L11. C. Diff Toxins

Question 1

What are the Major Toxins Produced by C. diff?

Answer 1

1. Toxin A (TcdA)
2. Toxin B (TcdB)
3. Binary toxin (only from some hypervirulent strains)

Question 2

What type of molecule are the two toxins (TcdA/TcdB) produced by C. diff

Answer 2

Both are single polypeptide chains

Question 3

What are the largest proteins produced by C. diff and what are their size?

Answer 3

1. toxin A - 308 kDa,
2. toxin B - 270kDa

Question 4

Describe the shared structure of the single polypeptide chain of Toxin A/B and the role of each section

Answer 4

> 1 to 2700 amino acids (Toxin B), 1 to 3080 amino acids (Toxin A)

> At N terminus, first 500 amino acids is enzyme activity

> 1300 amino acids in middle is involved in translocation (involved in getting the toxin into the cytoplasm of the cell it is going to target)

> At end is receptor binding bay

Question 5

What is the name of the pathogenicity locus of C. diff?

Answer 5

PaLoc

Question 6

What is a Pathogenicity locus?

Answer 6

A Pathogenicity Locus (PaL) is a specific region within a pathogen’s genome that contains genes critical for its ability to cause disease. These loci often encode various virulence factors

Question 7

What type of strains of C. diff is PaLoc found in?

Answer 7

Present in all pathogenic strains of C. diff mostly

Question 8

What does PaLoc contain?

Answer 8

Contains 5 genes: for TcdA and TdcB the 2 major toxins, and 3 regulatory or release genes (TcdD, TcdE, TcdC).

Question 9

What is the function of the 3 regulatory/ release genes in PaLoc?

Answer 9

1. TcdD (also known as TcdR)
   >An RNA polymerase sigma factor (directing it to promotors of tcdB/A/C genes)
   required for expression of tcdB and tcdA (and tcdD)
2. TcdE
   >Exact function is disputed; may be involved in release of toxins from bacterial cell, however expression of TcdE does not kill the cell so is different to a holin but does look like it is involved in toxin release.
   >Resembles a holin - phage protein that are cytolytic for bacteria (membrane pores which lyse bacterial cells)
3. TcdC
   >Putative negative regulator of toxin expression
   >TcdC is a membrane localised antisigma factor, Binds to sigma factors, sequesters them away from RNA polymerase, preventing transcription of TcdD, which in turn prevents TcdD from directing RNA polymerase from driving expression of the genes in this locus.

Question 10

What type of mutation is found in hypervirulent ribotype 027 strains and what does this do?

Answer 10

> TcdC has an 18 bp deletions found in hypervirulent ribotype 027 strains

> This deletion introduces a mutation into TcdC disrupting its function so it can no longer negatively regulate its expression of the toxins, accounting for the overexpression of the toxins in these ribotype 027 strains

Question 11

Describe an overview for how C. diff toxins lead to Pseudomembranous colitis

Answer 11

The toxins produced by C. diff in the lumen of colon, the toxins bind to epithelial surface, taken up into vacuoles, toxin enzymatic region released and acts on target within cell cytoplasm, leading to cell death breaking down epithelium, and inducing pro-inflammatory response where neutrophils are recruited (Pseudomembranous colitis is neutrophil infiltration into epithelial)

Question 12

What do toxins A and B do when inside the cell and what are the effects of this?

Answer 12

> Toxins both inactivate Rho GTPases inducing Rho, Rac and Cdc42 (what the toxins do within the cell)

> Inactivation of these 3 GTPases:
1. cytoskeleton changes
2. disruption of tight junctions
3. production of inflammatory mediators
from epithelial cells:
>accumulation of neutrophils
>Inflammation

Question 13

What is a difference between Toxin A and B mechanism of action?

Answer 13

TcdA seems to interact with epithelial cells at apical surface, while TcdB has to access the epithelium at basal membrane (at deeper tissue), so passes through gaps where TcdA disrupts tight junction

Question 14

What are the 3 domains of the Toxin A and B single polypeptide chain?

Answer 14

1. Receptor binding domain (C terminus)
2. Translocation domain
3. Enzymatic (catalytic) domain (N terminus)

Question 15

What is the structure of the receptor binding domain of Toxin A/B produced by C. diff?

Answer 15

> S shape

> Called “combined repetitive oligopeptides” (CROPS)- CROPs is made up of alternating short (SR, 18-24 aa) and long (LR, 31 aa) repeats creating this region

Question 16

What differs in the size of CROP domains in Toxin A and B and what could this reflect?

Answer 16

1. TcdA has 32 SRs and 7 LRs (longer CROPs domain)
2. TcdB has 18 SRs and 4 LRs

> Reflects evolution to binding to different receptors

Question 17

What is the role of the receptor binding domain (CROPS) in Toxin A/B and what is evidence for this?

Answer 17

> Binding of this region to cellular receptors promotes uptake of this toxin into epithelial cells

> Evidence is that antibodies to this domain inhibit binding of toxin to cells and inhibit uptake and inhibit uptake also genetic evidence where toxins lack CROPs domain which also inhibits uptake.

Question 18

What else differs in the CROPS region in Toxin A and B other than its size and what does this refelect?

Answer 18

> Differences in copy numbers and subtle differences in sequences

> Likely have different receptors and tissue specificity.

Question 19

Describe the structure of the Translocation domain in Toxin A/B

Answer 19

> Very large (1300 aa) compared to translocation domains from other toxins

> Contains a putative membrane spanning domain

Question 20

What is the role of the putative membrane spanning domain of the translocation domain in toxin A/B?

Answer 20

This putative membrane spanning domain may form a pore or channel in vesicle to allow toxin to enter cytosol

Question 21

What is the role of the Enzymatic domain in TdcA/B and what is the effect of this in human tissue?

Answer 21

> Role: glucosylate the small MW GTPases including Rho, Rac, Cdc42

> This glucosaylation reaction inactivates these 3 GTPases which causes cell rounding due to collapse of actin cytoskeleton as well as inducing pro-inflammatory cytokine response.

Question 22

Describe in 4 steps the binding and entry of TcdA/B into human epithelial cells (might include extra reading)

Answer 22

1. Binding of toxin to cell surface receptors via CROPs domain (green)
   >Toxin A binds to Sulfated Glycosaminoglycans on apical side of epithelial cells (extra reading)
   >Toxin B binds to Chondroitin Sulfate Proteoglycan 4 (CSPG4) on the basal side of epithelial cells (extra reading)
2. Entire toxin enters cell via clathrin-mediated endocytosis, making Toxin be inside vesicle in the cytoplasm
   >Clathrin forms pit under toxin, causing invagination of membrane and budding off into vesicle.
3. Vesicular ATPase mediates acidification of vesicles and this low pH induces refolding of translocation domain (black) to form a pore in the membrane
   >This acidification is a self-defence mechanism in all cells when undergoing endocytosis, where the contents inside the vesicle are destroyed via protons and proteases.
4. Catalytic (enzymatic) domain released into cytosol which modifies intracellular targets
   >This seeks out small GTPases and glucosylates them

Question 23

Where does the proteolytic cleavage of the enzymatic domain of Toxin A/B occur and what is the evidence for this?

Answer 23

> Cleavage of the enzymatic domain occurs in the cytosol not within the endosome it is taken up in

> Shown as the 60kDa fragment (N-terminal glucotransferase) reaches cytosol alone (without 200kDa fragment), this cleavage occurs in neutral pH conditions (so not in acidic vesicle, but neutral cytosol).

Question 24

What does the cleaving process of the enzymatic domain of Toxin A/B require, and how is this an adaptation?

Answer 24

Cleaving Processing requires Ins6P, inositol 6 phosphate, which is a compound found primarily in eukaryotic cells and not in bacteria or in normal bacterial environment so is a marker that the toxin is in the cytoplasm of a eukaryotic cell

Question 25

What type of protease is involved in the cleavage of the enzymatic domain of the Toxin A/B and what is the evidence for this?

Answer 25

> A cysteine residue Protease.

> Incubated toxin B with increased Ins6P concentrations, which showed increased cleavage (60kd fragments), when the 3 cysteine putative pathologic residues found in the cysteine protease were mutated it prevented the cleavage in the presence of Ins6P.

Question 26

What did the past literature say is to why both Toxins A and B are essential for Pathogenesis?

Answer 26

1. Toxin A (Enterotoxin):
   >First toxin discovered and defined as an enterotoxin.
   >Induces C. difficile pathology (diarrhea and death) in hamsters on its own.
   >Initially thought to be essential for pathogenesis due to its ability to reproduce disease symptoms independently.
2. Toxin B:
   >Similar to Toxin A in sequence, structure, and mode of action.
   >Initially not considered toxic to animals unless co-administered with Toxin A (based on one study).
   >Early studies’ limitations: Hamster model might not fully replicate human infection, and purification techniques in the 1980s may have been imprecise (potential contamination between toxins).

Question 27

What does the current literature say is to why both Toxins A and B are essential for Pathogenesis?

Answer 27

1. Importance of Toxin B:
   >Discovery of Toxin A-negative, Toxin B-positive (TcdA-ve, TcdB+ve) strains, known as “017 strains”, which still cause severe disease in humans.
   >No isolates found with Toxin A-positive, Toxin B-negative (TcdA+ve, TcdB-ve) configuration, indicating the essential role of Toxin B in disease.
2. Isogenic Mutant Strain Studies:
   >Lyras et al. (Nature 2009): Demonstrated Toxin B’s essential role in virulence. Knocking out Toxin B resulted in hamsters not getting sick, while knocking out Toxin A alone did not prevent illness.
   >Kuehne et al. (Nature 2010):Found that both Toxin A and Toxin B are required for full pathogenesis, though their roles were not as clear-cut as previously thought.

Question 28

Why did the Isonegic mutant strain studies, Lyras et al and Kuehne et al, two almost identical studies give different results?

Answer 28

1. Parental strains derived independently and the 2 groups used different derived strains.
2. Kuehne study used new genetic technique to make more stable mutants (ClosTron.com), while Lyras et al used unstable mutations involving suicide plasmids which is less stable mutant than Kuehne.
3. Different hamsters?
   >Same breed but different sources, Kuehne mice from UK while Lyras from US, so probably different microbiota
4. Small differences in how the models were run, and how pathogenesis were worked out
   >Different end-points, in US just let animals die in cages and find them the next day, this is illegal in the UK where end points measure body temp of animals and when It begins to drop (sign of death) they are put down; some of these animals could recover

Question 29

What is the overall conclusion for the importance of both toxins?

Answer 29

Overall both toxins are important, toxin B might have a greater role in virulence while Toxin A has an important role but not critical, but we just don’t know in humans