1. Variation of bacterial surface structures Flashcards
(98 cards)
1
Q
What is the purpose of bacterial surface variation?
A
To allow colonisation and hide from the immune system
2
Q
What species are we using as an example of surface variation?
A
The pathogenic Neisseria
1. N. meningitidis
2. N. gonorrhoeae
3
Q
How genetically similar are N. meningitidis and N. gonorrhoeae?
A
over 90% identical
4
Q
What is the major difference between N. meningitidis and N. gonorrhoeae?
A
- Nm has a polysaccharide capsule and Ng doesn’t.
- Ng doesn’t have the loci to encode a capsule but can still coat themselves with environmental molecules.
5
Q
Why do neisseriae pathogens present a challenge?
A
- They are human specific so are very well adapted to infect us.
- But it does mean that it is possible to eradicate it.
6
Q
What conditions does Neisseria require to grow?
A
- Lots of iron
- They are very fastidious
- They only really grow at 37oC and nothing else (very human specific)
7
Q
What is gonorrhoea?
A
- A collection of infections that cause inflammation at different sites.
- They are all different infections caused by the same organism.
- Most commonly infects urogenital sites.
- The main effect is inflammation.
8
Q
What is asymptomatic gonorrhoea?
A
- Gonorrhoea with no clinical signs.
- It creates an infectious reservoir.
- around 10% of infections in men and 30/40% of infections in women.
9
Q
What complications can gonorrhoea cause?
A
- Pelvic inflammatory disease
- Salpingitis (inflammation of the fallopian tubes)
9
Q
Why are there no vaccines for N. gonorrhoeae?
A
Due to its variation
10
Q
Is antibiotic resistance a problem in N. gonorrhoeae?
A
- It is a big problem.
- We have a limited amount of antibiotics that can treat N.gonorrhoeae.
- It is highly transmissible, so it can gain resistance quickly.
11
Q
What are the risk factors for N. meningitidis?
A
- No specific risk factors
- Low serum levels of antibodies can increase the risk.
12
Q
Who does N. meningitidis infect?
A
- Mostly neonates and adolescents
- Can still infect anyone at anytime.
13
Q
Can N. meningitidis be carried as a commensal?
A
- 5-30% of people carry it
- This can increase to up to 95% during outbreaks.
14
Q
What infections does N. meningitidis normally cause?
A
- Disseminated infections
- Mostly septicaemia
- Also meningitis
- These can occur at the same time or independently.
15
Q
Is the N.meningitidis capsule always expressed?
A
no
16
Q
How is N. meningitidis classified?
A
- Serogroups - by the capsule
- Different outer membrane components determine sub groups.
- Immunotypes are determined by LPS types.
17
Q
Is antibiotic resistance a problem in N. meningitidis?
A
- Not really
- This is weird due to the close relation to Ng.
18
Q
Why is N. meningitidis hard to treat?
A
Due to the rapid progression of the disease treatment is often too late.
19
Q
What do pathogens need to do in order to cause disease?
A
Grow to high enough numbers to colonise and obtain nutrients in order to overwhelm the immune system.
20
Q
What pathogens don’t need to colonise the host to cause disease?
A
Exotoxin secreting organisms
21
Q
What natural defence do bacteria need to overcome for colonisation?
A
- Cilia and mucus to attach to the surface.
- avoidance of the immune system like antibodies, T cells and phagocytes.
22
Q
What are the four main strategies bacteria use for effective colonisation?
A
- Expression of multiple adhesins through molecular redundancy
- Antigenic variation
- Phase variation
- Overcoming charge barriers
23
Q
What is molecular redundancy?
A
- Expression of different molecules that target the same receptor/cell/ECM target.
- OR Expression of 1 molecule that can target different host adherence molecules
- This allows adaptation and survival of a bacteria no matter what happens to a population.
24
How can the same molecule target different host molecules?
1. They contain different target motifs.
2. This gives the organism the ability to select the needed sequence.
25
What is antigenic variation?
1. Different variants of the same protein.
2. Generated by recombination or point mutations.
3. Very common way for bacteria to change surface structures.
26
What is phase variation?
1. Molecular switch of turning on and off gene expression.
2. Around 150 genes are subject to phase variation.
3. This means a daughter cell could appear very different to a parent.
4. The phase switch happens in about 1 in 100 divisions.
27
How do bacteria overcome the charge barrier?
1. All cells are negatively charged unless they are coated in something.
2. These negative charges repel each other.
3. Neisseria use pili as the first point of contact to overcome the charge barrier.
4. The pili are positively charged and then draw in the rest of the cell.
28
How can bacteria avoid host immune mechanisms?
1. Antigenic and phase variation of surface structures.
2. Sialylation of LPS and capsular coating on the surface.
3. Host mimicry
4. Diversion
29
What are the potential consequences of antigenic variation?
1. Changing a protein too much can prevent function.
2. This limits the amount of variation possible.
3. Trial and error process to produce a phenotype that escapes immune attack so the population can rebuild.
30
How does LPS sialylation help avoid the immune system?
1. Bacteria take sialic acid from the host and coat themselves in it.
2. This changes the appearance and charge of the bacteria.
3. This also allows them to coat themselves in complement inhibitors like factor H.
31
How do pathogens use host mimicry to avoid the immune system?
1. This is very common in human specific organisms.
2. The bacteria are seen as self so no effective immune response is mounted.
3. N. meningitidis capsule B looks like the neuronal sugar NCAM so no immune response is mounted.
32
How do pathogens use diversion to avoid the immune system?
1. They shed surface molecules in blebs or vesicles. These often contain LPS and other antigens.
2. This diverts antibodies away from the bacteria.
3. An excessive production of outer membrane causes this shedding.
4. The shedding means antibodies bind to the target but it is not attached to the bacteria so its a bit useless.
33
What mechanisms are happening simultaneously to allow bacteria to colonise the host?
1. Redundancy
2. Variation
3. Overcoming the charge barrier
4. Coating in host molecules
5. Host mimicry
6. Diversion and shedding
34
What non-inherited mechanism creates structural diversity in bacteria?
1. Environmental sensing changes gene expression based on whats available in the environment.
2. As the environment changes so does the protein expression.
3. This is an important consideration for vaccine development as it changes the expression of potential vaccine targets.
4. In response to things like iron levels or salt concentration.
35
What inherited mechanism creates structural diversity in bacteria?
1. Mutations
2. DNA rearrangement: RecA dependent
3. DNA rearrangement: RecA independent
36
How do random mutations that cause structural diversity in bacteria occur?
Due to error prone replication
37
What does N. meningitidis require for adhesion?
Pili
(capsular bacteria adhere really badly without pili)
38
What pili is the most common in Neisseria?
Type 4 pili
39
What is the structure of the type 4 pilus?
1. Encoded by 23 genes
2. Mainly made up of pilin which is encoded by pilE.
40
What is pilE?
The main protein subunit of the type 4 pilus.
41
How is the pilus formed?
1. The signal peptide is cleaved.
2. This triggers pilin to assemble into the pilus.
42
What can additional recombination in pilE lead to?
1. The generation of another peptide cleavage motif.
2. This causes the soluble head domain of pilin to be cleaved.
3. This is done by signal peptidase 1 (SP1).
4. the cleaved soluble pilin is never assembled into a pilus.
5. You can have s-pilin and a pilus generated at the same time.
43
What is the structure of pilin?
1. A long hydrophobic alpha-helical tail which forms the centre of the pilus.
2. A Globular ß domain that is exposed on the outer surface and is variable.
44
What are the properties of the N. meningitidis type 4 pili?
1. It is very strong and grows very quickly. Neisseria pili are the strongest machines in the world.
2. They can be retracted with the force of 150 piconewtons.
3. The fast retraction is due to being used to pick up DNA from the environment.
45
How highly expressed are the pili?
They are highly expressed on the bacteria's surface as they are very important to the bacteria.
46
Why do most disease strains of Neisseria have lots of variation in the pili?
1. They are constantly exposed to the immune response so have lots of variation and diversity to avoid the immune response.
2. They do this by adapting the amino acid sequence or phase variation.
47
What is the scale of diversity of meningococcal pili?
1. They can all be very different.
2. Point mutations can generate small changes in pili.
3. Large shifts in molecular weight on SDS-PAGE can show genetic recombination is occurring as mutations cannot make this change.
48
What is the structure of the type 4 pili genes?
1. A very conversed end domain.
2. Contains variable gene cassettes with small conserved regions between.
3. the variable regions can be swapped out via recombination to create variation.
49
What is the structure of the pilE gene?
1. It contains 1 conversed domain and 1 variable gene cassette domain.
2. The variable domain is what is switched during recombination.
50
What is pilS?
1. Silent pilE variable gene cassettes.
2. They recombine with pilE to cause variation in the pili.
3. This recombination occurs in about 1 in 100 divisions
4. pilS genes are not expressed unless they have recombined into pilE
51
How many pilS gene cassettes does N. Meningitidis have?
4/5 in 1 loci in the genome
52
How many pilS gene cassettes does N. gonorrheae have?
around 19 spread between 4/5 different loci.
53
How does recombination between pilS and pilE occur?
1. Homologous recombination
2. It uses the conversed domains either side of the cassettes as the regions of homology.
54
Are Neisseria naturally competent?
Yes
55
What does being naturally competent mean?
1. The bacteria can easily pick up extracellular DNA without stimulation of specialist uptake mechanisms.
2. This transforms bacteria
3. it is preferentially done by bacteria of the same species.
56
What is intergenomic recombination?
1. Recombination with genes from extracellular/ non-self DNA.
2. Mostly from lysed bacteria of the same genus
3. This leads to replacement of the pilS gene cassettes and an increase in diversity.
57
What is intragenomic recombination?
1. Recombination within the same chromosome/genome.
2. This leads to pilS recombining with pilE.
58
What happens to advantageous recombination?
They are quickly selected for
59
What happens to detrimental recombination?
they die out quickly
60
What is even recombination?
The same amount of DNA is exchanged. Eg one cassette swapped for 1 cassette.
61
What is uneven recombination?
1. Exchanging DNA of different lengths
2. Eg swapping 2 cassettes for 4 cassettes.
3. This can lead to the pilin protein being too different and not expressed.
4. It can lead to the signal peptide cleavage site being exposed to make soluble pilin so the pili is not expressed.
62
What is L-pilin?
1. Long pilin
2. Occurs after uneven recombination that is too different
3. the pili cannot be made so is phased off.
63
What is S-pilin?
soluble pilin
64
What does the pilE recombination need to be?
1. Balanced
2. It needs to be variable enough not to be detected by immunity but still needs to be functional.
65
What does recombination between variable pilE and pilS regions depend on?
RecA
66
What is RecA?
The enzymes that facilitates recombination and DNA repair via homologous recombination.
67
What is homologous recombination important for?
1. Maintaining genetic diversity
2. DNA repair
68
What could RecA potentially be?
1. A good drug target
2. A RecA inhibitor could lock a bacteria into a certain phenotype to make a vaccine or treatment more effective.
69
What controls the antigenic variation of Neisseria pili?
Recombination.
70
What can cause phase variation in Neisseria pili?
1. Aberrant recombination
2. This creates s-pilin or L-pilin
3. These forms of pilin cannot form a pili.
4. This means the pili is not expressed so it is phased off.
71
What other antigens in Neisseria can undergo phase and antigenic variation?
Opa and Opc
72
What is Opa's structure?
1. An 8 stranded ß-barrel
2. 4 exposed loops
3. 1 highly conserved loop so it is not seen by the immune system
4. 1 semivariable loop that is not really seen by the immune system
5. 2 hypervariable loops
73
What do the Opa hypervariable loops bind to?
1. They bind to CEACAM on the host. (carcinoembryonic antigen related cell adhesion molecules).
2. They are so variable they can look like different proteins
74
What phase variation occurs in Opa?
1. When Opa is switched on and expressed, Neisseria have on opaque phenotype.
2. When Opa is switched off they have a transparent phenotype.
3. They look like totally different species but the only difference is the phase variation of Opa.
75
What is the main difference between antigenic variation of PilE and of Opa?
1. With pilE only 1 gene copy is expressed
2. Opa can express all the Opa genes but normally only a couple at a time.
76
What is the antigenic variation of Opa in N. meningitidis?
1. There are 4 copies of Opa genes
2. All have hypervariable loops.
3. All can be phased on and off independently.
4. Usually 1 or 2 are phased on at the same time.
5. They can recombine with each other and rarely extracellular DNA.
77
What is the antigenic variation of Opa in N. gonorrhoeae?
1. there are 11-13 copies of Opa in the genome.
2. This is more variable then Nm.
3. They can all recombine with each other and rarely extracellular DNA.
4. All can be phased on or off.
78
How does homologous recombination occur in Opa?
By using the conserved regions either side of the variable regions as the sequences of homology.
79
What is slipped strand mispairing?
1. A mechanism of phase variation.
2. Uses the error prone nature of DNA replication to turn on and off gene expression.
80
How does slipped strand mispairing work in Opa proteins?
1. Opa gene contains a pentameric CTCTT repeat.
2. This repeat can be present in varying numbers.
3. If n is a multiple of 3 the gene is in the correct reading frame and can be expressed.
4. If n is not a multiple of 3, then the reading frame is wrong, and a premature stop codon is introduced.
5. This causes phase variation via antigenic variation.
81
Why does slipped strand mispairing occur?
1. Bacteria have error prone polymerases.
2. these polymerases struggle to copy repeated sequences correctly.
3. The polymerase can skip and few bases and the strand gets mismatched.
4. This causes one of the strand to then be nicked and repaired.
5. This can gain or lose DNA like complete CTCTT repeats.
82
How does slipped strand mispairing cause phase variation of Opa?
1. It changes the number of CTCTT repeats in the gene
2. This changes gene expression.
83
What type of gene expression control is slipped strand mispairing?
Translational control
84
What controls slipped strand misparing?
poor proof reading during replication
85
What is the mechanism of slipped strand mispairing?
1. During replication, DNA is unstable due to transient single-strand sections.
2. At points of repeats this is even more unstable.
3. A shift in the base pair binding can cause a loop of DNA to be formed.
4. This loop is nicked, removed and the DNA repaired.
5. This causes the gain or loss of repeats which can turn on or off gene expression.
86
What are homopolymeric tracts?
Regulatory regions that include long stretches of the same nucleotide.
87
What kind of gene expression control are homopolymeric tracts?
Translational or transcriptional, depending on where it is in the genome
88
How is pilC expression controlled by homopolymeric tracts?
1. This is a pilus associated protein.
2. PolyG tract
3. Single base pair gains or losses change the reading frame of the gene and, therefore, change the expression.
4. This is translational control.
89
What other method can cause phase variation in pilC?
Slipped strand mispairing
90
How is Opc expression controlled by homopolymeric tracts?
1. polyC tract in the promoter sequence.
2. Depending on the number of C you can get high, medium or no gene expression.
3. It is done and is reversible by errors and slipped strand mispairing during DNA replication.
4. Changing the number of C changes the binding efficiency of RNA polymerase binding.
5. This is transcriptional control.
6. Too many or too few Cs cause no expression as it is too different for the RNA pol to bind.
91
What is Opc?
1. An opacity protein
2. Transmembrane
3. Adhesin and invasin
92
What is antigenic drift?
1. The accumulation of point mutations over time.
2. This is caused by poor proof reading in bacteria during DNA replication.
3. Advantageous mutations are selected for.
4. The accumulation of mutations change the appearance of the antigen.
93
What bacteria is a good example of antigenic drift?
1. H. influenzae
2. Especially outer-membrane protein P2 and HifA in the Hi pili
94
What is important to remember about all the different bacterial antigens?
1. All the proteins are changing simultaneously and are not independent of each other.
2. When 1 specific antigen or form of antigen is expressed it could change the dynamic and become the dominant adhesin.
3. The dynamic bacteria have with our cells is ever changing and has changing impacts on cell signalling depending which factors are present.
4. The antigenic and phase variation determines the niche the bacteria can colonise and the receptors they can bind.
95
How are a large range of bacterial phenotypes created?
1. 1 in 100 division causes a change in phase variation.
2. This occurs in 100s of different genes.
3. This creates a complicated population within one strain of bacteria due to the large range of possible phenotypes
96
Can virulence factors co-operate with each other?
yes
97
What are some examples of N. meningitidis virulence factors co-operating?
1. When pili is expressed the toxicity mediated by LPS is increased.
2. When pili is expressed with Opc there is more invasion of the nervous system and crossing of the blood brain barrier.
