AGGREGATIBACTER ACTINOMYCETEMCOMITANS

AGGREGATIBACTER ACTINOMYCETEMCOMITANS is a bacterium with an array of diverse potential virulence characteristics, including multiple immune evasion mechanisms and novel mechanisms for binding to host matrices and invading host cells, any one of which may play a crucial role in the local tissue pathology of Localized aggressive periodontitis.

1. AGGREGATIBACTER ACTINOMYCETEMCOMITANS 1 DR.VIDYA VISHNU SENIOR LECTURER MALABAR DENTAL COLLEGE AND RESEARCH CENTRE

2. INTRODUCTION Periodontal diseases are infections caused by micro-organisms that colonizes the tooth surface at or below the gingival margin. Above 700 different species are capable of colonizing the mouth and any individual may typically harbor 150 or more different species . 2

3. The oral cavity should be considered as an ‘open growth system’ with uninterrupted ingestion and removal of microorganisms and their nutrients. Bacteria may attach to tooth surface, epithelium, CT and microbial surfaces. Tooth surface provides stable base for the microbes and held in immediate proximity to soft tissue of periodontium thus microbial colonization is facilitated. 3

4. ASSOCIATION OF PLAQUE MICROBES WITH PERIODONTAL DISEASES Susceptible host Presence of pathogenic species Absence /small proportion of beneficial bacteria Periodontal disease 4

5. PATHOGENIC BACTERIA ■ Bacteria accumulates on tooth surface as the organized structure called dental plaque. ■ Broadly the periodontal bacteria are classified into 1. those associated in health. 2. those associated with disease. 5

6. Associated with Disease ▪ (mostly Gm + ve , facultative species ) S.sanguis S. mitis A.viscosus A. naselundi ▪ (small proportion Gm – ve species) P.intermedia F. Nucleatum Capnocytophaga Nisseria & Veilleonella Beneficial species P seudomonas S . Sanguis V. Parvula C. Ochraceus A.Actinomycetemcomitans P.Gingivalis P.intermedia T. Forsythia Fusobacterium species Peptostreptococcus mutans Eubacterium species C.rectus Treponema species Enteric rods and Candida species Classification Micro-organisms Associated with Health 6

7. HISTORY The first isolates were recovered from cervicofacial actinomycosis , in 1912 by Klinger R. I nitially known as Bacterium actinomycetemcomitans . In 1921, the organism was referred to as Bacterium comitans by Lieske . 7

8. King & Tautam (1962) described the close phenotypic relationship of A.A. with H. aphrophilus . A.A. was subsequently reassigned to genus Hemophilus . ( Pott’s et al 1985) 8

9. First identified as periodontal pathogen in studies of LJP by Newman et al & Slots et al 1976 . Baehni et al 1979 important virulence factor- leukotoxin Genco et al 1980, Ebersole et al 1987 Elevated serum antibody response to the species 9

10. Slots & Rosling 1983 Species eliminated in successfully treated sites . Preus 1988, Mandell et al 1986 Treatment failures associated with failure to lower the number of organisms in treated sites. 10

11. Sreenivasan et al 1993 Invasion of A.a in gingival epithelial cells . Bueno et al 1998 s ubjects harboring JP2 clonal type A .a with 530 bp deletion in the leukotoxin operon were 22.5 times more likely to convert to LAP. 11

12. Scheiken et al 2000 Invasion of A.a in vascular endothelial cells Rudney et al 2001 Invasion of A.a in buccal epithelial cells in vivo. Named as ‘ Aggregatibacter actinomycetemcomitans ’ by Norskov , Lauritsen & Kilian in 2006. 12

13. 13

14. The name actinomycetemcomitans arises from its association with an actinomycete and frequent isolation from actinomycotic lesions along with Actinomyces israelii . The genus name refers to the star-shaped inner structure sometimes seen in colonies on selective medium. actin- star shaped bacillus- rod shaped 14

15. A conspicuous growth in broth as small granules adhering to walls of the test tubes was noted in the original description of Bacterium actinomycetemcomitans . Accordingly the name Aggregatibacter was proposed (rod shaped bacterium that aggregates with others ). 15

16. Taxonomy of A. actinomycetemcomitans Scientific classification Kingdom Bacteria Kingdom Bacteria Phylum Proteobacteria Class Gammaproteobacteria Order Pasteurellales Family Pasteurellaceae Genus Aggregatibacter Species A ctinomycetemcomitans 16

17. MORPHOLOGICAL CHARACTERISTICS Small gram-negative rod {0.4–0.5 μ m x 1.0–1.5 μ m in size} micro aerophillic & facultatively anaerobic ▪ Is Capnophillic requiring atmosphere containing 5-10% CO 2 for good growth . non sporulating, non-motile, non-hemolytic, saccharolytic , oxidase & catalase positive. 17

18. C losely related to members of the Pasteurellaceae Microscopically cultures appear predominantly bacillary with few coccal forms . Cells may appear coccobacillary , particularly if the colonies have been directly isolated from solid medium. 18

19. Longer forms may be seen in Gram stains of cultures older than 3 days, or from cells grown in a glucose-containing liquid medium . 19

20. ▪ Fermentative ability to utilize galactose , dextrin, maltose, mannitol and xylose permits the biotyping of this organisms, and to distinguish from other organisms of oral cavity. ( Zambon JJ 1985) Upon primary isolation, colonies are small approximately 0.5-1.0 mm in diameter, translucent /transparent with irregular edges & appear smooth, circular and convex. 20

21. The colonial morphology of fresh isolates is distinctive with the internal star-shaped or crossed cigar morphology form embedding in the agar that gives AA its name ( Zambon JJ 1985 ). Fresh colonial isolates are rough-surfaced. 21

22. ▪ Repeated subculture yields two types of colonial variants; one is smooth-surfaced and transparent , the other is smooth-surfaced and opaque . ▪ The colonial variation is associated with fimbriation. 22

23. BIOCHEMICAL CHARACTERISTICS ▪ King & Tatum provided the first detailed biochemical and serological description of A.A. ▪ A.A. is closely related to H. aprophilus than Actinobacillus lingeresii . ▪ However unlike the genus Hemophillus that require either Factor X ( hemin ), or factor V ( Nicotinamide adenin dinucleotide ), A.A . can grow in absence of these factors. (Slots J 1982) Periodontology 2000, Vol. 20, 1999, 14-52 23

24. ▪ AA can be distinguished from H. aprophilus by its ability to ferment starch, sucrose, trehalose and production of specific enzymes such as B- glucosidase & B- galactosidase ( Zambon 1985). 24

25. Decompose H 2 O 2 (catalase + ve ) - Benzidine + ve - Reduce nitrate to nitrite - Produce strong alkaline and acid phosphatases - Ferment fructose, glucose and mannose 25

26. SURFACE ULTRASTRUCTURE OF A.A. ▪ A significant feature of A.A. is its surface ultra structure Fimbriae Vesicles Extracellular amorphous material 26

27. The expression of these material appears to be a function of the strain as well as culture conditions, such as broth, agar, aerobicity , anaerobicity , or nutrients, and they helps in functioning of bacteria. Periodontology 2 000, Vol. 20, 1999, 136-167 27

28. Fimbriae It is small filamentous, cell surface appendage associated with bacterial colonization at host. Appears in peritrichous areas; may be more than 2µm in length and 5nm in diameter; often occurs in bundles. 28

29. Freshly isolated strains are fimbriated but in vitro subcultures results in organism to lack fimbriation. Fimbriated strains produce colonies with star shape - star positive. N on fimbriated strains - star negative . 29

30. Number of studies indicate that colonial variation and fimbriation are associated with adhesion of bacteria. The fimbriae associated protein gene is strongly expressed in fimbriated strain than non- fimbriated strain. Overall these observations demonstrate the positive correlation between fimbriation and adhesion. 30

31. 2. Vesicles Other prominent feature of surface is vesicles (blebs). originate from and are continuous with outer membrane protein. Almost all strains of Aa extrude membrane vesicles. Growth conditions alter the formation and morphology of the vesicles. Are lipopolysaccharides in nature, Also released in external environment in large number . Highly leukotoxic strains have an abundance of extracellular membranous vesicles in contrast to non- leukotoxic strains. (Lai C H 1981) 31

32. Other biologically active components of vesicles are: (1) leukotoxin (2) endotoxin (2) bone resorption activity & (3) bacteriocin ( actinobacillin ) It also shows the adhesive properties. These observations prompted that vesicles function as delivery vehicles of A.A. 32

33. 3.Extracellular amorphous material (EAM) Associated with the surface of certain AAC cells is an amorphous material that frequently embeds adjacent cells in a matrix. Some studies demonstrate that cells grown in liquid medium lacked this material. In contrast others did observe this material on cells grown in liquid culture, but not on all strains. 33

34. Production in part may be associated with growth in ‘ tryptone ’- based medium . The material is protein, most likely glycoprotein & has shown to exhibits both bone-resorbing activity and adhesive properties Can be removed by washing the cells with phosphate buffered saline. 34

35. Bacteria from which the amorphous material has been removed exhibit reduced adhesion to epithelial cells. A.A. strains, which normally exhibit low levels of adhesion, exhibit increased levels of adhesion when suspended in extracellular amorphous material, a phenomenon termed conveyed adhesion . 35

36. CLASSIFICATION King & Tatum 1962 : Distinguished 3 serogroups , from non-oral A. actinomycetemcomitans based on a heat stable component. Pulverer & KO 1972 : U sing tube agglutination assays, detected 24 groups within A. actinomycetemcomitans . 36

37. Three serotypes, a, b and c , were defined by Zambon et al . ( 1981 ). Taichman et al. (1982) described four serogroups based on surface antigens and proteinaceous leukotoxin . 37

38. The no of serotypes in A.a was later extended to:- 5 [ Mombelli & Van Winkelhoff 1999 ] to total 6 [ Kilian et al 2006 ]- a, b, c, d, e, f 38

39. According to serological specificity defined by structurally & antigenically distinct O-polysaccharide components of their respective lipopolysaccharide molecules the organism was classified into six serotypes a – f . ( Page et al.1991; Perry et al. 1996, Kaplan et al.2001) 39

40. Serotypes a and b were most common in the oral cavity, serotype c constituted 10% of the human oral isolates. ▪ The most frequently associated serotype with LAP – serotype b. 40

41. More commonly detected in adult periodontal diseases samples – serotype a. ▪ More common in periodontal health – serotype c 41

42. O rganism can be grouped into three major phylogenetic lineages comprising (i) serotype b strains, (ii) serotype c strains, and ( iii) serotype a, d, e and f strains 42

43. Most individuals harbour a single serotype that remains stable in the host over time, although some patients have been shown to harbour two or three serotypes of A.a . The distribution of A.a serotypes appears to vary according to the person’s geographical location and their ethnicity. Serotype alone does not appear to be a direct indicator of strain virulence. 43

44. Associations between a single serotype b clonal lineage (JP2 clone) and the aggressive form of localized periodontitis in adolescents have been the focus of much investigation. The JP2 clone of A.a has a 530 bp deletion in the leukotoxin promoter, which results in significant enhancement of leukotoxin production & alteration of iron acquisition mechanisms . 44

45. The JP2 clone shows a limited geographical & ethnic host range , predominating in subjects with an African lineage but absent from non- African populations from Northern Europe. It has been proposed that localized aggressive periodontitis actually comprises two different diseases. 45

46. In Northern European Caucasian populations, LAP is associated with a non-JP2 oligoclonal population of bacteria, resulting from indigenous A. a acting as an opportunistic pathogen, whereas in adolescents descended from North or West African populations, the JP2 clone has been suggested to be the main cause of LAP. T his interpretation has been challenged by Kaplan et al. 46

47. Subjects that harboured the JP2 clone alone had a greater relative risk of CAL compared to subjects that harboured either the JP2 clone in combination with a non-JP2 clone, harboured non-JP2 clones only, or who did not harbour A . a. The pathogenicity of the JP2 clone may be moderated in the presence of other bacteria . 47

48. These findings suggest that the presence or absence of A. a may not be the only indicator of disease risk, & that combinations of bacteria may also affect the clinical severity of periodontal diseases . Virulence phenotype of A . a is enhanced by growth in the presence of an oral streptococcus . 48

49. A.A. is unrelated to H. Influenza, the type specific genus of hemophilus and reclassification of A.A. is not favored by ICSB Subcommittee on Pasteurellaceae and related organisms ( Frederiksen 1987 ). Accordingly Norskov-Lauritsen & M. Kilian 2006 proposed Aggregatibacter as the generic name for the group. 49

50. The species coming under this genus are 1. Aggregatibacter actinomycetemcomitans Basonym : Actinobacillus actinomycetemcomitans (Klinger 1912) Topley and Wilson 1929. 50

51. 2. Aggregatibacter aphrophilus Basonym : Haemophilus aphrophilus Khairat 1940 3. Aggregatibacter segnis Basonym : Haemophilus segnis Kilian 1977 51

52. HOW IS AAC IMPLICATED IN DISEASE? Oral infections A.A. is an important pathogen in severe and recurrent forms of periodontitis. The prevalence is nearly 90% in LAP & 30-50% in severe adult periodontitis . Periodontology 2000, Vol. 20, 1999, 136-167 52

53. Localized Aggressive Periodontitis Microbiologic, clinical and immunological evidence as described below have been used to implicate this as causative agent: Association: - Large numbers of AAC are routinely isolated from localized juvenile periodontitis lesions, whereas the isolation of the bacterium from healthy sites is low. Elimination:- The eradication of AAC from diseased sites is usually correlated with recovery from clinical symptoms of disease. 53

54. Host response:- The presence of large numbers of A.A. in the periodontal pocket is correlated with a significant humoural immune response. Virulence Factors:- AAC produces a wide array of potent cell bound and secreted virulence factors that have been implicated in the pathogenesis of disease. Animal studies:- Induced disease in animal models like rats & mice. 54

55. Rapidly progressing periodontitis Severe and rapid loss of the bone is the hallmark of RPP, affecting 25 to 30yr age group adults. The isolation of A. actinomyceterncomitans , P gingivalis , B. capillus , P. intermedia , E. corrodens and Campylobacter rectus from disease sites has been reported either singly or in combination. Also has been associated with periodontal lesions that are refractory to standard therapy . 55

56. Extraoral infections It has been reported to cause serious infections at several sites outside the mouth. They have been reported from brain, meninges, septicemia, urinary tract infections, vertebral osteomyelitis and abscess of the abdomen, brain, face, hand, and thyroid gland. 56

57. Among these the endocarditis and soft tissue abscesses remain the most common infections, with the latter syndrome generally occurring in association with Actinomyces . AA-mediated endocarditis is a chronic syndrome with a sub-acute course including fever, chills, anorexia, weight loss, heart murmur and night sweats ( K aplan 1989). 57

58. Patients usually present clinical symptoms of hepatosplenomegaly and peripheral evidence of endocarditis. Laboratory investigation usually reveals anaemia , elevated ESR and microscopic hematuria. 58

59. Combination therapy using an aminoglycoside along with ampicillin has been most often used to treat endocarditis. Frequently identified complications of A. actinomycetemcomitans mediated endocarditis include embolism and congestive heart failure 59

60. 60

61. Three lines of evidence have led to conclusions that associate and implicate A.A. in oral and systemic diseases: Clinical and microbiological data linking A.A. to the initiation, progression and recurrence of disease in localized aggressive periodontitis. ( Zambon et al 1985). 61

62. Genetic and experimental data linking reputed virulence factors possessed by A.A. to pathogenic events known to occur in LAP. (Fives-Taylor PM, Meyer DH, Mintz KP, Brissette C. 1999) Periodontology 2000, Vol. 42, 2006, 114–157 62

63. Reports indicating that organisms of the HACEK group ( Haemophilus sp., A. A., Cardiobacterium hominis , Eikenella corrodens and Kingella kingae ), and A.A, in particular, are associated with systemic diseases distant from the oral cavity. ( Das M, Badley AD, Cockerill FR, Steckelberg JM, Wilson WR, 1997) 63

64. Contamination - first step Incubation - time required to reach the appropriate concentration -minimal concentration required for disease by A.A. 1 x 10 6 per ml. Prodromal period - subclinical infection, only detected by sophisticated tools. 64

65. Period of disease - frank tissue-destructive activity takes place & the host is compromised Recuperation period - In the best-case scenario, recuperation ends the cycle of disease activity and indicates that the host has successfully mounted an authentic immunological response. 65

66. A.a is also associated with periodontitis lesions of Papillon-Lefevre syndrome patients; virally mediated host defense impairment may cause overgrowth of subgingival A.a . 66

67. Modes of Transmission Vertical transmission: With the exception of one study ( Tinoco et al in 1998) in developing countries, the result agree that if children harbour A.A. the parent usually harbor the same species. 67

68. Horizontal Transmission Horizontal transmission may occur between siblings or between spouses. Siblings can harbor the similar strains of A.A. in their oral cavities. ( DiRenzo et al in 1994, Tinoco et al in 1998) Transmission of A.A is rather uncommon in between spouses in regard to their intimate cohabitance for several years. 68

69. Saliva is a likely transport vehicle for transmitting strains of A.A. Subgingival and salivary strains within the same individual were of the same serotype and same genotype. ( Asikainen et al 1997) These results suggest that saliva can be used as representative sampling material in searching for A.A. in transmission studies. 69

70. VIRULENCE FACTORS OF A.A . Virulence factors are attributes of a microorganism that enable it to colonize a particular environment in its host, overcome the host defenses and initiate a disease process. To act as a pathogen bacteria must colonize appropriate host tissue & cause destruction. 70

71. Process of tissue destruction results from interaction of bacteria with host cells. Organisms with many virulence factors are more pathogenic. 71

72. Virulence factors of A.A Factors that promote colonization & persistence in the oral cavity • Adhesins • Invasins • Bacteriocins •Antibiotic resistance Factors that interfere with host’s defenses • Leukotoxin • Chemotactic inhibitors •Immunosuppressive proteins • Fc -binding proteins Factors that destroy host tissues • Cytotoxins • Collagenase •Bone resorption agents •Stimulators of inflammatory mediators Factors that inhibit host repair of tissues •Inhibitors of fibroblast proliferation •Inhibitors of bone formation 72

73. A ACOMITANS P GINGIVALIS 73

74. ADHESION OF A. A The first challenge any microorganism faces is the ability to adhere tightly to a specific substrate. Without adhesion, the organism is quickly cleared from the surface . The oral cavity poses distinct problems for microbes, as eating, drinking, talking and salivary flow induce many shear forces that remove microorganisms not firmly attached. 74

75. The bacterial surface components involved in adhesion are called Adhesins . Proteinaceous structures found on the surface of the bacterial cell. 75

76. They interact and bind to very specific receptors in saliva, on the surface of the tooth, on extracellular matrix proteins and on epithelial cells. Attachment is favored by the surface structure - fimbriae, extracellular amorphous material and extracellular vesicles. 76

77. ATTACHMENT TO EPITHELIAL CELLS Most A.A. strains adhere strongly to epithelial cells. Binding occurs very rapidly, reaching saturation levels within 1 hour after infection. Fimbriae most probably function in adherence of rough variants, whereas non fimbrial components like vesicle are probably involved in adherence of smooth, highly invasive strains. 77

78. Trypsin and protease reduces ability to adhere, suggesting some adhesins in the outer membrane are protein in nature. A A also produces poly-N- acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation & detergent resistance. 78

79. Other components for adhesion are ‘extracellular amorphous protein’ and ‘surface vesicles’. Extracellular amorphous material has both a protein and carbohydrate moiety. 79

80. Attachment to Extracellular Matrix Proteins In order to initiate disease in extraoral sites, AA must bind to the extracellular matrix, the complex network of proteins & polysaccharides that is secreted by and underlies epithelial and endothelial cells and surrounds connective tissue. The major component of ECM is collagen. 80

81. Collagen types I, II, III, V and XI are predominant in CT. Type IV is the major component of the basement membrane. Less abundant, but equally important, are the noncollagenous glycosylated proteins, fibronectin and laminin . 81

82. AA binds to immobilized collagen types I, II, III and V but not to type IV collagen. Does not bind to any of the collagens in soluble form. Outer membrane proteins on the bacterial cell surface are essential for binding. 82

83. It also binds to fibronectin , but not to the plasma protein, fibrinogen. Binding is highly specific. Binding to the insoluble form of proteins aid the organism in its spread and colonization of oral sites and extraoral sites. 83

84. VIRULENT TRAITS RELEVANT TO COLONIZATION Autotransporter proteins Autotransporters constitute the largest known family of bacterial extracellular proteins ( Pallen MJ 2003). 84

85. Autotransporters are produced almost exclusively by members of the family Proteobacteria and are being increasingly recognized as virulence factors in pathogenic species (Ray SK 2002). 85

86. Bacterial autotransporters display a variety of effector functions, including adherence, invasion, proteolysis and serum resistance (Henderson 2001 ) Recent studies have shown that AA produces three autotransporter proteins: Aae , EmA and ApiA (also known as Omp100) All three autotransporters have been immuno localized either to the cell surface or to the outer-membrane cell fraction. 86

87. Surface components that may be involved in the colonization of host tissue Name Structure Functions Aae surface protein Attachment to epithelium ApiA surface protein Serum resistance Attachment to epithelium EmaA surface protein Attachment to collagen Attchment to epithelium PilA Pili Thin Pili DNA Uptake DNA Binding Flp-1 pili Bundle-forming pili Autoaggregation Attachment to abiotic surfaces Attachment to epithelium Biofilm formation PGA Surface polysaccharide Autoaggregation 87

88. Antibiotic Resistance Tetracyclines are frequently employed in localized aggressive periodontitis. Roe DE et al 1995 showed that 82% of 19 clinical isolates of AA were resistant to tetracyclines and carried the tetB resistance determinant. tetB determinant was capable of being transferred by conjugation to other AA and to H. influenzae . Antibiotic resistance in Aa is on the rise and likely to be responsible for treatment failures in the future. 88

89. Bacteriocins Bacteriocins are proteins produced by bacteria that are lethal for other strains and species of bacteria. Confer a colonization advantage for the bacterium by lessening the ecological pressures associated with competition by other organisms for both nutrients and space. 89

90. Actinobacillin , a bacteriocin that is active against S. sanguis , Streptococcus uberis and A. viscosus , has been identified and purified. Actinobacillin is associated with both the bacterial cell surface and extracellular vesicles. 90

91. Increase the permeability of the cell membranes of target bacteria. Leakage of DNA, RNA and macromolecules essential for growth. Actinobacillin may be responsible for the clinical observation that a reciprocal relationship occurs between AA and S. sanguis and/or A. viscosus in plaque and in patients with LAP. 91

92. Bone Resorption Aa stimulate bone resorption by several different mechanisms: Lipopolysaccharide (Kiley P, Holt SC 1980) Proteolysis-sensitive factor in microvesicles ( Nowotny A, Behling UH, 1982) & Surface -associated material. ( Meghji S, Henderson B, 1995) tumor necrosis factor-a, interferon-c interleukin-12, metalloproteinase 8 , matrix metalloproteinase II). 92

93. Surface-associated material has been identified as the molecular chaperone, GroEL . The chaperone appears to act in a direct way with the major bone-resorbing cell population, the osteoclast. 93

94. AA LPS are very effective bone resorption mediator, has been shown to cause the release of calcium from fetal long bones in the Ca 2+ - fetal bone resorption assay. The bone resorption activity is completely inhibited by dexamethasone and which states that probably it involves PG and IL-1. This is in direct contrast to GroEL , whose bone absorption activity is not inhibited by interleukin-1 receptor antagonist protein. 94

95. Generation and activation of osteoclasts is under the control of a tripartite cytokine signalling system. LPS of A A can promote osteoclast formation. The capsular-like polysaccharide material can induce osteoclastogenesis , inhibit osteoblast proliferation by inducing apoptosis and stimulate macrophage cytokine synthesis. 95

96. Collagenase A major feature of periodontal disease is a marked reduction in gingival collagen fibre density. Collagenases are enzymes that break the peptide bonds in collagen. Collagenase activity is associated with two important periodontal pathogens, Aa & Pg. Some of the reduction in collagen density may also be due to tissue collagenases induced in periodontal disease. 96

97. Fc-binding Proteins The Fc region of an antibody molecule is important in the binding of the antibody to specific receptors on polymorphonuclear leukocytes . If other proteins compete for binding to this region of PMN, binding of the antibody may be inhibited and, thereby, inhibit phagocytosis. Tolo & Hegland 1991, demonstrated that molecules on the surface of AA that are associated with capsular material and secreted into the medium bind to the Fc portion of IgG ; 97

98. The binding inhibits the ability of opsonizing antibodies to bind PMN and reduces phagocytosis by 90%. It is believed that the Fc receptors also play a role in complement activation. The ability of this organism to make proteins that bind to the Fc receptors would inhibit all of these functions. 98

99. LEUKOTOXIN This toxin is a 114-kDa protein produced by 56% of strains isolated from LAP patients . ( Zambon JJ 1983) Member of the RTX ( Repeats in Toxin ) family of toxins that produce pore-forming hemolysins or leukotoxins . ( Lally ET 1989) Name derives from the characteristic calcium-binding motif that is repeated in the carboxy terminal of such proteins ( Chenal A 2009) 99

100. LtxA can block the processes of bacterial uptake into phagocytes and the associated processes of bacterial killing. Killing of leukocytes has been shown to be due to induction of the processes of apoptosis. 100

101. LtxA induces apoptosis by the mitochondrial pathway, ( Korostoff 2000) involves activation of caspases . LtxA is the major factor of A.a that is able to induce interleukin-1 β synthesis ( Kelk P 2008). 101

102. 102

103. CYTOLETHAL DISTENDING TOXIN The most fascinating and well studied cell cycle-blocking bacterial toxin. First discovered in entero pathogenic E. coli Three genes, designated cdtA , cdtB and cdtC It was found to block eukaryotic cell division in G2 (1997) Cause cell distension and death The production of Cdt by AA was reported by Sugai et al (1998) 103

104. The AA protein dimerizes , and this is associated with rapid aggregation and loss of activity in buffered solution. The holotoxins bind to cholesterol within the cell membrane (Guerra 2005). The toxin is reported to be internalized through the Golgi complex, and then undergoes retrograde transport to the endoplasmic reticulum. 104

105. Role of Cdt in pathogenesis of periodontal diseases Shenker BJ in 1990 reported that A A produced an immunosuppressive factor that could block T-cell proliferation. This immunosuppressive factor was the Cdt B component of CDT, and was able to block cell-cycle progression in human T lymphocytes ( Shenker BJ 1999,2000) and induce apoptosis in these cells ( Shenker BJ 2001). 105

106. CDT may contribute to the immunosuppressive phenotype of A A , adding to, or synergizing with , the effects of AA leukotoxin . 106

107. ACTIONS OF CDT Deoxyribose nuclease activity immunosuppressive factor- able to block cell-cycle progression in human T lymphocytes and induce apoptosis in these cells. A A Cdt B has phosphatidylinositol-3,4,5-trisphosphate phosphatase activity. 107

108. CDT can stimulate the generation of a key osteoclast regulatory protein, receptor activator of NF- kB ligand(RANKL), in gingival fibroblasts and periodontal ligament cells ( Belibasakis GN et al 2008,2005 ) CDT inhibits nitric oxide production by activated macrophages. ( Fernandez 2008) 108

109. LIPOPOLYSACCHARIDE LPS (endotoxins) have a high potential for causing destruction of an array of host cells and tissues. It causes skin necrosis ( Schwartzmann reaction), bone resorption and platelet aggregation, and it activates macrophages. Low concentrations of AA LPS stimulate macrophages to produce IL (lα, lβ) and TNF, cytokines involved in tissue inflammation and bone resorption. 109

110. Macrophages that migrate to gingival sites of AA infection will be stimulated to produce these cytokines Both LPS & these cytokines, which are known to play an important role in atherosclerosis and coronary heart disease, also appear systemically, where they may function in initiating and/or exacerbating conditions associated with cardiac disease. 110

111. IMMUNOSUPPRESSIVE FACTORS Host defense mechanisms play a major role in controlling concentrations of bacterial communities in dental plaque. AA suppresses these host defense mechanisms. The organism produces a protein that inhibits DNA, RNA and protein synthesis in mitogen -activated human T cells. 60-kDa protein secreted shown to inhibit IgG and IgM synthesis by human lymphocytes. 111

112. AA affects IG production by activating B cells that downregulate the ability of B and T cells to respond to mitogens. Leukotoxin impairs ability of lymphocytes to respond to mitogens by inhibiting DNA, RNA, protein, IgG and IgM synthesis. 112

113. Ability of PMN to phagocytose the bacteria is reduced because PMN need AB's to ingest and kill AA. AA can suppress mitogen induced lymphoid proliferative responses. AB responses must be initiated before PMN can kill AA. The time required to produce AB's of the proper isotype , specificity and affinity may be prolonged by immunosuppressive factors elaborated by the microbe ( Shenker et al 1990). 113

114. Inhibitors of PMN Function The host’s first line of defense against invading bacteria is the recruitment of phagocytes to the area. The ability to disrupt chemotaxis permits the invading organism to survive this major challenge from the host. AA secretes a LMW compound that inhibits PMN chemotaxis . 114

115. Activity of PMN is the killing of bacteria by a wide variety of potent antibacterial agents that are gained when the PMN fuse with lysosomes . Organisms that can inhibit this fusion or resist the antibactericidal action of these agents are protected from this detrimental host activity. 115

116. AA is capable of inhibiting PMN from producing some of these compounds, and it is intrinsically resistant to others. A heat-stable protein in AA inhibits the production of H 2 O 2 by PMN and many strains are naturally resistant to high concentrations of H 2 O 2. Resistant to several of the cationic peptides, known as defensins that are found in neutrophils. 116

117. Penetration of Epithelial Cells Bacteria penetrate and survive within eukaryotic cells .( Finaly BB 1997) AA can penetrate the gingival epithelium AA was the first invasive periodontopathogen to be reported. Smooth colony variants of Aa invaded more efficiently than rough variants. 117

118. Meyer et al. reported that strain JP2, a high level leukotoxin producer, was not as invasive as SUNY 465, the invasion prototype. This uptake into cell confers an advantage to the bacterium. 118

119. It permits them to either transit the epithelial cell barrier or persist and grow in a protected cellular environment. Bacteria are protected from immune defenses and from antibiotics when they are within cells (Wilson 2002). 119

120. Studies showed that AA occurs in very specific intracellular locations and that it exhibits a definite pattern of penetration. AA invasion of epithelial cells is a highly dynamic complex process. (Mayer DH 1997) 120

121. attachment of organisms 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 cell-to-cell spread. 121

122. Meyer et al. 1997 suggest that the primary receptor for AA invasion is the transferrin receptor . Data suggest that integrins, transmembrane proteins involved in the adhesion of eukaryotic cells to the extracellular matrix, also mediate entry. Few isolates also enter via receptor-mediated endocytosis. 122

123. Invasion is an active process requiring protein synthesis and energy generation. Inhibition of either oxidative phosphorylation or glycolysis inhibits invasion, and it does not occur at 4°C. N ot inhibited by endosomal acidification in the host cells. Microbial uptake by non-professional phagocytes depends on the rearrangement of the host cytoskeletal network. 123

124. Invasion process is a rapid mechanism involving the formation of cell-surface craters or apertures with lip-like rims with bacteria appearing in the host cell cytoplasm within 30 min. Invasion was associated with protrusions from the host cells that formed connections between cells and harboured AA. It was postulated that entry of AA is rapidly followed by cell division and then transcytosis of bacteria via the protrusions. 124

125. Invasion by the majority of AA isolates is inhibited by cytochalasin -D, demonstrating a role for actin in the invasion process. AA is taken up in a host derived membrane bound vacuole, which it lyses and enters the cytoplasm. AA possesses phospholipase C implicated in vacuole lysis . 125

126. Intracellular AA is not quiescent. It transits through the cell to neighbouring cells via bacteria-induced protrusions that appear to be extensions of the host cell membrane. If neighbouring cells are lacking, bacteria are simply released from the host cell via rudimentary protrusions. Bacteria can be seen within these protrusions by scanning, transmission and fluorescent microscopy. 126

127. Microtubules are strongly implicated in the intra- and intercellular spread & its release into the extracellular environment. Microtubule inhibitors, Colchicine and Nocadozole , which bind to and inhibit microtubule polymerization and T axol , which binds to and stabilizes microtubules, increase the number of intracellular AA and prevent its release from the host cell. 127

128. IRON ACQUISITION There is an evolutionary struggle between bacteria and their hosts for the metal iron. Iron is essential for bacterial growth, excess iron uptake can produce toxicity, and iron uptake is consequently subject to rigorous control. The best known iron-responsive regulator is the ferric uptake regulator Fur. 128

129. A A has a LPS that binds to haemoglobin and could play a role in iron acquisition. AA can utilize haemoglobin as an iron source, and bacterial binding to haemoglobin depends on two cell-surface proteins with molecular masses of 40 and 65 kDa . Iron binding proteins of AA - a 35 kDa iron-repressible protein (termed a fuA ) that was located in the periplasm f bpA iron binding protein- expressed by AA 129

130. Another iron acquisition system used by A A is the a feABCD system . This gene system was present and expressed in three serotype strains of A A , including the JP2 clone. Under iron-limiting conditions, there is increased transcription and translation of all biofilm determinants. 130

131. A A & systemic diseases Organisms of HACEK group & A A in particular, are associated with systemic diseases distant from the oral cavity (Fine et al 2006). As A A is an organism that avidly attaches to both soft & hard tissues of tooth, & positions itself adjacent to permeable junctional & pocket epithelium, A A has been isolated from a no. of organs distant from the oral cavity & is capable of causing serious infections in humans. 131

132. Such infections include facial plane infection, heart infection, endocarditis, pericarditis, lung infection, necrotizing pneumonia, mediastinitis , mediastinal abcess , transdiaphragmatic infection, endophthalmitis , skin infection, vertebral osteomyelitis, cervical lymphadenitis, submandibular space abcess & UTI. (Fine et al 2006, Kaplan et al 1989). 132

133. Microbiologic tests for AAC ▪ CULTURE Selective Media: Are widely used to isolate pathogenic microorganisms from clinical specimens. Employs antimicrobial agents to suppress non-targeted microorganisms and thereby improve the detection limit. 133

134. MGB, a selective medium was one of the earliest media developed by ( Mandell / Socransky 1981). The medium consisted of Trypticase soy agar 40 gm /liter, bacitracin 128 μ g /ml, malachite green 8 μ g /ml and 5% defibrinated sheep blood. 134

135. This was followed by the development of a medium composed of trypticase soy agar and serum with bacitracin and vancomycin added as selective agents (TSBV) (Slots 1982 ). TSBV agar is an excellent primary selective medium for A.A. that can detect the microorganism in levels as low as 20 viable cells per ml ( Slots 1982). 135

136. Another medium was developed based on blown antibiotic susceptibility of periodontal flora. This medium known as ‘A’ medium is essentially TSBV supplemented with spiromycin , fusidic acid and carbenicillin (Holm et al 1987). 136

137. Defined Media Use to determine the precise nutritional requirement of microbe. Also useful in genetic analysis and isolation of metabolic mutants. A.A. is a fastidious organism; hence the development of the defined media is difficult. 137

138. More recent study utilized tissue culture medium as the defined medium ( Sreenivasan PK, Meyer DH, Fives-Taylor PM. 1993 ). RPMI- 1640 and Dulbecco's modified Eagle medium are commonly used tissue culture medium to support growth of A.A. 138

139. Effect of supplements Yeast extract - addition of yeast extract to trypticase soy broth enhances growth of most strains of A.A. ( Sreenivasan PK 1993). Cystine - Cystine and thiamine promoted growth of A.A . ( Sreenivasan PK 1993). Increased the growth of all strains of AA 3. Hormones - steroid hormones (estrogen, progesterone, and testosterone) enhanced growth of AA ( Sreenivasan PK, Meyer DH, Fives-Taylor 1993). 139

140. 4. pH – A.A. demonstrates optimal growth at pH 7.5 ( Sreenivasan PK1993). 5. Salt concentration – A.A. demonstrates optimal growth between 85.1mEq/L and 170mEq/L ( Sreenivasan PK1993). 6. Iron - AA expresses iron- binding proteins and has hemin -binding activity. A ddition of iron salts and protein-bound iron to trypticase soy broth did not influence the growth or protein profiles of A A . 140

141. Immunodiagnostic methods Employ antibodies that recognize specific bacterial Ag's to detect target microorganisms. The methods do not require viable bacteria and are less susceptible to variations in sample processing. less time-consuming and easier to perform than culture. 141

142. However, the accuracy of immunodiagnostic tests depends greatly on the quality of the reagents used. P oorer detection limits than nucleic acid probe or PCR assays. 142

143. Bonta et al. (1985) described an indirect immunofluorescence identification method of AA with a detection limit of 500 cells/ ml. 143

144. Evalusite Test is a commercially developed, antibody based sandwich enzyme-linked immunosorbent assay for the detection of A.a and P gingivalis 144

145. Radiolabelled DNA probes DNA probes entail segments of single-stranded nucleic acid, labeled with an enzyme or radioisotope, that can locate and bind to their complementary nucleic acid sequences with low cross-reactivity to non-target microorganisms. DNA probe may target whole genomic DNA or individual genes. 145

146. Whole genomic probes are more likely to cross-react with non-target organisms due to the presence of homologous sequences between different bacterial species. Based on 16S RNA genes, & showed a detection limit of 1000 cells with no evidence of cross reactivity (Dixit et al 1990). 146

147. A. actinomycetemcomitans cloned probe and P gingivalis whole genomic probe comprise the basis of the commercial DMDx detection method ( Omnigene , Cambridge, MA). 147

148. DNA–DNA hybridization assay Socransky et al (1994) developed a checker board, ‘DNA–DNA hybridization assay’ for the detection of oral bacteria. In this method, the sample DNA is released and immobilized on a nitrocellulose membrane. The membrane-bound DNA is then allowed to hybridize with either digoxigenin -labeled whole genomic DNA or 16s rRNA -based oligonucleotide probes. 148

149. The detection limit is approximately 1000 cells. Is able to simultaneously screen for the presence of upto 43 different bacterial species in a specimens using a single nitrocellulose membrane. P articularly suitable for large scale, microbiological studies of the subgingival microbiota . 149

150. PCR PCR involves a reiterate amplification of a region of DNA flanked by a selected primer pair specific for the target species . The presence of the specific amplification product indicates the presence of the target microorganism Displays the best detection limits, identifying as few as 3-5 cells and shows no cross reactivity under optimal condition. 150

151. EFFECTS OF PERIODONTAL TREATMENT ON AA AA may survive in untreated periodontal lesions for a considerable period of time. In 1983, Slots & Rosling showed that scaling and root planing alone was unable to remove AA from LAP periodontitis lesions. 151

152. Gunsolley in 1995 Nonsurgical therapy had the least effect on AA counts in heavily infected periodontal lesions, due to the ability of the organism to involve gingival tissue and thereby evade the effect of mechanical debridement and periodontal healing ( Saglie 1991). 152

153. AA cells in gingiva may constitute a reservoir for repopulating periodontal pockets. Modified Widman flap surgery may suppress AA to below detectable levels in about 50% of localized juvenile periodontitis lesions. 153

154. Tuan et al. (1999) found that an apically positioned flap with osseous recontouring is more effective than an apically positioned flap without osseous recontouring in reducing the pocket depth and levels of subgingival AA. Resective type of periodontal surgery is more effective than access flap surgery due to the excision of infected gingival tissue and to pocket depth reduction to levels permitting adequate cleaning by tooth brushing, flossing or other oral hygiene measures. 154

155. Systemic antibiotic therapy has the potential to eradicate AA residing in periodontal pockets and gingival tissue. Tetracyclines combined with scaling or root planning (Slots 1991) or with P.D. surgery ( Mandell 1986 ) may markedly suppress or eliminate subgingival AA in LJP. Systemic metronidazole has demonstrated good anti- AA activity in LAP patients ( Saxen / Asikainen 1993) but not in adult periodontitis patients ( Niemisen et al 1996). 155

156. Systemic use of Amoxicillin- Metronidazole has shown striking clinical results in the treatment of AA associated -LAP, adult periodontitis and refractory periodontitis even in the absence of other P.D. therapy (van Winkelhoff 1996). The antibiotic recommendation is a course of amoxicillin and metronidazole , 250-mg each, three times daily for 8 days (adult dosage). 156

157. Combination therapy is warranted in mixed periodontal infection because: 1) each drug is aimed at one or several important pathogenic microorganisms 2) delay emergence of microbial mutants resistant. 3) bactericidal synergism can be achieved reducing the dose required and shortening the course of therapy and avoiding toxicity. 157

158. Disadvantages of combination therapy: o Greater risk of adverse reaction. o Antagonism may take place. o Superinfection with resistant organism 158

159. Systemic antibiotics should only be administrated as an adjunct to mechanical debridement because in undisturbed subgingival plaque sample the target organisms are effectively protected. 159

160. Antibiotics have been used in essentially two ways: 1) In combination with intensive instrumentation over a short period of time after achievement of adequate plaque control in pre treatment motivation period. 2) As staged approach after completion of initial therapy. 160

161. CONCLUSION A A is a bacterium with an array o f diverse potential virulence characteristics, including multiple immune evasion mechanisms and novel mechanisms for binding to host matrices and invading host cells, any one of which may play a crucial role in the local tissue pathology of LAP. T echnologies have not really touched AA, although it is clear that this organism is capable of causing marked alterations in host as a result of its powerful toxins and its ability to adhere to host cells and to enter into them and travel through them. 161