Cox­iella In­ter­cepts Host Sig­nals!

by S. Mar­vin Fried­man

Queens­land scenery. Al­though Q fever was first dis­cov­ered in Queens­land, Aus­tralia, the "Q" ap­par­ently stands for "query" and not for the name of the place. Source

To the con­ster­na­tion of med­ical stu­dents and oth­ers who are obliged to learn such mat­ters for ex­ams, the num­ber of spe­cial at­trib­utes that dis­tin­guish one pathogen from the oth­ers is colos­sal. But to those who are gen­uinely in­ter­ested in the world of pathogens, each as­sort­ment of dis­tin­guish­ing prop­er­ties holds its own fas­ci­na­tion. Cox­iella bur­netii is a fine ex­am­ple of an agent rich in strik­ing traits. This is a γ‑proteobacterium, some­what akin to Le­gionella, that causes a zoonotic dis­ease, Q fever, which man­i­fests as an acute in­fluenza-like ill­ness. It can also es­tab­lish a chronic in­fec­tion in im­muno­com­pro­mised in­di­vid­u­als re­sult­ing in a po­ten­tially fa­tal en­do­cardi­tis that is no­to­ri­ously re­cal­ci­trant to an­tibi­otics. C. bur­netii is usu­ally dis­sem­i­nated via con­t­a­m­i­nated aerosols and live­stock work­ers are ex­posed to this pathogen while work­ing with in­fected an­i­mals. A 2007–2009 out­break of Q fever in Hol­land af­flicted over 4,000 peo­ple and claimed 11 lives. This is a highly in­fec­tious agent that has been in­cluded in the list of pos­si­ble can­di­dates for bioter­ror­ism. Clearly, we need to know more about this agent and how to con­trol it.

Q fever caused by C. bur­netii can be ac­quired from in­fected goats. Source: Wiki­me­dia

It had been thought that C. bur­netii is con­fined to liv­ing within host cells—a strict in­tra­cel­lu­lar par­a­site. In fact, it was re­cently cul­tured on it own, out­side cells, thanks to sharp in­sights into its me­tab­o­lism that al­lowed re­searchers to de­sign­spe­cial growth con­di­tions. We dealt with this topic here, in a piece that bore the happy ti­tle Cox­iella Es­capes Cell!

Among its other spe­cial at­trib­utes, C. bur­netii makes a some­what unique spore-like re­sis­tant tran­sit form, and—the topic of this article—it has evolved an un­usual strat­egy for in­tra­cel­lu­lar sur­vival. The or­gan­ism­repli­cates in­side alve­o­lar macrophages within a spe­cial­ized phago­some-like struc­ture that war­rants a dis­tinct name, the C. bur­netii-Replica­tive Vacuole (CRV, also known as the par­a­sitophorous vac­uole). The CRV is re­mark­able for its huge size, some­times oc­cu­py­ing nearly the whole cell vol­ume. Re­call that lyso­somes fuse with phago­somes to de­liver en­zymes toxic to pathogens, but the CRV de­lays this fu­sion, thus pro­tect­ing the pathogen from the on­slaught of these en­zymes and al­low­ing it to repli­cate re­peat­edly. For a dis­cus­sion of this spe­cial­ized struc­ture, see a re­view. But, in ad­di­tion to this par­tic­u­lar skill, C. bur­netii is an ex­pert at hi­jack­ing var­i­ous host sig­nal­ing path­ways to en­sure its own sur­vival and repli­ca­tion.

The C. bur­netii par­a­sitophorous vac­uole (CRV). Scan­ning elec­tron mi­cro­graph show­ing a cryo-pre­pared Vero cell in­fected with C. bur­netii for 4 days. The host cell cy­to­plasm (tan) con­tains a large CRV filled with C. bur­netii (or­ange) (Im­age cour­tesy of Beth Fis­cher, Rocky Moun­tain Lab­o­ra­to­ries). Source

As is the case with other in­tra­cel­lu­lar par­a­sites, it's in Cox­iella's in­ter­est to have the host cell re­main in­tact for a long time. One way is to help the cell steer clear of de­struc­tion by apop­to­sis. In­deed, Cox­iella accom­plishes just that, by hav­ing sev­eral anti-apop­totic mech­a­nisms in its ar­ma­men­tar­ium. One of them pre­vents the re­lease of cy­tochrome c from mi­to­chon­dria, which avoids the ac­ti­va­tion of the apop­to­sis-re­quired "ex­e­cu­tioner" cas­pases. The or­gan­ism has other anti-apop­totic mech­a­nisms, which I will dis­cuss here.

A re­cent study re­vealed that, dur­ing in­fec­tion, the host's anti-apop­totic ki­nases AKT and ERK 1/2 be­come ac­ti­vated by phos­pho­ry­la­tion, which leads to in­hi­bi­tion of apop­to­sis. An­tibi­otics that block Cox­iella tran­scrip­tion negate this ef­fect, sug­gest­ing that the anti-apop­totic ac­tiv­ity is in­deed trig­gered by the bac­te­ria. The cox­iel­las do this via a Type IV se­cre­tion sys­tem (T4SS) that translo­cates bac­te­r­ial ef­fec­tor pro­teins into the host cy­tosol. The role of these ki­nases was con­firmed by us­ing in­hibitors of the AKT ki­nase or an an­tag­o­nist of the ERK 1/2 ki­nase. In the pres­ence of these in­hibitors, the in­fected cells died, pre­sum­ably by apop­to­sis.

It gets more tan­gled. The anti-apop­totic ac­tiv­ity of C. bur­netii in­volves an in­ter­con­nec­tion be­tween apop­to­sis and au­tophagy (the se­ques­tra­tion of dam­aged cell ma­te­r­ial and in­vad­ing bac­te­ria into vesi­cles that even­tu­ally fuse with lyso­somes for de­struc­tion of the "cargo." See a pre­vi­ous post). Al­though au­tophagy is usu­ally at play in the de­struc­tion of in­tra­cel­lu­lar pathogens, in the case of C. bur­netii, it is ac­tu­ally re­quired for the for­ma­tion of the CRV. This or­gan­ism di­rects the or­ches­tra­tion be­tween au­tophagy and apop­to­sisvia the team­work of two pro­teins, the au­tophagy-re­lated Be­clin 1 and the anti-apop­totic Bcl‑2(please keep these two names apart – they are dif­fer­ent but in­ter­act­ing pro­teins). A study shows that soon af­ter in­fec­tion, Be­clin 1 is re­cruited to the mem­brane of the CRV, along with a pro­tein that is a marker for au­tophago­somes, LC3. Trans­fect­ing in­fected cells with a plas­mid that deletes en­doge­nous Be­clin 1 via a small in­ter­fer­ence RNA led to a sig­nif­i­cant de­crease in vac­uole num­ber and size.

Mod­u­la­tion of the au­tophagy and apop­to­sis path­ways by Cox­iella bur­netii (Cb). Cb is in­ter­nal­ized into the host cell af­ter a clas­si­cal phago­cy­to­sis. Au­tophagy path­way is in­duced, prob­a­bly through in­jected pro­teins through the Type IV Se­cre­tion Sys­tem of Cb. Thus, the com­part­ment con­tain­ing Cb fuses with autophago­somes, ac­quir­ing au­tophagic key pro­teins, such as LC3, Rab24 and Be­clin 1, which fa­vor the gen­er­a­tion of the large replica­tive vac­uoles in which Cb sur­vives and mul­ti­ples. More­over, Cb also es­tab­lishes a per­sis­tent in­fec­tion by means of in­hibit­ing apop­to­sis in in­fected cells. This anti-apop­totic ef­fect of Cb is due, at least in part, to the re­cruit­ment of the anti-apop­totic pro­tein Bcl‑2 to the mem­brane vac­uole. In con­cert with Be­clin 1, the bind­ing of pro-apop­totic pro­teins (e.g. BH3-only pro­teins) to Bcl‑2 is avoided and, there­fore, the trig­ger­ing of apop­to­sis is in­hib­ited. Source

The as­so­ci­a­tion of Be­clin 1 with Bcl‑2 has been known for some time but fur­ther de­tails have emerged. At early time points af­ter in­fec­tion, it has been shown that a mu­tant Be­clin 1 de­fec­tive in its abil­ity to bind to Bcl‑2 still gets re­cruited to the CRV mem­brane, but only few cells be­come in­fected. Af­ter longer time pe­ri­ods, both the num­ber and size of CRVs are re­duced. This and other data sug­gest that a proper ra­tio of Be­clin 1 and Bcl‑2 is re­quired for nor­mal CRV de­vel­op­ment. What re­sults from these trans­ac­tions is the for­ma­tion of a CRV in a cell able to avoid apop­totic de­struc­tion. Repli­ca­tion of the Cox­iella is now as­sured.

And there is more. A re­cent re­port de­scribes yet an­other host path­way ma­nip­u­lated by C. bur­netii to pre­vent apop­to­sis. It had been known that there are quite a few host ki­nases in­volved In CRV for­ma­tion, eleven in num­ber. The pa­per fo­cuses on one of them, PKA, a cAMP-de­pen­dent ki­nase. Pre­treat­ment with two PKA-spe­cific in­hibitors be­fore in­fec­tion re­sulted in smaller CRVsand lower vi­able bac­te­r­ial counts. Thus, PKA ac­tiv­ity is re­quired for op­ti­mal vac­uole for­ma­tion and thus bac­te­r­ial repli­ca­tion. Lev­els of phos­pho­ry­lated PKA were much higher in in­fected cells than in un­in­fected cells. Bad, a mi­to­chon­dr­ial apop­totic pro­tein that reg­u­lates the re­lease of cy­tochrome c, was phos­pho­ry­lated to a higher level in in­fected cells, thus also play­ing a role in pre­vent­ing apop­to­sis.

One more ad­di­tion to the story: as is the case with some other pathogens, actin is in­volved in the repli­ca­tion of C. bur­netii. In in­fec­tion, F‑actin be­comes re­arranged and is re­cruited to the ma­tur­ing CRV, a process re­quired for the vacuole's ex­pan­sion. This ac­tiv­ity is reg­u­lated by PKA. Two small GT­Pases, Rho A and Cdc42 that con­trol actin as­sem­bly, are also re­cruited to the vac­uole mem­brane shortly af­ter in­fec­tion. PKA has been shown to reg­u­late the ac­tiv­i­ties of both these ki­nases, which re­in­forces the role of PKA in poly­mer­iz­ing actin around the ma­tur­ing vac­uole.

C. bur­netii is not alone in pil­fer­ing host sig­nals to fur­ther its own ends. Sev­eral other in­tra­cel­lu­lar bac­te­r­ial pathogens are known to scav­enge their host's sig­nal­ing ma­chin­ery, e.g., Bru­cella suis, My­cobac­terium tu­ber­cu­lo­sis, He­li­cobac­ter py­lori, Neis­se­ria gon­or­rheae, Le­gionella pneu­mophila, Sal­mo­nella en­ter­ica and Chlamy­dia tra­choma­tis. These stud­ies on Cox­iella re­in­force the idea that the ma­nip­u­la­tion of host sig­nal­ing path­ways is a ma­jor theme in bac­te­r­ial patho­gen­e­sis. Such re­sults re­veal how sig­nal­ing ac­tiv­i­ties be­come ma­nip­u­lated in in­fected cells, thus ex­pos­ing some of the tricks used by pathogens to dis­rupt nor­mal cell func­tions. Each tac­tic that is un­cov­ered pro­vides us with one more po­ten­tial strat­egy for pre­vent­ing or cur­ing bac­te­r­ial dis­eases.

Ref­er­ences

Voth DE, Heinzen RA (2009). Sus­tained ac­ti­va­tion of Akt and Erk1/2 is re­quired for Cox­iella bur­netii an­ti­apop­totic ac­tiv­ity. In­fec­tion and im­mu­nity, 77 (1), 205−213. PMID 18981248

Vázquez CL, Colombo MI (2010). Cox­iella bur­netii mod­u­lates Be­clin 1 and Bcl‑2, pre­vent­ing host cell apop­to­sis to gen­er­ate a per­sis­tent bac­te­r­ial in­fec­tion. Cell Death and Dif­fer­en­ti­a­tion, 17 (3), 421−438. PMID 19798108

Mac­Don­ald LJ, Kurten RC, Voth DE (2012). Cox­iella bur­netii al­ters cyclic AMP-de­pen­dent pro­tein ki­nase sig­nal­ing dur­ing growth in macrophages. In­fec­tion and im­mu­nity, 80 (6), 1980−1986 PMID: 22473604

Mar­vin is Pro­fes­sor Emer­i­tus in the De­part­ment of Bi­o­log­i­cal Sci­ences at Hunter Col­lege of CUNY in New York City, and an As­so­ciate Blog­ger for Small Things Con­sid­ered.