Thomas P. Griener

In vivo supramolecular templating enhances the activity of multivalent ligands: A potential therapeutic against the Escherichia coli O157 AB5 toxins

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.

Human Serum Amyloid P Component Protects against Escherichia coli O157:H7 Shiga Toxin 2 In Vivo: Therapeutic Implications for Hemolytic-Uremic Syndrome

Shiga toxin (Stx) 2 causes hemolytic-uremic syndrome (HUS), an intractable and often fatal complication of enterohemorrhagic Escherichia coli O157:H7 infection. Here, we show that serum amyloid P component (SAP), a normal human plasma protein, specifically protects mice against the lethal toxicity of Stx2, both when injected into wild-type mice and when expressed transgenically; in the presence of human SAP, there was greatly reduced in vivo localization of Stx2 to the kidneys, suggesting a possible mechanism of protection. In humans, circulating SAP concentrations did not differ between patients with suspected enterohemorrhagic E. coli infection with antibodies to E. coli O157:H7 lipopolysaccharide and those without antibodies or between patients with HUS and those without it. However, the potent protection conferred by human SAP in the mouse model suggests that infusion of supplemental SAP may be a useful novel therapeutic approach to the treatment of this devastating condition.

The bundlin pilin protein of enteropathogenic Escherichia coli is an N‐acetyllactosamine‐specific lectin

Synthetic N‐acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc‐specific adhesin appears to be associated with the bundle‐forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp‐2 cells and binds to HEp‐2 cells. Recombinant bundlin also binds, with millimolar association constants (Kassoc), to synthetic LacNAc‐Benzene and LacNAc‐O(CH2)8CONH2 glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc‐BSA inhibits LA only of EPEC strains that express α bundlin alleles, suggesting putative locations for the LacNAc‐binding pocket in the α bundlin monomer. Collectively, these results suggest that α bundlin possesses lectin‐like properties that are responsible for LacNAc‐specific initial adherence of α bundlin‐expressing EPEC strains to host intestinal epithelial cells.

Recombinant Shiga Toxin B-Subunit-Keyhole Limpet Hemocyanin Conjugate Vaccine Protects Mice from Shigatoxemia

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.

N-acetyllactosamine-induced retraction of bundle-forming pili regulates virulence-associated gene expression in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi‐step process culminating in the intimate adherence of the organisms to the hosts intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle‐forming pili (BFP), plays an integral role. We previously reported that the major BFP structural subunit, bundlin, displays lectin‐like properties, which enables BFP to initially tether EPEC to N‐acetyllactosamine (LacNAc) glycan receptors on host cell surfaces. We also reported that incubating EPEC with synthetic LacNAc‐bearing neoglycoconjugates not only inhibits their adherence to host cells, but also induces BFP retraction and subsequent degradation of the bundlin subunits. Herein, we demonstrate that the periplasmic serine protease, DegP, is required for degrading bundlin during this process. We also show that DegP appears to act as a bundlin chaperone during BFP assembly and that LacNAc‐BSA‐induced BFP retraction is followed by transcriptional upregulation of the BFP operon and downregulation of the locus of enterocyte effacement operons in EPEC.

Assembly and Stability of the Shiga Toxins Investigated by Electrospray Ionization Mass Spectrometry

A systematic investigation into the assembly and stability of native and modified subunits of the Shiga toxins (Stx) in vitro is described. Analysis of the assembly of native and modified B subunits of Stx1 and Stx2 in solution, carried out using electrospray ionization mass spectrometry (ES-MS), suggests that the lower thermodynamic stability of the B subunit homopentamer of Stx2, compared to that of Stx1, is due to the presence of a repulsive interaction involving Asp70 of the Stx2 B subunit. In Stx1 B, the corresponding (spatially) residue is Arg. Using temperature-controlled ES-MS, it is shown that the Stx1 and Stx2 holotoxins exhibit differences in their resistance to temperature- and acid-induced dissociation. However, both Stx1 and Stx2 are fully assembled at pH >3.5 and 37 degrees C. This finding has several important biological implications. First, it argues against the likelihood that the difference in Stx1 and Stx2 toxicity arises from differential dissociation of the toxins during the intracellular trafficking steps of the cellular intoxication process. Second, it implies that the activation of the A subunits of Stx1 and Stx2 by enzymatic cleavage must occur while the A subunit is assembled with the B subunit homopentamer. It is, therefore, proposed that the differential toxicities of Stx1 and Stx2 reflect the relative efficiencies of intracellular activation of the A subunits.

Lipopolysaccharide Renders Transgenic Mice Expressing Human Serum Amyloid P Component Sensitive to Shiga Toxin 2

Transgenic C57BL/6 mice expressing human serum amyloid P component (HuSAP) are resistant to Shiga toxin 2 (Stx2) at dosages that are lethal in HuSAP-negative wild-type mice. However, it is well established that Stx2 initiates extra-intestinal complications such as the haemolytic-uremic syndrome despite the presence of HuSAP in human sera. We now demonstrate that co-administering purified Escherichia coli O55 lipopolysaccharide (LPS), at a dosage of 300 ng/g body weight, to HuSAP-transgenic mice increases their susceptibility to the lethal effects of Stx2. The enhanced susceptibility to Stx2 correlated with an increased expression of genes encoding the pro-inflammatory cytokine TNFα and chemokines of the CXC and CC families in the kidneys of LPS-treated mice, 48 hours after the Stx2/LPS challenge. Co-administering the glucocorticoid dexamethasone, but not the LPS neutralizing cationic peptide LL-37, protected LPS-sensitized HuSAP-transgenic mice from lethal doses of Stx2. Dexamethasone protection was specifically associated with decreased expression of the same inflammatory mediators (CXC and CC-type chemokines and TNFα) linked to enhanced susceptibility caused by LPS. The studies reveal further details about the complex cascade of host-related events that are initiated by Stx2 as well as establish a new animal model system in which to investigate strategies for diminishing serious Stx2-mediated complications in humans infected with enterohemorrhagic E. coli strains.

From alpha to beta : identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin

Bundle‐forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, α and β, based on their amino acid sequences. Alpha bundlins are also N‐acetyllactosamine‐ (LacNAc) specific lectins and bind to HEp‐2 cells, whereas β bundlins do not display these characteristics. The four surface‐exposed regions of amino acid sequence heterogeneity between α and β bundlin were therefore investigated as potential LacNAc‐specific carbohydrate‐binding domains in a bundlin. Mutation of one of these domains, 137‐GENNI‐141, in α1 bundlin to that of β bundlin (136‐SPDST‐140) resulted in BFP that no longer bound to LacNAc or HEp‐2 cells. Conversely, mutating the β3 bundlin gene to encode the α bundlin sequence at this domain resulted in the gain of HEp‐2 cell adherence. The importance of this domain in carbohydrate binding is supported by the finding that introducing the mutation GENNI→GENNT altered the α1 bundlin carbohydrate‐binding specificity from LacNAc to the Lewis X glycan sequence.

Impact of the Nature and Size of the Polymeric Backbone on the Ability of Heterobifunctional Ligands to Mediate Shiga Toxin and Serum Amyloid P Component Ternary Complex Formation

Inhibition of AB5-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin’s pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB5 complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor’s activity.

The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2. Of these, Stx2 appears to be linked to higher rates of HUS than Stx1. To investigate a possible reason for this, we exposed human macrophage-like THP-1 cells to Stx1 or Stx2 and then used the Luminex multiplex system to assess cytokine/chemokine concentrations in culture supernatant solutions. This analysis revealed that, relative to Stx1, Stx2 significantly caused increased expression of GRO, G-CSF, IL-1β, IL-8 and TNFα in macrophage-like THP-1 cells. This was determined to not be due to a difference in cytotoxicity since both Stx1 and Stx2 displayed similar cytotoxic activities on macrophage-like THP-1 cells. These observations indicate that, in vitro, Stx2 can provoke a greater pro-inflammatory response than Stx1 in macrophages and provides a possible partial explanation for higher rates of HUS in patients infected with EHEC strains expressing Stx2. To begin to determine a mechanism for Shiga toxin-mediated cytokine production, we exposed macrophage-like THP-1 cells to Stx1 or Stx2 A and B subunits. Luminex analysis of cytokines in cell culture supernatant solutions demonstrated that neither subunit alone induced a cytokine response in THP-1 cells.