Squids and Lu­mi­nous Bac­te­ria. A Tale of Twists and Turns

by Elio

Bac­te­ria are mas­ters at mak­ing  mul­ti­ple uses of their com­­po­nents. Thus, their con­stituents can serve a va­ri­ety of dis­sim­i­lar func­tions. An ex­am­ple is the li­po­po­ly­sac­cha­ride (LPS) of the Gram neg­a­tive cell outer mem­brane. This uniquely bac­te­r­ial con­stituent is im­por­tant sta­bi­li­zing the struc­ture of the mem­brane. In ad­di­tion, LPS pro­tects the mem­brane from harm­ful chem­i­cals, es­pe­cially hy­drophilic ones. Known also as en­do­toxin, LPS elic­its a strong in­nate im­mune re­sponse by bind­ing to re­cep­tors on var­i­ous im­mune cells and in­duc­ing them to re­lease in­flam­ma­tion-pro­duc­ing cy­tokines. Pep­ti­do­gly­can (PG), the main con­stituent of the bac­te­r­ial cell wall, is also such a multi-tal­ented mol­e­cule, sim­i­larly in­volved in struc­tural af­fairs and in elic­it­ing re­sponses from the host. It is fas­ci­nat­ing that such mol­e­cules, whose role seems to be prin­ci­pally me­chan­i­cal, should pos­sess such com­mu­ni­ca­tion skills. But then, mo­le­cules lo­cated on the sur­face of cells can be ex­pected to be se­lec­tively sensed by other cells. The at­trib­utes of bac­te­ria are cer­tainly not just skin-deep, but their skin mat­ters a great deal in their com­mu­ni­ca­tion with other liv­ing things.

Fig­ure 1. Ali­ivib­rio fis­cheri (for­merly Vib­rio fi­sche­ri ) show­ing bi­o­lu­mi­nes­cence. Source

To the well-known prop­er­ties of LPS and PG, we add their par­tic­i­pa­tion in a par­tic­u­larly ex­cit­ing sym­bio­sis, that be­tween a Hawai­ian squid and its lu­mi­nes­cent bac­te­ria, Vi­brio fis­cheri. We have pre­vi­ously touched on this sub­ject here and here. The bac­te­ria are lo­cated in a spe­cial struc­ture, the squid's light or­gan, where they pro­vide the host with a way to dis­cour­age preda­tors by shin­ing its light down­ward, which, on a moon­lit night, ren­ders the squid nearly in­vis­i­ble. Amaz­ingly, the squid gets rid of most of its lu­mi­nous bac­te­ria every day, to re­pop­u­late it­self with them at night so that bac­te­ria reach high pop­u­la­tion den­sities and emit light by a mech­a­nism that in­volves quo­rum sens­ing (QS).

Fig­ure 2. A Hawai­ian bob­tail squid (Eu­prymna sco­lopes). Source

The es­tab­lish­ment of the squid-vib­rio sym­bio­sis is quite elab­o­rate and en­tails con­sid­er­able mor­pho­ge­ne­sis on the part of the squid lar­vae. Briefly, the light or­gan of the newly hatched squid har­vests V. fis­cheri from sea­wa­ter by en­train­ing them via the beat­ing of cilia on ep­ithe­lial cells. As a re­sponse to the bacteria's PG, these cells man­u­fac­ture mu­cus that specif­i­cally cap­tures Gram-ne­­ga­ti­ves (which the vib­rios are). Next, the bac­te­ria make their way to the light or­gan by trav­el­ing through ducts to crypts, where they hap­pily mul­ti­ply. Once there, the bac­te­ria in­duce the de­ve­lop­men­tal changes that trans­form the or­gan from sim­ply a bac­te­ria-cap­tur­ing de­vice to one whose main func­tion is to emit light. To re­turn to LPS and PG, it is these two com­po­nents that act in syn­ergy to in­duce the de­vel­op­ment of the light or­gan. LPS prompts early apop­to­sis of the no-longer needed bac­te­ria-har­vest­ing cells, a process re­quired for the for­ma­tion of the ma­ture light or­gan. PG, on the other hand, pro­motes late stage apop­to­sis and the traf­fick­ing of the squid's phago­cytic blood cells, the he­mo­cytes. Re­mark­ably, the main stages of this mor­pho­gen­e­sis can be mim­ic­ked by adding pu­ri­fied LPS and PG monomers to the squid.

Now, the ques­tion be­comes, how are LPS and PG re­leased from the bac­te­ria to be de­liv­ered to the squid? As it hap­pens, these con­stituents are of­ten dis­charged into the medium, where they par­ti­ci­pate in var­i­ous kinds of host-bac­te­ria in­ter­ac­tions. One way that this takes place is by the de­tach­ment of mem­bra­nous vesi­cles from the outer mem­brane. Such outer mem­brane vesi­cles (OMV) are in­volved in path­o­genic mech­a­nisms in ver­te­brates and, alone, can trig­ger mor­pho­gen­e­sis of the light or­gan.

Fig­ure 3. Left, neg­a­tive-stained trans­mis­sion e­lec­tron mi­cro­graph (TEM) en­large­ment of a V. fi­scheri sheathed fla­gel­lum; white ar­row, area ex­pos­ing fla­gel­lar fil­a­ment; black ar­row, bleb form­ing on sheath; scale bar = 250 nm. In­set, TEM of three cells, with the fla­gel­lum in the area of en­large­ment (dot­ted box); scale bar = 1 μm. Right, TEM im­age of an OMV prepa­ra­tion; scale bar = 200 nm. Source

This begs the next ques­tion, does any­thing else fa­cil­i­tate the pro­duc­tion of OMVs? This re­quires yet an­other cu­ri­ous twist in the story. Oddly per­haps, some bac­te­ria make fla­gella that are en­cased in sheaths de­rived from the outer mem­brane. The rea­son for this strange ar­ran­ge­ment is not ob­vi­ous (me­chan­i­cal pro­tec­tion, mask­ing from an­ti­bod­ies, any­thing else, reader?). Find­ing this out is not easy be­cause no spe­cific mu­tants in this func­tion are as yet avail­able. Note that sheathed fla­gella are not unique to these vib­rios and are found in other vib­rios, He­li­cobac­ter and Bru­cella.

A re­cent study from the Mc­Fall-Ngai and Ruby labs pro­poses that the ro­ta­tion of sheathed fla­gella in­duces the re­lease of OMVs, thus lead­ing to the steps re­quired for light or­gan mor­pho­gen­e­sis. Pu­ri­fied OMVs work faster than whole bac­te­ria, per­haps be­cause the vesi­cles can dif­fuse faster into the squid's in­te­rior. Now, the au­thors asked, which com­po­nent of the OMV is in­volved here, the O anti­gen of LPS or some outer mem­brane pro­tein? Us­ing mu­tants, they could show that it is the lat­ter, specif­i­cally a pro­tein called OmpU, which is these vib­rios' ma­jor outer mem­brane pro­tein.

Fig­ure 4. Car­toon de­pict­ing OMV pro­duc­tion by dif­fer­ent V. fis­cheri strains; the pres­ence of shea­thed fla­gella and their abil­ity to ro­tate (red ar­rows), and the rel­a­tive pro­por­tion of OMV si­zes re­leased. The motB1 mu­tant has a non-ro­­ta­t­ing fla­gel­lum, the HS mu­tant is hy­per­fla­gel­la­ted. Source

The in­ves­ti­ga­tors fur­ther de­ter­mined that ro­ta­tion of the fla­gella pro­motes the pro­duc­tion of the OMVs. Mu­tants in fla­gella make fewer OMVs, fla­gella hy­per­pro­duc­ers make more. The sheath of the fla­gella mat­ter, as shown by the fact that in dif­fer­ent bac­te­r­ial species that vary in the num­ber of sheathed fla­gella per cell, their num­ber cor­re­lates with the for­ma­tion of OMVs. And these fla­gella in­flu­ence the size of the OMVs, which tend to be smaller in their pres­ence. Go fig­ure.

Now the ques­tion arises, how does the ro­ta­tion of whir­ling sheathed fla­gella con­tribute to OMV for­ma­tion? The au­thors say: "We also hy­poth­e­size that be­cause the cell's PG-linked outer mem­brane is con­tigu­ous with the rapidly ro­tat­ing mem­brane sur­round­ing the fla­gel­lar fil­a­ment, it may be that OMVs are pro­duced at this mem­brane-mem­brane in­ter­face when the bac­te­ria are motile."

So there you have it, mul­ti­func­tion­al­ity is all over the place. Con­stituents of the cell en­ve­lope, be­sides be­ing es­sen­tial struc­tural com­po­nents, act as de­vel­op­men­tal sig­nals in the squid and fla­gella do not just move the bac­te­ria around but also help them to shed some of their coat. A so­phis­ti­ca­ted world, that.