Ag­gre­gat­i­bac­ter actin­o­mycetem­comi­tans

A Unique Ex­o­toxin-Pro­duc­ing Oral Bac­terium

This ar­ti­cle, which ap­peared in Pedro's blog Mi­cro­bios, is trans­lated from the orig­i­nal with some mod­i­fi­ca­tions.

by Pe­dro Valero-Guil­lén

The pro­duc­tion and se­cre­tion of pro­tein tox­ins (ex­o­tox­ins) is a most com­mon strat­egy among mi­cro­bial pathogens. Yet, oddly, these vir­u­lence fac­tors are fairly rare among oral pathogens, with one ex­cep­tion, the Gram-neg­a­tive Ag­gre­gat­i­bac­ter actinomycetemco­mitans. For read­ers with an in­ter­est in tax­on­omy, this mi­crobe was orig­i­nally named Acti­nobacil­lus actin­o­mycetem­comi­tans and later re­clas­si­fied and re­named (you known tax­on­o­mists...). The new genus name refers to the microbe's ten­dency to ag­gre­gate with other bac­te­ria in the den­tal plaque, whereas the equally bom­bas­tic species name speaks to the fact that it was iso­lated to­gether with the com­mon mouth-dwelling Actin­o­myces. This mi­crobe makes pretty colonies that look like a flower. But, es­thet­ics aside, this mi­crobe has at­trib­utes that war­rant spe­cial at­ten­tion. It is found in about one third of the hu­man pop­u­la­tion and it is in­volved in a par­tic­u­larly rapid pro­gres­sive form of ado­les­cent pe­ri­odon­tal dis­ease, com­mon in those of African de­scent. It has also been im­pli­cated in in­fec­tive en­do­cardi­tis and other deep se­ri­ous in­fec­tions. Vi­able cells of A. actino­mycetem­comitans cell­shave been iden­ti­fied in ath­er­o­scle­rotic plaque. This is, with­out a doubt, a mi­crobe that knows how to cause trou­ble. But what makes it so promi­nent in the oral en­vi­ron­ment and such a suc­cess­ful pathogen?

Fig. 1. Colony of A. actin­o­mycetem­comi­tans, show­ing the char­ac­ter­is­tic flower-like mor­phol­ogy. From UCL East­man Den­tal In­sti­tute. Source

The Tox­ins of A. actin­o­mycetem­comi­tans

It has been known for quite some time that A. actin­o­mycetem­comi­tans makes two tox­ins, the leu­co­toxin (LtxA) and the cyto­lethal dis­tend­ing toxin (CDT), to trig­ger dis­ease. These tox­ins are pro­teins that the mi­crobe se­cretes and whose ac­tion is di­rectly re­spon­si­ble for the ag­gres­sive form of pe­ri­odon­ti­tis. The mi­crobe also se­cretes a third toxin, the cy­to­toxin CagE. How­ever, we know lit­tle about its mech­a­nism of ac­tion and role in patho­gen­e­sis, ex­cept that it shares ho­mol­ogy to a car­cino­genic fac­tor se­creted by an­other hu­man pathogen, He­li­cobac­ter py­lori.

Fig. 2. The crys­tal struc­ture of CDT at 2.4 Å res­o­lu­tion. Source

The Leu­co­toxin

The leu­co­toxin LtxA is a lipopro­tein be­long­ing to the RTX fam­ily of tox­ins that are found in var­i­ous Gram-neg­a­tive bac­te­ria. RTX stands for "repeat in toxin" and al­ludes to the many glycine-as­par­tate re­peats con­tained in the pro­tein. The gene en­cod­ing LtxA is part of the ltx­CABD operon, which en­codes the toxin and the pro­teins that con­trol its ex­pres­sion and ac­ti­va­tion, and, partly, its se­cre­tion. I say "partly" be­cause the se­cre­tion of LtxA is a com­plex process that in­volves the for­ma­tion of mi­crovesi­cles, the func­tion­ing of some trans­porta­tion sys­tems, the ac­tion of pro­teins in­volved in the in­tegrity of the cell en­ve­lope, and the avail­abil­ity of iron. In ad­di­tion, the se­cre­tion of the toxin does not fol­low the same path in all the strains. Some strains of A. actin­o­mycetem­comi­tans, es­pe­cially those di­rectly sub­cul­tured from oral biofilms, pro­duce rough, ad­her­ent colonies with cells con­tain­ing abun­dant fim­briae ap­pendages. The rough strains do not fully re­lease the toxin, which re­mains bound to the cell en­ve­lope. By con­trast, strains that pro­duce smooth colonies se­crete the toxin to the ex­ter­nal mi­lieu.

The lev­els of LtxA ex­pres­sion also dif­fer among strains. The high­est lev­els of pro­duc­tion have been re­ported for strain JP2 that car­ries a dele­tion of 530 bp in the toxin pro­moter. This strain also ap­pears to have a marked eth­nic predilec­tion, caus­ing ag­gres­sive pe­ri­odon­ti­tis es­pe­cially among North Africans and African-Amer­i­cans. At the core of this speci­ficity may be the abil­ity of LtxA to bind to in­te­grin LFA1, the trans­mem­brane re­cep­tor of cells of the im­mune sys­tem that me­di­ates cell at­tach­ment and signaling.Its bind­ing in­duces the for­ma­tion of pores on the mem­brane of mono­cytes, lym­pho­cytes, neu­trophils, and other myeloid and lym­phoid cells. Even­tu­ally, this leads to apop­to­sis and thus com­pro­mises the im­mune re­sponse of the host dur­ing in­fec­tion.

Fig. 3. CDT up­take and in­tra­cel­lu­lar trans­port in mam­malian cells. The CDT het­erotrimeric holo­toxin, con­sist­ing of the CdtA, CdtB and CdtC pro­tein sub­units, binds to a cell mem­brane re­cep­tor lo­cated within lipid rafts. It is rapidly taken up by en­do­cy­to­sis of the ac­tive CdtB pro­tein sub­unit in a clathrin-coated pit. The toxin is then trans­ported via the Golgi com­plex and en­do­plas­mic retic­u­lum (ER)into the nu­cleus us­ing nu­clear lo­cal­iza­tion sig­nals. This re­sults in DNA dou­ble strand breaks (DSB). Source

The Cy­to­lethal Dis­tend­ing Toxin

The sec­ond toxin pro­duced by A. actin­o­mycetem­comi­tans or CDT is sim­i­lar to cy­to­lethal dis­tend­ing tox­ins pro­duced by other pathogens such as E. coli and Campy­lobac­ter. This type of toxin blocks cell di­vi­sion and causes apop­to­sis in a va­ri­ety of eu­kary­otic cells. They are het­erotrimeric pro­teins com­posed of three sub­units (CdtA, CdtB and CdtC). The bi­o­log­i­cal ac­tiv­ity re­sides in CdtB, whereas CdtA's and CdtC's role is to an­chor CdtB to the mem­brane. CdtC is also be­lieved to func­tion as a chap­er­one of CdtB. The CdtB sub­unit fits well the clas­si­cal de­f­i­n­i­tion of geno­toxin, i. e., it dam­ages DNA thanks to its phos­phatase and DNA nu­cle­ase ac­tiv­i­ties, which have a sim­i­lar ef­fect on DNA as ion­iz­ing ra­di­a­tion. The toxin's ac­tion even­tu­ally leads to dis­ten­tion of the cy­to­plasm and the nu­cleus, phe­nom­ena that of­ten pre­cede apop­to­sis. As with LtxA, CDT can tar­get var­i­ous cells of the im­mune sys­tem, e. g., lym­pho­cytes, but seems to have a spe­cial predilec­tion for the oral ep­ithe­lium.

Fig. 4. Schematic rep­re­sen­ta­tion of func­tion­ally ac­tive pro­tein se­cre­tion sys­tems (Ss) present in A. actin­o­mycetem­comi­tans strain D7S. Source

The Ar­se­nal Of A Pathogen Re­vealed

In ad­di­tion to the LtxA and CDT tox­ins, A. actin­o­mycetem­comi­tans se­cretes nearly 200 pro­teins us­ing var­i­ous trans­port mech­a­nisms. This ar­se­nal of pro­teins is be­lieved to play a role in the im­munopathol­ogy of ju­ve­nile pe­ri­odon­ti­tis and some may help the mi­crobe sur­vive in the pe­ri­odon­tium. This is in­deed an im­pres­sive dis­play of po­ten­tial vir­u­lence fac­tors that high­light the com­plex­ity of this pathogen and the eti­ol­ogy be­hind the dis­eases it causes. Be­sides the mi­cro­bi­o­log­i­cal chal­lenges, it is worth em­pha­siz­ing that oral in­fec­tions are the most com­mon of all mi­cro­bial dis­eases in hu­mans. Thus, they de­serve more at­ten­tion. 

Elio adds a com­ment: Soon af­ter read­ing this ar­ti­cle, I saw my den­tist, a re­cent grad­u­ate from den­tal school. I asked him if he knew of A. actin­o­mycetem­comi­tans. To my plea­sure, he told me that it was em­pha­sized in school but that, in self-de­fense, it was re­ferred to as "AA."
 

Ref­er­ence

Zi­jnge V, Kiesel­bach T, Os­cars­son J (2012). Pro­teomics of pro­tein se­cre­tion by Ag­gre­gat­i­bac­ter actin­o­mycetem­comi­tans. PloS one, 7 (7). PMID 22848560

Pe­dro is Pro­fes­sor of Mi­cro­bi­ol­ogy at the Uni­ver­sity of Mur­cia, Spain.