Antibody response to lipopolysaccharides and recombinant proteins of Shiga toxin (STX)-producing Escherichia coli (STEC) in children with haemolytic uraemic syndrome in Poland.

Typical haemolytic uraemic syndrome (STEC-HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), is a serious, life-threating disease that mainly affects children. Bacteriological and genetic tests are commonly used in the routine laboratory diagnosis of STEC-HUS; however, serological methods have emerged as useful and reliable diagnostic tools, especially when bacterial isolation fails. In this study, we present the results of the serological investigation of 72 paediatric patients suspected for HUS, hospitalized during 2011-2019 at the Department of Pediatrics and Nephrology of Children's Hospitals in Poland. During the routine laboratory investigation STEC strains were isolated only from nine stool samples. However, serological investigations confirmed 45 cases of STEC infections in children with HUS. In this study, 22 (48·9%) paediatric patients were infected by E. coli serotype O26, 11 (24·4%) by serotype O145, 9 (20·0%) by serotype O157, and 3 (6·7%) by E. coli serotype O111. In the majority of these patients, in addition to a high level of IgA, IgG and IgM antibodies to lipopolysaccharide of particular E. coli serotypes, antibodies to recombinant proteins Tir, Stx2b and intimin were detected. Our results confirm that serological tests are useful in the diagnosis of STEC-HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that serological analysis greatly complements bacterial isolation and helps in the diagnosis and confirmation of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) infections. Serological tests can be performed to qualify the patient for the typical haemolytic uraemic syndrome (STEC-HUS). In Poland, STEC-HUS in children is mostly caused by the E. coli serotype O26, which indicates that there is an increasing number of non-O157 STEC infections associated with human diseases in Europe.

Development of support based on chitosan and cellulose nanocrystals for the immobilization of anti-Shiga toxin 2B antibody.

This work describes the development of membrane based on chitosan (CHI), cellulose nanocrystals (CNCs), and glycerol (GLY), and optimization of the formulation for immobilization of monoclonal anti-Shiga toxin 2B antibody (mAnti-stx2B-Ab) for E. coli O157:H7 detection. The effect of CHI deacetylation degree & viscosity, CNCs and GLY concentrations on Anti-stx2B-Ab immobilization efficiency was evaluated. Fractional factorial and Box-Behnken designs were applied to screen the effects of compounds interactions and optimize their concentrations for detection of Anti-stx2B-Ab. The results demonstrated that the use of 0.6 % (w/v) CNCs improved significantly the Anti-stx2B-Ab immobilization and the level of signal detection. The detection limit of Escherichia coli O157:H7 by developed optimized membrane is 1 log CFU/mL. The time needed for detection of E. coli O157:H7 was only 4 h of enrichment compared to 24 h with conventional methods. The developed immobilization support has potential for future pathogen detection in food and biomedical samples.

Oral immunization of mice with Lactococcus lactis expressing Shiga toxin truncate confers enhanced protection against Shiga toxins of Escherichia coli O157:H7 and Shigella dysenteriae.

Regardless of the communal impact of Shiga toxins, till today neither a specific treatment nor licensed vaccine is available. Lactococcus lactis (L. lactis), generally regarded as safe organism, is well known to provide a valuable approach regarding the oral delivery of vaccines. This study was undertaken to evaluate the protective efficacy of Stx2a1 expressed in nisin-inducible L. lactis, against Shiga toxins (Stx1, Stx2) in mouse model. Oral immunization of BALB/c mice with LL-Stx2a1 elicited significant serum antibody titer with elevated fecal and serum IgA, along with minimized intestinal and kidney damage resulting in survival of immunized animals at 84% and 100% when challenged with 10 × LD50 of Escherichia coli O157 and Shigella dysenteriae toxins, respectively. HeLa cells incubated with immune sera and toxin mixture revealed high neutralizing capacity with 90% cell survivability against both the toxins. Mice immunized passively with both toxins and antibody mixture survived the observation period of 15 days, and the controls administered with sham sera and toxins were succumbed to death within 3 days. Our results revealed protective efficacy and toxin neutralization ability of LL-Stx2a1, proposing it as an oral vaccine candidate against Shiga toxicity mediated by E. coli O157 and S. dysenteriae.

Nano-multilamellar lipid vesicles (NMVs) enhance protective antibody responses against Shiga toxin (Stx2a) produced by enterohemorrhagic Escherichia coli strains (EHEC).

Microlipid vesicles (MLV) have a broad spectrum of applications for the delivery of molecules, ranging from chemical compounds to proteins, in both in vitro and in vivo conditions. In the present study, we developed a new set of nanosize multilayer lipid vesicles (NMVs) containing a unique combination of lipids. The NMVs enable the adsorption of histidine-tagged proteins at the vesicle surface and were demonstrated to be suitable for the in vivo delivery of antigens. The NMVs contained a combination of neutral (DOPC) and anionic (DPPG) lipids in the inner membrane and an external layer composed of DOPC, cholesterol, and a nickel-containing lipid (DGS-NTA [Ni]). NMVs combined with a recombinant form of the B subunit of the Shiga toxin (rStx2B) produced by certain enterohemorragic Escherichia coli (EHEC) strains enhanced the immunogenicity of the antigen after parenteral administration to mice. Mice immunized with rStx2B-loaded NMVs elicited serum antibodies capable of neutralizing the toxic activities of the native toxin; this result was demonstrated both in vitro and in vivo. Taken together, these results demonstrated that the proposed NMVs represent an alternative for the delivery of antigens, including recombinant proteins, generated in different expression systems.

Eculizumab in the treatment of Shiga toxin haemolytic uraemic syndrome.

Haemolytic uraemic syndrome (HUS) remains a leading cause of paediatric acute kidney injury (AKI). Haemolytic uraemic syndrome is characterised by the triad of microangiopathic haemolytic anaemia, thrombocytopenia and AKI. In ~ 90% of cases, HUS is a consequence of infection with Shiga toxin-producing E. coli (STEC), most commonly serotype O157:H7. Acute mortality from STEC-HUS is now less than 5%; however, there is significant long-term renal morbidity in one third of survivors. Currently, no specific treatment exists for STEC-HUS. There is growing interest in the role of complement in the pathogenesis of STEC-HUS due to the discovery of inherited and acquired dysregulation of the alternative complement system in the closely related disorder, atypical HUS (aHUS). The treatment of aHUS has been revolutionised by the introduction of the anti-C5 monoclonal antibody, eculizumab. However, the role of complement and anti-complement therapy in STEC-HUS remains unclear. Herein, we review the current evidence of the role of complement in STEC-HUS focusing on the use of eculizumab in this disease.

Shiga toxin triggers endothelial and podocyte injury: the role of complement activation.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the offending agent in post-diarrhea-associated hemolytic uremic syndrome (HUS), a disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney failure, with thrombi occluding the renal microvasculature. Endothelial dysfunction has been recognized as the trigger event in the development of microangiopathic processes. Glomerular endothelial cells are susceptible to the toxic effects of Stxs that, via nuclear factor kappa B (NF-κB) activation, induce the expression of genes encoding for adhesion molecules and chemokines, culminating in leukocyte adhesion and platelet thrombus formation on the activated endothelium. Complement activation via the alternative pathway has been seen in patients during the acute phase of STEC-associated HUS. Experimental evidence has highlighted the role of complement proteins in driving glomerular endothelium toward a thrombogenic phenotype. At the glomerular level, podocytes are also an important target of Stx-induced complement activation. Glomerular injury as a consequence of podocyte dysfunction and loss is thus a mechanism that might affect long-term renal outcomes in the disease. New approaches to targeting the complement system may be useful therapeutic options for patients with STEC-HUS.

Single Locked Nucleic Acid-Enhanced Nanopore Genetic Discrimination of Pathogenic Serotypes and Cancer Driver Mutations.

Accurate and rapid detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for many fields such as food safety regulation and disease diagnostics. Current detection methods involve laborious sample preparations and expensive characterizations. Here, we investigated a single locked nucleic acid (LNA) approach, facilitated by a nanopore single-molecule sensor, to accurately determine SNPs for detection of Shiga toxin producing Escherichia coli (STEC) serotype O157:H7, and cancer-derived EGFR L858R and KRAS G12D driver mutations. Current LNA applications that require incorporation and optimization of multiple LNA nucleotides. But we found that in the nanopore system, a single LNA introduced in the probe is sufficient to enhance the SNP discrimination capability by over 10-fold, allowing accurate detection of the pathogenic mutant DNA mixed in a large amount of the wild-type DNA. Importantly, the molecular mechanistic study suggests that such a significant improvement is due to the effect of the single-LNA that both stabilizes the fully matched base-pair and destabilizes the mismatched base-pair. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, could be generalized for various applications that need rapid and accurate identification of single-nucleotide variations.

[Identification of Shiga toxin in patients with acute intestinal infections in the presence of mono- and mixed-O-antigens of pathogens]. / Vyiavlenie Shiga-toksina u bol'nykh ostrymi kishechnymi infektsiiami v prisutstvii mono- i mikst-O-antigenov vozbuditelei.

AIM: To investigate the time course of changes in the detection rates and levels of Shiga toxin antigen (STA) in their stool and middle-molecule circulating immune complexes (CICs) containing IgG (IgG CIC) in patients with acute intestinal infections (AIIs) in the presence of the body's circulation of mono- and mixed-LPS/O-antigens of intestinal pathogens. SUBJECTS AND METHODS: A total of 147 patients aged 15 to 55 years who had been hospitalized with AIIs were examined. The diagnosis was bacteriologically verified in 19% of the patients; in the others, it was confirmed by the detection of LPS/O-antigens of Shigella, Salmonella, Yersinia, and Campylobacter in their stool by means of the reaction of coagglutination (RCA) on glass slides. Plates for RCA displayed STA in the fecal and IgG CIC samples. RESULTS: Mono- and mixed infections were detected in 32 and 68%, respectively. The RCA plates exhibited STA in 25.2% of the fecal samples and in 90.5% of the IgG CIC ones from patients with AIIs and did not in those from donors. In monoinfection, the detection rates and levels of STA in the feces became lower in the course of the disease and remained unchanged in IgG CIC and the levels of STA also decreased in the feces, but increased in IgG CIC in mixed infection. CONCLUSION: In 25.2% of the patients with early AIIs, their stools show free STA; its detection rate and levels are significantly higher in mixed infections than those in monoinfection. The level of STA in serum IgG CIC was significantly higher in mixed infection, suggesting an active immune response to the pathogen. Given that the Shiga toxin-producing strains are present in patients with AIIs, caution should be exercised in the choice of an antibacterial drug to prevent horizontal gene transfer and to enhance toxin production and the body's intoxication. One of the advantages of RCA is the possibility of rapidly changing the spectrum of test systems, depending on the region of their application and the epidemiological situation.

Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA.

Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

Decreased Neutrophil Extracellular Trap Degradation in Shiga Toxin-Associated Haemolytic Uraemic Syndrome.

BACKGROUND: Neutrophil extracellular traps (NETs) can stimulate thrombosis, and their degradation is decreased in several autoimmune disorders. It was recently reported that some patients with haemolytic uraemic syndrome (HUS) also fail to degrade NETs and that neutrophils from Shiga toxin-associated HUS are primed to form NETs. METHOD: We used a well-characterized cohort of 74 thrombotic microangiopathy (TMA) patients, with a subset also providing follow-up samples, and 112 age-matched controls to investigate NET degradation and serum nuclease activity in TMA before, during and after treatment. RESULTS: We identified that in the cohort of TMA patients, 50% of patients with Shiga toxin-associated HUS displayed a decreased ability to degrade NETs. NET degradation correlated with serum nuclease activity, but not with autoantibodies against double-stranded DNA, which has been previously observed in some autoimmune disorders. Further, NET degradation negatively correlated with serum creatinine levels, suggesting that kidney function was negatively impacted by the low NET degradation ability. CONCLUSIONS: We revealed that decreased NET degradation is a common feature of Shiga toxin-associated HUS and that it is associated with decreased kidney function in these patients. It remains to be clarified whether improving NET degradation would be beneficial for the patient.

Tumor necrosis factor-α modifies the effects of Shiga toxin on glial cells.

Shiga toxin (STX) is one of the main factors inducing hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in infections with STX-producing Escherichia coli (STEC). Approximately 62% of patients with HUS showed symptoms of encephalopathy in the 2011 Japanese outbreak of STEC infections. At that time, we reported elevated serum concentrations of tumor necrosis factor (TNF)-α in patients with acute encephalopathy during the HUS phase. In the current study, we investigated whether TNF-α augments the effects of STX in glial cell lines and primary glial cells. We found that TNF-α alone or STX in combination with TNF-α activates nuclear factor-κB (NF-κB) signaling and inhibits growth of glial cells. The magnitude of the NF-κB activation and the inhibition of cell growth by the STX and TNF-α combination was greater than that obtained with TNF-α alone or STX alone. Thus, this in vitro study reveals the role of TNF-α in glial cells during STEC infections.

Induction of Neutrophil Extracellular Traps in Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Hemolytic uremic syndrome (HUS), a vascular disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure, is caused by enterohemorrhagic Shiga toxin (Stx)-producing bacteria, which mainly affect children. Besides Stx, the inflammatory response mediated by neutrophils (PMN) is essential to HUS evolution. PMN can release neutrophil extracellular traps (NET) composed of DNA, histones, and other proteins. Since NET are involved in infectious and inflammatory diseases, the aim of this work was to investigate the contribution of NET to HUS. Plasma from HUS patients contained increased levels of circulating free-DNA and nucleosomes in comparison to plasma from healthy children. Neutrophils from HUS patients exhibited a greater capacity to undergo spontaneous NETosis. NET activated human glomerular endothelial cells, stimulating secretion of the proinflammatory cytokines IL-6 and IL-8. Stx induced PMN activation as judged by its ability to trigger reactive oxygen species production, increase CD11b and CD66b expression, and induce NETosis in PMN from healthy donors. During HUS, NET can contribute to the inflammatory response and thrombosis in the microvasculature and thus to renal failure. Intervention strategies to inhibit inflammatory mechanisms mediated by PMN, such as NETosis, could have a potential therapeutic impact towards amelioration of the severity of HUS.

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Targeted Shiga toxin-drug conjugates prepared via Cu-free click chemistry.

The main drawback of the anticancer chemotherapy consists in the lack of drug selectivity causing severe side effects. The targeted drug delivery appears to be a very promising strategy for controlling the biodistribution of the cytotoxic agent only on malignant tissues by linking it to tumor-targeting moiety. Here we exploit the natural characteristics of Shiga toxin B sub-unit (STxB) as targeting carrier on Gb3-positive cancer cells. Two cytotoxic conjugates STxB-doxorubicin (STxB-Doxo) and STxB-monomethyl auristatin F (STxB-MMAF) were synthesised using copper-free 'click' chemistry. Both conjugates were obtained in very high yield and demonstrated strong tumor inhibition activity in a nanomolar range on Gb3-positive cells.

Expression, Purification and Immunological Characterization of Recombinant Shiga Toxin A Subunit.

Shiga toxin family comprises toxins belonging to two major groups, Stx1 and Stx2, produced by the bacteria Shigella dysenteriae and some strains of Escherichia coli. Shiga toxins are the leading cause of diarrhea associated with life threatening hemolytic uremic syndrome (HUS). StxA is a ribosome inactivating protein (RIP) which inhibits the protein synthesis in most species of prokaryotes and eukaryotes. An in vitro expression system has not been reported to produce full-length biological active StxA subunit; hence substantial progress has been hampered. In the present study, a DNA fragment (955 bp Gene Bank Accn No HM017965) encoding for subunit A of Stx was amplified from Shigella dysenteriae type 1 and subsequently cloned in pGEX-5X-2 vector. We successfully produced recombinant StxA as GST fusion protein in Escherichia coli using pGEX-5X-2-STXA construct under IPTG induction. For the purpose of immunization the GST-tag was removed by factor Xa mediated endoproteolytic cleavage from GST-StxA. Antisera raised against rStxA in mice reacted with recombinant purified protein of rStxA and lysate of Shiga toxin. It was shown that antisera produced against rStxA significantly recognized Stx producing strains of S. dysenteriae and E. coli. The antiserum produced effectively neutralized the Shiga toxin's cytotoxicity towards Vero cells.

Shiga toxin associated hemolytic uremic syndrome.

Shiga toxin associated hemolytic uremic syndrome (Stx HUS), a thrombotic microangiopathy, is the most common cause of pediatric acute kidney injury but has no direct treatment. A better understanding of disease pathogenesis may help identify new therapeutic targets. For this reason, the role of complement is being actively studied while eculizumab, the C5 monoclonal antibody, is being used to treat Stx HUS but with conflicting results. A randomized controlled trial would help properly evaluate its use in Stx HUS while more research is required to fully evaluate the role complement plays in the disease pathogenesis.

Activation of the alternative pathway of complement during the acute phase of typical haemolytic uraemic syndrome.

Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and acute renal failure. We studied the activation state of classical and alternative pathways of complement during the acute phase of Shiga toxin-associated HUS by performing a prospective study of 18 patients and 17 age-matched healthy controls to evaluate C3, C3c, C4, C4d, Bb and SC5b-9 levels. SC5b-9 levels were increased significantly in all patients at admission compared to healthy and end-stage renal disease controls, but were significantly higher in patients presenting with oliguria compared to those with preserved diuresis. C3 and C4 levels were elevated significantly at admission in the non-oliguric group when compared to controls. No significant differences were found for C4d values, whereas factor Bb was elevated in all patients and significantly higher in oliguric patients when compared to both controls and non-oliguric individuals. A positive and significant association was detected when Bb formation was plotted as a function of plasma SC5b-9 at admission. Bb levels declined rapidly during the first week, with values not significantly different from controls by days 3 and 5 for non-oligurics and oligurics, respectively. Our data demonstrate the activation of the alternative pathway of complement during the acute phase of Stx-associated HUS. This finding suggests that complement activation may represent an important trigger for the cell damage that occurs during the syndrome.

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies.

Shiga toxin 1 (Stx1) is a virulence factor of enterohaemorrhagic Escherichia coli strains such as O157:H7 and Shigella dysenteriae. To prevent entry of Stx1 from the mucosal surface, an immunoglobulin A (IgA) specific for Stx1 would be useful. Due to the difficulty of producing IgA monoclonal antibodies (mAb) against the binding subunit of Stx1 (Stx1B) in mice, we took advantage of recombinant technology that combines the heavy chain variable region from Stx1B-specific IgG1 mAb and the Fc region from IgA. The resulting hybrid IgG/IgA was stably expressed in Chinese hamster ovary cells as a dimeric hybrid IgG/IgA. We separated the dimeric hybrid IgG/IgA from the monomeric one by size-exclusion chromatography. The dimer fraction, confirmed by immunoblot analyses, was used for toxin neutralization assays. The dimeric IgG/IgA was shown to neutralize Stx1 toxicity toward Vero cells by assaying their viability. To compare the relative effectiveness of the dimeric hybrid IgG/IgA and parental IgG1 mAb, Stx1-induced apoptosis was examined using 2 different cell lines, Ramos and Vero cells. The hybrid IgG/IgA inhibited apoptosis more efficiently than the parental IgG1 mAb in both cases. The results indicated that the use of high affinity binding sites as variable regions of IgA would increase the utility of IgA specific for virulence factors.