Antiviral immunity in ancient archaea and possible origins of the progenitors of the eukaryotic innate immune system

Williams, Manon and Robinson, Nick and Unterholzner, Leonie (2023) Antiviral immunity in ancient archaea and possible origins of the progenitors of the eukaryotic innate immune system. Masters thesis, UNSPECIFIED.

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Abstract

Archaea are ancient microorganisms believed to be the precursors to modern eukaryotic cells, following the capture of an alphabacterium by an archaeon around 2.3–1.8 billion years ago. The recently discovered superphylum Asgardarchaeota has strengthened the notion that eukaryotes arose from an archaeal ancestor, due to their rich distribution of eukaryotic signature proteins, once thought to be exclusive to eukaryotes. Asgard archaea are currently believed to be the closest archaeal relatives of eukaryotes. Recent bioinformatic analyses of archaeal genomes have discovered the presence of cGAS homologues. In eukaryotes, the cGAS-STING (Cyclic GMP-AMP SynthaseStimulator of Interferon Genes) pathway is a vital component of the innate immune response. cGAS is a cytosolic DNA sensor responsible for the detection of foreign or damaged dsDNA, whereupon it initiates a signalling cascade leading to the production of Type I IFNs, which are responsible for preventing the spread of viral infection. In some archaea, the cGAS homologue is clustered in an operon alongside genes reminiscent of eukaryotic ubiquitination machinery: an E1E2 ubiquitin-activating and ubiquitin-conjugating fusion protein and a JAB/JAMM-family deubiqutylase, arranged in a Type II Cyclic oligonucleotide-based anti-phage signalling system (CBASS) operon. The role of these genes in relation to cGAS is unknown, and it is unclear whether they function as part of an archaeal ubiquitin modification pathway. In this project, cGAS homologues and potential associated signalling factors were cloned from two Archaeal species and expressed and purified in bacterial cells. Archaeal cGAS was also expressed in human cells to probe whether the second messenger system might be conserved between kingdoms. Significant progress was made in the expression and purification of these archaeal proteins, which in future studies will be analysed further to generate information about the interaction of these components. cGAS from one of the archaeal species was also expressed successfully in HEK293T cells, with some promising results indicating its ability to activate human STING. Further investigation will provide new insight into the evolution of the innate immune response and the evolutionary relationship between archaea and eukaryotes. Some pathogenic organisms in both eukaryotes and prokaryotes have evolved to antagonise cGAS signalling. Vaccinia (VacV) is a large, dsDNA brick-shaped poxvirus that belongs to the genus Orthopoxvirus, and tends to replicate entirely in the cytoplasm, leaving it vulnerable to detection by DNA sensors such as cGAS. Through a previous study, Vaccinia has been shown to antagonize the cGAS-STING pathway as well as various other components of the innate immune system, although not all of these mechanisms are known. It has been hypothesised that its key protease, I7L, is able to degrade cGAS, thereby inhibiting the innate pathway. To investigate this further and discern whether I7L could degrade cGAS directly, we aimed to perform in vitro experiments using recombinant I7L and G1L, another protease, as a control. I7L proved difficult to express in E.coli, and further work is needed to purify the recombinant protein. G1L was successfully expressed and purified and can be used for further research into the effects of VACV proteases on cGAS and other proteins.