Role of Actinobacillus (Aggregatibactor) Actinomycetemcomitans in periodontal diseases


This bacterium was first isolated from a cervicofacial actinomycotic lesion in 1912 and was named bacterium actinomycetemcomitans1. Epithet means “with actinomyces” i.e. its initial isolation was done with actinomyces species. Genus name refers to the star shaped internal structure. A. actinomycetemcomitans is a small nonmotile coccobacillus that grows singly, in pairs, or in small clumps and is variously described as fastidious, CO2-requiring, Gram-negative human pathogen in the γ subdivision of the Proteobacteria. As a member of the family Pasteurellaceae, it is related to the well-studied genera Haemophilus and Pasteurella.

Re-classification of Actinobacillus actinomycetemcomitans:

Re-classification of AAC has been done on the basis of recent studies that have shown a phylogenetic similarity of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus, Haemophilus paraphrophilus, and Haemophilus segnis suggesting the new genus Aggregatibacter for them2.

Morphological Structure:

ACC are small, short (0.4×1 µm) straight or curved rods with rounded ends3. Electron microscope studies have demonstrated membrane vesicles that appear to release from cells. On blood agar, colonies are small, gray to white, translucent, smooth and non-haemolytic. Growth is stimulated by addition of CO2 and species grows well in 5-10 % of CO4.  ACC ferments a range of sugars including glucose and fructose but not sucrose and lactose. Acid end products include lactate, succinate, acetate and propionate. ACC is grown in anaerobic cultures and from plaque it can be rapidly identified by fluorescent labelled anti-sera. It can also be identified by use of DNA probes.

When grown in culture, A. Actinomycetemcomitans forms small colonies approximately 0.5-1.0 mm in diameter. Colonies are translucent (or transparent) with irregular edges appear smooth, circular and convex. The colonial morphology of fresh isolates is distinctive with the internal star-shaped or crossed cigar morphology form embedding in the agar that gives A. actinomycetemcomitans its name3

Taxonomy of A. actinomycetemcomitans:

King and Tatum5 provided the first detailed biochemical and serological description of ACC. ACC is closely related to Haemophilus aphrophilus. Differentiation is done by its ability to grow in the absence of  haemin and factor V (Nicotinamide adenine dinucleotide). Serological studies have been attempted to determine the antigenic relationship between AAC and HA .Using agglutination studies, 24 different serogroups and 6 major agggutination antigens of ACC were identified.  A. actinomycetemcomitans strains are classified into six serotypes, a, b, c, d, e, and f 6-9, the specificity of which is defined by surface polysaccharides6-10. A new serotype i.e. serotype g of A. actinomycetemcomitans has recently been identified11. The predominant A. actinomycetemcomitans serotypes seem to be a, b, and c 12-18

Most leukotoxin strains are of serotype b. It is important to note that serotype b of AAC has been found more frequently and detected in higher numbers in active periodontitis lesions, whereas serotype a and c have a stronger association with periodontal health19. Taxonomic classification based biochemical characteristics include:

  1. Fermentation of different carbohydrates.
  2. Multivariate analysis of chemotaxonomic data.
  3. Cellular fatty acid composition.
  4. Composition of LPs.
  5. Analysis of total proteins.
  6. Multilocus enzyme electrophoresis.
  7. Respiratory quinones.

 Surface Ultrastructure of AAC:

  1. Fimbriae: They may exhibit fimbriae. 2 µm in length and 5nm in diameter. Fimbriated strains produce produce colonies with star shaped interior structure. Fimbriae are required for bacterium and host attachment.
  2. Vesicles: It is a prominent feature of AAC.These are structures LPs in nature and originate from and are continuous with outer membrane.AAC species with high leukotoxic production have abundant extracellular membrane vesicles. Vesicles per se exhibit leukotoxic activity .Other components of vesicles are: endotoxin, bone resorption activity, bacteriocin i.e , actinobacillin.
  3. Extracellular amorphous material: Certain AAC have an amorphous material that frequently embeds adjacent cell in matrix.

Virulence Factors of AAC 20:

Virulence means colonisation of organism in the host and overcoming the host defence and initiation of disease. This microorganism produces many virulent factors: leukotoxin as the most important, then bacteriocin, chemotaxis inhibiting factor, cytotoxic factors, Fc binding proteins, immunosuppressive factors, lipopolysaccharide collagenase, fibroblast inhibiting factor, antibiotic resistance determinants, adhesives, invasives and function inhibiting factor of polimorphonuclear leukocytes. These are discussed as follows,

Adhesion of AAC: It is executed by adhesions which are bacterial cell surface components. They interact and bind to very specific receptors in saliva, on surface of tooth, on extracellular matrix proteins and on epithelial cells. Distinct adhesions may be expressed by bacterium under different environment conditions21. Most of the adhesins are proteinaceous in nature.

Formation of dental plaque: Initially there is formation of acquired pellicle, then the initial colonizers (primarily streptococci) then finally AAC binds to them. Bacteria assert by means of aggregation, inter-generic and intra-generic coaggregation, interaction with distinct and bacteria specific salivary binding proteins.

Adhesion to epithelial cells: most of the AAC strains bind strongly to epithelial cells21. Binding is rapid reaching saturation within 1 hour22. Cell surface entities that mediate adhesion are fimbriae, extracellular amorphous material and extracellular vesicles21.

Binding to extracellular matrix proteins: In order to initiate disease AAC must bind to extracellular matrix. Major component of extracellular matrix is collagen23. The major fibres forming the extracellular matrix are collagen type I, II, III, V, XI. Less abundant but equally important are non collagenous glycosylated proteins fibronectin and laminin. Outer membrane proteins are important for binding of AAC to collagen and other proteins but binding is highly specific. After binding, AAC starts producing host destruction by virulence factors.

Bacteriocins: These are proteins produced by bacteria that are lethal for other strains and species of bacteria. AAC bacteriocins are active against S. sanguis, S. uberis and A. Viscosus24-25. Mode of action is that it increases the permeability of the cell membrane of the target bacteria leading to leakage of DNA, RNA and macromolecules essential for growth.

Bone resorption: AAC stimulate bone resorption in PD tissues by various mechanisms; 

  • Lipo-polysaccharides.
  • Proteolysis sensitive factor in micro vesicles.
  • Surface associated material.

 Surface-associated material has recently been identified as the molecular chaperone, GroEL. The chaperone appears to act in a direct way with the major bone resorbing cell population, the osteoclast26-28.

Collagenase: As collagen is the most abundant component of extracellular matrix, its destruction leads to the degradation of extracellular matrix. AAC produces a collagenolytic proteinase which can attack collagen. Collagenase is activity of 2 important periodontal pathogens A. actinomycetemcomitans and P gingivalis29-30.

Cytotoxins:  Fibroblasts are the most important source of collagen in extracellular matrix. cytotoxins act on fibroblasts and inhibit their proliferation. It has been identified as 50 kDa protein that inhibits DNA synthesis of fibroblast31.

Extracellular membrane vesicles: Almost all strains of AAC have these vesicles. Growth conditions alter the formation and morphology of vesicles. These vesicles contain leukotoxins, endotoxins, bone resorption activity and bacteriocin32-33.

Fc binding proteins: It binds to Fc portion of antibody and hence inhibits phagocytosis. Fc portion also activates complement system, so all these pathways are blocked.

Leukotoxin: The ability of A. actinomycetemcomitans extracts to cause death of leukocytes was first shown more than 30 years ago34-35.Leukotoxin is a member of RTX (repeat in toxin) family of toxins that produce pore forming haemolysis or leukotoxins36. The leukotoxin gene ( Ltx A) resides on operon consisting of 4 genes C, A, B and D37. Ltx B and Ltx D are involved in transporting the toxin to the surface of the cell while Ltx C post –transitionally activates the toxin. Expression appears to be regulated by O2 and is induced under anaerobic conditions38. The mechanism of leukotoxicity39 includes: 

  • Membranolytic activity producing pores in the target cell.
  • Phospholipids act as the receptor for the toxin whose activity result in a rapid influx of Ca2+ into the cell.
  • Necrosis and apoptosis.

Lipolysaccharides: These are endotoxin having the potential to modulate the host responses and contribute to tissue destruction. LPS causes tissue destruction by following mechanisms40 

  • Stimulation of in vitro bone resorption.
  • The production of IL-1, TNF and prostaglandin (PGE2) from macrophages.
  • Polyclonal activation of B-lymphocytes.

 The bone resorptive activities of this LPS41-42 are the result of stimulation of PGE2 , IL-I release from osteoblasts and other cells.Along with this AAC is also known to activate the complement cascade by the alternative pathway which in turn generates prostaglandins and this is the probable mechanism of bone resorption in case of periodontitis43.

Immunosuppressive factors: Organism produces proteins that inhibit DNA, RNA and protein synthesis by human lymphocytes. It is believed that it affects Ig production by activating B cells that down regulate the ability of B and T cells to respond to mitogens. In addition, leukotoxin impairs the ability of lymphocytes to respond to mitogens by inhibiting DNA, RNA, protein, IgG and IgM symthesis.

Inhibition of PMN function: AAC produces low molecular weight compounds that inhibit PMN leukocyte chemotaxis .The inhibitory activity is aborted by treatment with proteinase K, which means that the compounds are proteinaceous in nature. Along with this A. Actinomyceterncomitans has been shown to be capable of inhibiting PMNs from producing some potent antibacterial agents that are gained when the PMNs fuse with lysosomes20.

Penetration of epithelial cells: Research work has shown that AAC can penetrate the gingival epithelium cells44-45. Entry of the bacteria into the cell permits them to either transit the epithelial cell barrier or persist and grow in a protected cellular environment. Studies reported by Meyer et al (1997)46 suggest that primary receptor for AAC invasion is transferrin receptor. Invasion through integrins is the secondary pathway of entry. AAC invasion of epithelial cells is a highly dynamic complex process. It involves the attachment of organism to the host cell with initiation of some form of signalling, binding to a receptor, entry in a vacuole, escape from the vacuole, rapid multiplication, intracellular spread, exit from the cell and cell to cell spread47. 

Cytolethal distending toxin: It has been shown that A. actinomyctemcomitans also produces a 60-kDa protein, which down regulates both T and B-cell responsiveness through the activation of a subpopulation of B lymphocytes48..Cytolethal distending toxin is encoded by a locus of three genes, cdtA,B,C 49-50. It impairs the lymphocyte function by arresting its cell cycle. The active subunit, CdtB, exhibits DNase I activity. While the role of CdtA and CdtC is less clear, both proteins possess putative mucin-like carbohydrate-binding domains that predict interaction with the host cell surface.

Recent Research:

The function of systemic antibodies produced against the leukotoxin produced by Aggregatibacter actinomycetemcomitans has been studied recently in a study51. The study was conducted on Swedish population and results showed that these antibodies might contribute to limit systemic effects of the infection.

Detailed information regarding the Leukotoxin produced by Aggregatibacter actinomycetemcomitans has been given recently52-53. This article describes in detail the production and mechanism of action of Leukotoxins by Aggregatibacter actinomycetemcomitans and hence guides us to therapeutic strategies targeting the cellular and molecular inflammatory host response in periodontal diseases.


AAC has multiple mechanisms that are responsible for the destruction of periodontal connective tissues in periodontal disease progression. It is capable of attachment, colonization and most importantly by elaborating factors that interfere with the host’s defence system.  The ability of LtxA to cause death of all subsets of cells with hematopoetic origin might contribute to help the bacterium to survive the host immune response and also to release compounds essential for bacterial growth. AAC possesses many virulence factors but only few of them have been extensively studied. Further research is required to establish its genetic, molecular and biochemical features in detail. 


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Invasiveness of bacteria: Investigations have shown the bacterial invasion into cells 54-56. The immunofluorescence and immunohistochemical staining revealed the presence of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans and Actinomyces naeslundii within gingival tissues 57-61.with their presence being a characteristic of diseased sites.

The mechanism of cell invasion by P. Gingivalis has been explained as follows 62:

  1. P. gingivalis cells bind through adhesins such as fimbriae to surface receptors on gingival cells.
  2. Microtubules and microfilaments are rearranged to facilitate invagination of the membrane that results in the engulfment of bacterial cells.
  3. The P. gingivalis cells rapidly locate in the perinuclear area where they replicate.
  4. Calcium ions are released from intracellular stores and other signaling molecules such as the MAP-kinase family can be phosphorylated/dephosphorylated or degraded.
  5. Gene expression in the epithelial cells is ultimately affected.

Mechanism of invasion of AAC is different from P. Gingivalis 63  it has been described as follows:

  1. A.actinomycetemcomitans binds through adhesions such as fimbriae, outer membrane proteins, and extracellular vesicles and amorphous material, to surface receptors on gingival cells such as the transferrin receptor.
  2. The epithelial cell membrane ruffles and effaces, and invaginations engulf the bacteria which then become internalized within a membrane vesicle.
  3. Invasion can be actin dependent, resulting in an actin focus around the bacteria or actin-independent.
  4. The bacterial cells destroy the membrane vesicles possibly by secretion of phospholipase C releasing the bacteria into the cytoplasm where they grow and divide rapidly.
  5. Bacteria become localized at membrane protrusions through which they enter adjoining epithelial cells in a microtubule-dependent process.

Intracellular invasion has also been documented for Prevotella intermedia 64, and Fusobacterium nucleatum 65.



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