Field of Science

Questions about CRISPR-mediated phage immunity

Thursday's post described the hypothesis that bacteria might use gene transfer agent particles to inoculate other cells in the population with fragments of phage DNA, and outlined an experiment to test this.  Now I'm realizing that I need to know a lot more about the kind of immunity I should expect to see if this GTA-as-vaccine hypothesis is correct.

Simplistic outline of the experiment:
  1. Infect GTA-producer strain of R. capsulatus with phage under conditions where the infection is inefficient and few cells lyse.
  2. Remove cells and debris from the culture, to get a supernatant that will contain GTA particles and (unavoidably) some phage particles.
  3. Expose a new culture to the supernatant so cells obtain DNA from the GTA particles, again under conditions where successful phage infection will be minimized.
  4. Wash the surviving cells to remove phage (as much as possible).  Allow time for CRISPR formation if needed.
  5. Expose the cells to a titer of phage suitable for selecting resistant cells.  As a control, also expose cells not treated with GTA.  
  6. Plate to isolate colonies from surviving cells.
  7. Test the survivors for phage resistance.
  8. Compare the frequency of resistance in treated and control cultures.
  9. Test resistant colonies for CRISPR changes.

Things I should find out before I do the experiment:

1.  How efficiently do introduced DNA fragments give rise to CRISPR spacers?  If this efficiency is too low relative to the background rate of phage resistance, I won't be able to detect an effect.  This paper (Hynes et al. 2014, thanks to @AprilPawluk for pointing me to it) might let me estimate the  efficiency.  They exposed cells to a mixture of infectious and damaged phage (damaged by a restriction enzyme in the cell or by prior UV irradiation) at a multiplicity of infection (moi) of 0.1-0.2, and then examined the resulting confluently lysed lawns for phage-resistant colonies.  Unfortunately they only report relative changes in frequency of resistant cells (maxima 16-fold and 6 fold for restriction and irradiation respectively), but in their Methods they mention that the highest frequencies of resistance they observed were about 10^-6.  I don't know if this is for naive cells or pre-exposed cells, but even if it's for naive cells, the max frequency of CRISPR resistance I might expect is only about 10^-5.  This would not pose a detection problem, but it would limit the population-level benefits of the proposed vaccine system.

2.  What fraction of the survivors of a phage infection are genetically resistant, and what fraction of phage resistance arises by non-CRISPR mechanisms?  If most survivors are just lucky, then it might be a lot of work to identify the genetically resistant ones.  In the Hynes et al. experiments, all of the colonies were genetically resistant, and all had new CRISPR spacers.  However this might be quite different for different phages.  If most resistant cells have altered phage receptors rather than phage-specific CRISPR spacers, the effect of GTA-mediated CRISR resistance will be hard to detect.

3.  How quickly does CRISPR-mediated phage resistance arise after exposure to phage DNA?  I don't know.  Cells in the Hynes experiment might have had one or two lytic-cycle durations between being infected by the damaged phage and being infected by an infectious phage.

4.  What fraction of phage infections are abortive and thus could lead to CRISPR immunity to subsequent infection?  Inspired by the Hynes experiment, I can increase abortive infections by UV-irradiating the phage lysate.  (I know how to do this well from previous work.)

5.  How efficiently do phage spacers prevent phage infection?  April Pawluk (via Twitter) says they reduce infection by several orders of magnitude. In the Hynes work acquisition of a phage-derived CRISPR spacer enabled cells to form a colony in a sea of phage.

OK.  I have lysates of five sequenced R. capsulatus phages (from Dave Bollivar via Tom Beatty), and I have the R. capsulatus strain these phages were isolated on, as well as GTA-producing and recipient strains. Time to get to work!

No comments:

Post a Comment

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS