Putting Re­dun­dancy to Work

by Ka­t­rina Nguyen

Ge­netic re­dun­dancy, when two genes en­code for the same func­tion, is wide­spread among many or­gan­isms. Re­dun­dant genes con­fer an ad­van­tage: if one gene is lost, its part­ner can sub­sti­tute and the phe­no­type of the or­gan­ism will not change. It is un­clear how re­dun­dancy is main­tained dur­ing evo­lu­tion since se­lec­tion against un­needed genes should even­tu­ally wipe out a truly re­dun­dant gene. In ad­di­tion to pos­ing this evo­lu­tion­ary ques­tion, re­dun­dant genes also pose a tan­gi­ble prob­lem for re­searchers. It would be nice if knock­ing out a sin­gle gene re­sulted in a spe­cific phe­no­type that re­vealed the func­tion of the gene. How­ever, if a re­dun­dant gene is mu­tated or oth­er­wise knocked out, its col­league can still per­form the func­tion, and no phe­no­typic ef­fects will be seen. There­fore, as­sign­ing a func­tion to re­dun­dant genes is of­ten a real chal­lenge.

Fig­ure 1A. De­fec­tive in­tra­cel­lu­lar growth of L. pneu­mophila re­sult­ing from mu­ta­tions in Dot/Icm TS genes com­bined with de­ple­tion of host cell pro­teins as­so­ci­ated with the early se­cre­tory path­way. Cul­tured Drosophila cells treated with dsRNA were chal­lenged with a li­brary of L. pneu­mophila trans­po­son mu­tants. The rel­a­tive in­tra­cel­lu­lar growth un­der each de­ple­tion con­di­tion was de­ter­mined by TraSH. In­di­vid­ual genes are clus­tered into dis­tinct func­tional groups (red brack­ets, I to XIV) on the ba­sis of com­mon be­hav­ioral pat­terns across all host con­di­tions ex­am­ined.

A re­cent ap­proach to un­der­stand­ing host-pathogen in­ter­ac­tions pro­vides a so­lu­tion to the prob­lems of re­dun­dancy. O'Connor et al. de­vel­oped a novel ge­netic screen­ing method, called iMAD for inser­tional Muta­ge­n­e­sis And Deple­tion, which en­ables the in­ves­ti­ga­tion of com­pli­cated ge­netic in­ter­ac­tions. In iMAD, the bac­te­r­ial pathogen is mu­tated and a host func­tion needed to sup­port growth of the pathogen is de­pleted via RNA in­ter­fer­ence. The idea is to pro­duce an ag­gra­vat­ing ge­netic in­ter­ac­tion, where the com­bi­na­tion of de­fects in host and pathogen pro­duces a stronger phe­no­type than ei­ther would alone. Based on this in­ter­play, it is pos­si­ble to de­ter­mine which genes are re­dun­dant, as well as their func­tion (see here for a di­a­gram).

Fig­ure 2A. The Δwip­BΔl­idA mu­tant shows a growth de­fect in A. castel­lanii rel­a­tive to the WT and the cor­re­spond­ing sin­gle-dele­tion strain.

These re­searchers car­ried out iMAD with the bac­terium Le­gionella pneu­mophila, a hu­man res­pi­ra­tory pathogen and par­a­site of free-liv­ing amoe­bae. The bac­te­r­ial species was dis­cov­ered in 1977 af­ter a se­ri­ous out­break of pneu­mo­nia, later called Le­gion­naires' dis­ease, at a con­ven­tion of the Amer­i­can Le­gion. L. pneu­mophila is trans­mit­ted through aerosols and can live in most sources of stand­ing wa­ter, such as swim­ming pools, air con­di­tion­ing, cool­ing tow­ers, or fresh­wa­ter ponds.  L. pneu­mophila pro­duces over 270 ef­fec­tor pro­teins that are se­creted via the Dot/Icm Type IV se­cre­tion sys­tem. Some of these ef­fec­tor pro­teins may be in­volved in the repli­ca­tion of the bac­te­ria in­side amoeba or hu­man macrophages. For ex­am­ple, some of them are hy­poth­e­sized to pre­vent the de­liv­ery of bac­te­ria-con­tain­ing vac­uoles to the bac­te­ri­ci­dal com­pound-laden lyso­somes. Many of these pro­teins were sus­pected to be re­dun­dant, mak­ing them good can­di­dates for iMAD.

The re­searchers first cre­ated a li­brary of L. pneu­mophila trans­poso nmu­tants, as well as a set of Drosophila cells that were de­pleted of nec­es­sary fac­tors by the ac­tion of dsR­NAs. The Drosophila cells were then in­fected with the Le­gionella mu­tants and their in­tra­cel­lu­lar growth quan­ti­fied us­ing Trans­po­son Site Hy­bridiza­tion (TraSH). The re­searchers charted the bac­te­r­ial mu­tants against the de­pleted Drosophila cells and looked for ef­fects on growth of the bac­te­ria, not­ing ad­van­tages, dis­ad­van­tages, or no change com­pared to wild-type in­tra­cel­lu­lar growth (Fig­ure 1A).

Fig­ure 1F. The re­dun­dant re­la­tion­ships be­tween func­tional groups de­fied by ge­netic in­ter­ac­tion map­ping. The lines in­di­cate ag­gra­vat­ing ge­netic in­ter­ac­tions af­fect­ing in­tra­cel­lu­lar growth in un­treated Drosophila cells when two L. pneu­mophila genes, one from each func­tional group in blue let­ters), are deleted in com­bi­na­tion.

The genes that pro­duced sim­i­lar phe­no­types were clus­tered into groups based on their func­tion (Fig­ure 1F). Each clus­ter con­tains pro­teins that are part of the same com­plex or that par­tic­i­pate in the same process. The lines con­nect­ing the clus­ters in­di­cate re­dun­dancy. Mu­tat­ing one gene in con­junc­tion with a gene from a con­nect­ing group re­sulted in an ag­gra­vated ge­netic in­ter­ac­tion, which sug­gests that the two are re­dun­dant. Fur­ther re­dun­dan­cies could be in­ferred from such clus­ter­ing. For ex­am­ple, if gene wipB from group IX was shown to be re­dun­dant with lidA, a gene from XI, then all the genes from IX can be thought to be re­dun­dant with all the genes from XI.

Fig­ure 2C. The Δwip­BΔl­idA mu­tant is de­fec­tive for re­cruit­ment of the ER-tar­geted fu­sion pro­tein GFP-HDEL in the amoeba D. dis­coideum. Amoe­bas ex­press­ing GFP-HDEL were in­fected with L. pneu­mophila strains ex­press­ing the red flu­o­res­cent pro­tein td­Tomato for 4 hours, fixed, and vi­su­al­ized by flu­o­res­cence mi­croscopy. Ar­rows point to the bac­te­ria.

The re­sults of iMAD were then con­firmed ex­per­i­men­tally. In Fig­ure 2A, an ag­gra­vated phe­no­type due to in­ter­ac­tion be­tween wipB and lidA is seen in the in­tra­cel­lu­lar growth of the dou­ble mu­tant in the amoeba Acan­thamoeba castel­lanii, com­pared to wild-type and the sin­gle mu­tants. Flu­o­res­cent mi­croscopy (Fig­ure 2C) also showed that the dou­ble wipB/lidA knock­out was com­par­a­tively de­fi­cient in re­cruit­ing the tar­geted flu­o­res­cent fu­sion pro­tein GFP-HDEL, which acts as a proxy for en­do­plas­mic retic­u­lum pro­teins.

Dou­ble mu­tants of L. pneu­mophila in­fect­ing an­other amoeba host, Dic­tyostelium dis­coideum, as well as in macrophages from mouse bone mar­row sup­ported the iMAD-de­rived clus­ter­ing. How­ever, L. pneu­mophila grew in­tra­cel­lu­larly to a dif­fer­ent ex­tent in each host, which may re­flect dif­fer­ences in the hosts' se­cre­tory path­ways. This re­sult sup­ports the idea that L. pneu­mophila uti­lizes re­dun­dant path­ways in or­der to flour­ish in dif­fer­ent en­vi­ron­ments. The bac­te­ria are likely to re­quire a some­what dif­fer­ent set of vir­u­lence pro­teins for op­ti­mal fit­ness in each host.

By us­ing iMAD to de­ter­mine clus­ters of func­tion­ally sim­i­lar pro­teins, it has been pos­si­ble to iden­tify re­dun­dancy be­tween dif­fer­ent vir­u­lence pro­teins. A lim­i­ta­tion of the method is that iMAD re­quires that a pathogen have con­ver­gent bio­chem­i­cal path­ways that af­fect an eas­ily ma­nip­u­lated sys­tem of the host. Vari­a­tions of iMAD should be ap­plic­a­ble to a va­ri­ety of dif­fer­ent sys­tems as well. The screen is in­no­v­a­tive, in­ter­est­ing, and pro­vides a great deal of data that can eas­ily be con­firmed with ad­di­tional tech­niques. With iMAD, O'Connor et al. have made it pos­si­ble to over­come the ex­per­i­men­tal bot­tle­neck of ge­netic re­dun­dancy and to pin­point the role of cer­tain bac­te­r­ial pro­teins in patho­gen­e­sis. I ex­pect that this ap­proach will be widely used in the fu­ture with other pathogens and their host cells.

 

Ref­er­ence

O'Connor T, Boyd D, Dorer M, Is­berg R (2012). Ag­gra­vat­ing Ge­netic In­ter­ac­tions Al­low a So­lu­tion to Re­dun­dancy in a Bac­te­r­ial Pathogen. Sci­ence, 338 (6113), 1440−1444. DOI 10.1126/science.1229556

 

Ka­t­rina is a stu­dent in the 2013 Uni­ver­sity of Cal­i­for­nia at San Diego/San Diego State Uni­ver­sity In­te­gra­tive Mi­cro­bi­ol­ogy grad­u­ate course.