BIOLOGY & SCIENCE: article : Molecular pathogenicity of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans.

Publication: Annual Review of Microbiology

Publication Date: 01-JAN-03

Author: Henderson, Brian ; Nair, Sean P. ; Ward, John M. ; Wilson, Michael

* Abstract Periodontitis is mankind's most common chronic inflammatory disease. One severe form of periodontitis is localized aggressive periodontitis (LAP), a condition to which individuals of African origin demonstrate an increased susceptibility. The main causative organism of this disease is Actinobacillus actinomycetemcomitans. A member of the Pasteurellaceae, A. actinomycetemcomitans produces a number of interesting putative virulence factors including (a) an RTX leukotoxin that targets only neutrophils and monocytes and whose action is influenced by a novel type IV secretion system involved in bacterial adhesion; (b) the newly discovered toxin, cytolethal distending toxin (CDT); and (c) a secreted chaperonin 60 with potent leukocyte-activating and bone resorbing activities. This organism also produces a plethora of proteins able to inhibit eukaryotic cell cycle progression and proteins and peptides that can induce distinct forms of proinflammatory cytokine networks. A range of other proteins interacting with the host is currently being uncovered. In addition to these secreted factors, A. actinomycetemcomitans is invasive with an unusual mechanism for entering, and traveling within, eukaryotic cells. This review focuses on recent advances in our understanding of the molecular and cellular pathogenicity of this fascinating oral bacterium.

Key Words periodontitis, gingivitis, microbiota, cellular microbiology, bacterial virulence

CONTENTS INTRODUCTION THE PERIODONTAL DISEASES--MANKIND'S MOST COMMON BACTERIAL INFECTIONS A. actinomycetemcomitans and Localized Aggressive Periodontitis Extra-Oral Pathology Cansed by A. actinomycetemcomitans GENERAL CHARACTERISTICS OF A. ACTINOMYCETEMCOMITANS CURRENT THOUGHTS ON THE SYSTEMATICS OF A. ACTINOMYCETEMCOMITANS Serological Diversity of A. actinomycetemcomitans Clonal Diversity of A. actinomycetemcomitans and Evidence of Racial Tropism CLUES FROM THE GENOME ADHERENCE OF A. ACTINOMYCETEMCOMITANS IMMUNOMODULATION BY A. ACTINOMYCETEMCOMITANS Cytokine Induction by A. actinomycetemeomitans A. ACTINOMYCETEMCOMITANS AND IMMUNE SUPPRESSION/CELL CYCLE INHIBITION A. actinomycetemcomitans Leukotoxin Cytolethal Distending Toxin Other Immunomodulators/Cell Cycle Inhibitors CELLULAR MECHANISMS RESPONSIBLE FOR BONE DESTRUCTION HOST CELL INVASION BY A. ACTINOMYCETEMCOMITANS CONCLUDING REMARKS

INTRODUCTION

Ninety percent of the cells in the human body are prokaryotic (170). The study of the human microbiota has largely centered on those organisms that live in the gastrointestinal tract. However, in the past few decades increasing attention has been devoted to the oral cavity. We now realize that this extremely complex organ, which is the center of one of our senses (taste) and is responsible for our capacity to communicate, contains an immensely complex population of bacteria (138) every bit as diverse as that found in the gut--and much more accessible. As recently argued by Rehnan & Falkow (143), there is a pressing need to understand how our microbiota interacts with us. This is particularly true of the oral microbiota, which is the source of significant human pathology.

THE PERIODONTAL DISEASES--MANKIND'S MOST COMMON BACTERIAL INFECTIONS

The recent report of the Surgeon General has estimated that in the United States severe periodontal disease affects 14% of adults aged 45-54 and 23% of adults aged 65-74 (131a). Periodontitis is the result of the response of the periodontium to the presence of certain members of the oral microbiota. One of the most severe forms of periodontal disease is localized aggressive periodontitis (LAP), which is associated with the subject of this review.

A. actinomycetemcomitans and Localized Aggressive Periodontitis

Adult periodontitis can affect all the teeth. In contrast, localized juvenile periodontitis (LJP), which has been recently renamed LAP (4), affects only certain teeth--the incisors and premolars. This predilection may be the result of selective colonization of the teeth. The disease tends to afflict younger individuals and, as its name implies, is associated with rapid destruction of the periodontal ligament and alveolar bone, which support the teeth. Evidence has accrued to support the hypothesis that A. actinomycetemcomitans is one of the key organisms responsible for the pathogenesis of LAP (159). The prevalence of LAP is not uniform among the world's populations. In the United States the mean prevalence was 0.53% among adolescents of all racial origins. Adolescents of African-American descent, in contrast, were found to have a 15-fold-higher incidence of disease than Caucasian Americans (96). In Brazil 3.7% of 15- to 16-year-old adolescents examined had LAP (42), while in Nigeria a prevalence of 0.8% was found (46, 97).

The possibility that LAP is a genetic disease has been under consideration since the early 1980s (110). Defects in neutrophil function have been a recurring theme (20, 155), and decreased neutrophil chemotaxis to the bacterial chemoattractant formyl-methionine-leucine-phenylalanine (FMLP) was an early finding (101). This bacterial peptide is a ligand for the N-formyl peptide receptor (FPR), a G-protein-coupled receptor (110). Currently, genes encoding three FPR proteins have been identified in Homo sapiens (93). Single nucleotide polymorphism analysis of 30 patients with LAP revealed that 29 had one of two point mutations in the FPR gene (43). Both mutations are associated with almost complete loss of receptor signaling in response to FMLP (150). If these results are substantiated, they raise the key question of why this major defect in recognition of FMLP does not result in general susceptibility to bacteria.

Extra-Oral Pathology Caused by A. actinomycetemcomitans

A. actinomycetemcomitans is occasionally responsible for non-oral infections including endocarditis, bacteremia, pericarditis, septicemia, pneumonia, infectious arthritis, osteomyelitis, synovitis, skin infections, urinary tract infections, and abscesses (178). It has been estimated that approximately 0.6% of cases of infective endocarditis are caused by A. actinomycetemcomitans (17).

Currently there is great interest in the possibility that periodontal diseases may be a risk factor for development of cardiovascular disease (41); A. actinomycetemcomitans has been detected in 18% of atherosclerotic plaque samples (45).

GENERAL CHARACTERISTICS OF A. ACTINOMYCETEMCOMITANS

A. actinomycetemcomitans is a small nonmotile gram-negative coccobacillus that grows singly, in pairs, or in small clumps and is variously described as facultatively anaerobic, microaerophilic, and capnophilic. Growth is enhanced by the presence of 5% C[O.sub.2] (133) and the organism grows best at 37[degrees]C. The optimum pH range for growth is between 7.0 and 8.0 (163). In liquid media the organism forms isolated translucent granules that adhere to the sides and bottom of the tube with the broth itself remaining clear. On agar small translucent circular colonies (approximately 1-mm diameter after 2-3 days) with a slightly irregular edge are formed, and these tightly adhere to the agar surface. These "rough" colonies have a characteristic crossed-cigar or star-like appearance when viewed with a low-power microscope. Pitting of the agar occurs underneath the colony, resulting eventually in the colony becoming embedded in the agar medium. Ariel repeated subcultures the star-shape often disappears and the colonies become smooth and opaque and do not cause pitting of the agar. This transformation from rough to smooth phenotype is associated with the loss of fimbriae (62). Rough colony variants expressing fimbriae adhere better to hydroxyapatite and to saliva-coated hydroxyapatite than do the smooth colony variants (145).

The natural habitat of A. actinomycetemcomitans is the oral cavity of man and other mammals (5, 6, 8, 23). Within the human oral cavity A. actinomycetemcomitans has been isolated from a range of habitats including supragingival plaque, subgingival plaque, saliva, cheek mucosa, buccal mucosa, gingivae, tongue (dorsal and lateral surfaces), hard palate, and tonsils (5, 116). Furthermore, the organism can be detected in the oral cavity of periodontally healthy individuals as well as in those with periodontitis (57).

Significant advances have been made in understanding the molecular genetics of this bacterium. In a screening of wild-type isolates of A. actinomycetemcomitans for the presence of endogenous plasmids, only three plasmids were found out of 39 strains examined (94). The A. actinomycetemcomitans strain VT745 has the 25.42-kb plasmid pVT745, which has been completely sequenced (35). It contains 36 open reading frames (ORFs) and has a type IV secretion system, involved in conjugation of the plasmid, covering 12 kb of the plasmid (35, 130a). The plasmid carries no functional antibiotic resistance gene (it has an interrupted ROB [beta]-lactamase), hut derivatives with a cloned kanamycin resistance gene could transfer only to other A. actinomycetemcomitans strains. Such derivatives could mobilize the IncQ group plasmid pMMB67 into Escherichia coli. Derivatives of pVT745 with an integrated E. coli plasmid origin of replication could transfer to E. coli. The type IV secretion system from the plasmid is homologous to a type IV secretion system found on the chromosome of several A. actinomycetemcomitans strains (though not the parent strain from which pVT745 came), but it is not known what the chromosomally located type IV secretion systems can secrete.

The two other naturally occurring plasmids found in the screen of 39 strains were pVT736-1, which was only 2 kb, and pVT736-2, which was >30 kb. pVT736-2 is a rolling circle replicon, a type of plasmid more commonly associated with gram-positive bacteria (37-39). The small cryptic plasmid pVT736-1 has been made into shuttle replicons by fusing it to the E. coli plasmid ColEI(161), pUC, or p15a (120). A derivative of pVT736-1 with the E. coli plasmid p15a replicated in A. actinomycetemcomitans was used to propagate DNA from this bacterium, first in E. coli with subsequent transfer to A. actinomycetemcomitans (39). Structural instability was seen in a variety of the shuttle vectors, and those without an A. actinomycetemcomitans replicon or a broad-host-range replicon were lost after 100 generations of nonselective growth (39).

A. actinomycetemcomitans is a gram-negative bacterium, and as such, broad-host-range plasmids of the IncP, IncQ, and IncW group should replicate in it. Derivatives of the IncP group plasmid R995 were used to show that the IncC/KorB partition system of IncP group plasmids is effective in A. actinomycetemcomitans (157), and several reports have shown that IncQ group plasmids such as derivatives of pMMB67 (36, 39) replicate in A. actinomycetemcomitans.

A. actinomycetemcomitans is transformable by electroporation when using plasmid DNA (33, 162) and there is a natural competence system analogous to the extensively studied Haemophilus influenzae DNA uptake system (179). The natural competence system involves the cleavage and processing of linear DNA fragments during uptake and this has been manipulated to generate an efficient method of gene replacement for A. actinomycetemcomitans (179). Another useful tool for the manipulation of A. actinomycetemcomitans is the inducible transposon IS903[phi]kan on the IncQ plasmid pVJT128 (173) that has been used to create libraries of random insertion mutants. From insertion libraries using this transposon, a catalase-deficient mutant was isolated (173) and the tight adherence operon (tad) was defined (69). A more conventional kind of gene knockout was used to generate a recA mutant of A. actinomycetemcomitans, SUNY 465 (111). A recombination deficient strain will have uses in propagating cloned segments of the A. actinomycetemcomitans chromosome for complementation studies.

CURRENT THOUGHTS ON THE SYSTEMATICS OF A. ACTINOMYCETEMCOMITANS

Serological Diversity of A. actinomycetemcomitans

Currently six serotypes (a-f) are recognized (72). The immunodominant antigen is a high-molecular-mass O-polysaccharide of the lipopolysaccharide (LPS) (136). The O-polysaccharides of serotypes b, c, e, and f are the product of homologous gene clusters containing between 10 (serotype e) and 16 (serotype b) genes with highly conserved groups of genes at the proximal and distal ends and the central genes being unique to each cluster and containing a lower GC content (72). These serotype-specific gene clusters may have evolved from a common ancestral cluster by interspecific gene transfer from a...

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