UniProt functional annotation for P18177



	UniProt functional annotation for P18177

UniProt code: P18177.

Organism: Clostridioides difficile (Peptoclostridium difficile).

Taxonomy: Bacteria; Firmicutes; Clostridia; Eubacteriales; Peptostreptococcaceae; Clostridioides.

Function: [Toxin B]: Precursor of a cytotoxin that targets and disrupts the colonic epithelium, inducing the host inflammatory and innate immune responses and resulting in diarrhea and pseudomembranous colitis (PubMed:20844489, PubMed:24919149). TcdB constitutes the main toxin that mediates the pathology of C.difficile infection, an opportunistic pathogen that colonizes the colon when the normal gut microbiome is disrupted (PubMed:19252482, PubMed:20844489). Compared to TcdA, TcdB is more virulent and more important for inducing the host inflammatory and innate immune responses (PubMed:19252482, PubMed:24919149). This form constitutes the precursor of the toxin: it enters into host cells and mediates autoprocessing to release the active toxin (Glucosyltransferase TcdB) into the host cytosol (PubMed:10768933, PubMed:11152463, PubMed:12941936, PubMed:17334356, PubMed:20498856). Targets colonic epithelia by binding to the frizzled receptors FZD1, FZD2 and FZD7, and enters host cells via clathrin-mediated endocytosis (PubMed:27680706). Frizzled receptors constitute the major host receptors in the colonic epithelium, but other receptors, such as CSPG4 or NECTIN3/PVRL3, have been identified (PubMed:25547119, PubMed:26038560, PubMed:27680706). Binding to carbohydrates and sulfated glycosaminoglycans on host cells suface also contribute to entry into cells (By similarity). Once entered into host cells, acidification in the endosome promotes the membrane insertion of the translocation region and formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (PubMed:11152463, PubMed:12941936, PubMed:24567384). This activates the peptidase C80 domain and autocatalytic processing, releasing the N- terminal part (Glucosyltransferase TcdB), which constitutes the active part of the toxin, in the cytosol (PubMed:17334356, PubMed:27571750). {ECO:0000250|UniProtKB:P16154, ECO:0000269|PubMed:10768933, ECO:0000269|PubMed:11152463, ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:19252482, ECO:0000269|PubMed:20498856, ECO:0000269|PubMed:20844489, ECO:0000269|PubMed:24567384, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:25547119, ECO:0000269|PubMed:26038560, ECO:0000269|PubMed:27571750, ECO:0000269|PubMed:27680706}.

Function: [Glucosyltransferase TcdB]: Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases (PubMed:8144660, PubMed:7777059, PubMed:16157585, PubMed:17901056, PubMed:24905543, PubMed:24919149). Acts by mediating monoglucosylation of small GTPases of the Rho family (Rac1, RhoA, RhoB, RhoC, RhoG and Cdc42) in host cells at the conserved threonine residue located in the switch I region ('Thr-37/35'), using UDP-alpha-D-glucose as the sugar donor (PubMed:7777059, PubMed:16157585, PubMed:17901056, PubMed:24905543, PubMed:24919149). Monoglucosylation of host small GTPases completely prevents the recognition of the downstream effector, blocking the GTPases in their inactive form, leading to actin cytoskeleton disruption and cell death, resulting in the loss of colonic epithelial barrier function (PubMed:7777059, PubMed:24919149). {ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:17901056, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:7777059, ECO:0000269|PubMed:8144660}.

Catalytic activity: [Glucosyltransferase TcdB]: Reaction=L-threonyl-[protein] + UDP-alpha-D-glucose = 3-O-(alpha-D- glucosyl)-L-threonyl-[protein] + H(+) + UDP; Xref=Rhea:RHEA:64684, Rhea:RHEA-COMP:11060, Rhea:RHEA-COMP:16656, ChEBI:CHEBI:15378, ChEBI:CHEBI:30013, ChEBI:CHEBI:58223, ChEBI:CHEBI:58885, ChEBI:CHEBI:156085; Evidence={ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:17901056, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:7777059}; PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:64685; Evidence={ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:17901056, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:7777059};

Cofactor: [Toxin B]: Name=Zn(2+); Xref=ChEBI:CHEBI:29105; Evidence={ECO:0000269|PubMed:27571750, ECO:0000269|PubMed:31308519}; Note=Binds 1 Zn(2+) ion per subunit (PubMed:27571750, PubMed:31308519). Zn(2+) is required for autocatalytic cleavage (PubMed:27571750). {ECO:0000269|PubMed:27571750, ECO:0000269|PubMed:31308519};

Cofactor: [Glucosyltransferase TcdB]: Name=Mn(2+); Xref=ChEBI:CHEBI:29035; Evidence={ECO:0000269|PubMed:16054646, ECO:0000269|PubMed:27089365, ECO:0000269|PubMed:28433497, ECO:0000305|PubMed:31308519}; Name=Mg(2+); Xref=ChEBI:CHEBI:18420; Evidence={ECO:0000269|PubMed:27089365}; Note=Has higher activity with Mn(2+), but most likely uses Mg(2+) in host cells (PubMed:16054646, PubMed:28433497). Mn(2+) or Mg(2+) are required for glucosyltransferase activity (PubMed:27089365). {ECO:0000269|PubMed:16054646, ECO:0000269|PubMed:27089365, ECO:0000269|PubMed:28433497};

Activity regulation: [Toxin B]: Protease activity is activated upon binding to 1D-myo-inositol hexakisphosphate (InsP6), which induces conformational reorganization (PubMed:17334356). Inhibited by bezlotoxumab, also named Zinplava, a monoclonal antibody approved by the Food and Drug Administration (FDA), which specifically targets TcdB (PubMed:24821719). {ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:24821719}.

Biophysicochemical properties: [Glucosyltransferase TcdB]: Kinetic parameters: KM=6 uM for UDP-alpha-D-glucose {ECO:0000269|PubMed:16157585}; KM=4.3 uM for UDP-alpha-D-glucose {ECO:0000269|PubMed:17901056}; KM=960 uM for UDP-N-acetyl-alpha-D-glucosamine {ECO:0000269|PubMed:16157585}; Note=kcat is 2120 hour(-1) with UDP-alpha-D-glucose as substrate (PubMed:16157585). kcat is 11 hour(-1) with UDP-N-acetyl-alpha-D- glucosamine as substrate (PubMed:16157585). {ECO:0000269|PubMed:16157585};

Subunit: [Toxin B]: Interacts with host FZD1 (PubMed:27680706). Interacts with host FZD2; interaction promotes toxin entry into host cell and occupies the binding site for Wnt-adducted palmitoleate in FZD2, leading to prevent Wnt-binding and downstream Wnt signaling (PubMed:27680706, PubMed:31233493, PubMed:29748286). Interacts with host FZD7 (PubMed:27680706). Interacts with host CSPG4 (PubMed:25547119, PubMed:31233493). Interacts with host NECTIN3/PVRL3 (PubMed:26038560). {ECO:0000269|PubMed:25547119, ECO:0000269|PubMed:26038560, ECO:0000269|PubMed:27680706, ECO:0000269|PubMed:29748286, ECO:0000269|PubMed:31233493}.

Subcellular location: [Toxin B]: Secreted {ECO:0000269|PubMed:22685398}. Host endosome membrane {ECO:0000269|PubMed:11152463, ECO:0000305|PubMed:12941936}. Note=Secreted from C.difficile cell into the extracellular environment via help of holin-like protein TcdE/UtxA (PubMed:22685398). Binds to the cell surface receptors via the receptor-binding region and enters the cells via clathrin-mediated endocytosis (PubMed:20498856). Acidification in the endosome triggers conformational changes that promote the membrane insertion of the translocation region, allowing formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (PubMed:10768933, PubMed:11152463, PubMed:12941936). 1D-myo-inositol hexakisphosphate- binding (InsP6) activates the peptidase C80 domain and autoprocessing, generating the Glucosyltransferase TcdB form, which is released in the host cytosol (PubMed:17334356). {ECO:0000269|PubMed:10768933, ECO:0000269|PubMed:11152463, ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:20498856, ECO:0000269|PubMed:22685398}.

Subcellular location: [Glucosyltransferase TcdB]: Host cytoplasm, host cytosol {ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:15632438}. Host cell membrane {ECO:0000250|UniProtKB:Q46342}; Peripheral membrane protein {ECO:0000250|UniProtKB:Q46342}; Cytoplasmic side {ECO:0000250|UniProtKB:Q46342}. Note=Binding to phospholipids, such as phosphatidylserine and phosphatidic acid promotes localization to the inner face of the cell membrane close to its membrane anchored substrates (small GTPases). {ECO:0000250|UniProtKB:Q46342}.

Domain: [Toxin B]: Consists of 4 functional domains: (1) the N-terminal GT44 domain (glucosyltransferase, also named GTD), which mediates glucosylation of host small GTPases, (2) an autoprocessing region that catalyzes autoprocessing to release the N-terminal GT44 domain in the host cytosol, (3) the translocation region that forms a pore to promote translocation of the GT44 and peptidase C80 domains across the endosomal membrane and (4) the receptor-binding (CROPS) region that mediates binding to host cells and contribute to entry into cells. {ECO:0000269|PubMed:31308519, ECO:0000303|PubMed:29146177}.

Domain: [Toxin B]: The receptor-binding (CROPS) region is dynamic and can have open and closed conformations depending of the pH: has an open conformation at endosomal pH and a closed conformation at neutral pH. {ECO:0000269|PubMed:31308519}.

Domain: [Toxin B]: The cell wall-binding repeats bind carbohydrates, probably contributing to entry into cells. {ECO:0000250|UniProtKB:P16154}.

Domain: [Glucosyltransferase TcdB]: The four-helical bundle region mediates binding to phospholipids, such as phosphatidylserine and phosphatidic acid (PubMed:25882477). This promotes localization to the inner face of the cell membrane close to small GTPases (By similarity). {ECO:0000250|UniProtKB:Q46342, ECO:0000269|PubMed:25882477}.

Ptm: [Toxin B]: Undergoes autocatalytic cleavage to release the N- terminal part (Glucosyltransferase TcdB), which constitutes the active part of the toxin, in the host cytosol (PubMed:12941936, PubMed:17334356, PubMed:27571750). 1D-myo-inositol hexakisphosphate- binding (InsP6) activates the peptidase C80 domain and promotes autoprocessing (PubMed:17334356). {ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:27571750}.

Disruption phenotype: Cells lacking tcdB display virulence and cytotoxicity, because of the presence of TcdA (PubMed:20844489). Cells lacking both tcdA and tcdB display a strongly reduced virulence (PubMed:20844489). {ECO:0000269|PubMed:20844489}.

Similarity: Belongs to the clostridial glucosylating toxin (LCGT) family. {ECO:0000305}.

Annotations taken from UniProtKB at the EBI.


Organism:		Clostridioides difficile (Peptoclostridium difficile).
Taxonomy:		Bacteria; Firmicutes; Clostridia; Eubacteriales; Peptostreptococcaceae; Clostridioides.



Function:		[Toxin B]: Precursor of a cytotoxin that targets and disrupts the colonic epithelium, inducing the host inflammatory and innate immune responses and resulting in diarrhea and pseudomembranous colitis (PubMed:20844489, PubMed:24919149). TcdB constitutes the main toxin that mediates the pathology of C.difficile infection, an opportunistic pathogen that colonizes the colon when the normal gut microbiome is disrupted (PubMed:19252482, PubMed:20844489). Compared to TcdA, TcdB is more virulent and more important for inducing the host inflammatory and innate immune responses (PubMed:19252482, PubMed:24919149). This form constitutes the precursor of the toxin: it enters into host cells and mediates autoprocessing to release the active toxin (Glucosyltransferase TcdB) into the host cytosol (PubMed:10768933, PubMed:11152463, PubMed:12941936, PubMed:17334356, PubMed:20498856). Targets colonic epithelia by binding to the frizzled receptors FZD1, FZD2 and FZD7, and enters host cells via clathrin-mediated endocytosis (PubMed:27680706). Frizzled receptors constitute the major host receptors in the colonic epithelium, but other receptors, such as CSPG4 or NECTIN3/PVRL3, have been identified (PubMed:25547119, PubMed:26038560, PubMed:27680706). Binding to carbohydrates and sulfated glycosaminoglycans on host cells suface also contribute to entry into cells (By similarity). Once entered into host cells, acidification in the endosome promotes the membrane insertion of the translocation region and formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (PubMed:11152463, PubMed:12941936, PubMed:24567384). This activates the peptidase C80 domain and autocatalytic processing, releasing the N- terminal part (Glucosyltransferase TcdB), which constitutes the active part of the toxin, in the cytosol (PubMed:17334356, PubMed:27571750). {ECO:0000250\|UniProtKB:P16154, ECO:0000269\|PubMed:10768933, ECO:0000269\|PubMed:11152463, ECO:0000269\|PubMed:12941936, ECO:0000269\|PubMed:17334356, ECO:0000269\|PubMed:19252482, ECO:0000269\|PubMed:20498856, ECO:0000269\|PubMed:20844489, ECO:0000269\|PubMed:24567384, ECO:0000269\|PubMed:24919149, ECO:0000269\|PubMed:25547119, ECO:0000269\|PubMed:26038560, ECO:0000269\|PubMed:27571750, ECO:0000269\|PubMed:27680706}.



Function:		[Glucosyltransferase TcdB]: Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases (PubMed:8144660, PubMed:7777059, PubMed:16157585, PubMed:17901056, PubMed:24905543, PubMed:24919149). Acts by mediating monoglucosylation of small GTPases of the Rho family (Rac1, RhoA, RhoB, RhoC, RhoG and Cdc42) in host cells at the conserved threonine residue located in the switch I region ('Thr-37/35'), using UDP-alpha-D-glucose as the sugar donor (PubMed:7777059, PubMed:16157585, PubMed:17901056, PubMed:24905543, PubMed:24919149). Monoglucosylation of host small GTPases completely prevents the recognition of the downstream effector, blocking the GTPases in their inactive form, leading to actin cytoskeleton disruption and cell death, resulting in the loss of colonic epithelial barrier function (PubMed:7777059, PubMed:24919149). {ECO:0000269\|PubMed:16157585, ECO:0000269\|PubMed:17901056, ECO:0000269\|PubMed:24905543, ECO:0000269\|PubMed:24919149, ECO:0000269\|PubMed:7777059, ECO:0000269\|PubMed:8144660}.


Catalytic activity:		[Glucosyltransferase TcdB]: Reaction=L-threonyl-[protein] + UDP-alpha-D-glucose = 3-O-(alpha-D- glucosyl)-L-threonyl-[protein] + H(+) + UDP; Xref=Rhea:RHEA:64684, Rhea:RHEA-COMP:11060, Rhea:RHEA-COMP:16656, ChEBI:CHEBI:15378, ChEBI:CHEBI:30013, ChEBI:CHEBI:58223, ChEBI:CHEBI:58885, ChEBI:CHEBI:156085; Evidence={ECO:0000269\|PubMed:16157585, ECO:0000269\|PubMed:17901056, ECO:0000269\|PubMed:24905543, ECO:0000269\|PubMed:24919149, ECO:0000269\|PubMed:7777059}; PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:64685; Evidence={ECO:0000269\|PubMed:16157585, ECO:0000269\|PubMed:17901056, ECO:0000269\|PubMed:24905543, ECO:0000269\|PubMed:24919149, ECO:0000269\|PubMed:7777059};
Cofactor:		[Toxin B]: Name=Zn(2+); Xref=ChEBI:CHEBI:29105; Evidence={ECO:0000269\|PubMed:27571750, ECO:0000269\|PubMed:31308519}; Note=Binds 1 Zn(2+) ion per subunit (PubMed:27571750, PubMed:31308519). Zn(2+) is required for autocatalytic cleavage (PubMed:27571750). {ECO:0000269\|PubMed:27571750, ECO:0000269\|PubMed:31308519};
Cofactor:		[Glucosyltransferase TcdB]: Name=Mn(2+); Xref=ChEBI:CHEBI:29035; Evidence={ECO:0000269\|PubMed:16054646, ECO:0000269\|PubMed:27089365, ECO:0000269\|PubMed:28433497, ECO:0000305\|PubMed:31308519}; Name=Mg(2+); Xref=ChEBI:CHEBI:18420; Evidence={ECO:0000269\|PubMed:27089365}; Note=Has higher activity with Mn(2+), but most likely uses Mg(2+) in host cells (PubMed:16054646, PubMed:28433497). Mn(2+) or Mg(2+) are required for glucosyltransferase activity (PubMed:27089365). {ECO:0000269\|PubMed:16054646, ECO:0000269\|PubMed:27089365, ECO:0000269\|PubMed:28433497};
Activity regulation:		[Toxin B]: Protease activity is activated upon binding to 1D-myo-inositol hexakisphosphate (InsP6), which induces conformational reorganization (PubMed:17334356). Inhibited by bezlotoxumab, also named Zinplava, a monoclonal antibody approved by the Food and Drug Administration (FDA), which specifically targets TcdB (PubMed:24821719). {ECO:0000269\|PubMed:17334356, ECO:0000269\|PubMed:24821719}.
Biophysicochemical properties:		[Glucosyltransferase TcdB]: Kinetic parameters: KM=6 uM for UDP-alpha-D-glucose {ECO:0000269\|PubMed:16157585}; KM=4.3 uM for UDP-alpha-D-glucose {ECO:0000269\|PubMed:17901056}; KM=960 uM for UDP-N-acetyl-alpha-D-glucosamine {ECO:0000269\|PubMed:16157585}; Note=kcat is 2120 hour(-1) with UDP-alpha-D-glucose as substrate (PubMed:16157585). kcat is 11 hour(-1) with UDP-N-acetyl-alpha-D- glucosamine as substrate (PubMed:16157585). {ECO:0000269\|PubMed:16157585};
Subunit:		[Toxin B]: Interacts with host FZD1 (PubMed:27680706). Interacts with host FZD2; interaction promotes toxin entry into host cell and occupies the binding site for Wnt-adducted palmitoleate in FZD2, leading to prevent Wnt-binding and downstream Wnt signaling (PubMed:27680706, PubMed:31233493, PubMed:29748286). Interacts with host FZD7 (PubMed:27680706). Interacts with host CSPG4 (PubMed:25547119, PubMed:31233493). Interacts with host NECTIN3/PVRL3 (PubMed:26038560). {ECO:0000269\|PubMed:25547119, ECO:0000269\|PubMed:26038560, ECO:0000269\|PubMed:27680706, ECO:0000269\|PubMed:29748286, ECO:0000269\|PubMed:31233493}.
Subcellular location:		[Toxin B]: Secreted {ECO:0000269\|PubMed:22685398}. Host endosome membrane {ECO:0000269\|PubMed:11152463, ECO:0000305\|PubMed:12941936}. Note=Secreted from C.difficile cell into the extracellular environment via help of holin-like protein TcdE/UtxA (PubMed:22685398). Binds to the cell surface receptors via the receptor-binding region and enters the cells via clathrin-mediated endocytosis (PubMed:20498856). Acidification in the endosome triggers conformational changes that promote the membrane insertion of the translocation region, allowing formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (PubMed:10768933, PubMed:11152463, PubMed:12941936). 1D-myo-inositol hexakisphosphate- binding (InsP6) activates the peptidase C80 domain and autoprocessing, generating the Glucosyltransferase TcdB form, which is released in the host cytosol (PubMed:17334356). {ECO:0000269\|PubMed:10768933, ECO:0000269\|PubMed:11152463, ECO:0000269\|PubMed:12941936, ECO:0000269\|PubMed:17334356, ECO:0000269\|PubMed:20498856, ECO:0000269\|PubMed:22685398}.
Subcellular location:		[Glucosyltransferase TcdB]: Host cytoplasm, host cytosol {ECO:0000269\|PubMed:12941936, ECO:0000269\|PubMed:15632438}. Host cell membrane {ECO:0000250\|UniProtKB:Q46342}; Peripheral membrane protein {ECO:0000250\|UniProtKB:Q46342}; Cytoplasmic side {ECO:0000250\|UniProtKB:Q46342}. Note=Binding to phospholipids, such as phosphatidylserine and phosphatidic acid promotes localization to the inner face of the cell membrane close to its membrane anchored substrates (small GTPases). {ECO:0000250\|UniProtKB:Q46342}.
Domain:		[Toxin B]: Consists of 4 functional domains: (1) the N-terminal GT44 domain (glucosyltransferase, also named GTD), which mediates glucosylation of host small GTPases, (2) an autoprocessing region that catalyzes autoprocessing to release the N-terminal GT44 domain in the host cytosol, (3) the translocation region that forms a pore to promote translocation of the GT44 and peptidase C80 domains across the endosomal membrane and (4) the receptor-binding (CROPS) region that mediates binding to host cells and contribute to entry into cells. {ECO:0000269\|PubMed:31308519, ECO:0000303\|PubMed:29146177}.
Domain:		[Toxin B]: The receptor-binding (CROPS) region is dynamic and can have open and closed conformations depending of the pH: has an open conformation at endosomal pH and a closed conformation at neutral pH. {ECO:0000269\|PubMed:31308519}.
Domain:		[Toxin B]: The cell wall-binding repeats bind carbohydrates, probably contributing to entry into cells. {ECO:0000250\|UniProtKB:P16154}.
Domain:		[Glucosyltransferase TcdB]: The four-helical bundle region mediates binding to phospholipids, such as phosphatidylserine and phosphatidic acid (PubMed:25882477). This promotes localization to the inner face of the cell membrane close to small GTPases (By similarity). {ECO:0000250\|UniProtKB:Q46342, ECO:0000269\|PubMed:25882477}.
Ptm:		[Toxin B]: Undergoes autocatalytic cleavage to release the N- terminal part (Glucosyltransferase TcdB), which constitutes the active part of the toxin, in the host cytosol (PubMed:12941936, PubMed:17334356, PubMed:27571750). 1D-myo-inositol hexakisphosphate- binding (InsP6) activates the peptidase C80 domain and promotes autoprocessing (PubMed:17334356). {ECO:0000269\|PubMed:12941936, ECO:0000269\|PubMed:17334356, ECO:0000269\|PubMed:27571750}.
Disruption phenotype:		Cells lacking tcdB display virulence and cytotoxicity, because of the presence of TcdA (PubMed:20844489). Cells lacking both tcdA and tcdB display a strongly reduced virulence (PubMed:20844489). {ECO:0000269\|PubMed:20844489}.
Similarity:		Belongs to the clostridial glucosylating toxin (LCGT) family. {ECO:0000305}.