Bacteriology - biofilms, extracellular survival Flashcards
(166 cards)
1
Q
Dynamic biofilm environments
A
Physico-chemical gradients Enzymes Water channels Gene transfer Cell to cell communication
2
Q
EPS constituents
A
Polysaccharides, DNA, proteins, water, lipids and biosurfactants, minerals.
3
Q
Proteus mirabilis biofilm
A
Crystalline; cells in calcium apatite base.
4
Q
Biofilm formation steps.
A
Initial attachment –> multiplication –> a flat biofilm.
OR
Initial attachment leads to an aggregate formation, leading to a structured Biofilm 1 (motile cap) or a structured biofilm II (due to clonal growth).
EITHER
Then lead to dispersion or detachment.
5
Q
Pseudomonas stage III biofilm
A
LasI and RhlI are active, type IV pili are being made, extracellular DNA is found. GacA and rhlA are expressed.
6
Q
Pseudomonas stage IV biofilm
A
PQS active. Matrix contains PSL, PEL and DNA.
Denitrification genes active, rhlA expressed. Alginate made.
7
Q
Pseudomonas stage V biofilm
A
rhamnolipid (rhlA and rhlB). SadA expressed. Flagella made.
8
Q
Alginate
A
Scavenges free radicals, prevents phagocytosis, protects from defensins.
9
Q
Conversion to mucoidy in P. aeruginosa.
A
Stress reveals WVF on MucE.
WVF binds PDZ domain of AlgW, activating AlgW.
AlgW cleaves anti-sigma factor MucA, which releases AlgU which it has been sequestering.
AlgU activates alginate biosynthesis genes.
10
Q
Alginate synthesis regulation
A
By stress (leads to MucE/AlgW/MucA/AlgU) and high c-di-GMP.
11
Q
c-di-GMP and alginate synthesis
A
c-di-GMP binds Alg44 PilZ domain which helps co-ordinate alginate polymerisation and export.
12
Q
Importance of adhesion in UPEC.
A
Contributes to colonisation, biofilm formation, apoptosis and exfoliation.
13
Q
Biofilms in E. Coli.
A
Colanic acid rather than alginate.
14
Q
What is EPS?
A
Extracellular polymeric substances.
15
Q
Stabilisation of EPS matrix
A
Repulsive forces (e.g. between acidic groups) prevent collapse. Ionic/electostatic/hydrogen bonds/van der Waals stick it together.
16
Q
Biofilm formation pathway
A
reversible attachment, irreversible attachment, microcolony formation, mature biofilm, biofilm dispersal.
17
Q
Biofilms - pathology
A
Phagocytosis cannot occur, but phagocytic enzymes are released. Damage tissues around the biofilm.
Motile bacteria are released from the biofilms.
18
Q
Biofilm dispersal - swarming
A
Pseudomonas, Proteus mirabilis.
19
Q
Biofilm dispersal - swimming
A
Pseudomonas.
20
Q
Biofilm dispersal - clumping
A
Staph aureus, Mycobacterium tuberculosis
21
Q
Biofilm dispersal - rolling
A
Staph aureus
22
Q
Biofilm dispersal - sliding
A
Mycobacterium tuberculosis.
23
Q
Types of motility in Pseudomonas biofilms
A
Flagella based in early stages and in late dispersal.
Twitching important in complex biofilm structures - mutants make flat biofilms.
24
Q
HAP signalling pathways in biofilm formation
A
GacSA, sadARS.
25
Pseudomonas biofilms stage 1 and 2 as therapy targets.
Multicomponent vaccines, antibiotic therapies, quorum sensing inhibitors e.g. furanones. PREVENT FORMATION.
26
Pseudomonas biofilms stage 3 as a therapy target.
A few antibiotics still have effects.
Anti-inflammatories
Anti-biofilm agents.
27
Pseudomonas biofilms stage 4 as a therapy target. Alginate covered biofilm.
Alginate lyase, anaerobic growth inhibitors, DNase, novel antibiotics?
28
How do E. Coli colonise the host urinary tract?
Motility, adhesion, intracellular biofilms, extracellular biofilms, immune evasion.
29
How does E. coli damage the urinary tract?
Immune response, protein toxins.
30
UPEC motility
Flagella mediated, chemoattraction.
31
Phosphorylation pathway of chemoattraction
Empty receptor --> CheA --> CheY --> FliM --> tumble.
32
FliM
Switch motor protein in flagellar motor. Phosphorylation causes a switch to CW rotation.
33
Adhesion proteins expressed by UPEC
Curli pili
P-pili
Type 1 pili
S pili and Dr pili
34
Curli pili on E. coli
csgDEFG operon --> important in initial adhesion.
| CsgA is exported by CsgG
35
Exfoliation in UPEC
Exposes lower level of epithelium; FimH binds CD48 as well as other receptors; internalised in actin-bound antibiotic non-susceptible non-immunogenic vesicles.
36
Intracellular invasion by UPEC
Zipper mechanism. FimH binds integrins --> clustering --> signalling --> internalisation.
Hijacks the Rab27b fusiform vesicular trafficking pathway.
37
Signalling for internalisation in UPEC
1) FAK complexes with PI3K --> PIPs --> changed actin dynamics.
2) Activation of Rac1 --> α-actinin and vinculin --> local changes to actin.
38
Intracellular UPEC
Quiescent, expelled or replication.
39
Intracellular UPEC - expelled
Probably by Rab27b fusiform vesicular pathway.
40
Intracellular UPEC - quiescent
Form reservoir, resistant to antibiotics
41
Intracellular UPEC - multiplication
Has to escape vesicle.
Requires adhesion pili to form communities, otherwise distributed.
Differentiate into filamentous and flagellated forms.
42
Filamentous UPEC from IBCs
Less prone to phagocytosis.
43
Immune response to UPEC
LPS --> TLR4 --> NFkB --> neutrophil recruitment (lots of inflammation) and production of IL-6 and IL-8. Thought to make epithelial cells more resistant to invasion.
44
Immune evasion by UPEC
LPS; use different forms to decrease immunogenicity. Phase variation of FimH.
Filamentous form less prone to phagocytosis
Stabilise IkB to decrease NFkB activation. Alter immune response using HlyA.
45
UPEC; protein toxins
HlyA, CNF1, Sat and Vat.
46
HlyA
RTX protein toxin, associated with increased severity in UTIs.
47
HlyA - synthesis and export.
Two genes for synthesis, ditto for export.
Fatty acid chains attached by HlyC.
Exported by HlyB pump associated with TolC.
48
HlyA - effects (5)
Release of iron and other nutrients by cytolysis.
Promotes exfoliation.
Promotes expression mesotrypsin --l NFkB.
Fine tunes immune response by causing Ca++ fluxes in renal cells.
Immune cell dysfunction
49
CNF; binding, entry into cytosol, effects.
Binds laminin, uptake by endocytosis, transfer into cytoplasm with acidification, effects are aberrant Rho activation and subsequent Rho degradation.
50
Vat and Sat toxin delivery
Blebbing forms outer membrane vesicles which allow for concentrated burst of toxin delivery to the host cell.
51
Type 1 pilus structure
FimH (adhesin), FimG, FimF, then right hand helix of FimA attached to OM secretion system at base (FimD)
52
Pap pilus structure
PapG, PapF, PapE (5-10), PapK and PapA attached to PapC secretion system at base.
53
Adhesins; FimH/PapG
Pilin domain + lectin domain for binding to host surfaces. Phase variation leads to expression of adhesin switched on in the host.
54
EPEC initial adhesion
Due to bundle forming pili
55
bundle forming pili (EPEC)
long range plasmid associated Type IV pilus. Encoded by bfp operon.
56
Possible importance of A/E lesions
1) Efficient delivery of other proteins
2) Resistance to bulk flow
3) Antiphagocytic
57
Importance of biofilms
- Avoid predation by single cells (either immune or such as amoeba)
- Increased tolerance to antibiotics
- Some evidence that there is a subdivision of labour.
58
Bacterial motility in biofilm formation
Non-motile bacteria form microcolonies
• Cells migrate to form flat colonies.
• Formation of mushroom shaped colonies depends on migration of motile bacteria to colonise already established microcolonies.
59
Role of extracellular matrix
Scaffold, cell attachment, cell-to-cell interactions, antimicrobial tolerance.
60
Pel
Glucose rich and reported to be involved in formation of liquid-air pellicles.
61
Psl
important in cell-to-surface and cell-to-cell binding.
62
Type IV pili in biofilms
Adhesion. Also bind DNA and act as cross-linkers
63
Psl crosslinks with...
CdrA
64
Rhamnolipid
a biosurfactant produced in iron-limiting conditions which stimulates surface motility. Affects biofilm structure and formation.
65
LasI signal
3-O-C12-HSL
66
RhlI signal
C4-HSL
67
Subpopulations in biofilms
Secreted products may be of use to all bacteria: e.g. rhamnolipid, pyoverdine.
Some evidence that sub-populations have different roles e.g. in formation of mushroom-shaped colonies.
68
Mechanisms of biofilm dispersal
Controlled by low c-di-GMP. Biofilm dispersal locus makes BdlA which is a chemotaxis regulator affecting c-di-GMP levels.
RbdA decreases Pel and Psl synthesis, and increases rhamnolipid synthesis.
69
Antimicrobial tolerance in biofilms is due to ...
Restricted penetration.
Different physiological activity.
Presence of persisters.
Inducible anti-microbial resistance factors.
70
High c-di-GMP results in...
```
o Pel production
o Psl production
o Alg44 production
o Type IV pili
o Cup fimbriae in P. aeruginosa.
```
71
Controlling biofilms
Use quorum sensing
Use c-di-GMP
Use antibiotic combination therapies
Use implants which target antimicrobial surfaces.
72
Restricted penetration in biofilms
DNA is positive, shields from AMPs and cationic antimicrobials. Mucoidy.
73
Different physiological activity in biofilms.
Inner part of biofilm has low metabolic activity.
74
Inducible antimicrobial resistance factors in biofilms - P. aeruginosa
MexAB-OprM efflux pump. Pmr operon mediated LPS modification.
75
Identifying quorum inhibitors.
Chemical biology approaches
Structural based virtual screening
Isolated from food sources
76
c-di-GMP inhibitors
o Genetic manipulation suggests that this would work.
| o Novel benzimidazole agent.
77
Theory behind antibiotic combination therapies
o Targetting different subpopulations with different metabolic activity.
78
Extracellular survival general plan
* Avoid complement
* Avoid recognition
* Avoid effector arm
79
Complement initiation
Classical, MB-lectin and alternative
80
Complement initiation - classical
* Antigen:antibody
| * C1q, C1r, C1s, C4 and C2
81
Complement initiation - MB-lectin.
* Mannose binding lectin binds mannose on surfaces
| * MBL, MASP-1, MASP-2, C4, C2.
82
Complement initiation - alternative pathway.
* Pathogen surfaces
| * C3, Factor B, Factor D.
83
C3 convertase
C4b-C2a
84
C3 convertase effects
Cleaves C3 to give C3b, a C5 convertase important in MAC.
Binds complement receptors on macrophages
• Opsonise pathogens,
• Remove immune complexes.
85
Blocking C3 convertase activity
Blocked by Factor H and factor I.
Pathogens acquire regulators for resistance
• Factor H, FHL1, C4BP, CFHR.
86
IgA proportion of Ig in serum
15-20% total serum
87
IgA found in
mucus, saliva, milk
88
IgA structure
2 identical Fab regions bound by hinge to Fc, binds host cells
In secretions is dimerised by J chain and secretory component S-IgA1
89
IgA targetted by bacteria
Proteases. Zinc metalloproteases or serine proteases.
90
Staph aureus avoiding complement
Avoid initiation
Bind C3 to avoid conversion to C3b
Acquire complement regulators.
91
Staph aureus - binding C3
Efb, Sbi
92
Staph aureus avoiding initiation of complement
Aureolysin, or acquiring plasminogen from the host.
93
S. pyogenes anti-complement
GAPDH to bind C5a.
| SIC.
94
Strep IgA protease.
Specific for IgA1
Cleaved fragments compete with uncleaved.
Zinc metalloprotease
95
Proteases with specificity for IgA1
Cleaving at specific sites between Pro-Ser or Pro-Thr present in longer more flexible hinge region.
Fragments compete with IgA for binding to pathogens.
96
Zinc metalloproteases - structure
signal sequence, anchoring domain, autoproteolytic cleavage sites, and protease domain
97
Zinc metalloproteases - synthesis.
Synthesised, inserted into membrane, autoproteolytically cleaved, but protease domain remains associated with bacterial surface as non-covalently binds N-terminal domain.
98
B burgdorferi anti-complement
o CD59-like protein
| o CRASP-1
99
Neisseria - avoiding innate.
Capsule to avoid phagocytosis
Mimic or recruit host proteins to avoid complement.
Divert complement using blebs.
100
Neisseria importance of capsule
Major virulence determinant. meningococci rely on this partially for serum resistance and virulence. Unencapsulated are sensitive to killing by serum, but do not cause disease in complement deficient (encapsulated do); probably due to anti-phagocytic properties.
101
Importance of complement in Neisseia killing
o Deficiency (acquired or inherited) in complement leads to susceptibility to disease. (seminal study by Goldschneider showed importance of complement)
102
Neisseria avoiding complement - mimicing host proteins.
Avoid recognition - Group B capsule repeating unit is identical to NCAM, a host adhesion protein.
Carbs on LOS mimic host carbs.
C4BP inactivates C3 convertase.
103
Neisseria acquiring host proteins
Factor H
| Vitronectin
104
Neisseria acquiring factor H - proteins.
Gonococci: porin
meningococci: Factor H binding protein, Neisseria surface protein A ad sialylated lipo-oligosaccharide. Not PorB3 despite homology to porin.
105
Neisseria acquiring factor H; tropism.
Species specific complement evasion. Human pathogens' factor H binding proteins --> only binds chimpanzee factor H poorly, and rhesus macaque factor H barely detectably.
106
Neisseria acquiring factor H; convergent evolution.
• Factor H binding proteins in different Neisseria bind the same domains 6&7 as each other, which is the same as that used by host cell surface molecules – convergent evolution.
107
Role of factor H
H is the co-factor for factor I medicated cleavage of C3b to iC3b. Factor H also causes decay acceleration.
108
Gonococci factor H binding.
Uses porin.
Also uses sialylated LOS. Does not have serum resistance if LOS not sialylated for some strains. Decreases binding of antibodies and increases binding of fH.
109
Neisseria acquiring host proteins: vitronectin.
Inhibits the terminal complement complex at various stages; occupies metastable membrane binding site of nascent MAC. Can also block C9 polymerisation.
110
Neisseria: decreasing recognition
Antigenic variation --> phase variation and DNA sequence variation.
111
Phase variation
Switches on or off due to strand slippage at polyG or poly C tracts.
Genes, promoters, genes involved in biosynthesis, glycosyltransferases for LOS synthesis.
112
Phase variation: polyG strand slippage.
Variation in PilC and IgtA.
113
Phase variation: polyC strand slippage
Opa/Opc,
| SiaD
114
Neisseria: DNA sequence variation.
PilE silent genes being copied
| Pilin on/off
115
Neisseria PilE
Major pilin protein.
Silent genes lack a promoter, ribosome binding site and the first 35-43 codons.
Parts of these are copied into the PilE locus.
Unusual type of homologous recombination.
116
Neisseria pilin DNA variation - ways to switch off pilin synthesis.
Truncation - Some silent genes code stop codons in coding regions. Recombination.
Non-assembly - If PilC (tip adhesin) switched off in both alleles in gonococcus, then pilus is not assembled. Phase variation.
Non-export - Some variant sequences have poor interactions with the machinery, and do not form pilins. Recombination.
Non-export - Formation of L-pilin which is not exported across OM leads to phase variation by aberrent recombination.
117
Neisseria PilE
Silent genes lack a promoter, ribosome binding site and the first 35-43 codons.
Parts of these are copied into the PilE locus.
Recombination tracts are bordered by regions of microhomology.
118
Neisseria pilin DNA variation.
Some silent genes code stop codons in coding regions. If PilC switched off in both alleles in gonococcus, then pilus is not assembled.
Some variant sequences have poor interactions with the machinery, and do not form pilins.
119
Neisseria avoiding the effector arm of the adaptive immune response.
Cleave IgA
| Avoid phagocytosis with a slimy capsule.
120
Yersinia anti-complement activity.
YadA = fibrillar surface structure attachment to mammalian cells and ECM. Stalk does Factor H binding and serum resistance.
121
Yersinia adhesins
YadA
Inv
Ail
122
YadA
fibrillar surface structure attachment to mammalian cells and ECM.
Important for initial attachment before T3SS inserted into host membrane.
123
YadA structure
o Head performs neutrophil, collagen and autoagglutination.
o Stalk does Factor H binding and serum resistance.
124
Inv
promotes uptake into host cells
125
YadA and Inv
Both autotransporters.
126
T3SS for YOPS in yersinia
Similar to flagella synthesis. Basal body, needle, rings in membranes, ring for insertion into host membrane.
127
Yersinia T3SS needle
Needle made of YscF. Plugged by LcrV.
| Measured using molecular ruler.
128
Yersinia subverting macrophage function
Disrupt host cytoskeleton
| Trigger macrophage apoptosis.
129
Yersinia subverting macrophage function - disrupting host cytoskeleton.
Target Rho GTPases
| Use YopH.
130
Yersinia subverting macrophage function - Targetting Rho GTPases
o GAP mimic YopE
o GDI mimic YopO
o Cysteine protease YopT
131
YopH
• YopH is a tyrosine phosphatase that interferes with host cell adhesion and signal transduction.
Prevents cell survival by inhibiting PI3K-akt signaling.
132
Yersinia subverting macrophage function - triggering apoptosis.
YopJ and Yop H
133
YopJ
• YopJ inhibits MAPK and NFkB signaling, simulating apoptosis.
134
Autotransporters in OM. Crossing IM.
Via Sec.
135
Autotransporters in OM. In periplasm
* Chaperones like Skp and SurA bind passenger and B domain and DegP binds passenger domain.
* DegP = quality control mechanism.
136
Autotransporters in OM. Insertion into OM
* Targeted to B-barrel assembly machinery, Bam complex in OM. Inserts barrels into OM.
* Barrel then autotransports passenger domain extracellularly.
137
Capsules - protein
Bacillus anthracis. But usually capsules are carbohydrate.
138
Purpose of capsules
Protect from desiccation, act as adhesin, evasion of host defences.
139
LPS
Attached to OM of Gram -ive.
140
Difference between antigenic and phase variation.
Antigenic variation promotes change of surface antigens at a higher rate than normal.
Phase variation switches structures or proteins on or off.
141
Neisseria surface components
Capsule, type IV pili, Opa (gonococcus) or Opc (meningococcus), lipo-oligosaccharide.
142
Neisseria-host interactions
Pili for initial adhesion. Opa/Opc for tight adherence and cell tropism, invasion and transcytosis. Capsule prevents phagocytosis. Sialic acid element of capsule helps evasion.
143
Opa/Opc function
Neisseria. Tight adherence and cell tropism. Binds CD66. Meningococci have 3-4 opc genes, gonococci have 11 opa genes.
144
SiaD function
Neisseria sialylation enzyme for the capsule.
145
LgtA function
ligates lipo-oligosaccharide terminal sugars.
146
PilS
Partial gene copies of PilE (Neisseria). Silent. There are 17.
147
Neisseria homologous recombination in pilin genes.
Recombination tracts, or mini-cassettes, are bordered by regions of microhomology. Requires only short region of homology.
Naturally transformable - can use DNA from environment.
148
Mini-cassettes in Neisseria
Sequence variability doesn't perfectly match mini-cassettes; what is important is the functional conservation. There is a hypervariable area, mc2.
149
Neisseria: variation in pilin
Antigenic variation by homologous recombination.
| Formation of L-pilin which is not exported across OM leads to phase variation by aberrent recombination.
150
Neisseria - formation of S pilin
Signal sequence is normally removed before pilus assembly, but after aberrent recombination, it can be removed at +40, leading to soluble secreted S-pilin, which acts as a molecular decoy.
151
PilC polyG slippage
10 or 13 Gs = in frame
| 11 or 12 Gs = out of frame.
152
When does polyG slippage occur for phase variation in Neisseria?
During DNA repair.
| During replication.
153
How does polyG strand slippage occur in DNA repair?
Single strand break --> exonuclease degradation. Mispairing during DNA synthesis --> altered number of repeat units.
154
How does polyG strand slippage occur in DNA replication?
Parent strand loops out and repair process removes looped-out repeat. Daughter strand is shorter.
Lagging strand can slip back one repeat during synthesis. Daughter strand is longer.
155
Opa/Opc variation
Repeat sequence CTTCT. - Opc ON/OFF
In frame = multiples of 3. Out of frame = not multiples of 3 (2, 4, 5, 7 etc).
Opc polyC tract can act as a dimmer switch.Alters distance between -35 and -10 sequences, hence sigma factor binding and expression. 10 residues is off, 14 is on, and 12 is on with high expression.
156
Ways to apply phase variation (Neisseria).
ON/OFF.
| Dimmer switch.
157
Ways to apply recombination (Neisseria)
Variation.
ON/OFF - truncation, non-export.
Secretion of molecular decoy.
158
Ways to cause variation
```
Antigenic variation (Neisseria)
Phase variation by strand slippage (Neisseria).
Phase variation by invertible elements (E. coli and salmonella)
```
159
Phase variation by invertible elements
E. coli and salmonella, in fimbriae and flagella. Recombination sites within the promoter are in opposite orientation leading to switching of gene expression.
160
Ways to cause variation
```
Antigenic variation (Neisseria) - homologous recombination.
Antigenic variation - gene conversion (Borrelia).
Antigenic variation - segmental recombination (Borrelia).
Phase variation by strand slippage (Neisseria).
Phase variation by invertible elements (E. coli and salmonella)
Phase variation by BvgAS two component system.
```
161
Phase variation by invertible elements
E. coli and salmonella, in fimbriae and flagella. Recombination sites within the promoter are in opposite orientation leading to switching of gene expression. Temperature dependence
162
Phase variation by invertible elements temperature dependence
FimB and FimE recombinases are used. FimB switches either way, with higher activity at 37-40 degrees. FimE favours the OFF switch and is more active at lower termperatures, so fimbriae are switched on in hosts.
163
Segmental recombination B. burgdorferi.
In vlsE. 6 variable regions continuously recombine in surface lipoprotein VlsE in mammalian infections.
164
Gene conversion in Borrelia.
Structure = upstream homology sequence, coding region, variable gap and downstream homology sequence. Homology sequences contribute to expression switching, with order being determined by homology in UHS, and distance between coding sequence and DHS.
165
YopE
GAP activity decreases Rho activity. So YopE decreases polymerisation of actin cytoskeleton and hence decreases uptake.
166
YopO
Specifically targets Rac. Prevents Rac activation, although in vitro it can act on both Rac and Rho.
