Intracellular Neutralization of Shiga Toxin 2 by an A Subunit-Specific Human Monoclonal Antibody

ABSTRACT Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) is the leading cause of hemolytic-uremic syndrome (HUS). Stx2, one of two toxins liberated by the bacteria, is directly linked with HUS. We have previously shown that Stx2-specific human monoclonal antibodies (HuMAbs) protect mice and piglets from fatal systemic complications of Stx2. The present study investigates the mechanisms by which our most efficacious A- and B-subunit-specific HuMAbs neutralize the cytotoxic effects of Stx2 in vitro. Whereas the B-subunit-specific HuMAb 5H8 blocked binding of Stx2 to its receptor on the cell surface, the A-subunit-specific HuMAb 5C12 did not interfere with the toxin-receptor binding. Further investigations revealed that 5C12 did not block endocytosis of Stx2 by HeLa cells as both Stx2 and 5C12 colocalized with early endosomes. However, 5C12 blocked the retrograde transport of the toxin into the Golgi and the endoplasmic reticulum, preventing the toxin from entering the cytosol where the toxin exerts its cytotoxic effect. The endocytosed 5C12/Stx2 complexes appear to be rapidly transported to the plasma membrane and/or to the slow recycling perinuclear compartments, followed by their slow recycling to the plasma membrane, and release into the extracellular environment.

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

Infection and Immunity ◽

10.1128/iai.70.2.612-619.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 612-619 ◽

Cited By ~ 75

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.

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Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

Infection and Immunity ◽

10.1128/iai.00867-09 ◽

2010 ◽

Vol 78 (3) ◽

pp. 1376-1382 ◽

Cited By ~ 17

Author(s):

Donna E. Akiyoshi ◽

Abhineet S. Sheoran ◽

Curtis M. Rich ◽

L. Richard ◽

Susan Chapman-Bonofiglio ◽

...

Keyword(s):

Monoclonal Antibody ◽

Shiga Toxin ◽

Chinese Hamster Ovary Cells ◽

Human Monoclonal Antibody ◽

Chinese Hamster ◽

The Body ◽

Neutralization Activity ◽

Shiga Toxin 2

ABSTRACT 5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)2] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)2 fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

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Production and Characterization of Protective Human Antibodies against Shiga Toxin 1

Infection and Immunity ◽

10.1128/iai.70.10.5896-5899.2002 ◽

2002 ◽

Vol 70 (10) ◽

pp. 5896-5899 ◽

Cited By ~ 43

Author(s):

Jean Mukherjee ◽

Kerry Chios ◽

Dianne Fishwild ◽

Deborah Hudson ◽

Susan O'Donnell ◽

...

Keyword(s):

Shiga Toxin ◽

Immunoglobulin M ◽

B Subunit ◽

Significant Prolongation ◽

Uremic Syndrome ◽

A Subunit ◽

Light Chain Immunoglobulin ◽

Subunit B

ABSTRACT Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producing Escherichia coli (STEC). By using HuMAb-Mouse (Medarex) animals, human monoclonal antibodies (Hu-MAbs) were developed against Shiga toxin 1 (Stx1) for passive immunotherapy of HUS. Ten stable hybridomas comprised of fully human heavy- and light-chain immunoglobulin elements and secreting Stx1-specific Hu-MAbs (seven immunoglobulin M(κ) [IgM(κ)] elements [one specific for the A subunit and six specific for the B subunit] and three IgG1(κ) elements specific for subunit B) were isolated. Two IgM(κ) Hu-MAbs (2D9 and 15G9) and three IgG1(κ) Hu-MAbs (5A4, 10F4, and 15G2), all specific for subunit B, demonstrated marked neutralization of Stx1 in vitro and significant prolongation of survival in a murine model of Stx1 toxicosis.

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Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit

Toxins ◽

10.3390/toxins13050307 ◽

2021 ◽

Vol 13 (5) ◽

pp. 307

Author(s):

Laura Heinisch ◽

Maike Krause ◽

Astrid Roth ◽

Holger Barth ◽

Herbert Schmidt

Keyword(s):

Shiga Toxin ◽

Cell Receptor ◽

Molar Ratio ◽

Target Cells ◽

B Subunit ◽

Protein Toxins ◽

A Subunit ◽

Hct 116 ◽

Hct 116 Cells

AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.

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Blood group P1 antigen–bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013926 ◽

2020 ◽

Vol 295 (28) ◽

pp. 9490-9501 ◽

Cited By ~ 1

Author(s):

Kanta Morimoto ◽

Noriko Suzuki ◽

Isei Tanida ◽

Soichiro Kakuta ◽

Yoko Furuta ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Death ◽

Blood Group ◽

Shiga Toxin ◽

Retrograde Transport ◽

Host Cells ◽

Mammalian Host ◽

B Subunit ◽

Early Endosomes ◽

Hydrophobic Portion

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

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TSSC1 is novel component of the endosomal retrieval machinery

Molecular Biology of the Cell ◽

10.1091/mbc.e16-04-0209 ◽

2016 ◽

Vol 27 (18) ◽

pp. 2867-2878 ◽

Cited By ~ 14

Author(s):

David C. Gershlick ◽

Christina Schindler ◽

Yu Chen ◽

Juan S. Bonifacino

Keyword(s):

Plasma Membrane ◽

Affinity Purification ◽

Critical Role ◽

Gel Filtration ◽

Retrograde Transport ◽

Recycling Endosomes ◽

B Subunit ◽

Multiple Protein ◽

Early Endosomes ◽

Golgi Network

Endosomes function as a hub for multiple protein-sorting events, including retrograde transport to the trans-Golgi network (TGN) and recycling to the plasma membrane. These processes are mediated by tubular-vesicular carriers that bud from early endosomes and fuse with a corresponding acceptor compartment. Two tethering complexes named GARP (composed of ANG2, VPS52, VPS53, and VPS54 subunits) and EARP (composed of ANG2, VPS52, VPS53, and Syndetin subunits) were previously shown to participate in SNARE-dependent fusion of endosome-derived carriers with the TGN and recycling endosomes, respectively. Little is known, however, about other proteins that function with GARP and EARP in these processes. Here we identify a protein named TSSC1 as a specific interactor of both GARP and EARP and as a novel component of the endosomal retrieval machinery. TSSC1 is a predicted WD40/β-propeller protein that coisolates with both GARP and EARP in affinity purification, immunoprecipitation, and gel filtration analyses. Confocal fluorescence microscopy shows colocalization of TSSC1 with both GARP and EARP. Silencing of TSSC1 impairs transport of internalized Shiga toxin B subunit to the TGN, as well as recycling of internalized transferrin to the plasma membrane. Fluorescence recovery after photobleaching shows that TSSC1 is required for efficient recruitment of GARP to the TGN. These studies thus demonstrate that TSSC1 plays a critical role in endosomal retrieval pathways as a regulator of both GARP and EARP function.

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Targeting of Shiga Toxin B-Subunit to Retrograde Transport Route in Association with Detergent-resistant Membranes

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2453 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2453-2468 ◽

Cited By ~ 195

Author(s):

Thomas Falguières ◽

Frédéric Mallard ◽

Carole Baron ◽

Daniel Hanau ◽

Clifford Lingwood ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Shiga Toxin ◽

Secretory Pathway ◽

Retrograde Transport ◽

Toxin B ◽

B Subunit ◽

Early Endosomes ◽

Golgi Network ◽

Triton X

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to thetrans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

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Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin

Communications Biology ◽

10.1038/s42003-021-02068-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Miho Watanabe-Takahashi ◽

Masakazu Tamada ◽

Miki Senda ◽

Masahiro Hibino ◽

Eiko Shimizu ◽

...

Keyword(s):

Crystal Structure ◽

Shiga Toxin ◽

Crystal Structure Analysis ◽

Peptide Motif ◽

Systemic Complications ◽

X Ray ◽

B Subunit ◽

Major Virulence Factor ◽

Potent Inhibition ◽

A Subunit

AbstractShiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx.

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The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3289 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3289-3298 ◽

Cited By ~ 112

Author(s):

Wolfram Antonin ◽

Claudia Holroyd ◽

Ritva Tikkanen ◽

Stefan Höning ◽

Reinhard Jahn

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Subcellular Fractionation ◽

Immunogold Electron Microscopy ◽

Fusion Reactions ◽

Coated Pits ◽

Late Endosomes ◽

Early Endosomes ◽

Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

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