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Clostridioides difficile toxin B
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Clostridioides difficile toxin B
Clostridioides difficile toxin B (TcdB) is a cytotoxin produced by the bacteria Clostridioides difficile. It is one of two major kinds of toxins produced by C. difficile, the other being a related enterotoxin (Toxin A). Both are very potent and lethal.
Toxin B (TcdB) is a cytotoxin that has a molecular weight of 270 kDa and an isoelectric point, pl, of 4.1. Toxin B has four different structural domains: catalytic, cysteine protease, translocation, and receptor binding. The N-terminal glucosyltransferase catalytic domain includes amino acid residues 1–544 while the cysteine protease domain includes residues 545–801. Additionally, the translocation region incorporates amino acid residues from 802 to 1664 while the receptor binding region is part of the C-terminal region and includes amino acid residues from 1665 to 2366.
The glycosylation activity of toxin B occurs in the N-terminal catalytic region (residues 1–544). This region glycosylates substrates independent of any cytotoxic activity. However, a small deletion of the receptor binding region causes attenuation of toxin B activity. The translocation region contains a hydrophobic stalk-like structure, which may help residues 958–1130 in forming membrane spanning pores. The receptor binding region that includes the C-terminal repetitive region (CRR) increases the TcdB membrane interaction but does not participate in pore formation. In addition, cysteine protease and translocation regions both have complex structures that play an important functional role in translocation and receptor binding. However, deleting the translocation region of amino acids decreases the cytotoxic activity 4-fold. Both cysteine proteases and a majority of translocation regions harbor hydrophobic proteins, which show access to TcdB and other toxins crossing the cell membranes.
The C-terminal of TcdB (the green region of Fig. 2) contains a region known as the combined repetitive oligopeptides (CROPs) that contains amino acid residues 1831–2366. These CROPs make up 19–24 short repeats (SRs) of amino acids, roughly 31 long repeats (LRs) of amino acids, toxin A, and Toxin B. The TcdB CROPs region consists of 19 SRs and 4 LRs. This SRs and LRs region allows formation of cell wall binding motifs that help to bind sugar moieties of the cell surfaces.
In order to purify toxin B from C. difficile cell cultures, brain heart infusion broth is used because it promotes the synthesis of toxin B. The filtration method facilitates purification of toxin B from the supernatant of C. difficile. The toxin concentration of the supernatant is proportional to the organism cell count. It has been proposed by many studies that the majority of the toxins are released in either late log phase or early stationary phases, hence, toxin B is continuously secreted by cells. Although there are many methods employed by different studies in purifying toxin B, the majority of studies use methods involving concentrations of ultrafiltrated ammonium sulfate or precipitation, in lieu by either gel filtration or ion-exchange chromatography. In addition, the effectiveness of the ion-exchange chromatography method helps to differentiate between TcdA and TcdB.[citation needed]
When the catalytic threonine residue of glucosyltransferase deactivates a family of small GTPases,e.g. the Rho family; Rac, and Cdc42 inside the target cells disturb signal transduction mechanisms, which leads to dysfunctioning of actin cytoskeleton, cell-cell junction, and apoptosis (Fig. 5). Rho induces the activity of actin stress fibers. Rac proteins controls the activities of membrane ruffling and NADPH-oxidase neutrophil. Cdc42 regulates the F-actin filament formation in filopodia.[citation needed]
Several studies have demonstrated that the presence of TcdB in mammalian cells leads to rapid changes within cell morphology and cell signaling. Within a short period of time, cells have the appearance of plaque with small dosages of TcdB and TcdA. In addition, death of the cells is a major impact of these toxins after cells have been intoxicated. An investigation by Donta et al., forwarded that TcdB has serious impacts in other mammalian cells such as chinese hamster ovary cells, human cervical epithelial cells, mouse adrenal cells, rat hepatocytes and rat astrocytes (Fig.3).
The cytotoxic activity is based on cell types, which could range from 4-fold to 200-fold. Generally, when cells are infected with TcdB, they not only lose their structural integrity, but also diminutions of F-actin filaments. Cell roundings by TcdB take no longer than 2 hours (Fig. 4), but as far as cell death goes, it can take approximately 24 hours. With regard to C. difficile-associated diarrhea (CDAD), the effects of cytopathicity are more critical than actual cell death because once cells lose integrity of the cytoskeleton actin filament, they also lose its normal function.[citation needed]
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Clostridioides difficile toxin B
Clostridioides difficile toxin B (TcdB) is a cytotoxin produced by the bacteria Clostridioides difficile. It is one of two major kinds of toxins produced by C. difficile, the other being a related enterotoxin (Toxin A). Both are very potent and lethal.
Toxin B (TcdB) is a cytotoxin that has a molecular weight of 270 kDa and an isoelectric point, pl, of 4.1. Toxin B has four different structural domains: catalytic, cysteine protease, translocation, and receptor binding. The N-terminal glucosyltransferase catalytic domain includes amino acid residues 1–544 while the cysteine protease domain includes residues 545–801. Additionally, the translocation region incorporates amino acid residues from 802 to 1664 while the receptor binding region is part of the C-terminal region and includes amino acid residues from 1665 to 2366.
The glycosylation activity of toxin B occurs in the N-terminal catalytic region (residues 1–544). This region glycosylates substrates independent of any cytotoxic activity. However, a small deletion of the receptor binding region causes attenuation of toxin B activity. The translocation region contains a hydrophobic stalk-like structure, which may help residues 958–1130 in forming membrane spanning pores. The receptor binding region that includes the C-terminal repetitive region (CRR) increases the TcdB membrane interaction but does not participate in pore formation. In addition, cysteine protease and translocation regions both have complex structures that play an important functional role in translocation and receptor binding. However, deleting the translocation region of amino acids decreases the cytotoxic activity 4-fold. Both cysteine proteases and a majority of translocation regions harbor hydrophobic proteins, which show access to TcdB and other toxins crossing the cell membranes.
The C-terminal of TcdB (the green region of Fig. 2) contains a region known as the combined repetitive oligopeptides (CROPs) that contains amino acid residues 1831–2366. These CROPs make up 19–24 short repeats (SRs) of amino acids, roughly 31 long repeats (LRs) of amino acids, toxin A, and Toxin B. The TcdB CROPs region consists of 19 SRs and 4 LRs. This SRs and LRs region allows formation of cell wall binding motifs that help to bind sugar moieties of the cell surfaces.
In order to purify toxin B from C. difficile cell cultures, brain heart infusion broth is used because it promotes the synthesis of toxin B. The filtration method facilitates purification of toxin B from the supernatant of C. difficile. The toxin concentration of the supernatant is proportional to the organism cell count. It has been proposed by many studies that the majority of the toxins are released in either late log phase or early stationary phases, hence, toxin B is continuously secreted by cells. Although there are many methods employed by different studies in purifying toxin B, the majority of studies use methods involving concentrations of ultrafiltrated ammonium sulfate or precipitation, in lieu by either gel filtration or ion-exchange chromatography. In addition, the effectiveness of the ion-exchange chromatography method helps to differentiate between TcdA and TcdB.[citation needed]
When the catalytic threonine residue of glucosyltransferase deactivates a family of small GTPases,e.g. the Rho family; Rac, and Cdc42 inside the target cells disturb signal transduction mechanisms, which leads to dysfunctioning of actin cytoskeleton, cell-cell junction, and apoptosis (Fig. 5). Rho induces the activity of actin stress fibers. Rac proteins controls the activities of membrane ruffling and NADPH-oxidase neutrophil. Cdc42 regulates the F-actin filament formation in filopodia.[citation needed]
Several studies have demonstrated that the presence of TcdB in mammalian cells leads to rapid changes within cell morphology and cell signaling. Within a short period of time, cells have the appearance of plaque with small dosages of TcdB and TcdA. In addition, death of the cells is a major impact of these toxins after cells have been intoxicated. An investigation by Donta et al., forwarded that TcdB has serious impacts in other mammalian cells such as chinese hamster ovary cells, human cervical epithelial cells, mouse adrenal cells, rat hepatocytes and rat astrocytes (Fig.3).
The cytotoxic activity is based on cell types, which could range from 4-fold to 200-fold. Generally, when cells are infected with TcdB, they not only lose their structural integrity, but also diminutions of F-actin filaments. Cell roundings by TcdB take no longer than 2 hours (Fig. 4), but as far as cell death goes, it can take approximately 24 hours. With regard to C. difficile-associated diarrhea (CDAD), the effects of cytopathicity are more critical than actual cell death because once cells lose integrity of the cytoskeleton actin filament, they also lose its normal function.[citation needed]
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