L10. C. Diff Sporulation & Transmission

Question 1

What is the biggest risk factor for C. difficile infection?

Answer 1

Age is the biggest risk factor, with infections increasing from 60 onwards.

Question 2

Are C. difficile infections increasing only in older populations?

Answer 2

No, there are also increasing cases in younger populations and pregnant women.

Question 3

How are nosocomial C. difficile infections typically transferred?

Answer 3

They are hospital-transferred infections, but patients are often already colonized with strains that are not linked to the hospital; then antibiotics administered at hospital trigger the infection.

Question 4

What can mild diarrhoea caused by C. difficile lead to?

Answer 4

It can lead to potentially fatal inflammatory complications such as pseudomembranous colitis (PMC) and toxic megacolon (TMC).

Question 5

What type of anaerobe is C. diff and what does this mean?

Answer 5

C. difficile is an obligate anaerobe, meaning oxygen is toxic to it.

Question 6

What is a significant characteristic of C. difficile related to its survival and transmission?

Answer 6

C. difficile is a spore-former.

Question 7

Are all Clostridia species spore formers?

Answer 7

Yes, all Clostridia are spore-forming rods.

Question 8

Why is sporulation important for C. difficile?

Answer 8

Sporulation is needed for transfer through an oxygen environment.

Question 9

What is classed as the infectious form of C. diff and why?

Answer 9

Spores are the infectious form of C. diff (spore), as vegetive cell form would die to atmospheric oxygen

Question 10

Name 7 examples of what C. diff spores are resistant to

Answer 10

1. Oxygen
2. Bleach
3. UV
4. Gamma radiation
5. High temps
6. Alcohol (hand gels)
7. High salts

Question 11

What aspect of C. diff makes it persist in health care facilities?

Answer 11

As spores stay for months to years without very high temp and disinfection.

Question 12

Describe 6 ways the C. diff spore is adapted for survival in harsh environments

Answer 12

1. Highly cross-linked protein coats
   >Highly cross-linked protein coats are on the outside, forming self-assembling spores that create crystals and are further chemically cross-linked, requiring combinations of DTT, high temperature, urea, and detergent to be removed.
2. Cortex Peptidoglycan
   >The cortex peptidoglycan is very thick and chemically modified, consisting of alternating sugars with peptide stems that crosslink. This is more flexible than normal peptidoglycan, so can withstand mor mechanical pressure.
3. Two membranes
   >C. difficile spores have two membranes, despite being gram-positive, creating more protection.
4. Core:
   a. Dipocolinic acid (Ca-DPA) makes up 25% of the spore core, dehydrating it and making it enzymatically inert.
   b. It also reduces the water potential in the core, making the structures less susceptible to enzymatic or chemical damage, as these reactions often require water as a substrate.
   c. DNA is tightly packaged by small acid-soluble proteins, protecting it from UV damage.

Question 13

How can a C. diff spore differentiate between traditional peptidoglycan and cortex peptidoglycan when geminating?

Answer 13

When the cell comes to germinate, lytic hydrolase enzymes (which break down peptidoglycan) which specifically recognise this structure; so can break down cortex peptidoglycan without breaking down traditional peptidoglycan wall.

Question 14

What is the difference between cortex peptidoglycan and normal peptidoglycan, and what are the effects of this?

Answer 14

The basic repeating unit consists of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) with every second NAM modified to muramic δ-lactam (MAL). This new second beta-lactam ring (MAL) makes the peptidoglycan more flexible as a cross link cannot occur at the MAL subunit so a cross link occurs at every NAM (1 in 4 residues) compared to the more rigid cross link at every 1 in 2 residues found in normal peptidoglycan.

Question 15

What is an overview of what happens to C. diff in a) good environment b) stressful environment?

Answer 15

a) When environment is good, undergoes binary fission, cell elongates, septum is cnetred in the middle directly splitting into two cells (during infection)

b) If environment is stressful (not fully know for C. diff conditions), enter sporulation differentiation instead.

Question 16

Is Sporulation asymmetric division?

Answer 16

Not accurate to say its asymmetric cell division as gives idea that a single cell will give rise to two spore; it is cell differentiation as gives out 1 spore

Question 17

Describe a non-detailed overview of sporulation in 5 steps

Answer 17

1. Septum is placed using similar protein factors as cell division, but is closer to one pole, producing a larger mother cell compartment and a forespore
2. Mother cell engulfs forespore (like phagocytosis), membrane of mother cell stretched around, membrane on outside is mother cell, membrane on inside is spore fore
3. Between these two membranes, cortex peptidoglycan is synthesised
4. Around outside of membrane protein coat is lay down.
5. When spore is completed, mother spore lyses itself releasing spore release, until senses favourable environmental factor where it germinates and re-enters growth cycle.

Question 18

Is there much homology between how C. diff and B. subtilis (both gram +) sporulate?

Answer 18

Little homology in spore proteins between C. difficile and Bacilli/Clostridia

Question 19

Why do we have limited insights into how C. diff sporulate/ germinate?

Answer 19

Limited genetic tools so Regulation of sporulation/germination not fully understood

Question 20

How many genes do we know cause sporulation in C. diff and how do we know this?

Answer 20

798 genes, when disrupted causes no sporulation in C. diff

Question 21

What is the most important factor mediating sporulation and why?

Answer 21

> As we progress for sporulation, there are sequential activation of sigma factors which control the expression of all these genes

> The sequential activation of genes is important to maintain the correct order of events, ensuring that processes like cortex formation occur after engulfment.

Question 22

What controls the sequential activation of genes during the sporulation cascade in C. difficile?

Answer 22

The sequential activation is controlled by a sigma factor cascade, active in both the forespore and mother cell.

Question 23

Give an overview of how sigma factors regulate gene expression during sporulation in C. difficile?

Answer 23

Sigma factors control a subset of genes, and by controlling the order of their activation, they regulate the order of regulon expression and the sequence of events necessary for sporulation.

Question 24

What the first Spo gene and its significance in the sporulation process?

Answer 24

Spo0A is an example of a Spo gene and it acts as the master regulator, active at stage 0.

Question 25

What are the stages of sporulation in C. difficile?

Answer 25

Stage 0: Vegetative cell
Stage I: Onset
Stage II: Commitment, Asymmetric cell division
Stage III: Engulfment
Stage IV: Cortex formation
Stage V: Maturation, spore coat
Mother cell lysis: spore released

Question 26

In B. subtilis sporulation, which sigma factors are expressed in the forespore and mother cell, and what regulates them?

Answer 26

Sigma F is expressed in the forespore and Pro-Sigma E in the mother cell, both regulated by Spo0A.

Question 27

What is the sequence of sigma factor activation in B. subtilis sporulation?

Answer 27

Forespore Sigma F is needed for Sigma E, which is needed for Sigma G expression, which is needed for Sigma K.

Question 28

How many stages of gene expression are there in B. subtilis sporulation and what do they reflect?

Answer 28

There are four stages of gene expression reflecting the stages of spore development.

Question 29

How does C. difficile sporulation differ from B. subtilis in terms of Spo0A phosphorylation?

Answer 29

In C. difficile, Spo0A is phosphorylated, but it is not known in response to what.

Question 30

What does Spo0A lead to in C. difficile sporulation?

Answer 30

Spo0A leads to the expression of Sigma F in the forespore and proSigma E in the mother cell.

Question 31

How does Sigma F function differently in C. difficile compared to Bacillus?

Answer 31

Sigma F is not involved in the maturation of proSigma E to Sigma E in the mother cell, nor is it required for Sigma G expression in the forespore, leading to uncertainties in the timing of Sigma F and G.

Question 32

How does Sigma E function differently in C. difficile compared to Bacillus?

Answer 32

Sigma E in C. difficile is involved in the expression of Sigma K and can sometimes directly activate it without expressing proSigma K first, with minimal cross-talk between Sigma E and Sigma G.

Question 33

What does complex sporulation in C. difficile indicate about its evolutionary process?

Answer 33

It reflects a more primitive process compared to bacteria like Bacillus, but C. difficile has evolved for 2.2 billion years, becoming efficient at sporulation, the complexity reflects how crucial it is for its survival as there are more layers of feedback to fix errors.

Question 34

What happens when sigma factors are knocked out in C. difficile sporulation, give examples of specific Sigma factors?

Answer 34

> Knock out sigma factors, cascade gets to certain point then no further:

1. Sigma F knockout: Causes asymmetric septum forming, stopping sporulation progress.
2. Sigma E knockout: Results in two asymmetric septa, indicating changes in mother cell behavior.
3. Sigma K knockout: Leads to spores with two membranes and a cortex but no protein coat.
4. Sigma G knockout: Produces spores that do not mature properly due to lack of peptidoglycan, resulting in misshaped spores that do not survive well.

(Spores may not even form, and if they do they don’t survive well).

Question 35

What advantage does phase contrast microscopy provide in observing spores?

Answer 35

Phase contrast microscopy gives boosted contrast between similar refractive indices, making spores phase bright as they are dehydrated, transitioning a phase dark cell to bright.

Question 36

Why is the septum more brightly labeled than the outside of the cell in phase contrast microscopy?

Answer 36

The septum is more brightly labeled because there are two membranes present as the forespore and mother spore both pinch in.

Question 37

What makes the spore appear very bright in phase contrast microscopy?

Answer 37

The spore appears very bright because, when the mother cell engulfs the forespore, the spore has a double membrane.

Question 38

What is the role of DAPI staining in observing spores?

Answer 38

DAPI stains DNA, causing the forespore to have more fluorescence as all DNA is squeezed into a small space compared to the mother cell.

Question 39

What happens to C. difficile spores upon sensing a germinant within 90 minutes?

Answer 39

They produce a vegetative cell, transitioning from phase bright spores to phase dark cells through rehydration and outgrowth, forming new rod-like cells.

Question 40

What is the initial appearance of C. difficile spores before germination?

Answer 40

Phase bright spores.

Question 41

What triggers the transition of C. difficile spores to vegetative cells?

Answer 41

Germinants, which are chemical signals in the environment, usually nutrients, tell the spore to germinate.

Question 42

What is the primary germinant for C. difficile and what unusual amino acid does it contain?

Answer 42

The primary germinant is taurocholate, which contains the unusual amino acid taurine (found in Red Bull).

Question 43

Why is the correct environment crucial for the germination of C. difficile spores?

Answer 43

If germination occurs in the wrong place, the vegitive cells will die

Question 44

What is taurocholate and where is it synthesized?

Answer 44

Taurocholate is a primary bile acid synthesized in the liver.

Question 45

Where are primary bile salts stored and released, and what is their function?

Answer 45

Primary bile salts are stored in the gall bladder and released into the duodenum when eating. They solubilize and emulsify fats, which is critical for the uptake of fats and fat-soluble vitamins.

Question 46

How are secondary bile salts produced and where are they found?

Answer 46

Secondary bile salts are produced via bacterial metabolism in the colon as degradation products of primary bile salts in the intestine. Primary bile salts decrease in concentration as they move down the intestine.

Question 47

What are the two classes of primary bile acids produced by the liver and their main types?

Answer 47

The liver produces glycine/taurine conjugated bile acids. The four main types are glycocholate, glycochenodeoxycholate, taurocholate, and taurochenodeoxycholate.

Question 48

How are bile acids acted upon by microbiota in the intestine?

Answer 48

> Microbiota strip off amino acids (glycine or taurine), from primary bile acids, leaving cholate and chenodeoxycholate (secondary bile acids).

> These secondary bile acids can be further chemically modified by the enzyme 7-dehydroxylase to deoxycholate and lithocholate.

Question 49

Where are different bile acids found in the colon?

Answer 49

1. At the top: glycocholate, glycochenodeoxycholate, taurocholate, and taurochenodeoxycholate (primary bile acids).
2. In the middle: cholate and chenodeoxycholate (secondary bile acids).
3. At the end: deoxycholate and lithocholate (modified secondary bile acids).

Question 50

Why is taurocholate a primary germinant for C. difficile and where does it encounter it?

Answer 50

Taurocholate is encountered in the small intestine of large mammals, signaling a good place for germination past stomach acid, with abundant food, 37 degrees Celsius, and no oxygen.

Question 51

As well as the primary Germinant “Taurocholate”, what other products in the bile salt process impact C. difficile sporulation/germination?

Answer 51

> High concentrations of glycine (also found in the small intestine) act as a germinant.

> Cholate and deoxycholate can also induce germination, but less effectively.

> Some secondary bile acids, like chenodeoxycholate, inhibit germination and can block it when other germinants are present.

> Deoxycholate induces germination but can inhibit the growth of vegetative C. difficile cells.

Question 52

How does dysbiosis of the microbiome affect C. difficile infection and how does antibiotic treatment effect this?

Answer 52

> A healthy gut microbiome, high conc of Chenodeoxycholate as well as Deoxycholate in the small intestine as primary bile acids get processed very quickly, which inhibits germination (chemodeoxycholate does this) and if any cells do germinate the Deoxycholate inhibits growth of vegetive cells

> Antibiotics cause dysbiosis so this process happens more slowly, so Taurocholate hangs around for longer, and some is degradation occurs to Glycine still; both germinants for C. diff germination.

Question 53

How is the modulation of bile acids in the gut related to C. difficile infection in mice models?

Answer 53

Modulating bile acids in the gut can influence the outcome of C. difficile infection in mice, confirming the role of bile acids in infection dynamics.

Question 54

What agent is identified as responsible for the transmission of C. difficile?

Answer 54

The spore is identified as the agent of transmission.

Question 55

What experiment was used by Deakin et al (2012) to test that the spore is the agent of transmission for C. diff?

Answer 55

> The spore is the agent of transmission, shown by experiment with recipient (not infected but treated with vancomycin) and donor (infected with C. diff) mice having varying contacts with each other:

1. Mingling, 2 mice shared cage (eat each other’s faeces e.g.)
2. Contact Permeable barrier in between but can’t eat faeces (can groom)
3. Double barrier, with Airborne contact but not physical
4. Environmental contamination, cage which did contain donor mouse previously

Question 56

In the experiment with mingling mice (sharing a cage and eating each other’s feces), what was the transmission rate for a) wild-type (WT) b) spo0A gene knock out C. difficile strains and what did this show?

Answer 56

a) WT C. difficile strains transmitted 100% of the time.

b) The infection rate was significantly lower, with only 20% getting infected.

> Showed that spore transmission was important for the majority of transmission, and vegetative cells only from eating faeces which human’s don’t do.

Question 57

In the experiment with a permeable barrier (preventing coprophagy but allowing grooming), what was the transmission rate when spo0A was knocked out and what does this show?

Answer 57

> There was no transmission when spo0A was knocked out

> Which shows it is the spores needed as without eating faeces the 20% of infections seen by vegetive cell transmission has disappeared.

Question 58

What does the experiment with a double barrier (allowing airborne contact but preventing physical contact) indicate about C. difficile spores?

Answer 58

Infection occurred through airborne contact, but it stopped when spo0A was knocked out, indicating that spores can transmit airborne.

Question 59

What was observed in the environmental contamination experiment with cages that previously contained donor mice and what does this show?

Answer 59

> Without spo0A, C. difficile could not infect recipient mice through environmental contamination.

> Showing that spores are necessary for infection from surface contamination.

Question 60

What does the spo0A gene regulate in C. difficile in addition to spore formation? (Extra reading maybe, form Deakin et al (2012))

Answer 60

The spo0A gene may also negatively regulate toxin production and other functions related to persistence and transmission.

Question 61

How did the levels of the toxins TcdA and TcdB compare in spo0A mutants versus wild-type C. difficile strains? (Extra reading maybe, form Deakin et al (2012))

Answer 61

spo0A mutants produced elevated levels of the toxins TcdA and TcdB in vitro compared to wild-type parental strains (shows negative correlation between Spo0A and toxin production, as don’t need toxins when in spore).

Question 62

What is the role of Spo0A in C. difficile’s regulatory cascade?

Answer 62

Spo0A is at the top of the regulatory cascade, and spo0A mutants do not sporulate.

Question 63

What is observed in C. difficile cells when Spo0A is knocked out?

Answer 63

Spo0A knockout results in no spores (no red cells), but when the gene is reintroduced, spores return. This occurs across different strains.

Question 64

How is the persistence of C. difficile in the gut demonstrated using a relapse model with antibiotics?

Answer 64

Mice infected with C. difficile and treated with vancomycin experience a relapse of infection once the antibiotic treatment stops, indicating that spores are required for persistence.

Question 65

What is observed in the relapse model experiment with C. difficile and vancomycin treatment?

Answer 65

Mice infected with C. difficile and treated with vancomycin experience recurrent infection after stopping the treatment, repeatedly.

Question 66

What happens to the relapse infection in the relapse model when Spo0A is knocked out and what does this show?

Answer 66

When Spo0A is knocked out, the relapse infection does not occur after initial infection and vancomycin treatment, indicating that spores are crucial for re-infection.