After 5 years of work, including approximately 4 years dedicated to addressing reviewer comments from top journals and adding new experimental data, our paper describing the parasitic behaviour of Ca. Nha. antarcticus is finally out. The paper reports the first example of a predatory DPANN archaeon, Ca. Nha. antarcticus, that kills its’ host (Halorubrum lacusprofundi) as part of their interactions. Both organisms were isolated from a cold, hypersaline Antarctic lake. This work has spanned career stages (PhD – Post-doc: Josh, Post-doc – PI: Yan) and across continents (from Australia to Europe), overcoming the challenges of the global COVID-19 pandemic.
Both Yan and Josh completed PhDs in Prof. Rick Cavicchioli’s group at the University of New South Wales. At the time we started the project, Yan was already a Post-Doc in A/Prof. Iain Duggin’s group at the University of Technology Sydney. In 2019, Yan approached Rick to discuss potential collaborative projects, leading to the idea of using live fluorescence to track DPANN-host interactions. Although similar concepts had been discussed in Cavicchioli group, none of us had the necessary expertise to pull it off without help. Yan had gained significant experience in live imaging of halophiles since joining the Duggin lab, making her the ideal collaborator for this project.
We proposed the idea of purifying Ca. Nha. antarcticus and staining both it and the purified host with different non-cytotoxic dyes (Mitotracker dyes) before co-culturing, enabling us to track interactions in real time. When Yan put the first mixed samples under the microscope and imaged them, we were not expecting what we would end up observing. She found that Ca. Nha. antarcticus not only likely caused the lysis of its host Hrr. lacusprofundi, but the process appears to involve portions of the nanohaloarchaeon’s cell entering the host cell, which was both shocking and exciting. Early in our work on Ca. Nha. antarcticus when we were developing 16S rRNA fluorescence in-situ hybridisation (before the collaboration using live fluorescence was started) we observed what appeared to be an internalisation lifecycle for the nanohaloarchaeon. However, at the time the results were not reproducible, so we shifted our focus to other aspects of the work.
Figure 1 A slide from a presentation Josh gave in 2017 showing 16S rRNA FISH data suggesting internalisation of Ca. Nha. antarcticus
The data Yan generated represented the first reproducible evidence that the interactions between Ca. Nha. antarcticus and Hrr. lacusprofundi lead to host lysis and some morphological changes of host cells. However, the question remained whether the nanohaloarchaeon was truly entering the host cell or just making an invagination in the host membrane. To address this, we needed techniques capable of resolving individual membranes in 3-dimensional reconstructions, such as ultra-thin sectioning or cryo-tomography.
Initially, we attempted to develop a protocol for fixation, resin embedding, and ultra-thin sectioning. However, the high salt concentration from the samples posed challenges, including spontaneous polymerisation of the resin and incomplete infiltration, resulting in poor quality data. We also explored high-pressure freezing but again the salt concentration caused havoc, first by slowing the rate of freezing and then also inhibiting the freeze-substitution process. Ultimately, plunge-freezing followed by cryo-tomography was the only protocol we tested that consistently yielded reliable results, leading us to abandon the other techniques.
Figure 2 The best preservation we achieved with ultra-thin sectioning. The majority of cells were not as well preserved as this and the data was not considered publishable. Image collected by Joanna Biazik-Richmond.
Even with the improved sample preservation, the high salt concentration continued to cause issues by inducing internal diffraction of the electron beam and damaging the sample. At the University of New South Wales, the best cryo-electron microscope was a Talos Arctica with a 200 kV beam and no energy filter. To obtain the data quality we needed, we had to travel to Monash University in Melbourne, where we could access their Titan Krios with a 300 kV beam setup. Despite this improvement, the data still did not fully convince reviewers, and each review round required approximately 9 months of work to address comments, considering the high demand for the equipment and the logistics of sending samples across the country.
Figure 3 An early figure produced with the data from the Titan Krios at Monash University. Image collected by Hariprasad Venugopal, Georg Ramm, Nicholas Ariotti, Matt Baker, and Joshua Hamm
During this time, Josh completed his PhD and accepted a Post-doc position in the EvoSym group of Anja Spang in the Netherlands. Now the main authors were split across continents, and the logistics of the work became even more complicated. The quality of the electron microscopy data continued to be an concern raised by reviewers, prompting us to approach Andriko von Kügelgen and Tanmay Bharat at Oxford University (now Cambridge) to see if they could improve upon our results. Collaborating with them was logistically easier than continuing with facilities in Australia, especially as Rick’s lab was winding down and Josh had taken the cultures to the Netherlands to continue the work.
Andriko was able to significantly improve the quality of our tomograms, and this improvement, along with the addition of cryo-correlative light and electron microscopy, was eventually sufficient to get the paper through review. However, this still required an additional 3 years of work, much of which was complicated by the pandemic and associated lockdowns.
Now, the paper is out in Nature Communications, and our observations regarding the predatory behaviour of Ca. Nha. antarcticus are available to the scientific community. In summary, the nanohaloarchaeon is an aggressive predator that lyses large numbers of host cells in short periods. It also appears to either enter the host cell, or to induce formation of internal membrane bound structures as part of this process. This work is the product of a team of international collaborators working tirelessly over the course of 5 years. However, it began with the conversation between Josh, Yan, and Rick 5 years ago, when we decided to try something new and embarked on this journey.
Our journey is far from over. There are still unresolved questions to address, such as how widespread of this behaviour is among DPANN archaea and the molecular mechanisms involved in this process. We look forward to continuing this exploration and uncovering more of nature’s secrets.