REPORTING FROM AUGUST 2019; PRINCIPAL INVESTIGATOR MOVED TO UNIVERSITY OF WYOMING IN FALL 2019 and NEW GRANT 80NSSC20K0283 AWARDED (Ed., 7/24/2020)
The bulk of our research on NASA Grant NNX15AB44G that was conducted at UNC (the University of North Carolina) can be broken down into two categories: 1.) ground controls and experiments, and 2.) testing and validation for our flight experiment. Below are summaries of our research in both of these categories. Ground Controls and Experiments
Identification and functional assays to identify mediators of tardigrade desiccation tolerance: To prepare for comparison of changes in gene expression manifested by tardigrades (water bears) exposed to multigenerational spaceflight with ground-based stresses we have begun gathering transcriptomic datasets from terrestrial stresses. In addition we have performed functional experiments to assess which candidate genes from our transcriptomic datasets are functional mediators of desiccation (drying) tolerance.
Our major findings from these endeavors have been published in Boothby et al., 2017 (Boothby TC, Pielak GJ. Intrinsically disordered proteins and desiccation tolerance: Elucidating functional and mechanistic underpinnings of anhydrobiosis. Bioessays. 2017 Nov;39(11):700119. Epub 2017 Sep 13. https://doi.org/10.1002/bies.201700119 ; PubMed PMID: 28901557 ; Ed. Note 9/9/19: reported in August 2018 FY2018 Task Book report Bibliography ). These results will be summarized here briefly since detailed descriptions of the experiments, methods, and results are presented in publication.
To identify genes that might play a role in tardigrade desiccation tolerance, we extracted and sequenced RNA from tardigrades that had either been left unstressed in culture or desiccated. Comparison of transcript levels coming from each predicted gene was conducted and genes ranked based on fold change (how much expression of the gene increased during drying) and overall abundance (how many transcripts per million transcripts were coming from a particular gene).
The main takeaway from this comparison was that a class of tardigrade specific genes known as Cytosolic Abundant Heat Soluble (CAHS) genes are upregulated heavily during desiccation. We performed RNA interference experiments in tardigrades to reduce the level of expression of these genes and found that the animals no longer robustly survived drying when CAHS genes were targeted. We also found that expressing these genes in bacteria and yeast (which normally do not have these genes) led to up to two orders of magnitude increases (100X) in desiccation tolerance. Amazingly, when purified CAHS proteins were found to protect biological material (the enzyme lactate dehydrogenase) about an order of magnitude (10X) better than current FDA approved excipient trehalose and serum albumin.
Finally, we correlate the protective capabilities of these CAHS proteins to their ability to form vitrified (glass-like) solids, as opposed to crystalline solids. These finding may be of interest to NASA, as this presents an avenue for stabilizing and protecting biological material in a dry state without refrigeration. This might be useful for prolonged storage of biomaterials on the ISS or other spaceflight missions where freezer and refrigeration space is limited or logistically difficult.
Exploring cross-tolerance between desiccation and freeze tolerances in tardigrades:
Tardigrades survive an amazing number of abiotic stresses, and in some cases the severity of these stresses is well beyond that tardigrades would ever experience in nature (e.g., temperatures close to absolute zero, thousands of gray of radiation, the vacuum of outer space). The question therefore arises--how did tardigrades evolve tolerance to stresses they have never experienced? One hypothesis is that as tardigrades moved onto land from the ocean (where they originally evolved) they developed desiccation tolerance in response to their new, dryer, conditions and as a by-product became tolerant to other stresses. If this hypothesis is correct, then the mediators that tardigrades use to survive desiccation should in theory be the same mediators they use to survive other stresses. To assess if this is true we performed transcriptome sequencing on tardigrades that had been frozen, and compared this data to our previous datasets (desiccated and unstressed).
Surprisingly, we found that changes in gene expression between desiccated and frozen tardigrades are highly divergent. In fact, either stress condition is more similar to unstressed conditions than they are to each other. Most telling, we observed that expression of CAHS genes was not influenced by freezing conditions, and furthermore RNA interference targeting these genes did not result in statistical decreases in survival in tardigrades exposed to freezing conditions.
These results are presented in detail in Boothby et al., 2017 (Boothby TC, Pielak GJ. Intrinsically disordered proteins and desiccation tolerance: Elucidating functional and mechanistic underpinnings of anhydrobiosis. Bioessays. 2017 Nov;39(11):700119. Epub 2017 Sep 13. https://doi.org/10.1002/bies.201700119 ; PubMed PMID: 28901557 ; Ed. Note 9/9/19: reported in August 2018 FY2018 Task Book report Bibliography ).
We are now delving more into our frozen transcriptome to identify functional mediators of freeze tolerance in tardigrades using a similar approach to the one taken for our desiccation study.
Understanding to commonalities and differences between how tardigrades survive freezing and desiccation is an important facet of our overall strategy for this project, as spaceflight induced changes in gene response will ultimately be compared to ground-based stress responses. Comparing changes in gene expression for ground-based stresses now will help us understand the overlap with spaceflight induced changes later.
How do tardigrade CAHS proteins mediate desiccation tolerance?
To better understand of CAHS proteins protect tardigrades and other biological material and cells from the harmful effects of drying, and how these proteins might protect biological materials from other stresses (included spaceflight), we have been characterizing the biochemical and biophysical nature of these proteins.
We have discovered that these proteins behave in a very peculiar way. At room temperature and at concentrations greater than or equal to 30 g/L these proteins form reversible hydrogels. We have characterized the gel state of these proteins via cone plate rheometry as well as scanning electron microscopy. Both techniques clearly demonstrate that these proteins have classic gel-like behavior and morphology. In retrospect it makes sense that these proteins form hydrogels, as hydrogels are known to form vitrified solids when dried (see above).
We were curious if the gel state of these proteins is important for their protective capabilities. To probe this, we used 19-F NMR to look at the folded state of a test protein, SH3. SH3 is a ‘metastable’ protein, meaning that normally (in solution) SH3 is in a folded state ~50% of the time and in an unfolded state about ~50% of the time. This is easily measured using 19-F NMR. We first tested SH3 in solution, and as expected two clear peaks (folded and unfolded) were present. We then co-incubated SH3 with a CAHS protein (at increasing concentrations) and looked at the levels of folded and unfolded protein. We found that CAHS proteins had no noticeable effect on SH3 folding below 30 g/L. However, above 30 g/L of CAHS protein, there was a reduction in the level of unfolded SH3 protein, and a corresponding increase in folded SH3. Interestingly, 30 g/L is the concentration determined by cone plate rheometer as the gel point for these proteins. The hydrogels that CAHS proteins form are reverse and heat dependent. Therefore, we tested whether heating to induce the breakdown of the hydrogel influenced SH3 folding. We found when heated from 19C to 42C the CAHS proteins went back into solution (the gel state vanished) and there was a return of an SH3 unfolded population. Cooling this solution back down to 19C resulted in re-gelling of the CAHS proteins and a corresponding disappearance of the SH3 unfolded species and an increase in the SH3 folded species. Therefore, it appears that there is a strong correlation between the gelled state of CAHS proteins and their ability to stabilize proteins in a folded state.
We are now characterizing the sequence features of CAHS proteins at allow them to form gels. These studies are being conducted by making mutant versions of the proteins and testing their ability to form gels and protect biological materials.
Testing and Validation for Flight Experiment
The bulk of this effort has been made in preparing for and performing our Science Validation Test (SVT-1).
The main goal of SVT-1 was to compare the efficacy of culturing tardigrades (Hypsibius exemplasris) using the CellMax and PI Start Kit (PISK).
Syringes containing ~500 tardigrades or concentrated algae were prepared using the same stock cultures. Syringes were frozen at -80 degrees Celsius and either stored at this temperature or shipped on dry ice to NASA Ames.
On May 10th, tardigrades were injected into 3 PISK bioreactors (at NASA Ames) and 3 CellMax Bioreactors (at UNC).
Temperature control for PISK bioreactors was carried out using integrated temperature control – which varied between ~15 – 17 degrees Celsius. Oxygenation for PISK bioreactors was carried out via gas dosing using medical grade air.
Temperature control for CellMax bioreactors was carried out by placing the CellMax system in a controlled temperature chamber. The initial temperature used was 15 degrees Celsius, but was changed to 16 degrees Celsius to more closely mirror PISK temperatures. Oxygenation was not controlled, but rather relied on the passive transport of oxygen across the CellMax systems permeable tubing.
Every 7 days, up to day 21, subsamples (300 ul) were extracted from both PISK and CellMax bioreactors using syringes. These samples were stored at -80 degrees Celsius. At day 14, fresh algae (food source) was injected into each PISK and CellMax bioreactor. At day 28, the experiment ended and whole bioreactors were detached and frozen at -80 degrees Celsius. PISK subsample syringes and bioreactors were returned frozen on dry ice to the Principal Investigator's (PI) lab at UNC.
Upon receipt at the PI’s lab, samples were transferred from their shipping unit to a -80 degree freezer.
To compare the viability of culturing using the PISK, all samples were thawed. For each subsample (300 ul) the entire volume of thawed sample was analyzed by direct observation using a dissecting microscope. Total animal counts were taken and densities calculated. Similarly, for bioreactors, the entire contents of the bioreactor was thawed and transferred to a 15 mL tube. Three 50 ul samples were taken from each tube and total animal counts made and densities calculated.
The remaining bioreactor contents was fixed with RNAlater and processed for total RNA extraction.
Tardigrade Densities and Total Counts
Prior to testing our definition for success was achieving Day 28 animal densities in the PISK bioreactors that were above or within 20% of the densities from our CellMax bioreactors. Our average PISK animal density at Day 28 was 357.8 animals/mL where as our CellMax Day 28 animal density was slightly lower at 282.2 animals/mL. By our original definition, the PISK successfully competed with the CellMax system with regards to effectively culturing tardigrades over a 28 day period.
REPORTING IN AUGUST 2018
We have been preparing for our flight experiment. This has mostly manifested itself in the development of an SRD (Science Requirements Document), protocols, and coordinating with the engineering team who are doing some final tests on our bioreactor setup. Our next step will be to actually get our animals in the hardware and do ground tests.