Long, long ago, some two billion years, when earthlings were represented solely by prokaryotes, there existed dense microbial mats, bustling with activity and with metabolic sophistication to match the most advanced engineering of eukaryotic metropolises of today. Included in the mats were many types of bacteria alongside their "oddball" brethren, members of the Archaea family. Like any complex community of entities with varied interests, there was friction, there was war, there were truces, there was stealing, and sometimes there was cooperation.
In at least one of these microbial mats, an ancestor of the alpha-proteobacteria lineage (or maybe not) was in close communion with archaeal cells, perhaps like the γ-proteobacteria shown here (Fig. 1), basking in an exchange of metabolites. We can imagine that occasionally a bacterium was ushered into the archaeal cell's interior by endocytosis, and we can imagine further that the outcome of such an event was a slow and grisly death for the bacterium as it was degraded by enzymes. But on occasion a bacterium had some feature that allowed it to survive, or the host had some less than optimal enzymes, and the swallowed bug was able to continue to produce something of value to the host, as an endosymbiont.
The origin story of mitochondria described above represents a "rare but revolutionary" biological event – rarely successful, but wildly innovative in outcome when it succeeds. Here, adaptations in response to the bacterial endosymbiont likely spurred the evolution of key components of eukaryotic cells, such as a well-defined nucleus, decoupling of transcription and translation, and introns. The protistologist Lynn Margulis saw such genome-blending events as the very drivers of speciation – a dynamic that had eluded Darwin.
In vertebrates like us, we see plenty of close associations with bacteria in which we benefit from their metabolic expertise, for example, vitamin B12 synthesis. However, it is almost invariably bad news when one of our compatriots (congutriots?) bypasses our innate immune defenses and takes up residence inside our cells. Malaria, sleeping sickness, toxoplasmosis – outcomes of unwelcome parasites, all. An interesting exception, only recently discovered, involves algal cells living as more or less benign endosymbionts inside cells of salamander embryos.
The salamander Ambystoma maculatum (Fig. 2) lays its eggs among dead leaves in ponds, where oxygen supplies are often poor. Not the best environment for the developing embryos, but the salamander egg capsules carry a supplemental oxygen source – the green alga Oophila amblystomatis. Much like the dinoflagellates that live in coral (Fig. 3), the algal cells are photosynthesizers that provide periodic infusions of O2 to the salamander embryos, but as ectosymbionts, since they live within the egg capsule but outside of the salamander cells. Biologists have known about these ectosymbionts for over a hundred years, but the discovery of the same algae living within cells of the embryo occurred just a decade ago and has spurred a whole set of questions centered on a bigger question: Are the algae bona fide endosymbionts, or just run-of-the-mill pesky parasites?
The peripheral questions include: How do the algae adapt to intracellular life, and what metabolites do they provide to the salamander? How do they escape immune defenses, e.g. degradation by enzymes? Likewise, how do the salamander cells adapt to their new tenants? What might they provide to the algae? In order to find clues to these answers, a group of researchers used RNA-seq to characterize the transcriptome – the total set of genes being transcribed – in each cell type involved in this story (Fig. 4). They then compared genes being transcribed in intracapsular algal cells to those of intracellular ones, and likewise for salamander cells with and without algae inside. This type of analysis is known as differential gene expression, or just differential expression (DE), and can allow a peek into the physiological changes that accompany this endosymbiotic event.
First the numbers: of approximately 6726 known algal genes represented in the RNA-seq samples, 277 show significant DE in the intracellular algae compared with the intracapsular ones. Comparing salamander gene expression between embryo cells with and without algae living inside, 300 genes out of 46,549 show significant DE in the latter group. Already we might be impressed by the asymmetry in the expression changes. For the salamander, the transition to hosting an algal endosymbiont requires shifting expression, whether up or down, of just 0.6% of genes, whereas the algae cells have to shift expression of 4.1% of their genes to transition from an intracapsular to an intracellular lifestyle. The algae compromise a lot more than do the salamanders in this relationship!
In order to better understand what is going on, we need to know what these genes do, which is not as easy as it sounds: close to half of all the sampled transcripts fall into a black box: they represent never-before-seen genes, or genes conserved across related organisms but with as-yet-unknown functions. The remaining half, however, match sequences of known genes (homologs), and can therefore be grouped into functional categories that allow us to limn the biological changes occurring.
The picture that emerges shows rather stressed-out algae, consistent with their undergoing transcriptional changes affecting many genes. For the intracellular algae, the highest number of DE genes (compared with their intracapsular brethren) fall into the functional category of “stress response”. Genes for heat shock proteins and others related to oxidative and osmotic stress are upregulated when algae live inside the salamander embryos’ cells. Why so stressed? Remember that algae living outside the embryo cells but inside the egg capsule contribute O2 by photosynthesis from whatever small amount of light reaches the egg capsule. What happens when algae live inside embryo cells, where it is much darker? Decreased transcription for components of the electron transport chain and an increase in transcripts associated with a switch to fermentation. Some species of intracellular parasites derive energy through a fermentative process and, like the intracellular algae, show a stress response. Switching from oxidative to the less efficient fermentative metabolism is evidently no walk in the park.
How do the salamander cells respond to the algae they host? When vertebrate cells are infected by parasites, a common response by the host is an attempt to skip out on the deal by undergoing apoptosis. However, the transcripts of salamander cells with algae inside not only do not indicate apoptosis, but they include upregulation of genes for experimentally verified anti-apoptotic responses. Similarly, transcripts associated with the immune system show a limited and controlled immune response to the algae, at least at the cellular level. For example, some pro-inflammatory factors are upregulated, but not to the extent seen during parasitic interactions in amphibians, such as when frogs are infected with a skin fungus. The reseachers also saw upregulation of factors that are known to suppress NF-κB, a broad regulator of the immune response in vertebrates. While it's true that embryonic salamanders have generally immature innate immunity, these data suggest that the salamander cells are actively tolerating their algal roommates, much like the response of corals to their endosymbiotic dinoflagellates.
What might the salamander cells be gaining from the intracellular alga? First are clues to what they are NOT getting: salamander cells with algae inside produce lower numbers of transcripts for an acid phosphatase and trypsin-like proteins, suggesting that yolk platelets are not being used for nourishment in these cells. Additionally, the researchers found no clear evidence that the fermenting algae are biosynthesizing other potentially useful products, such as vitamins. So what ARE the salamander cells getting in this deal?
An informative category of transcripts in this regard is that of nutrient-sensing genes. Transcripts of these genes in salamander cells with intracellular algae suggest an alternation between the use of anabolic and catabolic pathways. Exactly which fermentative products are being used by the salamander cells is frustratingly unclear, however. Glycerol, perhaps? Another possible clue is found in the upregulation by intracellular algae of a gene similar to Neimann-Pick type C (NPC). Why is this interesting? NPC proteins have been implicated in lipid transfer in coral-dinoflagellate endosymbioses, though it is the corals and not the microbes that upregulate the expression of NPC, demonstrating that there is more than one way to pass the fat.
The transcription data show decreased expression of nitrogen and phosphate transporters in the intracellular algae compared with their intracapsular counterparts, suggesting that the former may benefit from increased concentrations of these products within the salamander cells. The researchers were able to test this assumption in vivo. While free-living species of O. amblystomatis are not found in nature, they can be cultured in the lab, where they use flagella to swim around in their dishes. When researchers raised the concentration of extracellular inorganic phosphate from that expected in the egg capsule microenvironment to that expected inside the salamander cells, the cultured algae downregulated the expression of a high-affinity phosphate transporter (PHT1-2). Similarly, when L-glutamine levels mimicked those expected in salamander cells, the alga responded by downregulating the expression of inorganic nitrogen transporters. Both findings are lovely pieces of evidence supporting what the RNA-seq data suggest: the intracellular algae benefit from the high levels of glutamine and nitrogen in the salamander cytoplasm.
Also interesting is the finding that the salamander cells increase expression of a receptor involved in endocytosis-triggering, hinting at how the algae enter the host cells. Complementing this finding, the invaded salamander cells express higher levels of an actin-modifying gene implicated in membrane ruffling and closure (like what is seen when Salmonella typhimurium invades intestinal cells.) Similar proteins are expressed at increased levels in malaria-infected liver cells, and predispose these cells to infection by allowing an entry path that bypasses the apoptotic response. Also increased is expression of genes homologous to virulence factors expressed in bacterial parasites that aid and abet in endosomal escape – evidence of how the algae shed the vesicle membrane in which they are likely endocytosed.
One additional gene category associated with salamander cells bears mentioning: transposable elements (TEs). In fact, this category topped the list in terms of transcript abundance among those differentially expressed. What could this mean? The salamander's genome is about ten times larger than ours, and much of this difference is due to the expansion of TEs, which are scattered abundantly throughout this genome. The thinking is that when a nearby gene is up- or downregulated, it may affect TEs that happen to share some regulatory features with those genes, resulting in a spillover effect.
You may have noticed by now that reading transcripts is only a little more informative than reading tea leaves. The data do not definitively describe the nature of the salamander/algae relationship, but they do point to productive hypotheses (Fig. 5) that can be tested by other means. And finally, for the "tail end" of this story: no one should be surprised to learn that genes involved with the structure and function of the flagella are downregulated in intracellular algae when compared with intracapsular algae. After all, a tail in densely packed cytoplasm is about as useful as a straw in the desert.