Aggregatibacter Actinomycetemcomitans – A Tooth Killer?

Strong evidence is available on Aggregatibacter actinomycetemcomitans (A.a) on its role as the causative agent of localised juvenile periodontitis (LJP), a disease characterised by rapid destruction of the tooth-supporting tissues. This organism possesses a large number of virulence factors with a wide range of activities which enable it to colonise the oral cavity, invade periodontal tissues, evade host defences, initiate connective tissue destruction and interfere with tissue repair. Adhesion to epithelial and tooth surfaces is dependent on the presence of surface proteins and structures such as microvesicles and fimbriae. Invasion has been demonstrated in vivo and in vitro. The organism has a number of means of evading host defences which include: (i) production of leukotoxin; (ii) producing immunosuppressive factors; (iv) divreting proteases capable of cleaving IgG; and (v) producing Fc-binding.

Periodontal disease results from the elaboration of noxious products by the entire plaque flora. Thus states the Non Specific Plaque Hypothesis which was proposed by Walter Loesche (1976). Later it was proposed in Specific Plaque Hypothesis, only certain plaque is pathogenic and its pathogenicity depends on presence of or increases in specific organisms. This was spurred by the recognition of Aggegatibacter actinomycetemcomitans as a pathogen in localized aggressive periodontitis.

Aggregatibacter actinomycetemcomitans (A.a) is an exogenous bacterium which causes true infections, transmissible among exposed individuals. It is associated with periodontitis in young individuals. It occurs in 90% of localised aggressive periodontitis and 30-50% in severe adult periodontitis. It has the ability to produce virulence factors.

This short review will introduce the reader to Aggregatibacter actinomycetemcomitans, an oral commensal which is also an opportunist pathogen with emphasis on its surprising but potential range of virulence factors and virulence mechanisms.

Bacterium actinomycetem comitans was described by Klinger as coccobacillary bacteria isolated together with Actinomyces from actinomycotic lesions of man [1]. It was reclassified as Actinobacillus actinomycetemcomitans by Topley & Wilson and as Haemophilus actinomycetemcomitans by Potts et al., [2,3]. Recent studies in 2006 involving multilocus sequence analysis by Nørskov-Lauritsen N and Kilian M have shown a phylogenetic similarity of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis. This resulted in the addition of the genus aggregatibacter (aggregate, to come together; bacter, bacterial rod; aggregatibacter, rod shaped bacterium that aggregates with others), to the family pasteurellaceae in 2006 to cover gram negative, non motile, facultatively anaerobic rods or coccobacilli that were previously known as haemophilus (actinobacillus or bacterium) actinomycetemcomitans (now aggregatibacter actinomycetemcomitans), haemophilus aphrophilus and hemophilus paraphrophilus (collectively known as aggregatibacter aphrophilus) and hemophilus segnis(now aggregatibacter segnis) [Table/Fig-1].

The genus aggregatibacter is taxonomically in the family pasteurellaceae, order pasteurellales, class gammaproteobacteria, phylum proteobacteria.

Five sero groups of Aggregatibacter actinomycetemcomitans were classified by Taichman based on surface polysaccharide located on the O-side chain of lipopolysaccharide using tube agglutination studies [5].

Serotypes a, b and c are most prevalent in the oral cavity. A particular clone of serotype b with enhanced leukotoxic activity is predominantly associated with cases of localized aggressive perioodontitis [4]. Serotype c is found in healthy subjects [5] [Table/Fig-2].

Aggregatibacter actinomycetemcomitans (actis, a ray; myces , a fungus; comitans, accompanying; actinomycetemcomitans, accompanying an actinomycete) is a gram negative coccobacillus measuring about 0.4 ± 0.1X 0.1 ± 0.4 micrometers in size [4] [Table/Fig-3].

Aggregatibacter actinomycetemcomitans possess fimbriae, vesicles and extracellular amorphous materials.

They are small filamentous cell surface appendages. They occur in a peritrichous array, measuring more than 2 micrometers in diameter and in bundles [6]. It forms two types of colonies; colonies with star shaped interior (star positive) or nonfimbriated strains (star negative). The most abundant protein in the fimbriae is 304-a with molecular mass 6.5k Da.

A.actinomycetemcomitans has numerous vesicles or blebs which are lipopolysaccharide in nature. Highly leukotoxic strains tend to have more vesicles. These vesicles contain endotoxin which has a bone resorption activitiy and a bacteriocin termed actinobacillin. These vesicles also exhibit adhesive properties and function as delivery vehicles for toxic materials [6].

ROBERT KOCH’S POSTULATES(1884) states that for an organism to produce a disease, it must (i) occur only in that disease, (ii) be able to grow in pure culture, (iii) be recovered from the host. Aggregatibacter actinomycetemcomitans did not meet these needs as it did not exist in isolation in the disease.

However, it met the criteria of a potential pathogen proposed by Sigmund Socransky which states that the pathogen must, (i) be associated with a disease by increase in number of organisms at sites, (ii) be eliminated in sites after treatment and (iii) demonstrate virulence factors to cause periodontal tissue destruction [7].

LJP is genetic or hereditary with a rapid destruction of tissues around incisors and first molars. Serotype b strains of A.actinomycetemcomitans are found associated with this disease [8]. Large numbers (97%) are isolated from LJP lesions whereas from healthy sites it is low. Large numbers found in periodontal pockets are related to low immune response in the host. A wide range of virulence factors are produced by the organisms. Rapid bone loss is the hallmark of the disease. Young adults (25-30 years) are affected. A.actinomycetemcomitans is isolated either singly or in combination with other bacteria [9].

Serious extra oral infections are caused by A.actinomycetemcomitans due to its ability to reduce oxygen in the tissues. Brain meninges, septicemia, UTI, osteomyelitis, endocarditis and abscesses are common [10].

A. actinomycetemcomitans mediated endocarditis is a chronic syndrome with fever, chills, anorexia, weight loss, heart murmur and night sweats. Aggregatibacter actinomycetemcomitans expresses a Human Shock Protein like molecule, HSP 60 that cross reacts with antibodies to human HSP 60 [11].

MGB (trypticase soy broth) with malachite green and bacitracin was the earliest media used to culture (A.a). It was then followed by medium with trypticase soy agar, serum with bacitracin and vancomycin (TSBV). Exclusive growth of A.a was found in a particular culture medium which contained TSBV, spiramycin, fucidic acid and carbencillin. RPMI – 1640 and Dulbecco’s modified Eagle medium are used now with a generation time of 246 and 346 min [12].

The putative virulence factors of A. actinomycetemcomitans can be subdivided into those that: (i) modulate inflammation, (ii) induce tissue destruction and (iii) inhibit tissue repair.

a. Virulence factors which modulate immune system

A. actinomycetemcomitans appears to employ multiple products to inactivate or evade immune defences. The most actively studied gene product of the organism is a leukotoxin and a member of the RTX (repeats in toxin) family whose cellular receptor is the integrin, LFA-1, thus accounting for its selective effect on leucocytes (although only those from primates) [13–15].

Almost all the RTX leukotoxins are secreted except LtxA toxin of A. actinomycetemcomitans which is thought to be entirely cell associated; either bound to cell surface-associated nucleic acids [16] or within membranous vesicles which bud from bacterium’s surface [17,18]. This affirms to the possibility that the bacterium itself is toxic to the target cells. The apoptosis of the target cells in response to A.actinomycetemcomitans leukotoxin is by a mechanism involving mitochondrial perturbation [19]. Injection of A. actinomycetemcomitans into mice has been claimed to induce immunosuppression and sonicates of this organism suppressed the IgG response to sheep red blood cells in mice [20,21].

It has also been proposed that A.actinomycetemcomitans can produce super antigens which have the ability to bring about T cell apoptosis by binding to T cell receptors [22,23]. A. actinomycetemcomitans has been reported to produce a number of, as yet unidentified, proteins with cell cycle-inhibitory activity causing arrest in the G2 phase of the cell cycle. These proteins range in molecular mass from the 8-kDa protein termed gapstatin to 60 kDa and all the way up to 80 kDa- [24–27]. One cell cycle-modulatory protein with immunosuppressive function that has recently been identified as being produced by A. actinomycetemcomitans is cytolethal distending toxin (CDT) [28,29].

Fc binding protein termed as Omp34 identical with OmpA of E. coli, a protein implicated in the virulence of this organism is another immunomodulatory virulence factor of A. actinomycemcomitans [30–31]. A. actinomycetemcomitans produces a 65-kDa macromolecule able to bind to the IL-10 receptor and henceforth can modulate monocyte/macrophage function as IL- 10 is considered to be a major macrophage de-activating cytokine [32].

A. actinomycetemcomitans has also been reported to produce a low molecular mass inhibitor of neutrophil chemotaxis to FMLP [33] [Table/Fig-4].

b. Virulence factors inducing tissue destruction

Tissue destructive virulence factors of A.actinomycetemcomitans include lipopolysaccaride(LPS) present on the bacterial cell wall and the secreted proteins like cell stress protein. LPS is reported to stimulate bone resorption in vitro and in vivo [34–36]. But it’s considered to be a less significant cytokine inducer than the secreted protein. A cell stress protein, chaperonin 60 is considered to b a potent bone degrading molecule bystimulating bone resorption by acting as an osteoclast ‘growth factor’ [37] [Table/Fig-4].

a. Adhesion

Bacterial adhesion which facilitates colonization is the key virulence mechanism [38]. Bacterial components involved in Adhesion are called adhesins. They are proteinaceous structures found on cell surfaces. They bind with specific receptors in the saliva, tooth, extra cellular matrix and epithelial cells. A.actinomycetemcomitans forms chains which cannot be broken up by sublethal sonication. Surface entities like vesicles mediate aggregation. A.actinomycetemcomitans adheres to the gingival crevice epithelium. Strains with fimbriae adhere three to four folds better. A.actinomycetemcomitans binds to collagen I,II,III and V but not IV. It also binds to fibronectin but not fibrinogen.

The tight auto-adhesion of A. actinomycetemcomitans has been described is due to the expression of long, bundled fibrils composed of a 6.5-kDa subunit protein,Flp-1 (fimbrial low-mol. wt protein) which has been reported to be glycosylated [39–46].

Bacteriocins are proteins produced by bacteria that are lethal for other strains and species of bacteria. These agents confer colonization by lessening ecological pressures. This is an advantage for the bacterium.

b. Invasion

It has been affirmed that many bacteria have the ability to invade host cells and A. actinomycetemcomitans is one among them [37]. Studies of invasion A. actinomycetemcomitans reveals that 25% of A.actinomycetemcomitans isolates are invasive [47,48] [Table/Fig-5]. A. Actinomycetemcomitans penetrate and survive within eukaryotic cells. They penetrate gingival epithelium. They occur in specific intracellular locations like the epithelial wall, enlarged intracellular pocket spaces and the epithelial side of basal lamina in connective tissue and alveolar bone. It has been observed that microfilaments and microtubules for intracellular movement [49]. The process of intracellular movement and the cell spreading could be inhibited by agents that interfered with microtubule dynamics, suggesting that this bacterium when internalized interacts closely with the microtubules of the host cell [50].

It has been suggested that the transferrin and integrin receptors are involved in the adhesion of the bacteria to host cells [51].

Unique aspects of the behaviour of A. actinomycetemcomitans are its rapid exit from cells after invasion, its ability to move from one cell to another and its capacity to divide rapidly within host cells [52].

A.a. is a highly non motile gram negative coccobacillus with a vast array of potential virulence factors and mechanisms. Though it was initially named as Actinobacillus actinomycetemcomitans, it was found that the bacterium is more similar to haemophilus than actinobacillus and hence it was reclassified under aggregatibacter as aggregatibacter actinomycetemcomitans. Scientific data clearly underlines its etiological role in localized aggressive periodontitis. This review also tries to throw light on the virulence abilities of this pathogen like immune evasion mechanisms like production of leukotoxin, cell cycle modulatory protein and immunomodulatory protein like Fc binding proteins. It also brings about tissue destruction by other novel mechanisms like binding to host matrices and invading host cells. Still, a lot is still to be understood and established. With the advent of newer technological methodologies and genome information, we would be able to understand not only how A. actinomycetemcomitans produces such profound but local pathology like periodontal infections but also its role in systemic pathology.

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