In the waters of volcanic hot springs, there is a heated arms race going on between hyperthermophiles, organisms which thrive at high temperatures and invading viruses. These fascinating single-celled organisms – archaea – have evolved diverse, intricate, CRISPR-Cas systems to eradicate invading viruses. In turn, the viruses have developed complex strategies to evade host defences and to overcome competing viruses. Here, we take a closer look at the research of Professor Xu Peng and Professor Roger Garrett from the University of Copenhagen, who have been exploring the intricacies of this microscopic battle for dominance.
Volcanic hot springs are a geological phenomenon famously found in locations such as Yellowstone National Park. These geothermal features are produced when water meets rock heated by magma in volcanic areas, reaching temperatures of over 100°C. These extreme temperatures are not the most welcoming of habitats, but some organisms, known as hyperthermophiles, are hardy enough to bear it and thrive. Microbial communities in hot springs are dominated by the fascinating archaea (single-celled organisms that are similar to, but distinct from, bacteria) and the archaeal viruses that infect them.
Water is not the only thing getting heated in these hot springs, however. There is a constant arms race between the archaea and archaeal viruses – and even between different competing archaeal viruses. This arms race is the result of many of years of adaptation, with archaea evolving to defend themselves against viral infection and viruses evolving to counteract these defences. The research of Professor Roger Garrett and Professor Xu Peng from Copenhagen University, Denmark, and their collaborators, has deepened our understanding of this struggle for dominance amongst these hyperthermophiles.
Archaea immunity: the CRISPR-Cas system
Archaea are single-celled organisms known as prokaryotes. They have evolved a range of anti-viral defence mechanisms. One such defence is CRISPR-Cas, an immune system that uses specialised molecular tools that not only recognise but cut up the genes of assaulting viruses. An acronym for Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR refers to specific regions of DNA in the genome of the prokaryote. CRISPR-Cas systems are divided into two classes, based on the types of Cas protein, and the mechanisms, they use to eradicate invaders. Class 1 contains types I, III, and IV; Class 2 contains types II, V, and VI. These six types of CRISPR-Cas system are further divided into 33 lettered subtypes.
The process of the CRISPR-Cas immune response against viruses takes place in three steps. First, short sequences (spacers) derived from the DNA or RNA of invading viruses are inserted into CRISPR regions which serve as a memory bank of previous infections. These are then transcribed into CRISPR RNA (cRNA), forming a complex with Cas proteins. Finally, these complexes recognise viruses with gene sequences complementary to the spacer, and can inactivate them by chopping up the virus genome.
Counteracting this threat, viruses have evolved a range of anti-CRISPR systems that block different stages of the CRISPR-Cas immune activity.
In an early study, Professors Garrett and Peng described an interesting phenomenon upon double virus infection of the native volcanic hot spring archaea – Sulfolobus islandicus. One co-infecting virus – Sulfolobus monocaudavirus 1 (SMV1) – was shown to activate the host’s CRISPR-Cas response against another competing virus. Interestingly, the competing virus – Sulfolobus tengchongensis spindle-shaped virus (STSV2), did not trigger a response from the archaeal host if it was infecting without SMV1.
While the hosts’ CRISPR-Cas system was directed against other viruses, SMV1 itself evaded detection. Indeed, archaeal cultures infected with another competing lipothrixvirus SIFV2 were insensitive to SMV1 infection. It was also observed that one mutant of SMV1 was susceptible to the host’s immune response. This mutant lacked a protein called ORF114, which the researchers theorised could protect the SMV1 DNA from CRISPR spacer selection.
Interestingly, not all viruses succumb to this tactic from SMV1. In a 2018 paper, the rudivirus Sulfolobus islandicus rod-shaped virus 3 (SIRV3) was shown to stably co-infect the same archaeon (Sulfolobus islandicus) over a period of 12 days. SIRV3 could do this even while the SMV1 virus induced CRISPR spacer acquisition against it. The CRISPR-Cas systems should have been effective at eradicating SIRV3, but SIRV3 has a trick up its sleeve: anti-CRISPR proteins.
Anti-CRISPR proteins (Acr) are small proteins, generally made up of less than 150 amino acids. They are a key method used by viruses to circumvent CRISPR-Cas systems in their archaeal hosts. They mostly work by inhibiting a single CRISPR-Cas subtype, binding to subunits of the effector complex to inhibit them. There are close to 50 known families of Acr, all of which are rapidly evolving in order to keep up with their archaeal host’s immune responses.
In a 2018 paper, Professor Peng’s team used elegant gene knockout strategies to find and characterise Acrs. ‘Knocking out’ the genes of Acrs means that they can no longer make their target proteins. In this way, the researchers confirmed that the viruses did not require Acrs in hosts that lacked CRISPR-Cas activity and were therefore only effective in response to this activity.
In another 2018 study from Peng’s group, Acrs from the rudiviruses SIRV2 and SIRV3 were structurally and functionally characterised. Deleting 4 kilobases from the genome of SIRV2 dramatically reduced infectivity in hosts with three CRISPR-Cas subtypes (I-A, I-D, and III-B). However, inserting a gene from SIRV3 restored infectivity in the S. islandicus hosts. This gene, gp02, encodes the protein AcrID1. It was shown that AcrID1 interacted directly with the protein Cas10d – needed for the interference stage in CRISPR-Cas subtype I-D.
The research team found that AcrID1 belongs to a family of proteins conserved in archaeal viruses, with 50 homologues found in four different archaeal virus families. One of the key characteristics of this protein family is its stability at extreme pH levels and temperatures – perfect for volcanic hot springs.
Another formidable strategy deployed by the rudivirus SIRV2 was revealed in a 2019 study and published in the renowned scientific journal Cell. The researchers discovered a new Acr belonging to the virus. This was called AcrIIIB1, coded by the gene gp48, and was found to inhibit the CRISPR-Cas subtype III-B. AcrIIIB1 is highly conserved in many different archaeal viruses. This accords with the fact that type III CRISPR-Cas subtypes are prevalent in archaea of the Sulfolobus family.
The study sheds light on the fact that AcrIIIB1 interacts with Cmr-α and Cmr-γ effector complexes of the CRISPR-Cas III-B subtype. These effector complexes make compounds that activate Csx1 – a protein associated with CRISPR-Cas. Csx1 cleaves single-stranded RNA. This mechanism shows how AcrIIIB1 inhibits the archaeal host’s immunity using the CRISPR-Cas III-B subtype.
This microscopic arms race between archaea and archaeal viruses has caused the systems of hosts and viruses to become more and more intricate as the process unfolds. More complex CRISPR-Cas immunity leads to more intricate Acrs to counteract them, and vice versa. This battle between hyperthermophiles in volcanic hot springs is a fascinating example of evolution at work.
- Erdmann, S., Le Moine Bauer, S., & Garrett, R. (2014). Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Molecular Microbiology, [online] 91(5), 900-917. https://doi.org/10.1111/mmi.12503
- He, F., Bhoobalan-Chitty, Y., Van, L., Kjeldsen, A., Dedola, M., Makarova, K. et al. (2018). Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity. Nature Microbiology, [online] 3(4), 461-469. https://doi.org/10.1038/s41564-018-0120-z
- Mayo-Muñoz, D., He, F., Jørgensen, J., Madsen, P., Bhoobalan-Chitty, Y., & Peng, X. (2018). Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses, [online] 10(12), 695. https://doi.org/10.3390/v10120695
- Papathanasiou, P., Erdmann, S., Leon-Sobrino, C., Sharma, K., Urlaub, H., Garrett, R., & Peng, X. (2018). Stable maintenance of the rudivirus SIRV3 in a carrier state in Sulfolobus islandicus despite activation of the CRISPR-Cas immune response by a second virus SMV1. RNA Biology, [online] 16(4), 557-565. https://doi.org/10.1080/15476286.2018.1511674
- Bhoobalan-Chitty, Y., Johansen, T., Di Cianni, N., & Peng, X. (2019). Inhibition of Type III CRISPR-Cas Immunity by an Archaeal Virus-Encoded Anti-CRISPR Protein. Cell, [online] 179(2), 448-458.e11. https://doi.org/10.1016/j.cell.2019.09.003
Professor Xu Peng and Professor Roger Garrett from Copenhagen University, Denmark, are researching the struggle for dominance between archaea and archaeal viruses.
Xu Peng acknowledges support from
The Novo Nordisk Foundation (Hallas-Moeller Ascending Investigator) and The Independent Research Fund Denmark, and the EU FP7 research program
- Yuvaraj Bhoobalan
- Fei He
- Susanne Erdmann
- Shiraz A Shah
Professor Xu Peng is the head of Microbial Immunity Group, Department of Biology, Copenhagen University
Roger Garrett is Professor Emeritus, Department of Biology, Copenhagen University
Address Department of Biology, Copenhagen University, Ole Maaloes Vej, 5, DK2200 Copenhagen
Prof Xu Peng
Prof Roger Garrett