Life in a Big Mac

by Merry

From the bac­te­r­ial point of view, a para­me­cium is a mi­crocosm of­fer­ing sev­eral tempt­ing niches for coloniza­tion. When you col­lect a para­me­cium from na­ture, it's likely to have hun­dreds – even thou­sands – of bac­te­r­ial en­dosym­bionts on board, in dif­fer­ent lo­ca­tions. Some will likely be in the cy­to­plasm, oth­ers in other cel­lu­lar com­part­ments, in­clud­ing the nu­clei. To re­mind you, the cil­i­ates evolved their own par­tic­u­lar ap­proach to nu­clear com­part­ments. They haveF two kinds of nu­clei, macro­nuclei (we'll call them Macs) and mi­cronu­clei (Mics), ty­pically two or more of each. The Macs carry out all the RNA tran­scrip­tion dur­ing nor­mal veg­e­ta­tive growth; the Mics trans­mit the germline genome to sub­se­quent ge­nerations. Both the Mics and the Macs are hosts to spe­cific sym­bionts. While lo­ca­tions in mi­to­chon­dria and nu­clei have been ob­served for bac­te­r­ial pathogens of an­i­mal cells, they are rel­a­tively un­com­mon.

IFs make it to the Mac. Im­age of P. cau­datum fed a mix­ture of prey bac­te­ria (green) and IFs of H. ob­tusa (yel­low). The IFs (ar­row) are in the Mac, the prey bac­te­ria in food vac­uoles. Bar: 25 μm. Source

So far, nine en­dosym­bionts are known to live within the nu­clei of para­me­cia, about half in the Mics, half in the Macs. These nine species, α‑proteobacteria all, have been lumped to­gether into the genus Holospora be­cause of their sim­i­lar cy­tol­ogy and lifestyle. Their clos­est known rel­a­tives are Rick­ettsia and Ehrlichia. They all ap­pear to be ob­lig­ate en­dosym­bionts, that is, are not known to live out­side their hosts. All are host-spe­cific and nu­clei-spe­cific.

Struc­ture of the IF of H. ob­tusa. Source

Life in a Mac has its perks, such as a goodly sup­ply of nu­trients and pro­tec­tion from cel­lu­lar de­fenses, in­clud­ing lytic en­zymes. But what's an en­dosym­biont to do when the host cell mates? Dur­ing this process, the Macs are di­gested along with the sym­bionts. New Macs formed af­ter con­ju­ga­tion are free of bac­te­ria. This may be one rea­son why, un­like the cy­to­plas­mic sym­bionts, all of these species are in­fec­tious, that is, they are ca­pa­ble of hor­i­zon­tal trans­mis­sion.

Quite a bit is known about Holospora ob­tusa, a sym­biont liv­ing in the Mac of P. cau­da­tum. Its life cy­cle in­cludes two stages. The re­pro­duc­tive form (RF) is a short rod, 1–3 μm by 0.5–1 μm, that grows and di­vides by bi­nary fis­sion within a Mac. Some RFs dif­fer­en­ti­ate into an in­fec­tious form (IF), long rods 5 to 20 μm in length. This is a dra­matic trans­for­ma­tion, in­volv­ing the re­place­ment of 60% of their pro­teins, along with marked changes in cell struc­ture.

Phase con­trast mi­cro­graph of IFs out­side the host. In­set: Af­ter the IFs en­ter a Mac, they un­dergo mul­ti­ple di­vi­sions to give rise to RFs. Three con­stric­tions can be seen in the IF on the left. Source

Both forms co­ex­ist, in­ter­min­gled, in the Mac un­til the host un­der­goes mi­to­sis. At that time, some of the RFs move to the poles of the di­vid­ing Mac, thus en­sur­ing that each daugh­ter nu­cleus will in­herit a share. Mean­while, the IFs are des­tined to be re­leased from the cells via a spe­cial, ded­i­cated struc­ture. They gather in a clus­ter in a cen­tral bridge that forms as the daugh­ter Macs pull apart. This struc­ture is seen only in sym­biont-bear­ing para­me­cia, not in "cured" ones. As the daugh­ter nu­clei sep­a­rate, the bridge breaks away from the Mac to form a mem­brane-bounded struc­ture that trav­els through the cy­to­plasm to the cy­to­proct (the quasi-anus of cil­i­ates), there to be dis­charged into the en­vi­ron­ment.

The IFs can re­main vi­able for weeks out­side a host cell. They have a unique struc­ture, the hall­mark be­ing a mas­sive periplasm filled with pro­teins re­quired for suc­cess­ful in­fec­tion. IFs are in­gested by para­me­cia along with other bac­te­r­ial prey. But in­stead of be­ing di­gested, they es­cape from the phago­some and jour­ney – periplasm tip first – through the cy­to­plasm. They ar­rive at the Mac 30 min­utes later. This process in­volves sev­eral fancy mem­brane fu­sions and evagi­na­tions. Host actin is also re­quired. Once in the nu­cleus, the IF con­stricts trans­versely, di­vid­ing to form 4–10 RFs. The cy­cle is then re­peated.

A re­cently in­gested IF es­cap­ing from a phago­some that is filled with other prey bac­te­ria. Bar: 1.0 μm. Source

Many ob­ser­va­tions sug­gest a long shared his­tory be­tween H. ob­tusa and P. cau­da­tum, the in­ti­mate commu­nications be­tween them dur­ing in­fec­tion be­ing one. Here are some oth­ers. The as­so­ci­a­tion seems to be quite spe­cific. If you ex­per­i­men­tally in­fect a dif­fer­ent species of para­me­cium with H. ob­tusa, the IFs make it into the Mac, but within 24 hours they are de­ported to the cy­to­plasm, of­ten in frag­ments, and "shat out" via the cy­to­proct. Al­though P. cau­da­tum can get along just fine in the lab when cured of their en­dosym­bionts, some nat­ural iso­lates have H. ob­tusa on board, es­pe­cially when col­lected in cold cli­mates. In the lab, para­me­cia with H. ob­tusa en­dosym­bionts do bet­ter in colder tem­per­a­tures and also bet­ter han­dle rapid heat­ing from 25 °C to 35 °C.

The sym­bionts al­ter host gene ex­pres­sion, up-reg­u­lat­ing sev­eral genes and down-reg­u­lat­ing oth­ers. Re­pro­duc­tion of the RFs in the Mac re­quires some pro­teins made by the host. Are these en­dosym­biont genes that have been trans­ferred to the host genome? Ini­tial es­ti­mates in­di­cate a re­duced genome size for the en­dosym­biont, about 1.7 Mb, but ev­i­dence of gene trans­fer must await ge­nomic data.

En­dosym­bioses have a way of fas­ci­nat­ing us. The very idea! Bac­te­ria liv­ing within the or­ganelles in­side cells? When sketch­ing a eu­kary­ote cell, we draw a mem­brane around it, and around the nu­cleus and the other or­ganel­lar com­part­ments. These mem­branes mark se­ri­ous bound­aries. But en­dosym­bionts re­mind us that such bound­aries can be door­ways – if you know the right in­can­ta­tion.