Trans-Translation Part 2: Of Vibrios and Squids

by Janie
 

Figure 1. Hawaiian bobtail squid hatchlings (note the plastic recycling symbol on the container for size comparison). Source: Ruth Isenberg (Mandel Lab). Thank you, Ruth, for the great photo!

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.
 

Figure 2. (Adapted from Fig. 1 of the paper) Top row: Localization of the SsrA transcript in the host crypt cells, with wild-type V. fischeri cells in green and SsrA transcripts in magenta. Bottom row: a close-up. SsrA transcripts (magenta) are found in the cytoplasm of the host's crypt cells. Source.

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.
 

Figure 3. (From Fig. 4 of the paper) D: A schematic depicting the location of the light organ and yolk sacs in a juvenile bobtail squid, and a confocal microscopy image of a hatchling's yolk sacs. D': A comparison of yolk sac area between squids immediately after hatching (“Hatch”) and squids colonized by wild-type or ΔssrA symbionts. E: Representative SEM images of yolk sacs from panel D'. Source.

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.