Molecular and Leucotoxicity Studies of Actinobacillus actinomycetemcomitans

Downes, Evelyn Anne (1995) Molecular and Leucotoxicity Studies of Actinobacillus actinomycetemcomitans. PhD thesis, University of Glasgow.

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Actinobacillus actinomycetemcomitans is a Gram-negative rod, which has been implicated in the aetiology of periodontitis especially, juvenile periodontitis and also with non-periodontal diseases e.g. endocarditis, brain abscesses and soft tissue abscesses. The aims of the study described in this thesis, were to (i) investigate the strain diversity of a collection of A.actinomycetemcomitans held at Glasgow Dental Hospital and School and (ii) develop a rapid and accurate method to screen A.actinomycetemcomitans strains for the production of leucotoxin which is regarded as an important virulence factor. The A.actinomycetemcomitans collection consisted of 96 A.actinomycetemcomitans isolates (85 clinical strains and 11 reference strains). The clinical isolates were collected between 1985 and 1990 and included multiple strains from a number of individuals and reflect a variety of clinical conditions, including juvenile periodontitis, chronic periodontitis and prepubertal periodontitis. The majority of these strains were stored as freeze dried ampoules and had been used in experimental studies including adhesion, hydrophobicity and leucotoxicity activity. Strain diversity was investigated in two ways. The entire collection was screened for the presence of plasmids and demonstrated that 47% (40 out of 84) of clinical isolates and 9% (1 out of 9) of reference strains harboured plasmids. The number of plasmids found in plasmid positive strains varied between 1 and 3; 31% of all strains contained only a single plasmid. These results contrast with several published studies which have reported that very few strains of A.actinomycetemcomitans harbour plasmids. A subset of 17 strains was selected, on the basis of leucotoxic status, single or multiple isolates from single individuals and plasmid profile and investigated in detail. The strain diversity of the 17 strains of A.actinomycetemcomitans was further studied by the determination of whole cell DNA restriction endonuclease fragmentation patterns (REFPs) with 3 different 4 base restriction enzymes - HinfI, Sau3AI and HaeIII. Type strains isolated in America were shown to be genetically very similar to each other. Additionally, these American Type strains showed very little genetic similarity to the Danish Type strain and to the clinical strains isolated in Glasgow, which suggested a different clonal origin for the American and European strains. In addition, the REFP results showed that it was possible for an individual to be infected by at least two different strains of A.actinomycetemcomitans. The second aim of the investigation was to produce a rapid method to screen strains of A.actinomycetemcomitans for leucotoxin production. Two methods were compared, one of which had not been previously used to investigate the toxicity of A.actinomycetemcomitans. The first involved the exposure of HL-60 cells (derived from human monocytes), to whole and sonicated cells of A.actinomycetemcomitans. Cell death was measured by trypan blue exclusion. The second, novel, method was a chemiluminescence assay, which had been used to previously investigate the activity of the adenylate cyclase toxin of Bordetella pertussis. The toxins from both A.actinomycetemcomitans and B.pertussis are known to disrupt the cell membranes of human polymorphonuclear leucocytes (PMNLs). Healthy PMNLs will normally phagocytose bacteria with the production of a burst of light, which can be enhanced and measured. However, when exposed to toxin producing bacteria, which kills or disrupts the PMNL membrane, the cell can no longer phagocytose and therefore little chemiluminescent activity results. The chemiluminescence and cytotoxicity assays were compared and in both cases, the same five strains of A.actinomycetemcomitans were shown to be toxic. However, variation was seen in the rank order of toxicity. When outer membrane protein preparations of A.actinomycetemcomitans were probed with a monoclonal antibody (MCA 9D4) to the related toxin, adenylate cyclase toxin produced by B.pertussis, the presence of a protein with a molecular weight approximately equivalent to that of the A.actinomycetemcomitans toxin was found. The overall conclusions of the thesis are : (i) there was no association of plasmid status with either the production of a toxin or a particular form of periodontal disease. (ii) analysis of whole cell DNA REFPs, suggested a distinct clonal origin for American Type strains, compared to European Type and wild strains. (ii) the novel chemiluminescent assay is as accurate a method of determining toxicity status as the older method of HL-60/trypan blue exclusion assay. Although the two methods are very similar, when large numbers of strains have to be screened, the chemiluminescence method is faster than and the results obtained comparable to the trypan blue exclusion assay (iii) the monoclonal antibody to the B.pertussis toxin, recognises an outer membrane protein with approximately the same molecular weight as the A.actinomycetemcomitans leucotoxin despite the low (less than 50%) homology between the two toxins. This result suggests the view that the leucotoxin is present in the outer membrane and supports the findings of other groups.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: T W MacFarlane
Keywords: Microbiology
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-74951
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:01
Last Modified: 27 Sep 2019 15:01

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