This New Year story begins with newly hatched baby bobtail squids. Each squid is translucent and barely the size of a grain of barley. As they swim around, the six pores on their mantles ringed with mucus-coated cells snag passerby Vibrio fischeri from the water. The bacteria then travel down ducts into what will become the light organs, finessing their way past squid chemical defenses that nix any non-fischeri intruders. Their final destination: little nooks within epithelial cell folds (the "crypts"). It's in these spaces that the Vibrios show off their famous light-making moves.
In an earlier post, we featured a Twitter exchange on tmRNA, a multitalented molecule that enters jammed ribosomes first in the tRNA-mimic mode, and then switches into the mRNA mode to append a degradation tag onto the incomplete polypeptide. To start off the new year, we wrap up that cliffhanger! Here is a look into the unexpected role of tmRNA in establishing the squid-Vibrio light-organ symbiosis.
Moriano-Gutierrez et al. first found that adult squid hemolymph – the invertebrate equivalent of blood – contains RNA sequences that unexpectedly map to the V. fischeri genome. This meant that RNA originating from the light-organ denizens was circulating throughout the rest of the squid – like humans, squids have a circulatory system comprised of a meshwork of blood vessels, except with added oomph of two extra hearts. Upon closer inspection of these RNAs, which were mostly a mix of run-of-the-mill tRNAs and rRNAs, a special 4% were small noncoding RNAs. Of this fraction, a further 70% were SsrA transcripts (aka tmRNA; encoded by the ssrA gene, which stands for "small stable RNA"). Here, the tmRNA enters the story.
SsrA transcripts were also the most abundant of noncoding RNAs ferried by the Vibrios' outer membrane vesicles, where they were found at similar levels as in the hemolymph. This suggested that the symbionts release OMVs into the surrounding crypt space, that then find their way into squid-wide circulation. An OMV-delivered RNA free-for-all! It appeared that its significance to the bacteria depended on the stage of symbiosis: V. fischeri with ssrA deletions had no problem getting into the crypts, but once there, they were less able to persist than the wild-type.
There is another piece to this SsrA localization surprise. With FISH staining, SsrA unexpectedly lit up in the cytoplasm of crypt epithelial cells. The RNAs were therefore not only inside and around their bacterial originators, but had also found their way into host squid cells. Moriano-Gutierrez et al. figured that the RNA transcripts are continuously delivered by OMVs of live symbionts.
But how does this OMV delivery of SsrA affect the symbiosis? The initiation steps appeared to be unaffected, since ΔssrA mutant Vibrios triggered the normal early patterns of hemocyte trafficking and epithelial apoptosis in their squid hosts. What follows, however, is a string of oddities. The most readily visible defect is the drop in light production: squids colonized with mutants only emitted 20% of the wild-type level of luminescence. This SsrA-dependency of the light show is evidently itself dependent on the localization of the bacteria. The ΔssrA mutants were just as bright as their wild-type counterparts when free-living, but within the squid light organs, they did not live up to their full bioluminescent potential.
Less flashy changes induced by the Vibrios included the abnormally early swelling of crypt epithelial cells, plus the upregulation of a handful of immune response genes within the light organs. One of these genetic red flags encoded laccase-3, an oxidase enzyme involved in invertebrate response to pathogens. When the authors tracked down the whereabouts of these overexpressed laccase-3 transcripts, they found that the FISH signals lit up around crypt epithelia touching the ΔssrA symbionts. Lack of SsrA is clearly the culprit. This could explain the symbiosis's diminished luminescence: the increased laccase-3 concentration in these abnormally colonized crypts eats up the ambient oxygen, siphoning it away from Vibrio light production.
All this is to say, the absence of SsrA throws a hapless squid and its symbiotic companions very much out of whack. The need for SsrA in their tag-team light production was apparent at this point; the remaining question was the mechanism. As we covered in our previous Twitter-ful post, the known function of SsrA aka tmRNA is to rescue stalled ribosomes. To deduce whether the importance of SsrA in the symbiosis also depends on this molecular safeguarding, the authors deleted the V. fischeri gene for the required chaperone protein. In a plot twist, these ΔsmpB mutants functioned fine, setting off all the right host responses. Thus, the RNAs were up to something more unusual than ribosome-rescue.
The authors also found that hatchling squids colonized by ΔssrA symbionts had decreased survival rates. The revved-up immune response in ΔssrA-colonized squids, indicated by laccase-3 overexpression, is an energy sinkhole – macrophages snap up ATP like no-cell's business. Surely enough, only squids colonized by ΔssrA symbionts lost significant weight between hatching and four days post-colonization. They also had shrunken yolk sacs, which are the nutrient reservoirs that provide energy for youngsters still learning the ropes at hunting prey. No SsrA is bad news for a baby squid.
So, the symbiosis hinges upon the presence of SsrA in the host crypt epithelial cells. It follows then that there must be a host tactic for detecting the RNAs. Using readily cultured hemocyte cells as a proxy for finicky crypt epithelial cells, the authors found that only ΔssrA OMVs induced a significant uptick in expression of a cytosolic RNA sensor, RIG-I, which has been implicated in bacterial RNA recognition in other systems. RIG-I may be involved in the detection of the V. fischeri SsrA and the downstream response, but a more detailed mechanism amid the labyrinth of immune response pathways now waits to be puzzled apart. Apparently, tmRNAs are molecules that wear even more hats than previously thought.
With this squid-Vibrio SsrA/tmRNA story, the roles of interkingdom noncoding RNA exchange in symbioses become more mysterious than ever. What other ncRNAs are shipped out via OMV? Elio previously covered the role of OMVs in the symbiosis between Paracatenula marine flatworms and their bacteria here. Other symbioses with possible OMV transfer include the tag-team efforts between Gram-negative bacterial symbionts and people, plants, insects, and nematodes. Perhaps the enigmatic tmRNAs are also sneaking around and about these cells.