this exceptionally virulent strain.102 The features of E. coli O104:H4 stunned the experts, since enteroaggregative E. coli are a well known group of human pathogens. Actually, enteroaggregative E. coli are a frequent cause of persistent watery diarrhea in infants and children in developing countries105 and have been involved in rare outbreaks in adults,97 always without inducing renal involvement. The lesson to scientists and Public Health Authorities arising from this severe outbreak is two-fold: first, the extreme plasticity of E. coli genome can allow other less virulent strains to acquire the genes encoding for Stxs; second, Stxs emerge as pivotal toxins in human pathology. In this light, to reduce the threat in the near future, it will be of paramount importance to set up international prevention programs aimed at rapidly identifying new emerging pathogenic bacteria producing Stxs.


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Ribosome-inactivating Proteins (Ricin and Related Proteins) || Shiga Toxins