C. difficile Flashcards

Question

When is faecal transplant used in UK? Risks?

Answer 1

Used as an emergency therapeutic in UK hospitals Potential risks such as pathogens which we haven’t identified before and cant screen for - Some cases where faecal transplant recipient have become severely ill and also died due to a pathogen being introduced

Answer 2

In future, this crude treatment will be replaced with defined microbiome transplantation - Mixture of bacterial species that have been characterised and grown in the lab can be introduced as a treatment

Answer 3

Resist antibiotics, harsh chemicals, UV radiation, desiccation etc. Immune to the effects of oxygen - Can persist for long periods of time in an oxygenated environment C. difficile ability to form spores is critical to its ability as a pathogen; Needs it to transmit from one patient to the next

Answer 4

Multi-layered structure Highly cross-linked protein coats (cysteine rich for di-sulphide bonding); Multiple concentric layers of 2D crystals of protein Thick cortex PG layer; Modification where every 2nd NAM/MurNAc converted to MAL residue - Only every 2nd disaccharide unit has a peptide stem; PG is less cross-linked than normal Original cell wall; Distinct from modified cortex PG – Specific PG hydrolases produced that only digest cortex, not original cell wall - When germinating, they remove the cortex but leave the cell wall 2 membranes; Unusual for gram-positive Core: - Dehydrated; Chemical reactions don’t really happen - 25% dipicolinic acid (Ca-DPA) creates dehydrated conditions - DNA bound and protected by small acid soluble proteins (CASPs)

Answer 5

Standard vegetative growth; Cell increases in length and forms a septum in the centre of the cell when it reaches an appropriate length Synthesises PG at the septum and the 2 cells divide

Answer 6

Process is largely governed by availability of nutrients Also initiated randomly, even under ideal growth conditions - Random sporulation at low levels is likely a insurance mechanism, in case of a sudden exposure to oxygen, killing many of the cells - Genetic advantage ensures some of the population survives as spores

Answer 7

Asymmetric septum is formed toward one pole of the cell; Forms the mother cell (larger) and the forespore Mother cell engulfs forespore compartment and it is invaginated to form 2nd membrane around spore Spore matures in the cell, building various layers Once spore is fully developed, the mother cell lyses and release the spore - This is cell differentiation

Answer 8

Spo0A is the master regulator - Transcription factor Phosphorylation of Spo0A and the subsequent gene regulation is what commits a cell to sporulation pathway

Answer 9

Differ in the orphan kinases upstream of Spo0A that regulate its phosphorylation - B. subtilis; 5 histidine kinases that phosphorylate Spo0A and trigger sporulation - C. difficile; No homologues of histidine kinases; Several regulators that feed into Spo0A expression – Don’t know what happens to phosphorylate Spo0A

Answer 10

Formation of asymmetric septum Regulates activation of SigF in the forespore and simultaneous expression of SigE in the mother cell - Followed by SigG expression in the forespore; Responsible for expression of 2nd wave of genes in the forespore - Also followed by SigK expression; Leads to final set of genes expressed in mother cell

Answer 11

Sigma factors in each compartment required for processes in the other - SigF is required for activation of SigE - SigE is required for activation of SigG - SigG is required for activation of SigK

Answer 12

2 sigma factors which sequentially activate in the forespore, and also in the mother cell Each of these has a regulon of genes that must be activated in the right sequence to create the spore in the right order; Build layers of protection in correct order 4 sigma factors are conserved However, the sequential control is probably different and doesn’t seem to be as tightly regulated

Answer 13

Very similar phenotypes to what wesee in B. subtilis ∆SigE – Asymmetric septation but no spore formation ∆SigF – Beginnings of forespore engulfment, but no spore layer assembly ∆SigG – Coat layers built but no cortex is generated; Pore misshapen ∆SigK – Cortex and layers undeath are built correctly, but spore lacks protein coats; Viable but less resilient

Answer 14

Spore is dehydrated so it has a different index to the microscopic media around it; Its ‘phase bright’ FM4-64 fluorescently stains membranes - Can see asymmetric septum - Can see membrane begin to curve as it’s engulfed DAPI stains DNA - Very bright in forespore as its DNA is very compacted

Answer 15

Combining cholic acid and taurine; This bile salt is the most common one in the intestine

Answer 16

Primary - Made by liver and secreted Secondary - Bacterial metabolism of primary bile salts

Answer 17

Taurocholate Glycine Cholate

Answer 18

Chenodeoxycholate

Answer 19

Stimulates C. difficile germination, but directly kills C. difficile vegetative cells

Answer 20

Balance of these enzymatic reactions is altered Changes the pool of available secondary bile salts as the species diversity that digests the various molecules has been altered; Alters colonisation protection provided by the microbiome

Answer 21

Without spo0A, mutants wouldn’t sporulate Spores not present in ∆spo0A, but vegetative cells are By complementing spo0A back in, spores are again seen

Answer 22

Sporulation defective C. difficile can infect, but once cleared with vancomycin it cannot relapse In the WT, infection is cleared with vancomycin, but once treatment is stopped, the infection immediately reappears

Answer 23

Recipient (treated with antibiotics) and donor (infected with C. difficile) mice were caged in 4 different arrangements - Some able to contact, and some blocked by different numbers of barriers - Single – Can still groom each other; Double – Can’t touch but airflow between Transmission is very high in strains that can form spores Transmission is massively reduced in strains which cannot form spores

Answer 24

Golden Syrian hamsters C57BL/6 Mice

Answer 25

Acute phase of C. difficile infection where toxins are a key virulence determinant Hamster model isn't very good - Doesn’t accurately reflect disease we see in humans; Far more severe in hamsters - Severe disease means many ethical complications - Was used for many years as it was the only robust model and method available Does mimic human infection to a certain extent as both require disturbed microbiome

Answer 26

- Cheaper - Wide range of available reagents for assays and research - Access to knock-out mutant mouse lines These mice don't get very sick (e.g. no diarrhoea) Do become colonised with C. difficile the way we see in humans

Answer 27

High number of spores being shed in the faeces, referred to as a super-shedder state After pausing of antibiotic treatment the number of C. difficile spores in the faeces decreases - Upon subsequent dose of clindamycin, animals return to the super-shedder state – C. difficile is retriggered

Answer 28

Small number of spores left in intestines after they resolve infection - Over time, particularly with antibiotic treatment, these spores can germinate and induce further infections Vary the intensity of the infection; Use cocktails of 6-7 antibiotics and the infection can become lethal - Can exploit this to model different aspects of human disease

Answer 29

TcdA (Toxin A) TcdB (Toxin B) Most strains produce both; Some weird linages that only produce toxin B

Answer 30

Single polypeptide chains A - 308 kDa B - 270 kDa (members of Large Clostridial Cytotoxin Family) Enzymatic domain that does the damage; At N-Terminus Large translocation domain in the middle Receptor binding domain of peptide repeats; At C-Terminus

Answer 31

TcdD (a.k.a TcdR) TcdB TcdE TcdA TcdC

Answer 32

Encodes alternate RNA polymerase sigma factor; Can direct transcription by RNAP to a different set of promoters – This directs it to TcdD itself; Positive feedback loop Required for expression of tcdB and tcdA (and tcdD)

Answer 33

Exact function is disputed; May be involved in release of toxins from bacterial cell Resembles a holin – Phage protein that are cytolytic for bacteria (makes pore in membrane); Co-opted from phage to allow release of large toxins, but controlled so it doesn’t kill the cell

Answer 34

Putative negative regulator of toxin expression Membrane localised – Anti-sigma factor, binding the positive regulator of TcdD 18 bp deletions found in hypervirulent ribotype 027 strains; Greater toxin secretion Only one encoded on opposite strand

Answer 35

When vegetative C. difficile is present in dysbiotic lumen Bind receptors on epithelial cells lining GI tract - TcdA enters cell via apical surface (lumen exposed) - TcdB enters basolaterally (bottom of cell)

Answer 36

TcdA causes initial damage that opens gaps in epithelial layer, allowing TcdB to get into deeper tissue and intoxicate cells

Answer 37

Both inactivate Rho GTPases - Cytoskeleton changes; Cells lose structure - Disruption of tight junctions; More toxins access deeper tissues - Production of inflammatory mediators is induced - Accumulation of neutrophils in the tissues - Some neutrophils enter epithelium and cause inflammation (pseudo-membranes)

Answer 38

Begins a vicious cycle; Production of more pro-inflammatory cytokines, greater inflammation of the site of infection If unchecked this can lead to generalised inflammation of the intestine (e.g. toxic megacolon)

Answer 39

“Combined Repetitive Oligopeptides” (CROPs) Made from 2 different protein repeat sequences which alternate - Short (SR, 18-24 aa) and long (LR, 31 aa) repeats TcdA has 32 SRs and 7 LRs, TcdB has 18 SRs and 4 LRs; The difference in number of repeats is what causes most of the difference in length between toxins (TcdA slightly bigger) Antibodies to this domain inhibit binding of toxin to cells and inhibit uptake Sequences of TcdA and TcdB RBDs are related, but distinct Likely have different receptors

Answer 40

Very large (1300 aa) Contains putative membrane spanning domain; Hydrophobic looking regions in protein sequence Hydrophobic regions may form a pore or channel in vesicle to allow toxin to enter cytosol - Never seen this pore and don't know how it would form it

Answer 41

Toxin catalytic domains very similar; Likely do the exact same thing Glucosylate (transfer a glucose moiety) small-MW GTPases including Rho, Rac, Cdc42; Inactivates them Cause cell rounding due to collapse of actin cytoskeleton

Answer 42

Binds receptors on epithelial cells via the CROPs domain Induces uptake into vesicle via clathrin-mediated endocytosis Vesicular ATPase pumps proton into lumen of vesicle; Attempt to kill potential pathogen from cell TcdA and TcdB are conformationally changed by this acidification; This low pH structure results in insertion of the translocation domain into the vesicle membrane - Enzymatic domain (red) passes through this pore and is released into the cytoplasm

Answer 43

Cleavage shown to occur primarily in the cytosol fraction - At neutral pH - i.e. not within the endosome Processing requires Ins6P; Abundant in eukaryotic cells, but not found in bacteria - Only activated once in a host cell

Answer 44

Cysteine protease activity was identified In absence of InsP6, there is a single band for full toxin; No breakdown By increasing InsP6 concentration, there is increasing degradation of full length protein into different domains

Answer 45

When given to hamsters, Toxin A reproduced pathology of infection Toxin B not toxic to animals unless co-administered with toxin A (one study only) A-ve, B+ve strains (017 strains) causes severe disease in humans - Potentially have other virulence factors? To resolve this, isogenic mutant strains are created and tested in animal models - Ability to take individual toxins away to see how this affects infection

Answer 46

2009 - Mutant strains that can't make TcdB were not virulent - Mutant strains that can't make TcdA were fully virulent - Toxin B more important 2010 - Concluded that both toxins were required for virulence - TcdB mutation had greater reduction in virulence compared to TcdA mutation

Answer 47

Parental strains derived independently; Genetic differences between bacterial strains (2009) Used suicide plasmids; Insert entire plasmid sequence into target genome – Not a great way of making mutants as they aren’t stable - Plasmid can get kicked out and you get mixed phenotypes (2010) used new genetic technique to make more stable mutants; Used a type II integron which inserts into target genes and is much more stable and reliable Different hamsters; Same breed but different sources so probably different microbiota; Differences in microbiome and C. difficile interactions could account for the differences we see Different end-points; Experiments in US are allowed to go to a natural conclusion (succumb to infection) whereas in UK hamster temperature is monitored and they are euthanised when their internal body temperature drops (this indicates they are succumbing to infection) - Possible that in UK model, maybe some would actually survive if you allowed infection to run its course (not ethical)

Answer 48

Overall it looks like toxin B is slightly more important than toxin A We can’t say for sure

Answer 49

S (surface) layer 2D para-crystalline (not perfect crystal) protein array surrounding the cell ≈600,000 protein subunits per C. difficile cell; All the same protein Very expensive structure for cell to make; Large proportion of energy required

Answer 50

SlpA (s-layer protein A); Decorated with up to 28 additional CWPs (cell wall proteins) Between species, S-layer proteins are not closely related First cleavage removes N-terminal signal peptide that signals secretion of the protein from the cytoplasm to outside of cell Second cleavage breaks protein into 2 halves - Low molecular weight (LMW) - High molecular weight (HMW)

Answer 51

High affinity heterodimer complex that forms basic subunit of S-layer Complex self-assembles on surface, with HMW anchored to PS-II (cell wall sugar Post-secretion cleavage by Cwp84 (cysteine protease)

Answer 52

Dedicated ATPase, SecA2; Present in all bacteria Made from a membrane complex of 3 proteins (SecYEG) and a motor ATPase called SecA (provides energy for secretion across membrane) C. difficile has 2 copies - SecA1; Housekeeping set that is responsible for majority of protein secretion - SecA2; Dedicated for secretion of S-layer proteins

Answer 53

29 members with SlpA being the most common SlpA makes up bulk of lattice that holds the whole thing together These other proteins can be substituted in at different points to add functionality to the cell surface

Answer 54

All have 3 copies of Pfam04122 domain in HMW Cell wall binding domain; These 3 domains interact with PS-II in cell wall to anchor complex on outside of the cell

Answer 55

Used to construct 3D model of protein subunit structure Shows that Slp subunits come together and form small pores

Answer 56

Research hampered by inability to make an S-layer mutant Probably isn’t a conserved function that they all serve 10-20% of all cellular energy goes towards making it Tn-Seq of C. difficile gave no transposon insertions found in SlpA or SecA2; These genes are essential

Answer 57

Bacteriophage that infects C. difficile Bacteriophage could be a potential therapeutic to kill this bacteria

Answer 58

New protein nanomachine therapeutic that targets S-Layer and kills C. difficile Binds target cell and sheath contracts and shortens, driving needle through the cell envelope, collapsing pmf and killing cell - Use phage targeting systems to recognise C. difficile

Answer 59

Avidocins don’t have a capsid with phage DNA Difficin produced by C. difficile to kill other competing C. difficile strains - Narrow killing spectrum Difficin engineered to improve stability and widen spectrum --> Avidocin-CD - Swap receptor binding proteins on ends of legs for equivalent of receptor binding proteins from bacteriophage that identify S-layer

Answer 60

Used Avidocins that we knew targeted C. difficile S-layer and repeatedly exposed large C. difficile populations to large amounts of these Avidocins With repeated regrowth and re-exposure, eventually resistant C. difficile strain developed through evolution

Answer 61

No S-layer for Avidocin to target Gel shows that FM2.5 and FM2.6 strain doesn’t have either LMW or HMW Slp Can easily repair/complement the mutation to restore the S-layer; “Revertants”/RW strains

Answer 62

1 bp insertion or a substitution; Both cause a frame-shift and introduce a premature stop codon – Truncated protein “Water marked” the revertants; Introduce synonymous mutation that we can see when we sequence, but doesn’t change protein coding

Answer 63

Lysozyme and LL-37 LL-37 is a human cathelicidin antimicrobial peptide produced by nearly every cell in the GI tract These insert into bacterial membrane and form pores

Answer 64

If treated with a concentration of lysozyme they would encounter in the human body, C. difficile continues to grow; Completely resistant S-layer mutant treated with lysozyme slowly starts to die and slowly recovers Complemented strain has restored resistance to lysozyme

Answer 65

WT and complemented is same as in lysozyme Mutant strain is completely killed; Very sensitive to this peptide

Answer 66

Pores in S-layer might be small enough to exclude lysozyme and molecules of this size of bigger However, LL-37 is tiny; Still don’t understand the mechanism of how the S-layer protects against this peptide – Not size exclusion We think the surface of the S-layer has evolved to be quite ‘sticky’ for small peptides like LL-37; Peptide gets stuck on surface of S-layer

Answer 67

Hamsters infected with WT die within days S-layer mutant avirulent in hamsters; None died and didn’t get sick at all Complemented mutants restored lethality Colonisation is still normal in mutant strains; S-layer not required for colonisation Hamsters faeces barely contained toxins from C. difficile, despite being heavily colonised

Answer 68

Developed mutant with a single deletion in LMW domain; LMWΔD2 Mutant lacking the outermost domain of LMW still forms an S-layer It causes significantly less severe disease in mice Mice don’t die, but get diarrhoea; Mice actually lose weight, then recover microbiome and regain weight If infected with mutant strain, they still lose weight, but it is a lot less severe when compared to WT C. difficile

Answer 69

Colonises similarly and produces toxin to normal levels WT S-layer is required for full disease severity; Not just toxins

C. difficile Flashcards

(93 cards)