Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo, Leading to Production of Biologically Active Stx2

ABSTRACTShiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagicEscherichia coli(EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfectedin vitrowith thestx2gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2in vivoafter pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressedin vivofrom the wildstx2gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection.IMPORTANCEEnterohemorrhagic Shiga toxin (Stx)-producingEscherichia coli(EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.

Download Full-text

Cognitive deficits found in a pro-inflammatory state are independent of ERK 1/2 signaling in the murine brain hippocampus treated with Shiga toxin 2 from enterohemorrhagic Escherichia Coli

10.1101/2020.08.24.264929 ◽

2020 ◽

Author(s):

Clara Berdasco ◽

Alipio Pinto ◽

Mariano Blake ◽

Fernando Correa ◽

Nadia A. Longo Carbajosa ◽

...

Keyword(s):

Escherichia Coli ◽

Central Nervous System ◽

Nervous System ◽

Hemolytic Uremic Syndrome ◽

Signaling Pathway ◽

Shiga Toxin ◽

Enterohemorrhagic Escherichia Coli ◽

Shiga Toxin 2 ◽

Uremic Syndrome ◽

The Brain

AbstractShiga toxin 2 (Stx2) from enterohemorrhagic Escherichia coli (EHEC) produces hemorrhagic colitis, hemolytic uremic syndrome (HUS) and acute encephalopathy. The mortality rate in HUS increases significantly when the central nervous system (CNS) is involved. Besides, EHEC also releases lipopolysaccharide (LPS). Many reports have described cognitive dysfunctions in HUS patients, the hippocampus being one of the brain areas targeted by EHEC infection. In this context, a translational murine model of encephalopathy was employed to establish the deleterious effects of Stx2 and the contribution of LPS in the hippocampus. Results demonstrate that systemic administration of a sublethal dose of Stx2 reduced memory index and produced depression like behavior, pro-inflammatory cytokine release and NF-kB activation independent of the ERK 1/2 signaling pathway. On the other hand, LPS activated NF-kB dependent on ERK 1/2 signaling pathway. Cotreatment of Stx2 with LPS aggravated the pathologic state, while dexamethasone treatment succeeded in preventing behavioral alterations. Our present work suggests that the use of drugs such as corticosteroids or NF-kB signaling inhibitors may serve as neuroprotectors from EHEC infection.Author SummaryShiga toxin (Stx) from enterohemorrhagic Escherichia coli (EHEC) is one of the most virulent factors responsible for hemolytic uremic syndrome (HUS). Stx2, the endemic variant targets the brain, among other organs, thus inducing encephalopathies. Central nervous system (CNS) compromise was the main predictor of death in patients with HUS. Stx2 may exert a direct action in the CNS, by disrupting the neurovascular unit. In this context, we investigate the molecular signaling triggered by Stx2 in the murine brain hippocampus involved in inflammatory mechanisms that altered hippocampal-related cognitive behaviors. The present data underscore that the use of drugs such as dexamethasone or those blocking the cascade by preventing NF-kB translocation to the nucleus may serve as effective neuroprotectors with potentially beneficial use in the clinic.

Download Full-text

Steroid Pulse Therapy for Severe Central Nervous System Involvement in Shiga Toxin-Producing Escherichia coli-Related Hemolytic Uremic Syndrome

Case Reports in Pediatrics ◽

10.1155/2021/5587050 ◽

2021 ◽

Vol 2021 ◽

pp. 1-4

Author(s):

Chiara Rosazza ◽

Alberto M Cappellari ◽

Cristiano Gandini ◽

Elisa Scola ◽

Gianluigi Ardissino

Keyword(s):

Escherichia Coli ◽

Central Nervous System ◽

Nervous System ◽

Hemolytic Uremic Syndrome ◽

Shiga Toxin ◽

Pulse Therapy ◽

Steroid Pulse Therapy ◽

High Dose ◽

Steroid Pulse ◽

Uremic Syndrome

We report on the case of a 7-year-old boy with Shiga toxin-producing Escherichia coli-related hemolytic uremic syndrome (STEC-HUS), initially presenting with abdominal pain as the only clinical feature and thus requiring differential diagnosis with a surgical emergency. Diagnosis of STEC-HUS was made with the appearance of bloody diarrhea and renal function impairment, and the clinical picture rapidly progressed to multiorgan failure. Relatively late and severe central nervous system (CNS) involvement was present, characterized by subacute encephalitis progressing to coma, which became apparent when the acute phase of thrombotic microangiopathy was resolving. Therefore, neurologic manifestations were thought to be related to reperfusion damage to the CNS and high-dose IV steroid pulse therapy was empirically administered. Following this therapeutic scheme, neurologic involvement resolved with no sequelae. This case offers several points of discussion on the clinical presentation and the diagnostic approach to STEC-HUS, on the related neurologic complications, and on a novel approach to their management.

Download Full-text

Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.01231-17 ◽

2017 ◽

Vol 83 (18) ◽

Cited By ~ 8

Author(s):

Peter C. H. Feng ◽

Sabine Delannoy ◽

David W. Lacher ◽

Joseph M. Bosilevac ◽

Patrick Fach ◽

...

Keyword(s):

Escherichia Coli ◽

Hemolytic Uremic Syndrome ◽

Shiga Toxin ◽

Severe Illness ◽

Content Type ◽

Virulence Potential ◽

Short Palindromic Repeat ◽

Uremic Syndrome ◽

Severe Infections ◽

Genetic Profiles

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identical or nearly identical to those of the clinical O91:H21 strains that have caused severe diseases. Multilocus sequence typing and clustered regularly interspaced short palindromic repeat analyses showed that the O91:H21 strains clustered within the STEC 1 clonal group but the other O91 serotype strains were phylogenetically diverse. IMPORTANCE This study showed that food and environmental O91:H21 strains have similar genotypic profiles and Shiga toxin subtypes and are phylogenetically related to the O91:H21 strains that have caused hemolytic-uremic syndrome, suggesting that these strains may also have the potential to cause severe illness.

Download Full-text

AB5Preassembly Is Not Required for Shiga Toxin Activity

Journal of Bacteriology ◽

10.1128/jb.00918-15 ◽

2016 ◽

Vol 198 (11) ◽

pp. 1621-1630 ◽

Cited By ~ 7

Author(s):

Christine A. Pellino ◽

Sayali S. Karve ◽

Suman Pradhan ◽

Alison A. Weiss

Keyword(s):

Escherichia Coli ◽

Enzymatic Activity ◽

Hemolytic Uremic Syndrome ◽

Shiga Toxin ◽

Target Cells ◽

Content Type ◽

B Subunit ◽

Uremic Syndrome ◽

A Subunit ◽

B Subunits

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5complex is not required for cellular toxicity orin vivotoxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenicEscherichia coliis considered to be an AB5heterohexamer; however, no known mechanisms ensure AB5assembly. Stx released byE. coliis not in the AB5conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5assembly is a potential treatment for Stx-associated illnesses.IMPORTANCEComplications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.

Download Full-text

Serogroup-Specific Bacterial Engineered Glycoproteins as Novel Antigenic Targets for Diagnosis of Shiga Toxin-Producing-Escherichia coli-Associated Hemolytic-Uremic Syndrome

Journal of Clinical Microbiology ◽

10.1128/jcm.02262-14 ◽

2014 ◽

Vol 53 (2) ◽

pp. 528-538 ◽

Cited By ~ 21

Author(s):

Luciano J. Melli ◽

Andrés E. Ciocchini ◽

Ana J. Caillava ◽

Nicolás Vozza ◽

Isabel Chinen ◽

...

Keyword(s):

Escherichia Coli ◽

Hemolytic Uremic Syndrome ◽

Shiga Toxin ◽

Diagnostic Procedures ◽

Stool Culture ◽

Human Infection ◽

Healthy Children ◽

Content Type ◽

E Coli ◽

Uremic Syndrome

Human infection with Shiga toxin-producingEscherichia coli(STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure.E. coliO157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157E. colilipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis.

Download Full-text

A Novel Tail-Associated O91-Specific Polysaccharide Depolymerase from a Podophage Reveals Lytic Efficacy of Shiga Toxin-Producing Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00145-20 ◽

2020 ◽

Vol 86 (9) ◽

Cited By ~ 2

Author(s):

Yibao Chen ◽

Xiangmin Li ◽

Shuang Wang ◽

Lingyu Guan ◽

Xinxin Li ◽

...

Keyword(s):

Escherichia Coli ◽

Hemolytic Uremic Syndrome ◽

Proinflammatory Cytokines ◽

Shiga Toxin ◽

Hemorrhagic Colitis ◽

Content Type ◽

Increased Risk ◽

Specific Polysaccharide ◽

Life Threatening ◽

Uremic Syndrome

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic foodborne pathogens, causing diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. However, antibiotic treatment of STEC infection is associated with an increased risk of HUS. Therefore, there is an urgent need for early and effective therapeutic strategies. Here, we isolated lytic T7-like STEC phage PHB19 and identified a novel O91-specific polysaccharide depolymerase (Dep6) in the C terminus of the PHB19 tailspike protein. Dep6 exhibited strong hydrolase activity across wide ranges of pH (pH 4 to 8) and temperature (20 to 60°C) and degraded polysaccharides on the surface of STEC strain HB10. In addition, both Dep6 and PHB19 degraded biofilms formed by STEC strain HB10. In a mouse STEC infection model, delayed Dep6 treatment (3 h postinfection) resulted in only 33% survival, compared with 83% survival when mice were treated simultaneously with infection. In comparison, pretreatment with Dep6 led to 100% survival compared with that of the control group. Surprisingly, a single PHB19 treatment resulted in 100% survival in all three treatment protocols. Moreover, a significant reduction in the levels of proinflammatory cytokines was observed at 24 h postinfection in Dep6- or PHB19-treated mice. These results demonstrated that Dep6 or PHB19 might be used as a potential therapeutic agent to prevent STEC infection. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen worldwide. The Shiga-like toxin causes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. Although antibiotic therapy is still used for STEC infections, this approach may increase the risk of HUS. Phages or phage-derived depolymerases have been used to treat bacterial infections in animals and humans, as in the case of the “San Diego patient” treated with a phage cocktail. Here, we showed that phage PHB19 and its O91-specific polysaccharide depolymerase Dep6 degraded STEC biofilms and stripped the lipopolysaccharide (LPS) from STEC strain HB10, which was subsequently killed by serum complement in vitro. In a mouse model, PHB19 and Dep6 protected against STEC infection and caused a significant reduction in the levels of proinflammatory cytokines. This study reports the use of an O91-specific polysaccharide depolymerase for the treatment of STEC infection in mice.

Download Full-text

Genome Sequence for Shiga Toxin-Producing Escherichia coli O26:H11, Associated with a Cluster of Hemolytic-Uremic Syndrome Cases in South Africa, 2017

Genome Announcements ◽

10.1128/genomea.00989-17 ◽

2017 ◽

Vol 5 (38) ◽

Cited By ~ 5

Author(s):

Anthony M. Smith ◽

Kalule J. Bosco ◽

Mark P. Nicol ◽

Jackie Kleynhans ◽

Mignon McCulloch ◽

...

Keyword(s):

Escherichia Coli ◽

South Africa ◽

Hemolytic Uremic Syndrome ◽

Genome Sequence ◽

Foodborne Pathogens ◽

Shiga Toxin ◽

Sequence Data ◽

Content Type ◽

Genome Sequence Data ◽

Uremic Syndrome

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are primarily foodborne pathogens that may cause diarrheal outbreaks and are associated with severe complications, specifically hemolytic-uremic syndrome (HUS). We report here genome sequence data for STEC O26:H11, which is associated with a cluster of cases of HUS, a rarely described syndrome in South Africa.

Download Full-text