Go­ing Next Door With­out Get­ting Your Feet Wet

by Elio

In­tra­cel­lu­lar life has its perks. In­side host cells, bac­te­ria are pro­tected from neu­trophils, com­ple­ment, an­ti­bod­ies, some an­tibi­otics, and the other un­pleas­ant things float­ing around in tis­sue flu­ids. In ad­di­tion, here they have ac­cess to am­ple food. But there is a catch, namely how to in­fect other cells and other hosts.

In­tra­cel­lu­lar life has an­cient and dis­tin­guished ori­gins. When uni­cel­lu­lar eu­kary­otes first arose, not only did they arise be­cause some­one (who­ever it was) in­gested a prokary­otic cell, but soon there­after the emerg­ing pro­tists must have learned to go af­ter the abun­dant food mi­cro­bial around them. The prey, in turn learned to cope with this and to sur­vive in­side the preda­tor cells, some­thing some bac­te­ria do suc­cess­fully to this very day. Con­sider how these over­come the de­struc­tive forces of phago­cytes and other cells. The reper­toire of ways they do it is im­pres­sive in­deed. Of course, not all bac­te­ria live in­tra­cel­lu­larly, but all viruses must do so at some stage, so this sub­ject is old hat to vi­rol­o­gists.

Fig­ures 16 and 17. (16) Thin sec­tion of a por­tion of the sur­face of a macrophage in­fected for 4 h with Lis­te­ria. These macro­phages were fixed in situ in the dish in which they were grow­ing, a, Lo­cated at the tip of a pro­jec­tion from the macrophage cell sur­face is a sin­gle Lis­te­ria and be­hind it a long, fine, fil­a­men­tous tail. b, The fine, fil­a­men­tous tail at higher mag­ni­fi­ca­tion. Note that the fil­a­ments are ran­domly ori­ented rel­a­tive to each other, some in trans­verse sec­tion (dots), oth­ers in oblique and lon­gi­tu­di­nal sec­tion. (17) Macrophages were in­fected with Lis­te­ria for 4 h, then ex­tracted with Tri­ton X‑100 and in­cu­bated with S1. This sec­tion is taken of the same re­gion as Fig. 16. Basal to the Lis­te­ria at the end of this pseudo­pod is the fine, fil­a­men­tous tail whose com­po­nent fil­a­ments are dec­o­rated with S1. The small ar­row in­di­cates the po­lar­ity of sev­eral dec­o­rated fil­a­ments. The large ar­row in­di­cates resid­ual mem­brane that has not been sol­u­bi­lized. Source

For both bac­te­ria and viruses, the in­tra­cel­lu­lar lifestyle presents a prob­lem. How do they in­fect their next host? How do they es­cape from their "prison cell"? One way out is to lyse the cell, but this ex­poses them to the vi­cis­si­tudes of ex­tra­cel­lu­lar life. A safer tac­tic is to move di­rectly to an ad­ja­cent cell. Lis­te­ria, Shigella, and Rick­ettsia do just that, and in an in­ge­nious way, by bor­row­ing some of the actin from their eu­kary­otic host cell. The bac­terium uses an actin-bind­ing pro­tein, ActA, to poly­mer­ize the actin so as to form a comet tail ex­tend­ing out­wards from one bac­te­r­ial pole. Why not at both poles? This would lead to the dilemma of Dr. Doolittle's two headed llama, with not much in the way of di­rec­tional move­ment. The two poles of the bac­terium are not the same. ActA is present at higher con­cen­tra­tion near the old pole— the one present in the mother cell—so that pole has first dibs in bind­ing actin. The bac­te­ria are im­pelled for­wards by the poly­mer­iza­tion of actin fibers be­hind them. The only thing that the bac­terium needs to make this hap­pen is its lo­cal­ized ActA. If you coat small poly­styrene beads with ActA and place them in an actin-rich cell ex­tract and sup­ply ATP, they move quite well. Fun can be had by watch­ing movies from Julie Theriot's lab (click here and don't miss the "Prime Time En­ter­tain­ment" sec­tion). In­ter­est­ingly, vac­cinia viruses also use this kind of motil­ity.

The in­tra­cel­lu­lar life cy­cle of Lis­te­ria. The bac­te­ria en­ter the host cell and are con­tained within vac­uoles where they se­crete a pore-form­ing pro­tein, lis­te­ri­olysin O (LLO), al­low­ing bac­te­r­ial es­cape and en­try into the cy­tosol where they can grow and repli­cate. Here, the bac­te­ria use host actin to move and pro­pel them­selves into neigh­bor­ing cells. Fig­ure from Tilney and Port­noy. Source

Sooner or later, bac­te­ria that move in­side a cell, such as Lis­te­ria, will run up against the cell mem­brane. Some­how, this in­duces the for­ma­tion of host cell protrusions—long fil­a­ments that are phago­cy­tosed and taken up by the ad­ja­cent cell. A bac­terium po­si­tioned at the tip of this struc­ture is now in a good po­si­tion to in­vade this ad­ja­cent cell. To do this, it must tra­verse the mem­brane of its orig­i­nal home cell, then pass through that of the phago­some in­side the new cell. But keep in mind that Lis­te­ria are good at cross­ing mem­branes, us­ing a pore-form­ing pro­tein called lis­te­ri­olysin.This story was elu­ci­dated in 1989 by Tilney and Port­noy.

Ex­cit­ing though this may be, this mech­a­nism must now share the glory with a re­cently dis­cov­ered mech­a­nism for in­ter­cel­lu­lar travel. First, some­thing about the or­gan­ism in­volved. It is Burk­holde­ria thai­lan­den­sis, a rel­a­tive of the-rel­a­tively-bet­ter known B. pseudo­ma­llei, the agent of me­lioi­do­sis (a se­ri­ous and some­times lethal hu­man dis­ease) and of B. mallei, (the agent of the equine dis­ease glan­ders). One rea­son for work­ing with B. thai­lan­den­sis is that the other two are con­sid­ered pos­si­ble bioter­ror­ism agents and there­fore re­quire high level biosafety con­tain­ment (BL3). B. thai­lan­den­sis is less vir­u­lent and can be ma­nip­u­lated us­ing fewer pre­cau­tions (BL2). The re­searchers, led by Jeff F. Miller at UCLA, dis­cov­ered a new mech­a­nism by which these in­tra­cel­lu­lar bac­te­ria trans­fer to new cells. Motil­ity in this case is due to the ac­tion of fla­gella (al­though these or­gan­isms also have the actin-based mech­a­nism). I be­lieve this may be the first time that fla­gel­lar mo­tion has been de­scribed for a bac­terium in­side a host cell, but I could be wrong. In any case, this is an ef­fec­tive way of mov­ing about, com­pa­ra­ble to the tra­di­tional actin sys­tem.

Model of the in­tra­cel­lu­lar life cy­cle of Burk­holde­ria. In­vasins, which have yet to be iden­ti­fied, fa­cil­i­tate actin- de­pen­dent in­ter­nal­iza­tion into a pri­mary en­do­some. The ac­tiv­ity of T3SSBsa is re­quired for es­cape from the en­do­some and en­try into the cy­to­plasm. Ei­ther one of two in­de­pen­dent motil­ity sys­tems, fla­gel­lar (Fla2) or actin based (BimA), is re­quired for ef­fi­cient cell–cell spread. T6SS‑1 fa­cil­i­tates in­ter­cel­lu­lar spread and mult­i­n­u­cle­ate gi­ant cell for­ma­tion. Mem­brane in­ter­ac­tion fa­cil­i­tated by motil­ity and T6SS‑1 can also lead to cell death. The life­cy­cle model for the Burk­holde­rias con­trasts with that of Lis­te­ria and Shigella be­cause it does not re­quire en­gulf­ment of mem­brane pro­tru­sions with bac­te­ria at their tips,
ob­vi­at­ing the need for ly­sis of dou­ble-mem­brane vesi­cles for cell–cell spread. Source

One more thing. These bac­te­ria in­duce the fu­sion of ad­ja­cent cells into gi­ant mult­i­n­u­cle­ated syn­cy­tia. This way, the bugs do not have to go to the trou­ble of cross­ing two mem­branes in or­der to reach a new host cell. This sit­u­a­tion does not last for­ever be­cause, in time, the gi­ant cells are de­stroyed. In­volved here is a se­cre­tion sys­tem, Type VI (T6SS), which is wide­spread among Gram-neg­a­tives. The fac­tors se­creted by this par­tic­u­lar T6SS par­tic­i­pate in the fu­sion of the cells  and their killing. Cell fu­sion fol­lowed by killing prop­a­gates cen­trifu­gally, re­sult­ing in plaques in cell cul­tures com­pa­ra­ble to those formed by lytic viruses.

The pa­per also probes an­other question—how to de­ter­mine if vir­u­lence fac­tors act at late stages of in­fec­tion. Specif­i­cally, in the case of Burk­holde­ria it has been known that a par­tic­u­lar Type III se­cre­tion sys­tem (T3SS_Bsa) plays a key role early in in­fec­tion. The ques­tion has arisen, does this sys­tem also come into play later on? The dif­fi­culty here is that in­fec­tion by T3SS_Bsa mu­tants are de­fec­tive in an early stage of patho­gen­e­sis, which makes it hard to de­ter­mine if T3SS_Bsa also works later on. What to do? The au­thors em­ployed  a new de­vice, called the pho­tother­mal nanoblade, to in­tro­duce bac­te­ria into the cy­to­plasm of a host cell. This gad­get con­sists of a cap­il­lary coated with ti­ta­nium. If placed next to a cell and sub­jected to a laser flash, the ther­mal ex­ci­ta­tion re­sults in a nanome­ter-sized va­por bub­ble that makes a small, tem­po­rary in­ci­sion in the cell mem­brane, al­low­ing trans­fer of the cap­il­lary con­tents. The cargo thus delivered—ranging in size from mol­e­cules to bacteria—enters the cell with­out im­pair­ing its vi­a­bil­ity.

Plaque for­ma­tion on HEK293 mono­lay­ers. (B) Fol­low­ing in­fec­tion. Bar = 1 cm. (D) Fol­low­ing nanoblade de­liv­ery. Bar = 1 cm. (E) Plaques in D stained for bac­te­ria (red) and actin (green). Bar = 500 μm. (F) Mag­ni­fied edge of plaques in E. Bar = 20 μm. Source

This is ex­cit­ing stuff, and these re­searchers put it to good use. What they found out is that the T3SS_Bsa se­cre­tion ma­chin­ery is only needed early in in­fec­tion, specif­i­cally for the exit of the bac­te­ria from their phago­cytic vesi­cles. This clar­i­fies de­tails of one im­por­tant step in the patho­gen­e­sis of Burk­holde­ria. (We dis­cussed this pa­per in a re­cent pod­cast of This Week in Mi­cro­bi­ol­ogy. Click here for TWIM #12 with more of our thoughts about this work.)

The au­thors thus com­bined in one ex­cit­ing pa­per the use of an in­ge­nious tech­nique to by­pass early stages in patho­gen­e­sis in which mu­tants are de­fec­tive. with the dis­cov­ery  of a novel mech­a­nism for how Burk­holde­ria spreads be­tween host cells. Slowly, but surely, the ways Burk­holde­ria causes dam­age are be­gin­ning to be un­der­stood. To­gether with what is known about model or­gan­isms, such as Lis­te­ria , this will help elu­ci­date the com­plex chore­og­ra­phy of other in­tra­cel­lu­lar pathogens. The reper­toire of strate­gies such or­gan­isms em­ploy to sur­vive and thrive in host cells is im­pres­sive in­deed. With this pa­per, it has be­come even more so.

Ref­er­ence

French CT, Toesca IJ, Wu TH, Tes­laa T, Beaty SM, Wong W, Liu M, Schröder I, Chiou PY, Teit­ell MA, Miller JF. (2011). Dis­sec­tion of the Burk­holde­ria in­tra­cel­lu­lar life cy­cle us­ing a pho­tother­mal nanoblade. Proc Natl Acad Sci USA, 108 (29), 12095−12100. PMID 21730143