Phase variation and phage resistance

This is an exercise aimed at helping you visualise phase variation, as well as consider the role of phase variation in phage resistance of Campylobacter jejuni. Before starting this tutorial, you should read up on C. jejuni on the pathogens and disease page as well as the phase variation page.

Note: you can download the simulator and activity work sheets from the 'all infection resources' page.

Background: bacteriophage

Bacteriophage are viruses that specifically infect and replicate within bacterial cells. In order to infect a target bacterial cell, the cell must express on its surface a molecule (receptor) complementary to the viral anti-receptor. Secondly, the surface molecule most have been modified suitable to be recognised by the phage anti-receptor. Only then can the phage infect the cell and begin to replicate (see diagram below).

phage life cycle

Note: further information on phage biology can be found on the 'bacteriophage' page of the VGEC.

With antimicrobial resistance becoming ever more a huge problem, scientists are exploring alternative therapies for the treatment of bacterial disease. The specificity of phage to bacterial cells makes them amenable for use in such treatments.

Resistance of Campylobacter to phage infection

The Campylobacter jejuni capsule is extremely diverse. Both components of the capsule and enzymes that modify key residues are prone to phase variation by SSM. This produces massive capsular diversity, producing 47 different serotypes.

Bacteriophage F336 specifically infects C. jejuni and has previously been explored as a potential phage therapy for the treatment of Campylobacter induced gastroenteritis. Phage F336 infects C. jejuni cells using components of the bacterial capsule as an anti-receptor.

In order for phage F336 to recognise and invade the host cell, its capsule must be appropriately modified. Phage resistant strains of C. jejuni however have since been characterized. One partially charecterised mechanism for this resistance is the phase variation of genes encoding capsule-modifying enzymes. The diagram below shows the modifications that confer phage resistance or phage sensitivity.

 

phage res 2

Above -  Phage sensitivities of cells expressing different capsular modifications. In the table, the first, second and third numbers represent genes one, two and three respectively (see below). The number 1 indicates an ON state, whereas 0 indicates and OFF state.

Introduction to the simulator

Accompanying this activity you have been provided with a simulator programme. This simulator will model phase variation of C. jejuni genes.

When you start the simulator you will begin with a single bacterial cell.  This cell has three phase variable genes each encoding a capsular modification enzyme.

The cells will begin to divide, and some of them will mutate at a given rate. The cells will continue to divide until there are too many on the screen to continue growing.

In the simulator, each gene is represented by a number (I.E gene 1 is represented by the first number, gene 2 the second number and so on).

If a gene is switched on, its corresponding number will be 1; if it is switched off the number will be 0 (For example, if expression of all 3 genes is switched on, the number will be 111). Running totals of each genotype are provided next to their assigned colour.

Genes in the simulator-

  • Gene 1 cj1421
  • Gene 2cj1422
  • Gene 3cj1426

Simulator

 

 

 

Note: many browsers require you to enable Java to use the simulator. Failing this you can download the simulator file and accompanying activity from the 'all infection resources' page.

Exercises

Exercise 1

Run the simulator on the 'very high' setting a few times, making a note of what the final population looked like each time.


• Did you get the same results on both runs?
• Why do you think this is?

Comments on exercise 1 (opens in new window)

Exercise 2

Look at the different capsular compositions and their respective resistance phenotypes.

• Which combination of modifications are essential for phage binding?
• Which genes encode the enzymes for each modification? As a result, which phasotype matches to which modification (A-E)?
• Using the answers above, what is the phenotype of the 1-0-0 phasotype?

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Exercise 3

Run the simulator at three different mutation rates and jot down how the final population differs.

• How does varying the mutation rate affect the proportion of cells capable of resisting phage infection?
• How does varying the mutation rate affect the genetic diversity of the final population?
• What could an advantage of phase varying these genes, given that phage infection is undesirable?

Comments on exercise 3 (opens in new window)

Exercise 4

Scroll back through the simulation, noting what happens to cells that mutate earlier, and later on.

• Do mutations that occur early in the simulation have more or less effect on the population structure than mutations that occur later?                                              
• Look at the cells with the highly resistant (1-1-1 specifically) phasotype. Which cells do they emerge from most often? Is this always the case?

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Exercise 5

Now lets consider the use of simulators in researching PV and phage resistance in real life bacteria.

• How realistic do you think the simulator is?                                              
• What advantages do you think using a simulation can have over real experiments?                                                                                                                                           • Do you think phage therapy is a suitable treatment option for Campylobacter infection? What further research may be required?

Comments on exercise 5 (opens in new window)

Take home messages from the tutorial

Campylobacter jejuni is a huge burden on human health, particularly in the developing world due in part to the limited availability of therapeutics and poor hygiene. C. jejuni can be infected by bacteriophage which recognise and bind modifications to the organisms capsule. Genes encoding enzymes facilitating these modifications undergo phase variation, allowing C. jejuni to evolve enormous diversity in bacterial populations. This diversity allows C. jejuni to respond to rapidly changing environments.

Further information on Campylobacter jejuni phage infection and phase variation can be found in the paper by Sørensen et al. (2012)

 

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