Imagine you were surrounded by enemies, and had a choice of two defenses. You could send out a targeted laser to kill each bad guy, or instead, put in a bit more effort to form a clever disguise so that none of them could find you. How many baddies would there have to be to make the disguise worth the extra effort?
I recently read an article; The effect of phage genetic diversity on bacterial resistance evolution. The authors deliberately evolved viruses that infect bacteria, to see if exposure to a more diverse group of these viruses would result in a change to the bacteria’s defense strategies.
Pseudomonas aeruginosa (pronounced “Soo-da-moan-us orig-in-osa”) is a bacteria that causes disease in both plants and animals, including humans. It’s important for us to know more about it because it can have a negative impact on agricultural industries, and it’s able to form multi-cellular structures called biofilms to develop chronic infections on medical devices like catheters. It’s an opportunistic pathogen, and one of the most common species associated with infections of immune-compromised or vulnerable patients such as those with cystic fibrosis.
The ‘bad guys’ in this story are bacteriophages, or “phages” for short. Phages are viruses that specifically infect bacteria. They do this by attaching to the outside of the bacterial cell and injecting their DNA, before using the cell’s own machinery to replicate themselves. The newly formed phage then burst out of the cell to each infect their own targets, killing the host cell in the process. Click here to see a fun video explaining how this happens & why studying phage is so important, as well as a sneaky peek at a P. aeruginosa chronic infection that was treated with phage therapy!
A phage called DMS3vir was used in this study and is specific to P. aeruginosa, but bacteria can employ a variety of different strategies to protect themselves from infection.
One of these strategies is a general resistance mechanism called surface modification. Bacterial cells can make changes to the receptor proteins of their surface (which phages use to recognise the cell), to mask their identity as a target and evade infection. There are also more targeted strategies where the cell actively damages the phage particle, such as the CRISPR-Cas adaptive immune system.
CRISPR-Cas (or CRISPR) is utilised when infecting phage are recognised by the bacteria; the cell uses an enzyme called Cas to cleave the viral DNA, and a short sequence is then incorporated into a special region of the bacterial cell’s genome (known as CRISPR). This short sequence is called a spacer, and bacteria can accumulate them over time. Spacers can be used to help the bacteria recognise and disable infecting phage sooner, by creating a guide to allow Cas enzymes to become targeted towards the phage which have that specific sequence. This becomes a type of immune memory to allow for a more precise and prompt immune defense. You can learn more about CRISPR-Cas and how this targeted mechanism is being developed for gene editing here.
While CRISPR is an effective targeted strategy, the spacers acquired are specific to a particular phage’s DNA, and will only work against phage that also have that sequence. So bacteria have to accumulate multiple spacers to have a broader range of immunity against different phage, and the cost of using this defense increases with each spacer they accumulate. By contrast, surface modification has a fixed cost, as one change to the receptors on the cell surface will prevent recognition by a variety of phage. This could make it a very efficient system when there are a lot of different phage to defend against.
P. aeruginosa has been shown to readily evolve CRISPR-based immunity to phage DMS3vir in the lab, but the communities of bacteriophages we can isolate from the environment are likely to be far more diverse, which can impact the way that bacteria protect themselves. The authors of this article wanted to know whether phage diversity has an effect of the development of CRISPR-based immune defense by the host bacteria, and hypothesised that surface modification will be favoured in conditions when phage diversity is high.
They started by evolving the phage DMS3vir to be more diverse. To do this, they mixed the phage with a culture of host bacteria which lacked a DNA repair gene; they thought that if the bacteria was more likely to mutate, then the phage resulting from infections of that bacteria may be even more diverse than those evolved using a ‘normal’ or wild-type strain of P. aeruginosa.
After letting the phage infect the bacteria for 24 hours, they killed and removed any remaining bacterial cells before using the new collection of phage to infect a fresh bacterial culture. Each time they did this, the resulting population of phage was able to accumulate mutations and become more diverse. They repeated this for 17 days using 12 separate replicate experiments. At the end of the evolution experiment, they also took one purified phage (referred to as clonal phage) from each diverse population for use as a control to show that it was the diversity of phage bringing about any change in the bacterial response, not the evolved state of the phage alone.
They then obtained DNA sequences for each of these 12 diverse populations and clonal phage, as well as the ‘ancestral’ phage that they started with, to determine how many single-nucleotide polymorphisms (changes), or SNPs, had occurred as a result of the phages evolving. As shown in Figure 1A, the diverse phage populations had a much higher frequency of SNPs than the clonal phage, indicating that each type of phage within the diverse populations is likely to have different SNPs to each other.
They then tested each of these phage against bacteria that had some CRISPR-based immunity already. For the bacteria, they used lab strains that each had a unique spacer against phage DMS3vir (1-12 in Figure 1B), as well as 6 strains that each had 2 spacers & would be harder for the phage to infect (combined, ‘2sp BIM’), and one surface-modified mutant (‘SM’). While none of the phage could infect the 2-spacer strains or the SM mutant, the diverse phage had a greater infection capability than its paired clonal isolate against each of the single-spacer strains.
The authors wanted to see how phage diversity affects which defense strategy the bacteria will develop. For this, they used wild-type bacteria with no existing CRISPR spacers for the DMS3vir phage. After exposing this bacteria to infecting phage for 3 days to allow bacterial defenses to come into play, they then added them to both a wild-type phage and a modified phage that had a gene to disable CRISPR-based immunity. If the bacteria was resistant to both of these phages, the authors assumed it had developed a surface modification, whereas resistance to the wild-type but not the anti-CRISPR modified phage was assessed to be due to a CRISPR-based immunity. Figure 2B shows that bacteria challenged by a diverse phage population utilised surface modification (SM) more often than when challenged by clonal phage, whereas bacteria challenged by a clonal phage population almost always developed a CRISPR-based immunity.
It was also observed that when bacteria challenged by a diverse population did develop CRISPR-based immunity, they were more likely to acquire multiple rather than single spacers. As shown in Figure 3A, the majority of bacteria challenged by one type of phage (clonal phage) only acquired a single spacer, as that was all that was required, whereas exposure to a more diverse population of phages more often resulted in multiple spacers being accumulated by P. aeruginosa.
Overall, this study showed that an increased phage diversity promoted surface modification as a form of generalised resistance to the phage. It was also demonstrated that when CRISPR-based immunity was developed, a diverse phage population increased the likelihood of multiple spacers being acquired in the bacterial host.
While I found the research interesting, I found the ‘scoring’ for type of bacterial resistance to be based on a huge assumption that other strategies were not coming into play. Bacteria have many different ways to protect themselves against phage, and the authors don’t really mention how they eliminated other mechanisms as possible explanations for what they observed.
I also don’t feel that a lab-evolved population of bacteriophages is ever going to be an accurate representation of the diversity in an environmental sample; while this team had a readily available stock of phage DMS3vir in the lab, I would have been interested to hear their justification for not isolating P. aeruginosa bacteriophages from the environment to use in testing the effect of phage diversity either alongside or instead of their lab-evolved phage.
What do you think?
1. Broniewski, J.M., et al., The effect of phage genetic diversity on bacterial resistance evolution. The ISME Journal, 2020. 14(3): p. 828-836.
2. Wu, W., et al., Chapter 41 – Pseudomonas aeruginosa, in Molecular Medical Microbiology (Second Edition), Y.-W. Tang, et al., Editors. 2015, Academic Press: Boston. p. 753-767.
3. Khatoon, Z., et al., Bacterial biofilm formation on implantable devices and approaches to its treatment and prevention. Heliyon, 2018. 4(12): p. e01067.
4. Bassetti, M., et al., How to manage Pseudomonas aeruginosa infections. Drugs in context, 2018. 7: p. 212527-212527.
5. White, H.E. and E.V. Orlova, Bacteriophages: Their Structural Organisation and Function, in Bacteriophages – Perspectives and Future, R. Savva, Editor. 2019.
6. Westra, Edze R., et al., Parasite Exposure Drives Selective Evolution of Constitutive versus Inducible Defense. Current Biology, 2015. 25(8): p. 1043-1049.
7. Westra, E.R., et al., Evolution and Ecology of CRISPR. Annual Review of Ecology, Evolution, and Systematics, 2016. 47(1): p. 307-331.