Abstract
For a long time, a whole domain of life was overlooked. Archaea emerged as extremophiles with hybrid features between bacteria and eukaryotes, and only relatively recently have their diversity, ecological importance and widespread occurrence been investigated in detail. Like all cellular life, the archaea harbour their own viruses, which constitute an extraordinarily diverse group with exotic morphologies and unique features.
Prokaryotes possess a variety of defence mechanisms. The CRISPR-Cas adaptive immune system is of great importance for archaea –84% of them possess it, compared to 45% for bacteria–, and constitute the dominant form of antiviral immunity in thermophiles. This system has an extraordinary impact in shaping the interactions of archaea and their viruses. It consists of a versatile RNA-guided protein complex that detects and destroys invader genetic elements in the cell, and can generate immune memory by inserting in its own genome short invader-derived DNA fragments forming a database –the CRISPR locus. Little was known about this system until recent years, and the generation of immune memory has been the most elusive step.
In this work, the interactions of the spindle-shaped monocaudavirus STSV2 and its host Sulfolobus islandicus REY15A were studied. This interaction produced, after several days, de novo CRISPR adaptation – that is, without any previous memory that can act as a trigger. We employed transcriptome sequencing to characterise the long-term progression of this interaction, as well as investigating the influence of medium composition on the virus life cycle and host defence. Comparative analysis with previous work on archaeal virus infections allowed us to define a set of virus-responsive systems, such as the replication and division machinery, the basal transcriptional factor and toxin-antitoxin pairs. Surprisingly, the DNA-targeting cas modules, and not the cmr, which target different DNA or RNA substrates, were activated by the infection, showing the specialised function of CRISPR-associated genes. We also provided the first evidence for antisense transcripts arising from type I cas gene modules.
In a second step, combinations of crenarchaeal viruses that were inducing CRISPR adaptation were analysed. Transcriptomes revealed that these viruses have different vulnerabilities to the host immune system, and confirmed that STSV2 stimulates CRISPRCas adaptation.
Further work was performed in vitro on a cluster of putative transcriptional regulators encoded in the genome of STSV2.
Prokaryotes possess a variety of defence mechanisms. The CRISPR-Cas adaptive immune system is of great importance for archaea –84% of them possess it, compared to 45% for bacteria–, and constitute the dominant form of antiviral immunity in thermophiles. This system has an extraordinary impact in shaping the interactions of archaea and their viruses. It consists of a versatile RNA-guided protein complex that detects and destroys invader genetic elements in the cell, and can generate immune memory by inserting in its own genome short invader-derived DNA fragments forming a database –the CRISPR locus. Little was known about this system until recent years, and the generation of immune memory has been the most elusive step.
In this work, the interactions of the spindle-shaped monocaudavirus STSV2 and its host Sulfolobus islandicus REY15A were studied. This interaction produced, after several days, de novo CRISPR adaptation – that is, without any previous memory that can act as a trigger. We employed transcriptome sequencing to characterise the long-term progression of this interaction, as well as investigating the influence of medium composition on the virus life cycle and host defence. Comparative analysis with previous work on archaeal virus infections allowed us to define a set of virus-responsive systems, such as the replication and division machinery, the basal transcriptional factor and toxin-antitoxin pairs. Surprisingly, the DNA-targeting cas modules, and not the cmr, which target different DNA or RNA substrates, were activated by the infection, showing the specialised function of CRISPR-associated genes. We also provided the first evidence for antisense transcripts arising from type I cas gene modules.
In a second step, combinations of crenarchaeal viruses that were inducing CRISPR adaptation were analysed. Transcriptomes revealed that these viruses have different vulnerabilities to the host immune system, and confirmed that STSV2 stimulates CRISPRCas adaptation.
Further work was performed in vitro on a cluster of putative transcriptional regulators encoded in the genome of STSV2.
| Originalsprog | Engelsk |
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| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
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| Antal sider | 159 |
| Status | Udgivet - 2015 |