C. difficile

Question 1

What are the characteristics of C. diff?

Answer 1

Clostridioides difficile is a gram positive, spore forming, and anaerobic bacterium. Causes a spectrum of diseases collectively known as CDAD (C.diff associated disease). Killed by atmospheric oxygen.
C. difficile is the leading cause of hospital acquired infection worldwide. Most commonly seen in the elderly but increasingly seen in the community and younger populations (incl. children, pregnant women). Also in animals (pigs, cattle, horses, chickens). Costs the United States 3.2b a year and Europe €3b a year.

Question 2

How does C. diff disease progress?

Answer 2

Patient receives antibiotics -> Alterations in normal gut flora -> Infection with C.diff -> Spores germinate in gut forming vegetative cells -> Cells multiply, produce toxins and sporulate. Spores excreted. The spores are an absolute requirement for transmission between patients. Toxins for C.diff include TcdA and TcdB

Question 3

Where were the hyper virulent strains of C. diff?

Answer 3

Georgia, Pennsylvania, Maine, New Jersey, Oregon and Pennsylvania 2000- 2003 and Quebec 2003.
Strain: NAP1 (North American Pulse-field electrophoresis Type 1), Ribotype 027.

Question 4

Why were the C. diff strains hyper virulent?

Answer 4

027/NAP1 strains are not new but were previously rare and are now fluoroquinolone resistant suggesting recent acquisition. Characteristics: Produces more spores; Produces more toxin in vitro; Produces a third toxin – binary toxin (CDT); Contain a 18 bp deletion in tcdC (anti-Sigma factor that negatively regulates toxin expression). Canadian and USA strains arose from ancestral strain independently – separate acquisition of fluoroquinolone resistance.
Epidemic ribotype 027 has a point mutation in treR -> >500 fold more sensitive to trehalose (manufactured sugar found in a lot of processed food). Deletion of treA attenuates Ribotype 027, and supplementation with trehalose enhances virulence.

Question 5

What is the relationship between C. diff and the microbiota?

Answer 5

Most C. difficile infections follow treatment with antibiotics.
Dysbiosis a prerequisite for CDI. Microbiota recovers as infection is cleared.
We can potentially exploit this to treat infections by recruiting therapeutics such as faecal microbiota transplantation. Over 99% effective in most studies.

Question 6

What are the two C. diff models used?

Answer 6

Hamster model and mouse model.

Question 7

What is the Syrian golden hamster model?

Answer 7

Most have stopped being used globally with labs either switching to non-animal forms or to the mouse model. Clindamycin treated hamsters are challenged with C. difficile spores. C. difficile infection is invariably lethal within a few days. Model used to study the ACUTE phase of C. difficile infection where toxins are key virulence determinant. Model has gone out of use due to ethical reasons (all animals used die), also does not replicate what happens in humans (humans don’t die after 3 days) and hamsters are not commercially used so no commercial reagents available.

Question 8

What is the C57BL/6 mouse model?

Answer 8

Cheaper, availability of reagents and knock-out animals. Model for C. difficile COLONISATION: Clindamycin treated mice are challenged with C. difficile spores. High number of spores being shed in the faeces, referred to as a super-shedder state. After cessation of antibiotic treatment the number of C. difficile spores in the faeces decreases, but if a subsequent dose of clindamycin animals return to the super-shedder state. Model variations which are more extreme by giving animals antibiotic cocktails to give more colonisation. Can change the cocktail to tweak the outcome and severity of the infection.

Question 9

Why are spores pivotal in transmission?

Answer 9

• Highly infectious
• Resistant to various chemical and physical insults
• Persist in health care facilities for long periods of time

Question 10

What is the structure of C. diff spores?

Answer 10

Spores contain a highly cross-linked protein coat, cortex peptidoglycan, original cell wall, two membranes and a core containing 25% dipicolinic acid (Ca-DPA) which drives out water to dehydrate the spore, making the core enzymatically inert. Cortex has more flexible peptidoglycan layer due to the presence of Muramic delta lactam which does not have side chain amino acids present.

Question 11

How does sporulation occur in C. diff?

Answer 11

A single cell gives rise to a single spore. Little homology in spore proteins between C. difficile and Bacilli/Clostridia. Regulation of sporulation/germination not fully understood. Few germinants known (taurocholate, glycine). Little mechanistic insight into sporulation/germination – limited genetic tools.

Question 12

Why is the sporulation cascade complex?

Answer 12

Due to the sequential activation of other genes. This is controlled by a sigma factor cascade in the fore-spore and mother cell. Initial signal to cause sigma factor cascade and ultimately sequential activation of other genes is the phosphorylation of Spo0A

Question 13

What is the sporulation cascade?

Answer 13

Stage 0 = Vegetative cells
Stage 1 = Onset
Stage 2 = Commitment asymmetric cell division
Stage 3 = Engulfment
Stage 4 = Cortex formation
Stage 5 = Maturation of spore coat
Stage 6 = Mother cell lysis and spore release

Question 14

What is the use of bile salts?

Answer 14

Primary bile salts are synthesised in the liver. Stored in the gall bladder and released into the duodenum. Emulsify fats – critical for uptake of fats and fat-soluble vitamins. Secondary bile salts produced via bacterial metabolism in the colon.
These induce germination.

Question 15

What is the main bile salt produced in the liver?

Answer 15

Taurocholate is one of the main bile salts produced by the liver and is one of the main germinants. When in the intestine, bacteria act upon it to strip amino acids and can be further chemically modified to convert it into lithocholate and deoxycholate.
Cholate and deoxycholate can also induce germination but they are less effective as they have been modified. Deoxycholate can also inhibit vegetative growth.

Question 16

What is the role of C. diff spore?

Answer 16

Anaerobe – transmission will involve exposure to oxygen. Spo0A is at the top of the regulatory cascade. spo0A mutants don’t sporulate. Sporulation-defective C. difficile can infect (via gavage) but once cleared with vancomycin cannot relapse. Can conclude spores are the reason for relapse.

Question 17

How was sporulation tested?

Answer 17

To test transmission used different mice housing arrangements - mingling, contact, airborne and environmental. Saw that when mice are mingling, wild type transmit with 100% efficiency but knocking spo0A mutants decreases transmission. Should be that mice get infections via eating faeces and vegetative cells.
Results show spores can move via air transference.

Question 18

What are the two major toxins of C. diff?

Answer 18

TcdA (toxin A) and TcdB (toxin B).

Question 19

What is the structure of C. diff toxins?

Answer 19

These are single polypeptide chains and members of the Large Clostridial Cytotoxin Family. They are closely related to each other and share a common structure. Third toxin called binary toxin but its contribution to virulence is disputed.

Question 20

What is involved in PaLoc (operon)?

Answer 20

TcdD (also known as TcdR) - an RNA polymerase sigma factor and required for expression of tcdB and tcdA (and tcdD).
TcdE - exact function is disputed; may be involved in release of toxins from bacterial cell, resembles a holing - phage protein that are cytolytic for bacteria.
TcdC - putative negative regulator of toxin expression that is membrane localised – anti-sigma factor to prevent TcdD activating polymerase. 18 bp deletions found in hypervirulent ribotype 027 strains, mutation allows for overexpression of toxins.
Toxin A and B both inactivate Rho GTPases. They cause cytoskeleton changes causing disruption of tight junctions. Also cause production of inflammatory mediators, accumulation of neutrophils and inflammation.

Question 21

What are the three domains involved in C. diff toxins?

Answer 21

Receptor binding domain, translocation domain and enzymatic domain.

Question 22

What is involved in the toxin receptor binding domain?

Answer 22

Called “combined repetitive oligopeptides” (CROPs). Alternating short and long repeats. TcdA has 32 SRs and 7 LRs, TcdB has 18 SRs and 4 LRs. Antibodies to this domain inhibit binding of toxin to cells and inhibit toxin uptake. Sequences of TcdA and TcdB RBDs are related, but distinct highlighting maybe different receptors.

Question 23

What is involved in the toxin translocation domain?

Answer 23

Very large compared to translocation domains from other toxins. Contains a putative membrane spanning domain that may form a pore or channel in vesicle to allow toxin to enter cytosol.

Question 24

What is involved in toxin enzymatic domain?

Answer 24

Toxin catalytic domains very similar. Glucosylate small MW GTPases including Rho, Rac, Cdc42. This glucosyaltion activates the GTPases which cause cell rounding due to collapse of actin cytoskeleton.

Question 25

What is the process of binding and cell entry for C. diff toxins?

Answer 25

1. Binding to cell receptors
2. Entire toxin enters cell via clatherin-mediated endocytosis
   Vesicular ATPase mediates acidification of vesicles.
3. Low pH induces refolding of translocation domain forming pore in membrane
4. Catalytic domain released into cytosol modifying the intracellular target.

Question 26

How is the enzymatic domain released?

Answer 26

Toxin B is cleaved by incubation with cultured animal cells. 60 kDa fragment identified as Nterminal glycotrasferase. This fragment reaches cytosol - the 200 kDa does not. Cleavage shown to occur primarily in the cytosol fraction – at neutral pH - i.e. not within the endosome. Protease not identified. Processing requires Ins6P.
Bioinformatic analysis identified potential aspartate or cysteine protease sequences adjacent to the enzymatic domain. Eventually a cysteine protease activity was identified – mutations in any of the 3 catalytic residues prevented cleavage. Autocatalytic cleavage event so it is within the toxin itself.

Question 27

What was the background with toxins in C. diff?

Answer 27

Toxin A was the first toxin to be discovered. It is an enterotoxin and when given to hamsters, it reproduced pathology of infection - thought to be essential for pathology.
Toxin B was then discovered being very similar to toxin A in sequence, structure and mode of action but not toxic to animals unless co-administered with toxin A (one study only).
HOWEVER Toxin A-ve, toxin B+ve strains “017 strains” cause severe disease in humans (no toxin A+ve, toxin B-ve strains isolated to date). To resolve this, isogenic mutant strains are created and tested in animal models.

Question 28

What were the two results gotten from studies by Lycras et al and Kuehne et al?

Answer 28

Lycras et al and Kuehne et al gave contrasting results. Lycras stated tcdB mutant was avirulent in hamsters, tcdA mutant fully virulent. Kuehne stated both toxins are required for full virulence in hamsters.

Question 29

Why did the two almost identical studies give different results?

Answer 29

Parental strains have been derived independently. Kuehne study used new genetic techniques to make more stable mutants. Could be due to different hamsters being used – same breed but different sources so probably different microbiota. Small differences in how the models were run. Different end-points.

Question 30

Which toxin is the main virulence factor in C. diff?

Answer 30

Toxin B

Question 31

What is the S layer?

Answer 31

This is the paracrystalline array surrounding the cell – 2D crystal of protein. Made of S layer proteins with the majority being glycosylated. Not found on a lot of model species such as E.coli, Staph. Aureus etc. but these are the odd ones out.

Question 32

What is the structure of the S layer in C. Diff?

Answer 32

Approx 600,000 protein subunits and very expensive. C. Diff has both glycosylated and non-glycosylated SLPs with non being predominant. Main constituent SlpA (S layer protein A) but decorated with up to 28 additional cell wall proteins (CWPs).

Question 33

What is the structure of S layer proteins?

Answer 33

SlpA contains a single polypeptide required for translocation across the membrane, low molecular weight protein and high molecular weight protein. The two weighted proteins form a heterodimer becoming the 2D crystals.
The complex self-assembles on surface, anchored to PS-II. LMW and HMW form high affinity heterodimer. Post-secretion cleavage by Cwp84. Secretion via dedicated ATPase, SecA2. SecA2 is dedicated to the secretion of S layer.

Question 34

What protein family is Slp a part of?

Answer 34

CWP. These are anchored in the same way but do not form crystal arrangement. All members of this family have 3 copies of cell wall binding domain (Pfam04122) which interacts with sugar in PS II. Only a few of these CWPs have been characterised.

Question 35

Why is research into C. Diff S layer hampered?

Answer 35

Due to by the inability to make a mutant in the SlpA gene (until 07/05/2024 who made one using CrispR technology).

Question 36

What is the work of AvidBiotics?

Answer 36

Created a new therapeutic that targets the S-layer and kills C. difficile – “Avidocin”.

Question 37

What is the mechanism of Avidocins?

Answer 37

Recognises specific receptor on outside of the cell. Upon binding, the contractile sheath pushes the needle inside into the cell envelope, killing the target cell. Has an open channel from cell cytoplasm to normal environment so lots of things freely move out. Lose proton motive force meaning no voltage gradient and no ATP can be made

Question 38

What else has Avidbiotics engineered along with Avidocins?

Answer 38

Myroviridae phage is produced by C. diff to kill other strains. It has a narrow killing spectrum and has been engineered to improve stability and widen the spectrum -> Acidocin-CD.

Question 39

What evidence using the S layer is there that show bacteria are better at molecular biology?

Answer 39

There are two resistant mutants with no S layer found with bacteria (not been engineered by humans). 1 bp insertion or a substitution -> both introduce a premature stop codon. Can easily repair the mutation to restore the S layer – revertants. The revertants are watermarked to easily distinguish.

Question 40

What is the effect of Lysozyme and LL-37 on bacterial mutants with no S layer?

Answer 40

Mutants are sensitive to two immune effectors – lysozyme and LL-37. LL-37 is a human cathelicidin antimicrobial peptide produced by nearly every cell in the GI tract. With the S layer, growth can occur as normal in the presence of lysozyme and LL-37.

Question 41

How does the S layer prevent lysozyme and LL-37 action?

Answer 41

Lysozyme is 14 kDa – so about 2 nm Pores in S-layer might be small enough to exclude lysozyme by acting as a physical barrier for anything above 10kDa. LL-37 is tiny so don’t know how the S layer prevents LL-37 activation. Current hypothesis is LL-37 preferentially binds to binding sites on outside of the S layer and so it doesn’t enter. The mechanism of LL-37 is still unclear.

Question 42

What was the role of S layer mutants in mice?

Answer 42

S-layer mutant avirulent in hamsters but colonises normally. However, exact role in virulence confounded by toxin production defect.

A mutant lacking the outermost LMW domain of LMW still forms an S-layer but causes significantly less severe disease in mice. It colonises similarly and produces toxin to normal levels.

Shows a wild type S layer is required for full disease severity.