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Bacterial Pathogenicity > C. difficile > Flashcards

C. difficile Flashcards

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1
Q

What is the prerequisite for C.diff infection

A

Dysbiosis
- disruption of the healthy normal microbiota

Often caused by antibiotics

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2
Q

Koch’s Postulates for microbiota studies

A

Identify all species in both healthy and diseased individuals

Isolate, purify and culture relevant bacterial species

Intervene and ameliorate

Detect and reisolate those species

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3
Q

What type of bacteria is C.difficile?

A

gram-positive (monoderm)

strict anaerobe

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4
Q

C.difficile niche

A

large intestine of mammals
particularly humans

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5
Q

Name for the spectrum of human diseases caused by C.difficile

A

C.diff associated diseases (CDAD)

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6
Q

CDADs

A

Diarrhoea

Pseudomembranous colitis

Toxic megacolon

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7
Q

Common symptom in C.difficile infection

A

Colitis
- inflammation of colon

Accumulation of neutrophils in tissue

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8
Q

Diarrhoea

A

simplest form of C.diff infection

majority of cases are self-limiting

recurring in small number of patients

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9
Q

Pseudomembranous colitis

A

neutrophils penetrate epithelium causing neutrophil filled blisters
on epithelium lining of colon

if untreated can lead to perforation of large bowel, causing sepsis

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10
Q

Treatment for pseudomembranous colitis

A

Treatment involves surgical removal of infected part of bowel

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11
Q

Toxic megacolon

A

Neutrophil accumulation in tissue releases more pro-inflammatory cytokines
- vicious cycle
More dangerous; less common
Inflammation of entire large bowel

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12
Q

What percentage of CDAD deaths are associated with inflammatory complications?

A

90%

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13
Q

What is the leading cause of hospital inquired infection?

A

C.difficile

MRSA affects only 1/5 of amount of patients that C.diff does

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14
Q

C.diff disease progression

A

Patient received antibiotics

Alteration in normal gut flora (dysbiosis)

Infection with C.diff spores

Spore germinate in gut forming vegetative cells

Cells multiply, produce toxins and sporulate

Spores excereted

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15
Q

Affect of antibiotics on microbiota

A

Changes species that make up biomass but not biomass itself

Kill susceptible bacteria
- their place in ecosystem will be replaced by other bacteria
- proliferate to fill same ecological niche

Left with more resistant species and strains

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16
Q

C.diff introduction to microbiome

A

Spores introduced via faecal-oral route

If introduced at the right time during dysbiosis it can proliferate and thrive

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17
Q

North American C.diff epidemic (2003)

A

Caused by two separate branches of 027 strain
Hypervirulent strain that is resistant to fluoroquinolone

Appeared in Philadelphia and Quebec

In 2004, epidemic strains appeared in UK

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18
Q

078 strain

A

In 2008, appeared in hospitals
Hypervirulent strain

Previously associated with animal infection

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19
Q

History of C.diff strains

A

(2001) 001 strain was dominant in UK
(2004/2005) outbreaks attributable to 027 strains
(2007) >50% infection in UK due to 027 strains

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20
Q

Measures put in place by NHS for control 027 outbreaks

A

Reintroduction of soap and water on wards in UK
- spores immune to killing by alcohol

Banned jewellery, watches and ties

Deep cleaning of beds, hospital wards

Isolation of rooms after cases for 24 hrs
- bleach every surface

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21
Q

What does the large diversity of C.diff ribotypes ciruclating suggest?

A

Source of infection if not transmission within hospitals

If C.diff transmission was primarily confined to hospitals you would expect to see a limited number of dominant ribotypes

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22
Q

Characteristics of Ribotype 027

A

Hypervirulent and fluoroquinolone resistant

Form complex, structured biofilms
Produce more spores
Produces more toxin in vitro
Produces third toxin (CDT)
- binary toxin
Contain 18bp deletion in tcdC (negative regulator of toxin expression)

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23
Q

Two branches of 027 strain in North America outbreak

A

Fluoroquinolone resistance had been acquired independently

Lineage FQR1 and FQR2

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24
Q

Lineage FQR1

A

Largely affecting hospitals in USA and in Korea

Didn’t appear anywhere else
- not as good penetrance as other lineage

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25
Lineage FQR2
Found in North American hospitals - spread to Quebec, Australia and all over Europe Not sure why more successful - very very small genetic differences between lineages
26
Trehalose
Two glucose molecules stuck together - used in ice cream to reduce crystallisation Use of trehalose increased due to enzymatic synthesis
27
FQR2 and Trehalose
FQR2 is able to metabolise trehalose very easily If take away ability to metabolise trehalose it loses virulence - affects Toxin B production
28
How does intact microbiota prevent colonisation by C.difficile?
Microbiome - competition - production of toxic compounds - reduction of benefical compounds by metabolism - production of antimicrobial products (bacteriocins) Host - production of secretory IgA - production of antimicrobial peptides
29
How long does germination take?
approx 90 mins
30
Principle germinant for C.difficile
Taurocholate - bile salt
31
Name C. diff germinant receptor
CspC
32
Faecal microbiota transplantation
Best source of human colonic microbiome Used as emergency therapeutic 94% effective with a single treatment at cleaning C.diff infection
33
Problems with faecal transplantation
Discomformt with idea from patients and doctors Potential risk - can't screen for every pathogen
34
Replacement for faecal transplantation
Defined microbiome transplantation - consortia of bacterial species that have been characterised and grown in lab
35
C.diff spore
Highly infectious metabolically inert forms of cells Resistant to various chemical and physical insults Persistant in any environment
36
C.diff spore structure
Multi-layered: Highly cross-linked protein coats - cysteine-rich proteins - act as single coherent protein in outside of cell Two PG layers - original cell wall - thick cortex PG Two membranes - original membrane - second membrane outside of cortex Core
37
Cortex PG
Unique modification to structure Every second NAM is converted to muramic delta-lactam (MAL) - less cross-linking
38
Significance of modification to cortex PG
Chemically distinct from other cell wall - can be digested without breaking down primordial cell wall bacteria produce specific PG hydrolase
39
C.diff spore core
Highly dehydrated due to high concentration of Ca-DPA Very little water means chemical reactions can't happen (metabolically inert)
40
When does C.diff undergo sporulation?
When environment becomes challenging BUT also stochastically
41
C.diff sporulation
Produces asymmetric septum Mother cell engulfs forespore compartment - membrane envagination from behind forespore compartment Endospore maturation - various layers being layed down concentrically Mother cell lysis
42
Sporulation cascade
SpoA phosphorylated - leads to asymmetric separation Simulataneous activation of SigF in forespore and SigE in mother cell SigF leads to expression of SigG SigE leads to expression of SigK
43
Role of SigG and SigK
Responsible for downstream gene expression in forespore (G) and mother cell (K)
44
Sp0A
Master regulator in both B.subtilis and C.diff When phosphorylated becomes active DNA binding protein Regulates expression of number of genes This phosphorylation event is conserved between B.subtilis and C.diff
45
How is Sp0A phosphorylated in B.subtilis vs C.diff?
In B.subtilis there are five histidine kinases that phosphorylate Sp0A No homologues for these kinases in C.diff
46
Knockout SigE
Asymmetric septation occurs BUT spore doesn't develop any further
47
Knockout SigF
Asymmetric septation occurs Beginning of forespore engulfment BUT don't get assembly of different layers
48
Knockout SigG
Coat formation on outside BUT not cortex synthesis Not viable spore
49
Knockout SigK
get dehydrated core and cortex synthesis BUT lack protein coats - still viable - less resilient
50
Primary bile salts
Synthesised in the liver - stored in gall bladder and released into duodenum when you eat Help solubilise fat - critical for uptake of fats and fat-soluble vitamins i.e. taurocholate
51
Secondary bile salts
Produced by bacterial metabolism of primary bile salts Involves removal of aa (taurine of glycine) Generally detergent molecules i.e. cholate, deoxycholate
52
Activity of primary and secondary bile salts
Primary bile salts induce germination Secondary bile salts can induce or inhibit germination
53
Chenodeoxycholate
inhibitory for germination
54
Deoxycholate
stimulates germination but also directly kills vegetative cells
55
How does dysbiosis affect production of secondary bile salts?
Changes the enzymatic reactions in intestine Changes the proportion of primary to secondary bile salts Changes the species present to digest various molecules Alters colonisation protection
56
How did they prove Sp0A was required for C.diff sporulation?
Took two strains of C.diff stained for cells and spores in WT - revealed presence of both in both strains sp0A knockout shows only vegetative cells - no spores
57
How did they prove spores were required for C.diff infection?
Infected mice with WT and spoA knockout strains In knockout, when treated with vancomycin C.diff goes away In WT, causes recurrent infection C.diff comes back when vancomycin is taken away
58
In vivo models of C.diff infection
Golden Syrian Hamsters C57BL/6 Mice
59
Golden syrian hamsters as model
first model that exhibited same symptoms as humans - diarrhoea and colitis used to study acute phase of C.diff infections where toxins are key virulence determinants not a very good model - much more severe infection in hamsters than humans - ethically poor due to severity
60
C57BL/6 Mice
Most common mouse line used in biomedical research Cheaper Wider availablity of reagents and knockout animals Used to study C.diff colonisation Mice do not get sick but do get colonised in same way human does Resolves over time and C.diff levels drop Antibiotics trigger infection again
61
C.diff toxins
TcdA - Toxin A (308 kDa) TcdB - Toxin B (270 kDa) Hypervirulent lineages produce a third toxin (CDT)
62
B+ A- C.diff strains
Dominant strain in ireland and japan Causes 1-5% cases worldwide
63
Third toxin (CDT)
Produced by hypervirulent lineages - 027 and 078 Binary toxin Consists of binding component (CDTb) and enzymatic domain (CDTa) Induces actin depolymerization
64
Structure of C.diff toxins
Enzymatic domain - responsible for damage Cysteine Protease - cleaves toxin to release enzymatic domain Translocation domain - transfer toxin across membrane Receptor binding domain - combined repetitive oligopeptides (CROPs) - alternating short and long repeats
65
Receptor binding domain of C.diff toxins
TcdA - 32 SRs and 7 LRs TcdB - 18 SRs and 4 LRs Toxin B slightly smaller (270 kDa) SRs and LRs in both toxins are very similar but different enough that they have different receptors
66
Translocation Domain of C.diff toxins
very large (1300 aa) contains putative membrane spanning domain forms pore or channel in vesicles
67
Enzymatic Domain of C.diff toxins
Glucosylate small MW GTPases i.e. Rho, Rac Causes cell rounding due to collapse of actin cytoskeleton and release of pro-inflammatory cytokines
68
Role of C.diff toxins
Inactivates RhoGTPases
69
Consequences of RhoGTPase inactivation
Cytoskeleton changes Disruption of tight junctions between cells - allows toxins and lumen contents into deeper tissue Leads to pro-inflammatory cytokines release Leads to accumulation of neutrophils in tissue
70
C.diff toxin binding and entry to cells
Toxins bind receptors via CROPs domain Whole toxins enter cell via clathrin-mediated endocytosis Toxin A binds receptors on apical surface Toxin B binds receptors on basal-lateral surface
71
What happens to toxins inside vesicle?
Vesicular ATPase pumps H+ into vesicle - acidification to kill vesicle contents Acidification leads to refolding of translocation domain - inserts into vesicular membrane Enzymatic domain passes through pore into cytoplasm - is cleaved off
72
C.diff pathogenicity locus (PaLoc)
tcdA and tcdB in same locus PaLoc contains 5 genes - tcdA - tcdB - tcdD - tcdE - tcdC
73
tcdD
Encodes RNA polymerase sigma factor Can direct transcription to different set of promoters directs expression of itself (+ve feedback loop) Required for expression of TcdA and TcdB
74
tcdE
Encodes a protein that resembles a holin - phage protein that are cytolytic for bacteria - insert into membrane and create a pore Involved in release of toxin from cell BUT does not cause cell lysis somehow
75
tcdC
Anti-sigma factor for TcdD - binds and sequesters TcdD to prevent expression of toxins TcdC is membrane localised Site of 18bp deletion in hypevirulent strains (027)
76
Release of C.diff toxin enzymatic domain
Happens at neutral pH Requires inositol-6-phosphate (abundant in eukaryotic cells) Cleaved by cysteine protease in toxin sequence
77
Are both toxins essential for C.diff pathogenesis?
Knockouts revealed Toxin B more important for virulence BUT this is disputed amongst papers AND there are B+ and A- strains but no known vice versa
78
Why did two studies get different results about importance of C.diff toxins?
Parental strains derived independently - some genetic differences Stability of mutants - more stable and reliable in mutagenesis compared to suicide plasmids Different hamsters - same breed but different location - sourced from different places Different end points - UK vs US
79
S-layer
Surface layer Paracrystalline arrray; 2D crystal protein made up to 600,000 protein subunits per cell
80
Paracrystalline array
Can't perfectly wrap around due to 2D crystal structure Slightly imperfect srystals
81
S-layer is very expensive to make
10% of all cell energy used 10% of all proteins made by cell are in S-layer
82
What is the main consistuent of S-layer?
SlpA (S-layer protein A) But is decrated with 28 additional cell wall proteins (CWPs)
83
Cleavage of SlpA
Undergoes two cleavage events 1. Removes N-terminal signal peptide that directed it for secretion from cell 2. Protein broken into LMW SLP and HMW SLP - cleaved by CWP84 (cysteine protease)
84
How is SlpA transported across membrane out of cell?
SEC system - SecY, SecE, SecG and SecA Conserved protein secretion system present in nearly all bacteria
85
SecA
Motor protein (ATPase) Provides energy for secretion of proteins across membrane Two copies: SecA1 and SecA2
86
Role of SecA1 and SecA2
A1 is housekeeping - responsible for majority of protein secretion A2 is dedicated to secretion of SLPs
87
PS-II
Secondary cell wall sugar HMW SLP anchors S-layer on outside of cell through interaction with PSII
88
LMW and HMW complex
Form a complex on outside of cell Form high affinity heterodimer HMW bound to cell wall LMW surface on outside
89
CWPs in S-layer
Substituted in at random points in S-layer to add functionality Every CWP has three copies of 04122-Pfam domain - cell wall binding domains
90
Examples of CWPs in S-layer
Amidase (PG hydrolase) CWP20 (beta-lactamase)
91
Proof of S-layer signifance for bacterial cell
Uses 10% of cell energy to make TraDIS using 75,000 mutants showed no mutants in gene for SlpA or SecA2 - essential genes
92
Avidocin
Protein nano-machine Uses phage-targeting systems to recognise C.diff cells Related to contractile myovirus phage - just no DNA
93
Structure of Avidocin
Tail fibres - act as legs Receptor binding proteins on end of fibres Contractile sheath - contains nanotube core (needle)
94
Avidocin action
Binds target cell Sheath contracts and shortens; drives internal needle through cell envelope Allows ions to escape, destroys pmf across membrane Cell can't make energy --> dies
95
Experiment used to make S-layer mutants
Small experimental evolution Big population of C.diff + avidocin Produced C.diff derivative strains that could grow in presence of Avidocin 1bp insertion or substitution - introduced a premature stop codon - no longer produces SlpA
96
S-layer function
SlpA -ve mutants are sensitive to lysozyme and LL-37 - S-layer protective against effectors of innate immune system
97
LL-37
Anti-microbial peptide Produced in large quantities in GI tract
98
Regulatory mechanisms used by C. diff
PaLoc - toxin expression Sp0A cascade of sporulation Germination control by bile salts

Bacterial Pathogenicity (4 decks)

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