scholarly journals Two Distinct Cytotoxic Activities of Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli

2006 ◽  
Vol 75 (1) ◽  
pp. 488-496 ◽  
Author(s):  
Naoko Morinaga ◽  
Kinnosuke Yahiro ◽  
Gen Matsuura ◽  
Masaharu Watanabe ◽  
Fumio Nomura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Serine Protease ◽  
Catalytic Triad ◽  
Cytotoxic Activities ◽  
B Subunit ◽  
A Subunit ◽  
Subtilase Cytotoxin

ABSTRACT Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 μg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was ∼1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.

scholarly journals In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli

Vaccine ◽  
2009 ◽  
Vol 27 (32) ◽  
pp. 4302-4308 ◽  
Author(s):  
Shuang Liang ◽  
Kavita B. Hosur ◽  
Hesham F. Nawar ◽  
Michael W. Russell ◽  
Terry D. Connell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
B Subunit ◽  
Heat Labile

scholarly journals A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

2009 ◽  
Vol 16 (5) ◽  
pp. 712-718 ◽  
Author(s):  
Leticia V. Bentancor ◽  
Marcos Bilen ◽  
Romina J. Fernández Brando ◽  
María Victoria Ramos ◽  
Luis C. S. Ferreira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Vaccine ◽  
Protective Immunity ◽  
Host Cells ◽  
Therapeutic Interventions ◽  
28S Rrna ◽  
A Subunit ◽  
B Subunits

ABSTRACT Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A1 peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A2 peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A2 sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

scholarly journals Human Stx2-Specific Monoclonal Antibodies Prevent Systemic Complications of Escherichia coli O157:H7 Infection

2002 ◽  
Vol 70 (2) ◽  
pp. 612-619 ◽  
Author(s):  
Jean Mukherjee ◽  
Kerry Chios ◽  
Dianne Fishwild ◽  
Deborah Hudson ◽  
Susan O'Donnell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Shiga Toxin ◽  
Cerebral Lesions ◽  
Systemic Complications ◽  
E Coli ◽  
B Subunit ◽  
Significant Prolongation ◽  
A Subunit

ABSTRACT Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and κ light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1κ A-subunit-specific and 3 IgG1κ and 1 IgG3κ B-subunit-specific antibodies. Six IgG1κ A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1κ B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of >95% activity of 1 ng of Stx2 in the presence of 0.04 μg of HuMAb in vitro and significant prolongation of survival of mice given 50 μg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7.

scholarly journals A New Family of Potent AB5 Cytotoxins Produced by Shiga Toxigenic Escherichia coli

2004 ◽  
Vol 200 (1) ◽  
pp. 35-46 ◽  
Author(s):  
Adrienne W. Paton ◽  
Potjanee Srimanote ◽  
Ursula M. Talbot ◽  
Hui Wang ◽  
James C. Paton
Keyword(s):  
Escherichia Coli ◽  
Bacterial Toxin ◽  
Virulence Plasmid ◽  
B Subunit ◽  
New Family ◽  
Uremic Syndrome ◽  
A Subunit ◽  
K 12 ◽  
Ab5 Toxins ◽  
Subtilase Cytotoxin

The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB5 toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12–expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.

scholarly journals Covariance of Complementary rRNA Loop Nucleotides Does Not Necessarily Represent Functional Pseudoknot Formation In Vivo

2000 ◽  
Vol 182 (20) ◽  
pp. 5671-5675 ◽  
Author(s):  
Natalya S. Chernyaeva ◽  
Emanuel J. Murgola
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Site Directed Mutagenesis ◽  
23S Rrna ◽  
Directed Mutagenesis ◽  
Bacterial Cells ◽  
Domain I

ABSTRACT We examined mutationally a two-hairpin structure (nucleotides 57 to 70 and 76 to 110) in a region of domain I ofEscherichia coli 23S rRNA that has been implicated in specific functions in protein synthesis by other studies. On the basis of the observed covariance of several nucleotides in each loop inBacteria, Archaea, and chloroplasts, the two hairpins have been proposed to form a pseudoknot. Here, appropriate loop changes were introduced in vitro by site-directed mutagenesis to eliminate any possibility of base pairing between the loops. The bacterial cells containing each cloned mutant rRNA operon were then examined for cell growth, termination codon readthrough, and assembly of the mutant rRNAs into functional ribosomes. The results show that, under the conditions examined, the two hairpins do not form a pseudoknot structure that is required for the functioning of the ribosome in vivo and therefore that sequence covariance does not necessarily indicate the formation of a functional pseudoknot.

scholarly journals Mucosal Adjuvanticity and Immunogenicity of LTR72, a Novel Mutant of Escherichia coli Heat-labile Enterotoxin with Partial Knockout of ADP-ribosyltransferase Activity

1998 ◽  
Vol 187 (7) ◽  
pp. 1123-1132 ◽  
Author(s):  
Marzia Monica Giuliani ◽  
Giuseppe Del Giudice ◽  
Valentina Giannelli ◽  
Gordon Dougan ◽  
Gill Douce ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Wild Type ◽  
Ileal Loop ◽  
Heat Labile ◽  
A Subunit ◽  
Reduced Toxicity ◽  
Adp Ribosyltransferase

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala → Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 μg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose–response experiments in mice, 20 μg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

scholarly journals Development of DNA Vaccines against Hemolytic-Uremic Syndrome in a Murine Model

2003 ◽  
Vol 71 (7) ◽  
pp. 3971-3978 ◽  
Author(s):  
Alejandra V. E. Capozzo ◽  
Virginia Pistone Creydt ◽  
Graciela Dran ◽  
Gabriela Fernández ◽  
Sonia Gómez ◽  
...  
Keyword(s):  
Hemolytic Uremic Syndrome ◽  
Humoral Response ◽  
Host Cells ◽  
Newborn Mice ◽  
B Subunit ◽  
Adult Mice ◽  
Uremic Syndrome ◽  
A Subunit

ABSTRACT Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2ΔA). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.

Sign in / Sign up

Export Citation Format

Share Document