Salmonella's Ex­clu­sive In­testi­nal Restau­rant

by Elio

In­ter­est­ing, how we get car­ried away by ex­cit­ing con­cepts. I am think­ing about how the study of pathogens has fo­cused so much on the mi­crobes' vir­u­lence fac­tors, by which we mean nasty sub­stances such as tox­ins, ad­hesins, and in­vasins that par­tic­i­pate di­rectly in the dis­ease process. Their study has dom­i­nated mi­cro­bial patho­gen­e­sis for many decades. With­out doubt, un­der­stand­ing how they func­tion is es­sen­tial, but in the process we may have tended to over­look an ob­vi­ous fact: for sur­vival, pathogens have to find nour­ish­ment in the body of the host.

Trans­mis­sion elec­tron mi­cro­graphs of S. en­ter­ica strains grown on ethanolamine as car­bon and en­ergy source. The ar­row points to one car­boxy-some-like struc­ture termed a metabolo­some. Source

Let's make a dis­tinc­tion here be­tween broad nu­tri­tional strate­gies that are com­mon to many bac­te­ria, path­o­genic or not, and those that are quite spe­cific to pathogens. We can ex­pect pathogens to have evolved their own in­di­vid­ual kind of me­tab­o­lism matched to con­di­tions in their host. A widely known ex­am­ple that comes to mind is the way many bac­te­ria have in­vented sys­tems to en­hance the sup­ply of iron within the host. But there is much more to this. Food for thought, no? An en­light­en­ing man­i­festo was pub­lished re­cently with the chal­leng­ing ti­tle Are Path­o­genic Bac­te­ria Just Look­ing for Food? Me­tab­o­lism and Mi­cro­bial Patho­gen­e­sis.

The struc­ture of tetrathion­ate ion. Source

It's ob­vi­ous that an­i­mal hosts are full of good­ies that mi­crobes can read­ily uti­lize. Our bod­ies are re­plete with small mol­e­c­u­lar weight com­pounds such as amino acids and sug­ars, and our macro­mol­e­cules can be cut to size by the mi­crobes' ex­tra­cel­lu­lar en­zymes. But if this sim­ply led to a feed­ing frenzy, there would be lit­tle in it for the pathogens, as the com­men­sals would do just as well. Here I will dis­cuss a par­tic­u­larly in­ter­est­ing case of a spe­cific meta­bolic strat­egy evolved by one of the best stud­ied of all path­o­genic bac­te­ria, Sal­mo­nella.

Schematic of ethanolamine uti­liza­tion and tetra-thion­ate res­pi­ra­tion in the in­flamed gut. Source

Here's the story. S. Ty­phimurium (I won't bother  fur­ther with its full name, Sal­mo­nella en­ter­ica serovar Ty­phimurium) causes acute in­flam­ma­tion in the gut of mice but man­ages to sur­vive it quite well. It was re­cently found that the se­lec­tive ad­van­tage for these or­gan­isms lies in their be­ing able to use a some­what un­usual sul­fur-rich com­pound, tetrathion­ate, for their en­ergy me­tab­o­lism un­der anaer­o­bic con­di­tions. Read­ers with ex­pe­ri­ence in the clin­i­cal mi­cro­bi­ol­ogy lab will re­mem­ber that Tetrathion­ate Broth is in­deed help­ful for the ini­tial en­rich­ment of sal­mo­nel­lae from fe­cal sam­ples. It was in­tro­duced in the clin­i­cal lab­o­ra­tory in 1927 and is used to this day. And there is a very good rea­son for this: by us­ing anaer­o­bic res­pi­ra­tion with tetrathion­ate as a ter­mi­nal elec­tron ac­cep­tor, the or­gan­isms ob­tain more en­ergy than their neigh­bors, which are lim­ited to fer­men­ta­tion.

Bio­chem­i­cal model of the tetrathion­ate re­duc­tase in S. ty­phimurium. The en­zyme is com­prised of the three sub­units TtrC, TtrB and TtrA. TtrC is pre-dicted to con­tain 9 trans­mem­brane he­lices. TtrC is likely in­volved in the trans­fer of elec­trons from the quinone (Q) pool to other com­po­nents of the re­duc­tase com­plex, while at the same time re­leas­ing pro­tons (H+) into the periplasm. TtrB is pre­dicted to con­tain an iron-sul­fur clus­ter (FeS) bind­ing do­main (pfam00037) and may link trans­port of elec­trons from TtrC to TtrA, the sub­unit that ac­tu­ally re­duces tetrathion­ate. TtrA con­tains a pre­dicted iron-sul­fur clus­ter-bind­ing do­main and be­longs to the molyb­dopterin (MPT) bind­ing su­per­fam­ily (cl09928) which also in­cludes var­i­ous mem­bers of ox­i­dore­duc­tases such as bac­te­r­ial ni­trate re­duc­tases, di­methyl-sul­fox­ide re­duc­tases, thio-sul­fate re­duc­tases and ar­sen­ite ox­i­dase. Al­though molyb­dopterin co-fac­tor con­tain­ing en­zymes typ­i­cally catal­y­ses re­ac­tions that in­volve the trans­fer of an oxy­gen atom or the cleav­age of a C–H bond, molyb­dopterin is as part of the ac­tive cen­ter of TtrA di­rectly in­volved in the re­duc­tion of tetrathion­ate. Source

But where does tetrathion­ate come from? It turns out that this is a good story. It in­volves a trio of par­tic­i­pants: the in­testi­nal tis­sue, the nor­mal bac­te­r­ial flora, and the sal­mo­nel­lae. Bac­te­ria in the in­testines make abun­dant hy­dro­gen sul­fide. But H₂S is quite toxic to tis­sue, prompt­ing the in­testi­nal ep­ithe­lial cells to detox­ify it to thio­sul­fate. When the sal­mo­nel­lae in­duce in­flam­ma­tion, neu­trophils mi­grate into the in­testi­nal lu­men, where they gen­er­ate re­ac­tive oxy­gen species that fur­ther ox­i­dize thio­sul­fate to tetrathion­ate. The abil­ity of the sal­mo­nel­lae to use this last com­pound as an elec­tron re­cep­tor in anaer­o­bic res­pi­ra­tion gives them a com­pet­i­tive edge in the en­vi­ron­ment of the in­tes­tine. No­tice that un­less the sal­mo­nel­lae caused in­flam­ma­tion, thio­sul­fate would not be ox­i­dized to tetrathion­ate and these or­gan­isms would lose their se­lec­tive ad­van­tage. Pretty in­ge­nious. The sal­mo­nel­lae drive the host to pro­vide them with a sub­stance that al­lows them to out­grow their com­peti­tors. If you won­dered how Tetrathion­ate Broth works, now you know.

The five genes in­volved in salmonella's tetrathion­ate res­pi­ra­tion are all lo­cated on the SPI2 path­o­genic­ity is­land. Three of them en­code the en­zyme anaer­o­bic tetrathion­ate re­duc­tase, the other two a two-com­po­nent reg­u­la­tory sys­tem. Note that these genes are needed when there is in­flam­ma­tion, thus they may help en­sure the main­te­nance of this ge­nomic is­land. In­ter­est­ing is that both "stan­dard" vir­u­lence genes and meta­bolic genes are lo­cated on the same mo­bile ge­netic el­e­ment. In a broad sense, the prod­ucts of the meta­bolic genes can also be con­sid­ered to be vir­u­lence fac­tors. In a re­cent pod­cast, This Week in Mi­cro­bi­ol­ogy #27, my col­league Michael Schmidt pro­posed that such at­trib­utes should be lumped to­gether un­der the term Fit­ness Fac­tors. Re­mem­ber, you read it here first.

It turns out that be­ing able to carry out anaer­o­bic res­pi­ra­tion has an­other ad­van­tage. An abun­dant sub­strate in the gut is ethanolamine, de­rived from the mem­brane lipid phos­phatidylethanolamine. Ethanolamine can­not be fer­mented and can only be uti­lized via res­pi­ra­tion. In­ter­est­ingly, me­tab­o­lism of ethanolamine re­quires a sac-like struc­ture akin to car­boxysomes, but how this works is still un­der study. For a re­view on the uti­liza­tion of ethanolamine by pathogens, go here. Fig. 3 Since other (fer­men­ta­tive) or­gan­isms in the gut can­not uti­lize ethanolamine, Sal­mo­nella alone is ca­pa­ble of ox­i­diz­ing it un­der these anaer­o­bic con­di­tions. The suc­cess of this or­gan­ism, there­fore, de­pends on its abil­ity to do two things, cause in­flam­ma­tion and use for its en­ergy source a spe­cific com­pound that is not ser­vice­able for other mi­crobes. I am be­gin­ning to think that I am start­ing to un­der­stand some­thing about Sal­mo­nella, or at least how it out­com­petes the huge num­ber of other or­gan­isms in the large in­tes­tine of mice.

Sal­mo­nella, the host and its mi­cro­biota. S. Ty­phimurium uses its vir­u­lence fac­tors (fla­gella, T3SS‑1 and T3SS‑2) to in­vade the ep­ithe­lium and sur­vive in mononu­clear cells. The en­su­ing in­flam­ma­tory re­sponse re­sults in the ep­ithe­lial re­lease of an an­timi­cro­bial (lipocalin‑2) that se­questers iron chela­tors (en­ter­obactin) pro­duced by the mi­cro­biota, but not an iron chela­tor (salmoche­lin) pro­duced by S. Ty­phimurium. ROS gen­er­ated by neu­trophils mi­grat­ing into the in­testi­nal lu­men ox­i­dize an en­doge­nous sul­fur com­pound (thio­sul­fate) to gen­er­ate a res­pi­ra­tory elec­tron ac­cep­tor (tetrathion­ate) that en­ables S. Ty­phimurium to edge out the fer­ment­ing mi­cro­biota, thereby en­hanc­ing trans­mis­sion of the pathogen. Source

Just don't ask me why other bac­te­ria have not learned the trick of me­tab­o­liz­ing ethanolamine us­ing tetrathion­ate for res­pi­ra­tion. But Sal­mo­nella is not alone in hav­ing this sort of skill and other ex­am­ples of meta­bolic savvy can be cited. The re­view men­tioned above de­scribes how Vib­rio cholerae in­duces the cleav­age of sialic acid from a gan­glio­side on the sur­face of ep­ithe­lial cells and uses it for its nu­tri­tion, as well as how He­li­cobac­ter py­lori man­ages to ob­tain the nickel it needs for the ac­tion of its ure­ase by pos­sess­ing a high affin­ity trans­port sys­tem for this metal. Per­haps the les­son here is that every pathogen must have its own meta­bolic reper­toire that al­lows it to out­com­pete other or­gan­isms and thrive in its host niche. If I were in a preachy mood, I'd say, go back and brush up on the cen­tral me­tab­o­lism of all mi­crobes. It may pay to do this if you want to un­der­stand the causes and ef­fects of pathogen ac­tion. Amy Vollmer made a case for me­tab­o­lism in a post Hello Again, Me­tab­o­lism in this blog a cou­ple of years ago. And you thought that you could es­cape from bio­chem­istry!

Ref­er­ence

Win­ter SE, Thi­en­nim­itr P, Win­ter MG, But­ler BP, Huseby DL, Craw­ford RW, Rus­sell JM, Bevins CL, Adams LG, Tso­lis RM, Roth JR, Bäum­ler AJ (2010). Gut in­flam­ma­tion pro­vides a res­pi­ra­tory elec­tron ac­cep­tor for Sal­mo­nella. Na­ture, 467(7314), 426–429. PMID 20864996