It's Time for Sal­mo­nella

by Su­san Golden

If one were to ask, "What hap­pens when Sal­mo­nella in­fects the gut?" it might not be ob­vi­ous that you'd also need to ask, "What time is it?" But in­deed you'd need to know the lat­ter to ap­pro­pri­ately ad­dress the for­mer. An in­vad­ing Sal­mo­nella, in­tent on set­ting up camp, is try­ing to pitch its tent on a land­scape that is chang­ing through­out the day. The mam­malian gut, like most eu­kary­otic and some prokary­otic cells, uses an in­ter­nal tim­ing de­vice to con­trol its phys­i­o­log­i­cal func­tions. The cir­ca­dian, or 24‑h (circa diem, about a day) clock em­ploys an en­doge­nous os­cil­la­tor to con­trol gene ex­pres­sion, me­tab­o­lism, and other cel­lu­lar processes in such a way that in­di­vid­ual genes, path­ways, and their com­po­nents ebb and flow in ac­tiv­ity with dis­tinc­tive daily pro­grams, mesh­ing to­gether to ex­e­cute the en­ter­prise of the cell. Among cir­ca­dian reg­u­lated processes are as­pects of the im­mune sys­tem, in­clud­ing com­po­nents of the in­flam­ma­tory re­sponse. In­cluded are changes in the ex­pres­sion of sev­eral in­flam­ma­tion-re­lated ("proin­flam­ma­tory") cy­tokines by macrophages not only in the an­i­mal but also ex-vivo. Not co­in­ci­den­tally, Sal­mo­nella thrives in the in­flamed gut.

Bel­let and col­leagues set out to in­ves­ti­gate the rel­e­vance of cir­ca­dian rhythms of the mam­malian im­mune re­sponse dur­ing in­fec­tion by us­ing a mouse model. In this pro­to­col, an­i­mals are pre­treated with strep­to­mycin, overtly to rid the mouse of much of its in­testi­nal mi­cro­biome (this is known as the "col­i­tis model"). Pre­treat­ment with the an­tibi­otic changes the way the mice re­spond to Sal­mo­nella en­ter­ica serovar ty­phimurium, which would oth­er­wise mount a sys­temic in­fec­tion more sim­i­lar to ty­phoid in hu­mans. The handy mouse col­i­tis model frees re­searchers from the re­stric­tive and ex­pen­sive need of us­ing the other es­tab­lished model: cows. The mice pre­treated with strep­to­mycin, once in­fected with Sal­mo­nella, mount a mas­sive in­flam­ma­tory re­sponse in the gut, as do hu­mans.

Fig­ure 1. Rep­re­sen­ta­tive im­ages (10× mag­ni­fi­ca­tion) of ce­cal in­flam­ma­tion in WT mice in­fected (Sal­mo­nella) or not at day or night. Source

The au­thors asked whether the mag­ni­tude of the re­sponse and the abil­ity of the bac­te­ria to col­o­nize the gut are in­flu­enced by the time of day when mice are in­fected, and/or the time of day when the mea­sure­ment is taken—even when the elapsed time of in­fec­tion is equiv­a­lent. Such an out­come would be likely if a tem­po­rally reg­u­lated in­flam­ma­tory re­sponse is at work. They found that both these as­pects are time-de­pen­dent. Mice in­fected in the morning—the be­gin­ning of the rest time for these noc­tur­nal creatures—showed sig­nif­i­cantly higher lev­els of Sal­mo­nella col­o­niza­tion and higher de­gree of in­flam­ma­tion than those in­fected 12 hours later in the day. The dif­fer­ence in in­flam­ma­tion be­tween mice in­fected at 10 AM and 10 PM was es­pe­cially ev­i­dent at 72 h post-in­fec­tion (Fig. 1). As we will see be­low, the in­flam­ma­tion-re­lated genes show a cir­ca­dian pat­tern of gene ex­pres­sion with a peak dur­ing the day. There­fore, the time of mea­sure­ment is im­por­tant, no mat­ter when the mice are in­fected. How­ever, the over­all mag­ni­tude of ex­pres­sion of in­flam­ma­tion-re­lated genes is higher in day-in­fected than night-in­fected mice. Mice that carry an al­tered al­lele of the Clock gene, or are miss­ing this gene en­tirely, are per­turbed in their cir­ca­dian rhythms of the im­mune re­sponse and do not show this day/night dif­fer­ence in re­sponse to Sal­mo­nella in­fec­tion.

The au­thors next in­ves­ti­gated the po­ten­tial link be­tween cir­ca­dian reg­u­la­tion of an­timi­cro­bial pep­tides in the in­flamed gut, to which Sal­mo­nella is re­sis­tant, and im­proved col­o­niza­tion by Sal­mo­nella dur­ing the day. Lipocalin‑2 is one such pep­tide that sup­presses growth of most bac­te­ria by bind­ing up iron and de­priv­ing the bac­te­r­ial cells of this nu­tri­ent. It is ex­pressed to a much higher level dur­ing the day than at night. Sal­mo­nella, how­ever, has a high-affin­ity iron chela­tor, salmoche­lin, that over­comes the ef­forts of Lipocalin‑2. Hence, Sal­mo­nella should have a growth ad­van­tage over com­pet­ing mi­crobes dur­ing the day. The au­thors rea­soned that the iroN mu­tant of Sal­mo­nella, lack­ing the re­cep­tor for salmoche­lin, would be out­com­peted by wild-type Sal­mo­nella dur­ing the day, but that the two strains would be re­cov­ered at equal lev­els when in­fected dur­ing the night. Their ex­per­i­ments sup­ported this pre­dic­tion.

Fig­ure 2. Heat di­a­gram show­ing changes in gene ex­pres­sion de­tected by mi­croar­ray analy­sis in the ceca of un­in­fected WT and Clock mu­tant mice at day (10:00 AM) and night (10:00 PM). Rel­a­tive in­crease (red) or de­crease (green) of mRNA level is shown. A list of the most rep­re­sented sub­cat­e­gories of genes, the num­ber of genes in­cluded in each sub­cat­e­gory, and the rel­a­tive P value (solid lines) are shown.

These work­ers iden­ti­fied clus­ters of mouse genes whose ex­pres­sion changes un­der dif­fer­ent con­di­tions: day vs. night, a nor­mal host vs. a cir­ca­dian mu­tant, with and with­out in­fec­tion. In many cases, com­pu­ta­tional analy­sis sug­gested that tran­scrip­tion fac­tors that con­trol groups of im­mune func­tion genes are reg­u­lated by the cir­ca­dian clock. This analy­sis pro­vides in­sights into the con­nec­tions be­tween the clock and sus­cep­ti­bil­ity and patho­gen­e­sis. In the words of the au­thors: "The re­sults of our analy­sis point to con­nec­tions of the cir­ca­dian clock to other func­tional sys­tems, in­clud­ing meta­bolic and im­mune, dur­ing in­fec­tion and will be in­stru­men­tal for fu­ture stud­ies fo­cused on elu­ci­dat­ing these mech­a­nisms."

These ex­per­i­ments all fo­cused on the role of the mam­malian cir­ca­dian sys­tem in re­spond­ing to in­fec­tion by Sal­mo­nella, but the work also showed that the in­flu­ence goes both ways. While the host cells are tem­po­rally mod­u­lat­ing the hos­pitabil­ity of the en­vi­ron­ment of the in­vad­ing Sal­mo­nella, the bac­te­ria are also in­flu­enc­ing cir­ca­dian rhythms in the host. The au­thors re­ported sup­pres­sion or at­ten­u­a­tion of cir­ca­dian rhythms of ex­pres­sion in me­tab­o­lism and clock-mech­a­nism genes at the site of in­fec­tion. They pro­pose that rhythms in im­mune fac­tors are self-re­in­forc­ing, but the pow­er­ful sig­nal of an im­mune chal­lenge dis­rupts cir­ca­dian co­or­di­na­tion, un­cou­pling some cir­ca­dian out­puts and re­sult­ing in less co­he­sive and, hence, lower am­pli­tude rhythms. Mi­cro­bi­ol­o­gists ap­pre­ci­ate that bac­te­ria are so­phis­ti­cated life forms, as are cells lin­ing the in­tes­tine, and that the in­ter­ac­tion be­tween the two adds ad­di­tional lay­ers of com­plex­ity. Here, the au­thors show that the sit­u­a­tion be­comes even more in­ter­est­ing when a bac­terium sets its sights on mam­malian cells that also watch the clock.

 

Ref­er­ence

Bel­let MM, De­riu E, Liu JZ, Grimaldi B, Blaschitz C, Zeller M, Ed­wards RA, Sa­har S, Dan­dekar S, Baldi P, George MD, Raf­fatellu M, Sas­sone-Corsi P (2013). Cir­ca­dian clock reg­u­lates the host re­sponse to Sal­mo­nella. Proc Natl Acad Sci USA, 110 (24), 9897−9902. PMID 23716692

 

Su­san is Dis­tin­guished Pro­fes­sor of Mol­e­c­u­lar Bi­ol­ogy at the Uni­ver­sity of Cal­i­for­nia at San Diego.