The Serine 31 Residue of the B Subunit of Shiga Toxin 2 Is Essential for Secretion in Enterohemorrhagic Escherichia coli

ABSTRACT Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) include Shiga toxin 1 (Stx1) as well as Shiga toxin 2 (Stx2). Stx1 is cell associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxB-H). Although neither StxB1-H nor StxB2-H was secreted in StxB-H-overexpressed EHEC, StxB2-H-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, StxB1-H-overexpressed EHEC showed no alteration of Stx2 secretion. B-subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of StxB2 that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in StxB2-H enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in StxB1-H caused the partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed an isogenic mutant EHEC (Stx1B subunit, N32S) strain and an isogenic mutant EHEC (Stx2B subunit, S31N) strain. Although the mutant Stx2 was cell associated in isogenic mutant EHEC, mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotoxins, the overexpressed mutant Stx1 was found in the supernatant fraction, and the overexpressed mutant Stx2 was found in the cell-associated fraction in mutant holotoxin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.

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Enterohemorrhagic Escherichia coli O157 subclade 8b strains in Chiba Prefecture, Japan, produced larger amounts of Shiga toxin 2 than strains in subclade 8a and other clades

PLoS ONE ◽

10.1371/journal.pone.0191834 ◽

2018 ◽

Vol 13 (1) ◽

pp. e0191834 ◽

Cited By ~ 2

Author(s):

Shinichiro Hirai ◽

Eiji Yokoyama ◽

Taku Wakui ◽

Taichiro Ishige ◽

Masaki Nakamura

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Enterohemorrhagic Escherichia Coli ◽

Escherichia Coli O157 ◽

Chiba Prefecture ◽

Shiga Toxin 2

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Effects of Antibiotics on Shiga Toxin 2 Production and Bacteriophage Induction by Epidemic Escherichia coli O104:H4 Strain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06315-11 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3277-3282 ◽

Cited By ~ 112

Author(s):

Martina Bielaszewska ◽

Evgeny A. Idelevich ◽

Wenlan Zhang ◽

Andreas Bauwens ◽

Frieder Schaumburg ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Therapy ◽

Shiga Toxin ◽

The Other ◽

Emerging Pathogen ◽

Outbreak Strain ◽

Content Type ◽

Shiga Toxin 2 ◽

Uremic Syndrome

ABSTRACTThe role of antibiotics in treatment of enterohemorrhagicEscherichia coli(EHEC) infections is controversial because of concerns about triggering hemolytic-uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated for some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction ofstx2-harboring bacteriophages,stx2transcription, and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increasedstx2-harboring phage induction and Stx2 production in outbreak isolates (Pvalues of <0.001 to <0.05), while fosfomycin, gentamicin, and kanamycin insignificantly influenced them (P> 0.1) and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased them (P≤ 0.05). Ciprofloxacin and chloramphenicol significantly upregulated and downregulatedstx2transcription, respectively (P< 0.01); the other antibiotics had insignificant effects (P> 0.1). Meropenem, azithromycin, and rifaximin, which were used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither inducedstx2-harboring phages nor increasedstx2transcription or Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options for patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

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Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Applied and Environmental Microbiology ◽

10.1128/aem.01906-07 ◽

2007 ◽

Vol 74 (1) ◽

pp. 67-72 ◽

Cited By ~ 44

Author(s):

Alexander Mellmann ◽

Shan Lu ◽

Helge Karch ◽

Jian-guo Xu ◽

Dag Harmsen ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Integration Site ◽

Hot Spot ◽

Blot Hybridization ◽

Enterohemorrhagic Escherichia Coli ◽

Biologically Active ◽

E Coli ◽

Shiga Toxin 2

ABSTRACT Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx 2-positive isolates fliC H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2-positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2-positive and stx-negative isolates demonstrated that yecE was occupied in stx 2-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx 2-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2-harboring phages. The ability to recycle stx 2, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

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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.

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