by Januka S. Athukoralage, University of St Andrews

Abstract

Viruses can infect archaea and bacteria (collectively called prokaryotes) and just like humans prokaryotes have powerful adaptive immune systems, called CRISPR systems, that target the nucleic acids of these invading organisms. An adaptive immune system is one which is able to form genetic memories of an infection so as to mount a rapid and more efficient defence upon reinfection. This article discusses the events that follow a viral infection and reveals how an extended period of self-targeting, a side-effect that is otherwise associated with immunity, is minimised so as to avoid cell death.

Article

Sniffling, I like to imagine that archaea and bacteria (prokaryotes) are also burdened with their equivalent of the ‘flu’ season. Whether or not that is the case, it is certainly true that, just like humans, these organisms are armed with an adaptive immune system. In prokaryotes, protection against viruses is provided by the CRISPR system, which is able to form genetic memories and target viral invaders. CRISPR is an abbreviation for Clustered Regularly Interspaced Palindromic Repeats and is taken from when it was first identified in prokaryotic DNA as repetitive sequences of unknown function. It was later discovered that genetic memories of infection were inserted between these repeats which, together with a host of proteins called Cas proteins, form the basis of adaptive immunity in prokaryotes. Currently six types of CRISPR system are known, but CRISPR type III, the focus of this article, is believed to be the most ancient and is arguably the most complex. The type III CRISPR complex is a multi-subunit protein complex capable of degrading both DNA and RNA that is identified to be of viral origin. RNA is a short-lived copy of DNA that is decoded by cells to make proteins necessary for growth and survival.  However, the real prowess of type III CRISPR lies in its ability to string together 4 to 6 molecules of ATP; the fundamental energy molecule of life, and synthesise thousands of powerful signalling rings called cyclic oligoadenylates. These signalling rings, which vary in size between organisms, bind to and activate powerful non-selective RNA cleaving enzymes (ribonucleases) in order to initiate a potent anti-viral response within cells (Fig.1).

Figure 1. The type III CRISPR complex detects and degrades viral RNA while simultaneously synthesising anti-viral signalling rings to activate RNA cleaving enzymes (ribonucleases).

But targeting RNA is not without consequence. There is equal likelihood of targeting the product of one’s own genes, essential for growth and survival, as there is of shredding the genetic material of the invader. This is especially true when the virus is cleared from the cell, at which point ribonucleases will solely degrade self RNA. This can significantly decrease metabolism and hamper cell recovery, ultimately leading to cell death (Fig.2). This problem arises because the signalling rings produced during infection persist in the cell and continue to activate ribonucleases. Therefore, an off-switch; an enzyme to degrade these rings must exist.

Figure 2. Signalling rings activate non-selective RNA cleaving enzymes (ribonucleases) with potential for self-harm.

Recently, to find this off-switch, we looked at all the proteins found within the archaeal organism Sulfolobus solfataricus. Using column chromatography, a method by which proteins can be separated based on desired characteristics, we purified and identified the enzyme responsible for degrading the signalling ring. In addition, computational biology enabled us to identify another less potent ring degrading enzyme in S. solfataricus, as well as to confirm the presence of these enzymes across the prokaryotes.  In respect of their unique function, we named this novel class of enzyme ‘ring nuclease’.

Ring nucleases first bind and linearise the signalling ring, and then cleave it into products that no longer activate ribonucleases involved in immunity (Fig.3). Hence, these enzymes function to restore the cell to its pre-anti-viral state and are likely essential for cell recovery and avoiding cell death.

Figure 3. Ring nucleases degrade signalling rings to deactivate RNA cleaving enzymes (ribonucleases) and minimise self-harm.

The discovery of ring nucleases poises a unique opportunity to set a prokaryotes immune system against itself. Many pathogenic bacteria harbour functional type III CRISPR systems and inhibiting the activity of ring nucleases, while stimulating ring activated ribonucleases with unbreakable rings, may enable us to kill these organisms by harnessing the destructive power of their immune systems. Thus, the “Achilles heel” of type III CRISPR may now give rise a new generation of antimicrobials for targeting multi-drug resistant bacteria.

Further reading:

• Athukoralage, J. S., Rouillon, C., Graham, S., Grüschow, S. & White, M. F. Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate. Nature In press, (2018).
• Rouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S. & White, M. F. Control of cyclic oligoadenylate synthesis in a type III CRISPR system. Elife 7, 1–25 (2018).
• Niewoehner, O. et al. Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543-548 (2017).
• Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G. & Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605–609 (2017).

About me

I am a second year PhD student supervised by Professor Malcolm White at the University of St Andrews. My research includes mechanistic aspects of type III CRISPR immunity and CRISPR anti-viral signalling. My aim is to further our understanding of the consequences of unregulated CRISPR anti-viral signalling and develop this into a genetic tool for medical and industrial applications.