A “Fire Amoeba” Takes First Place for Eukaryotic Heat Tolerance

Temperature has a huge effect on living organisms because of its impact on the stability and function of enzymes and biological systems. Thermophiles, or organisms that live at or above 45°C, possess a variety of unique adaptations to survive increased heat. While some thermophilic bacteria and archaea have been found to grow at temperatures exceeding 100°C, the limit for eukaryotic survival has been estimated at 60°C based on previous sampling.^1-4

Now, researchers recently discovered an amoeba that regularly lives at 63°C, breaking the record for the most heat-tolerant eukaryote. In a study published in Cell, scientists described how the single-celled organism, which they dubbed Incendiamoeba cascadenis, roughly translating to “fire amoeba from the Cascade mountain range,” survives and thrives in hot springs in Lassen Volcanic National Park.⁵ The findings redefine upper heat limits for eukaryotes. As a potentially eukaryotic thermophile, I. cascadensis could offer unique heat-stable proteins and other molecules for biotechnology.

To better characterize heat tolerant eukaryotes, Angela Oliverio, a microbial ecologist at Syracuse University, and her team collected water, biofilm, and sediment samples from different locations of a hot spring tributary in Lassen Volcanic National Park. The water ranged between 49–64°C but was largely neutral, not dropping below a pH of six.

When the team cultured these samples in the lab to study the eukaryotic communities present, they noticed that one amoeba thrived at 57°C, a temperature previously thought to be the upper limit for these organisms. Instead, when the researchers increased the temperature, the amoeba, later named I. cascadensis, remained active and continued to divide up to 63°C.

The team sequenced the extreme microbe, created a phylogenetic tree from 102 genes, and saw that this new amoeba fell in the same clade as the genus Echinamoeba, the hottest-growing amoeba previously known. The most genetically similar species to I. cascadensis was Vermamoeba vermiformis, which can grow at room temperature and up to 45°C. The researchers used this species as a comparison for their morphology studies.

Using light microscopy and scanning electron microscopy, Oliverio and her team saw that I. cascadensis had an elongated, worm-like form and a compact, amoeboid shape with pseudopodia. This was similar to the characteristics of V. vermiformis, but the researchers noted that I. cascadensis attached to surfaces for shorter periods of time and with less area than its sister species, pointing to molecular or biophysical differences.

A photograph of an amoeba during cell division against a black background. The two cells’ membranes are grey and vertically stacked on the right of the image. A blue circle in the middle of each cells marks the nucleus, while one cytoskeleton protein is labeled in pink and extends between the two dividing cells. — *Incendiamoeba cascadensis* maintains its ability to grow and replicate up to 63°C. Angela Oliverio and her team confirmed this with the help of confocal microscopy, watching a cell duplicate its nucleus (DNA stained in blue) and extend one cytoskeleton protein (pink) during replication.
Felix Mikus

Using growth curve studies, Oliverio and her team showed that I. cascadensis grew between 42–63°C, with its peak activity and replication at 57°C. This suggests that the new amoeba is an obligate thermophile as opposed to only tolerating higher temperatures. At 64°C, the amoeba began to encase itself in a cyst, but if it was returned to more suitable temperatures, I. cascadensis resumed its growth and activity. It was able to do this up to 70°C, after which it was unable to survive.

Next, the researchers used long-read sequencing and compared the genes from I. cascadensis to V. vermiformis and other closely-related thermophiles. Overall, I. cascadensis had 6,919 genes, representing more than 50 percent of the amoeba’s anticipated genes, that were unique to it. They also saw that pathways and proteins involved in heat tolerance, such as calcium signaling and heat shock proteins, were more enriched in I. cascadensis than the other amoebas.

To study how high temperatures regulated gene expression, the team grew I. cascadensis at 48°C and 61°C. At the higher temperature, the amoeba upregulated genes involved in chaperone proteins, the ubiquitin system, DNA repair and maintenance pathways, and proteins involved in membrane trafficking. Meanwhile, the researchers saw that genes involved in energy and biosynthesis were downregulated at higher temperatures. These findings are similar to previously observed strategies for thermal tolerance.^6,7

Finally, the researchers compared the biophysical properties of the most abundant I. cascadensis proteins to 13 other thermophiles and their orthologous proteins. They saw that the proteins from I. cascadensis have less hydrophobic amino acids on their surface, possibly helping to prevent aggregation because of the inherent clumping of these residues. Additionally, positively-charged surface residues were more enriched in proteins from this amoeba compared to amino acids with negative charges. Other thermophiles also have this feature, suggesting that it could be an example of convergent evolution.

“This finding pushes the bounds of what we thought was possible, which is incredibly exciting,” Oliverio said in a press release. “There could be more eukaryotes that can survive at even higher temperatures than we know of.”

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