Lecture 5 - evading immune system, phase variation, experimental approaches to understand regulatory mechanisms, effects of phage an antigenic variation

Question 1

Give some examples of some physical chemical and anatomical barrier to resisting infection

Answer 1

• Normal flora compete with pathogens
• Removal of particles including microorganisms by rapid passage of over over cillia in the nasopharynx
• Flushing of the urinary tract prevents colonisation
• Stomach acidity (pH=2) inhibits microbial growth

Question 2

What are the features of helicobacter pylori?

Answer 2

• Colonises gastric mucosa
• only active at low pH
• causes stomach ulcers and gastritis
• asjust the pH of their microoenvironment by the pH dependent activity of urease (hydrolyses urea to ammonia, raising the pH)

Question 3

What is the chemical reaction for the hydrolysis of urea to ammonia?

Answer 3

H₂N-C(=O)-NH₂ + 3H₂O -> 2NH₄⁺ + 2OH^- + CO₂

Question 4

What pathogens other than helicobacter pylori utilise urease and why?

Answer 4

To exploit nitrogen sources

• Proteus mirabilis: inducible (urea)
• Klebsiella sp: inducible (nitrogen starvation)
• Precipitates, kidney stones

Question 5

What strategies do bacteria use to evade the host defenses?

Answer 5

• Hide, enter cell and stay intracellular
• Modify, blok, resist host defences
• Evade innate immune response
• Evade adaptive immune response
• Molecular mimicry
• Phase vairation
• Antigenic variation
  *

Question 6

Once infected, how does the human body respond to infection?

Answer 6

If have no innate immunity -> disease

Otherwise leads to adaptive immunity, following recovery and reinfection the specific immunological memory is triggered so that no disease

Question 7

What is the involvement of pathogen PAMPs in innate immunity?

Answer 7

• All pathogens that enter our body have unique pathogen associated molecular patterns (PAMPs)
• Related to the innate immune resonse
• Macrophages, dendritic cells survey for PAMPs
• Non-specific approach to dealing with the pathogen
• e.g. peptidoglycan of gram- bacteria

Question 8

How does the capsule protect against phagocytosis?

Answer 8

Blocks recognition of PAMPs by the innate immune system

e.g. The Vi capsular polysaccharide enables salmonella enterica serovar typhi to evade the immune system

Question 9

How do pathogens use mimicry to evade the immune system

Answer 9

• mimic host cells
• either by host protein function mimicry or surface decoration to mimic the host

e.g. Human neutrophils recognise sialic acid as self.

• GroupB Streptococci sialylate capsule
• Haemophilus influenzae sialylates LPS (LPS major PAMP, TLR4)

Question 10

What are the basic features of the adaptive immune system?

Answer 10

• Highly specific and retains memory
• Antibodies are raised in response to specific antigens
• T Cells: regonise fragments of pathogens (particular epitopes trigger immune response - if antigen looks different or is no longer present there is no longer an antigenic response, by translational fusion)
• B cells: recognise intact pathogen molecules

Question 11

Why can we not say that all cells in a clonal population are identical even thoug derived from same parent?

Answer 11

Stochastic variation (due to weak promoter, some cells may express and some may not)/threshold responses

Antigenic and phase variation (digital repsonses)

Question 12

How can you experimentally determine whether a gene is being expressed?

Answer 12

Use reporter genes

Transcriptional fusion: place the reporter gene downstream of rbs

Translational fusion: place the reporter next to the rbs so that the proteins are fused together

This experiment is dependent on the regulatory information, can determine whether regulation occurs at the mRNA or the translational level

Question 13

What reporters can be used in a reporter fusion experiment?

Answer 13

LacZ: enzymatic assay, liquid (population average); enzymeatic reaction, colony

Gfp: microscopy or flow cytometry (individual cells) fluorimeter (population average)

Question 14

What are the features of heterogeneous expression in a population and how is it identified experimentally?

Answer 14

Experimentally

• Uses reporter fusion (transcriptional reporter), and at the intermediate level of inducer the bacterial (e.g. A.tumefaciens) gene (e.g. VirE) is differentially expressed in a population

Features

• Stochastic
• Not heritable
• Not phase variation

Question 15

What is antigenic variation and how is it used to evade the immune system?

Answer 15

• Variants of a protein each with differnet antigenicity
• Allow bacteria to retain function as different antigens ellicit different immune response
• Often cell surface structures/proteins

Question 16

Describe the involvement of antigenic variation in escaping the immune system?

Answer 16

1. Immune system recognises one protein and begins to clear the infection
2. The population is 1/2 one protein and 1/2 its antigenic variant
3. Once the first is gone the second dominates

Pathogens can have thousands of different antigenic variants for evading the immune system, need a large population for this to be possible

Question 17

Give an example of antigenic variation in evading immune system

Answer 17

Borrelia hermsii

• Uses gene conversion on surface lipoproteins
• Leads to relapsing fever
• Spirochaete species of the borrelia genus

1. Antibody mediated clearance
2. Recombination between expression plasmid and storage plasmid (lots of variation)

Question 18

What structures does antigenic variation affect?

Answer 18

Mostly antigenic surface structures including:

• flagella
• outer membrane proteins
• LPS (O-antigen)
• fimbriae

Also true for phase variation

Question 19

What are the features of phase variation?

Answer 19

• Heritable but reversible expression states
• On to Off; Off to On
• Very common in some species
• Mostly surface proteins/structures

Question 20

When does stochastic variation occur?

Answer 20

• Happens at certain frequencies without any regulation
• But environmental factors may impose regulation, although bacteria may not respond in the same way to the same environment

Question 21

How can phase variation be experimentally demonstrated?

Answer 21

Reporter fusion on a plate

1. Have a reporter of a phase variable gene e.g. with LacZ, get blue white colonies
2. Streak from blue/white colonies
3. May be taken for contamination when actually phase variation

Question 22

What are the molecular mechanisms of phase variation?

Answer 22

• Reversible
• Frequency higher than normal mutation rate
• DNA sequence change
  
  • Gene conversion (antigenic variation)
  • Site specific recombination (PV)
  • Short sequence repeats (PV) - can be identified by genome sequencing
• or DNA methylation state change (PV) at specific sites in the genome

Question 23

What are the features of short sequence repeats of phase variation?

Answer 23

• Mono-, di-, tri-, tera-nucleotide repeats
• Slipped strand mispairing/role of mismatch repair
• Recombination
• Varies between species, tract length (if repeats are long enough, the DNA polymerase will stutter and make mistakes, repeats have the flexibility to expand/contract slightly, important where the difference occurs)
• Can have transcriptional or translation effect

Question 24

What is the process of phase variation by short sequence repeats which affects transcription?

Answer 24

1. Spacing is key when the activator need to touch RNA polymerase for gene expression
2. -10 to -35 spacing very important for binding site
3. If the number of SSR changes (slipped strand mispairing/mismatch repair/recombination) won’t get transcription

Question 25

How do short sequence repeats and slipped strand mispairing lead to phase variation by translational effect?

Answer 25

Have an area followng the promoter made up of lots of single nucleotide repeats. If one is deleted this could lead to the formation of a premature stop codon and the formation of a truncated protein.

Question 26

What is the purpose of the O antigen in v.cholera?

Answer 26

Protects bacteria against bacteriophage

Question 27

How is phase variation controlled by site specific recombination? (SSR)

Answer 27

Get recombination between inverted repeat recombinase recognition sequences (IRR and IRL) results in an inversion of a fragment of DNA, for example in a region with a promoter e.g. lyseria Need a short piece of DNA that can flip on the genome. Need enzymes that clearly recognise sequence and then flip DNA around. Affects expression e.g. Fim operon

Question 28

Give an example of site specific recombination in phase variation What are the proteins that allow the switch frequency to change?

Answer 28

**Type I fimbriae (E.coli) phase variation** * 2 site specific recombinases FimB and FimE (tyrosine recombinase) * 9bp inversion repeats * FimE promotes on to off, Fim B equal * Ratio of the protein allows switch frequency to change Proteins which allow the switch frequncy to change: environmental/regulatory input, IHF, Lrp, Fis, sialic acid, temperature, ppGpp, RcsB (biofilm), SlyA (virulence)

Question 29

Give an example of phase viration in virulence

Answer 29

1. Used murine model bladder system with indepenent innoculations of strains into different mice. 2. WT E.coli strain F11 and phase locked variants F11-ON or F11-OFF (Fim expression 3. Lock on (by destroying recombinase sites) mutant as good at colonising as the WT, Lock off mutant less good However this does not demonstrate phase variation, instead... 1. Coinnoculations with F11 and each phase locked mutants 2. Mice were transurethrally coinnoculated with a bacterial suspension (E.coli WT strain F11 and F11-ON at 1:1 ratio) (E.coli F11 and F11-OFF at 1:1 ratio) 3. Lock off mutant does worse compared to WT, On does equally well If phase variation was important, would see both same effect in both (becuase phase variation has been turned off) **Conclusion:** Type I fimbriae is important for bladder colonisation not PV itself in this assay

Question 30

What are the features of methylation dependent phase variation?

Answer 30

* Epigenetic mechanism * Identified in E.coli, salmonella * Involves: * DNA binding proteins * Deoxyadenosine methylase (Dam) * methylates adenine in GATC sequences not part of the restirction modification system * Reversible but heritable * No enzyme to demethylate DNA * Needs DNA synthesis to switch from On to Off

Question 31

What are the features of the enzymes Dam?

Answer 31

* Deoxyadenosine methyltransferase * Methylates adenine GATC sequenes in DNA * Processive enzyme - both hemi and unmethylated DNA is a substrate * No de-methylating enzyme in E.coli (until replication) * Integral part of E.coli biology and not part of the restriction/modification system * Always present (100-300 molecules per cell)

Question 32

What are the basic principles of methylation dependent phase variation?

Answer 32

1. DNA methylation-dependent binding of regulatory protein 2. Binding of the regulatory protein blocks Dam, site stays unmethylated. no expression * If Dam binds first, the DNA binding protein cannot bind, expression continues

Question 33

Give an example of phase variation by methylation

Answer 33

**Phase variation by Lrp and Dam: Pap operon** Two binding sites for Lrp, both with one GATC. Mutually exclusive binding of LRP between two sites. If LRP binds DAM binds to the other site and methylates it - no expression of papI if Dam binds to GATC-1 site by exclusion of the GATC-II site by LRP expresssion of papB/pilin and papI ON if DAM binds first to the GATC-II site Non-reversible (no enzyme to demethylate DNA) **Phase variation of CDS** OxyR binding to GATCs prevents methylation and there is no expression Dam binding to GATCs methlates DNA opening it up for synthesis if OxyR is deficient

Question 34

How is pap methylation dependent phase variation random but regulated?

Answer 34

1. PapI binds to Lrp increasing its affinity for GATC-I and allowing it to compete with Dam. PapI required for LRP binding at 12,3 ON phase 2. GATC is Dam methylatoin site 3. PapB (positive for PapI) binds near the promoter for PapI and activates PapI transcription, forming a positive feedback loop 4. cAMP-CAP essential second activiator 5. Environmental factors contribute to variation: glucose level and temperature, ensure that Pap doesn't get made even if it should have been acording to its epigenic state

Question 35

What were the methods of Khandige to show the model of pap fimbriae phase variation?

Answer 35

* Hfq - protein that is required for the stability of all sRNAs * LacZ as a reporter for expression (and some Gfp fusion) * FLAG tag = alternative to His tag * Anti-fimbrial antibodies, stained (to stain bacteria) * Flow cytometry * In vitro model system (bladder and kiney cell lines) * Attachent and cell invasion * alllowed cells to attach and invade, count bacterial numbers on the outside and inside. cells + for gentamycin, kills bacteria but doesn't kill internalised cells and plate up * Distinguish between the role of Type I and Pap using a competetion for binding with mannose (type I) by using the Fim present on yeast surface

Question 36

What did Khandige show about P-fimbriae phase variation?

Answer 36

1. **Hfq impact on bladder cell lines**: shows sRNAs important as when there is Hfq the levels of CFU per well decreases in adhesion and in invasion. Complemented strain (with FLAG tag) works 2. **Heme- and yeast cell agglutination with and without the addition of alpha-D-mannose:** Yeast has a receptor for bacteria so get clumping. Used a with plasmid strain, vector control strain with PapR, recomplemented strain - plasmid strain: shows clumping resistance to mannose therefore not type I dependent, PapR affects PAP regulation. In absence of PapR get clumping of RBC. Clumping in yeast cells with the receptor can be competed out with mannose. Type I substrate. 3. **PD07i bladder cells and IMCD3 kidney collecting duct cells:** strains were either left untreated or treated with 3% alpha-D-mannose and used for infection. Bacterial adhesion measured. See a decrease in adhesion in certain conditions. More PapR = less adhesion, PapR is a negative regulator for adhesion. Stats significant. 4. **Flow cytometry:** immunolabelled with alpha-PapA and apha-Fim antibodies used to detect the extent of P- and type 1 fimbriated cells. Results suggested PapR decreases expression of PAP fimbriae. 5. **PapR mediated PapI repression:** beta-galactosidase assays, papA-lacZ transcriptional fusion. Showed as soon as PapR was expressed get a decrease in PapI, showed if locked on strain is not dependent on PapI, no effect from PapR. 6. Western, northern blots show PApI GFP mRNA affected Conclusion: new layer of complexity of PapR-mediated modulation of P-fimbriae phase variation PapR regulation

Question 37

What is the second role of PapB?

Answer 37

increase cross talk to the Sfa operon (also Dam-dependent PV) decrease cross talk to type I Environmental input directly into pap, sfa (glucose, cAMP-CAP) and into type I

Question 38

What is the sensitivity of sfa and pap to glucose?

Answer 38

When high levels of glucose, neither are made. Once depleted, allows expression of Type I

Question 39

What are the complex regulatory negworks of PV down to?

Answer 39

Multiple binding sites on closely related operons

Question 40

What is the biological significance of phase variation?

Answer 40

Depends also on the role of the protein * evade the immune system * PV of adhesins (potentially to facilitate dispersal) * Alters phage-bacterial interactions (LPS, OMP) * Althers host-pathogen interactions

Question 41

What structures does phases variation affect?

Answer 41

Mostly antigenic surface structures including: * flagella * outer membrane proteins * LPS (O-antigen) * fimbriae