Clostridioides difficile toxin B
Structure of C. difficile glucosyl transferase Toxin B showing UDP and glusose from PDB entry 2BVM.[1]
Identifiers
Organism	Clostridioides difficile
Symbol	toxB
Alt. symbols	tcdB
Entrez	4914074
PDB	2BVM
RefSeq (Prot)	YP_001087135.1
UniProt	P18177
Other data
EC number	2.4.1.-
Chromosome	genome: 0.79 - 0.8 Mb
Search for Structures Swiss-model Domains InterPro

Clostridioides difficile toxin B (TcdB) is acytotoxin produced by the bacteriaClostridioides difficile. It is one of two major kinds oftoxins produced byC. difficile, the other being a relatedenterotoxin (Toxin A). Both are very potent and lethal.^[2][3]

Toxin B (TcdB) is acytotoxin that has a molecular weight of 270kDa and anisoelectric point, pl, of 4.1.^[4] Toxin B has four different structural domains:catalytic,cysteine protease,translocation, andreceptor binding.^[5] TheN-terminalglucosyltransferase catalytic domain includes amino acid residues 1–544 while thecysteine 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 theC-terminal region and includes amino acid residues from 1665 to 2366.^[5]

Theglycosylation activity of toxin B occurs in theN-terminal catalytic region (residues 1–544). This region glycosylates substrates independent of any cytotoxic activity.^[6] However, a small deletion of the receptor binding region causes attenuation of toxin B activity.^[6] The translocation region contains a hydrophobic stalk-like structure, which may help residues 958–1130 in forming membrane spanningpores.^[5] The receptor binding region that includes theC-terminal repetitive region (CRR) increases the TcdB membrane interaction but does not participate in pore formation.^[7] In addition,cysteine protease and translocation regions both have complex structures that play an important functional role in translocation and receptor binding.^[8] However, deleting the translocation region ofamino acids decreases thecytotoxic activity 4-fold. Bothcysteine proteases and a majority of translocation regions harborhydrophobic proteins, which show accessto TcdB and othertoxins crossing thecell membranes.^[8]

TheC-terminal of TcdB (the green region of Fig. 2) contains a region known as the combined repetitiveoligopeptides (CROPs) that containsamino acid residues 1831–2366.^[9] 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.^[9][10] 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.^[9]

In order to purify toxin B fromC. difficile cell cultures,brain heart infusion broth is used because it promotes the synthesis of toxin B.^[11] The filtration method facilitates purification of toxin B from thesupernatant ofC. difficile. The toxinconcentration 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 latelog phase or earlystationary phases, hence, toxin B is continuously secreted by cells.^[2] Although there are many methods employed by different studies in purifying toxin B, the majority of studies use methods involving concentrations of ultrafiltratedammonium sulfate orprecipitation, in lieu by eithergel filtration orion-exchange chromatography. In addition, the effectiveness of theion-exchange chromatography method helps to differentiate between TcdA and TcdB.^{[citation needed]}

When thecatalyticthreonine residue ofglucosyltransferase deactivates a family ofsmall GTPases,e.g. theRho family;Rac, andCdc42 inside the targetcells disturbsignal transduction mechanisms, which leads to dysfunctioning ofactincytoskeleton,cell-cell junction, andapoptosis (Fig. 5).^[12][13][14]Rho induces the activity ofactin stressfibers. Racproteins controls the activities ofmembrane ruffling andNADPH-oxidaseneutrophil.Cdc42 regulates theF-actin filament formation infilopodia.^{[citation needed]}

Several studies have demonstrated that the presence of TcdB inmammalian cells leads to rapid changes withincellmorphology andcell 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 beenintoxicated. An investigation by Donta et al., forwarded that TcdB has serious impacts in other mammalian cells such aschinese hamster ovary cells, human cervicalepithelial cells, mouseadrenal cells, rathepatocytes and ratastrocytes (Fig.3).^[15][16]

Thecytotoxic 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 ofF-actinfilaments.^[17] Cell roundings by TcdB take no longer than 2 hours (Fig. 4), but as far ascell death goes, it can take approximately 24 hours.^[15] With regard toC. difficile-associated diarrhea (CDAD), the effects ofcytopathicity are more critical than actual cell death because once cells lose integrity of thecytoskeletonactin filament, they also lose its normal function.^{[citation needed]}

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The cause ofcytotoxic activity by TcdB within the host cell is mainly mediated via receptorendocytosis^{[citation needed]}.Acidicendosomes allow toxin B to enter thecytosol. This phenomenon takes place by abinding receptor region, which enables toxin to enter host cells^{[citation needed]}. Through the accessibility of the host cells'cytosol, TcdB deactivates thesmall GTPases (Fig. 5), e.g. theRho family membersRac andCdc42 by the process ofglycosylation ofthreonine 35 inCdc42 and Rac, andthreonine 37 in Rho.^[18][19] TheseRho GTPases are found ubiquitously in thecytosol ofeukaryotic cells that are responsible for the organization of theactincytoskeleton because the toxins in thecytosol cause condensation ofactin filaments as a consequence of cell rounding and membrane blebbing (Fig. 3), which ultimately leads toapoptosis.^[20][21] TcdB causes critical changes to cell dynamics andmorphology. Figure 3 shows the probable effect of toxin B on a cell's surface; membrane blebbing (black arrows).^[22] In addition, TcdB inactivates Rho GTPases. As a consequence, cell-cell junctions are disrupted, which enhances epithelial permeability of toxin B and fluid accumulation in the lumen. This is one of the main causative agents in contractingC. difficile-associated diarrhea (CDAD)(Fig. 5).^[23][24]

Furthermore, the rate ofhydrolysis by TcdB ofUDP-glucose is approximately five-fold greater than TcdA.^[25] Several studies have indicated that Rho exhibitsposttranslational modification throughprenylation and carboxymethylation, which occurs in thecytoplasmic side of theplasma membrane, hence, the exchange ofGTP toGDP.^[26] When TcdB binds to Rho and othersmall GTPases,GTPhydrolyzes toGDP, which leads to GTP-bound (active) to GDP-bound (inactive) (Fig. 5). In addition, this interchange activity is regulated byguanine factors in the cell's cytosol.^[27]

The cellular regulation of Rho,Rac, andCdc42 has effects outside the vicinity of theactin filaments of the cytoskeleton (Fig. 4),^[17] Thesesmall GTPases are incorporated in thecell cycle that regulates signals viamitogen-activated protein kinase kinases (MAPKKs).^[28] Some physiological parts of the cells that are not involved inactin filaments, may not cause cell rounding orcell death right away, but in downstream pathway activity, may lead to the deterioration ofactin filaments and finally,cell death.^[17]

In 1993, a study conducted by Shoshan et al., showed that cells with TcdB changedphospholipase A2 activity. This was an independent event from disruption of theactincytoskeleton.^[29] Shoshan et al., also showed that TcdB inhibited the receptor signaling activity by deactivating the Rho proteins viaphospholipase D.^[29]

TcdB accesses the interior of the cell throughclathrin-mediated endocytosis,^[30] When toxin B is part of thecytosol, theglucosyltransferase passes through theendosomal membrane, which decreases pH, inducestranslocation and finally leads tomorphological changes of translocation region residues (958–1130).^[31] Thehydrophobic regions are embedded in the host membrane to form pores that allowglucosyltransferase domains to pass through.^[31] When cells are infected with TcdB in an acidic environment, it attenuates toxins and causes shape rearrangements (Fig. 6).^[31] As a consequence of acidic pH, TcdB displays clear differences in original fluorescence oftryptophan, susceptibility ofproteases, andhydrophobic surfaces.^[31] Another group has shown that acidification leads to conformational changes of the toxin and, more importantly, helps to form pores.^[7] A putativetranslocation region ( Fig. 2) makes up approximately 801–1400 amino acids, of which residues 958–1130 arehydrophobic and are responsible for the formation of transmembrane pores.^[20] A majority of the studies used TcdB strain 630 to show the pore formation activity ofC. difficile toxins.^[31]

To see whether effects ofproteolytic cleavage of TcdB takes place at the cell surface or inacidicendosomes, studies usedBafilomycin A1, which is known to block thev-type H⁺-ATPases of endosomes. This reduces the acidity in endosomes.^[31] Thephysiological uptake pathway of TcdB preventscytopathic activity by TcdB.^[31] When cells were inacidic conditions (pH 4.0) for 5 minutes after binding TcdB to the cell surface at 37 degree Celsius, the shape rearrangements and rounding were observed. However, when rounded cells were incubated for an additional hour in neutral pH (7.0) with similar parameters, no cell rounding was observed.^[15][31] Both studies showed that toxin B has a property ofproteolytic cleavage, which is critical for access to thecytosol.^[7][15][31] Having an acidic endosome pH leads to topological alterations of TcdB (Figure 6).^[7]

The gene that encodes the TcdB protein,tcdB, is located within thechromosomal region of 19.6kb. This is known as thelocus ofpathogenicity or PaLoc (Figure 2).^[32][33] Theopen reading frame (ORF) for tcdB is 7,098nucleotides in length.^[17] It is important to mention that—besides the major toxin genes in the PaLoc region—there are three other accessorygenes that encode in the PaLoc region:tcdR (L),tcdC (R) andtcdE in the middle. These genes help to regulate TcdA and TcdB expression. They also help to secrete or release thetoxins from the cell.^[17] The encodinggenetcdE, located between tcdB and tcdA, is analogous toholin proteins, thus, it is suggested that tcdE works as a facilitatorgene that enhances the release or secretion of TcdA and TcdB consequently increasing thepermeability of the hostcell membrane.^[17]

There are differentplasmid sizes ofC. difficile. The detected molecular weights range from 2.7x10⁶ to 100x10⁶, butplasmid sizes show no correlation withtoxicity. In order to detect the toxin B level inC. difficile, clinicians extensively usecell culture assays derived from stool specimens from patients withPMC.^[2][3] The cell culture assay is regarded as a "gold standard" for detectingtoxicity inC. difficile because a small quantity of toxin B is capable of causing cell rounding (Fig. 4), thus, it is a major advantage of clinical laboratories to make correlations with the CDAD caused by TcdB.^[2][3] Although cytotoxic activity of large clostridial toxins (LCTs) was found in PMC patient stool specimens, toxin B activity had more detrimental cytotoxic effects in comparison with toxin A.^[2] Therefore, the activity of toxin A is attenuated when it is not isolated from toxin B.^[2][3] The detection ofC. difficiletoxicity is extremely sensitive, however, using thecell culture assay allows clinical laboratories to overcome the challenge; using doses as little as 1 pg/mL of toxin B is enough to causes cell rounding.^[2][3] This is the major advantage in using the culture tissue assay to detecttoxicity inPMC patients.^[2] Even though clinical laboratories have tried to use an assay microtiter plateenzyme-linked immunosorbent assay (ELISA) and other techniques to detect thecytotoxic activity of toxin B in the feces ofPMC patients, the results are not as accurate as those wherecell culture assays were used.^[2][3][34]

By addingantimicrobial, e.g.clindamycin, into the culture growth medium, studies have shown that thecytotoxic activity inC. difficile cultures increases by 4–8 fold.^[35][36] Moreover, knowing the role ofantibiotics on the causes of PMC, many earlier studies focused on the effects ofantimicrobials production of toxins. As a result, studies were able to conclude that the subinhibitory nature ofvancomycin andpenicillin levels were increasing the toxin production in cultures ofC. difficile.^[37] The amounts of toxin production were correlated with the usage of growth medium for the organisms. Another study illustrated that the high levels of toxin production of TcdB were observed in complex mediums such as brain and heart infusionbroth.^[38][39] High levels of toxins were produced with isolation of highlyvirulent. Conversely, low levels of toxins were produced with isolation of weaklyvirulent. Thus, it shows that the productions of toxins were co-regulated. Although the mechanism behind the environment's involvement in modulating the signals expressing the toxins is not understood, invitro studies have shown that expression of toxin is strengthened bycatabolite repression and stress, e.g.antibiotics.^[40][41][42] Another study has shown that limitingbiotin in well-characterized medium increases the production of TcdB by 64-fold and TcdA by 35-fold. This was done withC. difficile and doses ofbiotin as small as 0.05nM.^[41] Several other early studies have argued against the theory that the production of toxin has anything to deal with stress or catabolite repression of either toxin TcdA or TcdB.^[42] Also, many studies say the main reason for the differences among other studies is due to toxin production not occurring with all isolates ofC. difficile.^{[citation needed]}

Many early studies have suggested that toxin A (also known as TcdA) is the major toxin protein causingantibiotic-associated diarrhea (AAD); however, research scientists within the last decade or so have shown that Toxin B (or TcdB) plays a more important role in disease than anyone had forecasted. With this knowledge, Toxin B has been identified as the majorvirulence factor that causes the opening oftight junctions ofintestinal epithelial cells, which enables toxin to increasevascular permeability and inducehemorrhaging. Hence, this leads totumor necrosis factor α (TNF α) andproinflammatoryinterleukins being established as the major causative agents ofpseudomembranous colitis (PMC) and antibiotic-associated diarrhea (AAD).^[2][3][43]

The involvement of toxin A and—most importantly—toxin B is the key element that determines the disease caused byC. difficile. Clinical laboratories have identified these toxins in patients' stool based onantibody andcytotoxicity assays.^[44] These bacterial toxins have been shown to be associated withClostridium sordelliihemorrhagic toxin (TcsH), lethal toxin (TcsL), andClostridium novyi alpha toxin (Tcn α), thus, making this cohort to be the large family of toxin clostridial.^[17] Because of similarities of these toxins with others, researchers have classified them as the family of large clostridial toxins (LCTs).^[9]

Bezlotoxumab is a human monoclonal antibody designed for the prevention of recurrence of Clostridium difficile infections. By x-ray crystallized structure of N-terminal of TcdB, the toxin is identified to consist of three domains: a glucosyltransferase domain (GTD), a cysteine protease and a combined repetitive oligopeptide (CROP) domain. Bezlotoxumab specifically binds to two homologous sites within the CROP domain of TcdB. Structural analysis by X-ray crystallography indicates that antibody binding partially occludes putative carbohydrate binding pockets. Consistent with this idea, Bezlotoxumab blocks binding of TcdB to mammalian cells.^[45]

In early stages of the disease ofPMC, many studies have speculated that TcdA is more potent than TcdB. This has been deduced from in vivo experiments where toxin productions of TcdA were more severe than TcdB with antibiotics cecitis.^[38][46] Later, several studies showed that TcdB plays a major role in the disease ofPMC and ADD. The study demonstrated that even thoughC. difficile did not produces TcdA, it still showed symptoms for the disease.^[47] Furthermore, later studies have shown that a purified form of TcdB is a more lethalenterotoxin in comparison to TcdA, and also, that intestinal epithelium is severely damaged and causes an acute inflammatory response.^[48] With better understanding of the toxin, researchers were able to state that TcdB is the majorvirulence factor that causes CDI over TcdA. However, when TcdA is present in the gut, it helps to facilitate TcdB's activity to have broader impacts, consequently, affecting multiple organ systems.^[49] In addition, when hamsters were vaccinated against TcdA, it showed hamsters were not fully protected from theC. difficile disease and this lead studies to conclude that TcdB is very lethal and potent.^[50] Furthermore, injecting a small dose of TcdA with a lethal dose of TcdBintravenously orintraperitoneally proved sufficient in causing the death of an animal. Therefore, TcdA works as a facilitator of TcdB exiting from the gut.^[50]

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