Lima, F.L; Farias, F.F.; Campos, P.C.; Totola, A.H.; Tavares, C.A.P.; Costa, J.E.; Farias, L.M.; Carvalho, M.A.R.,
Laboratório de Microbiologia Oral e Anaeróbios, Departamento de Microbiologia/ICB/UFMG - Belo Horizonte, Minas Gerais, Brasil.

Actinobacillus actinomycetemcomitans is a Gram-negative cocobacillus, capnophilic, that has been associated to periodontal diseases. One of the virulence factors of this bacterium is a toxin that specifically lyses human polimorphonuclear lymphocytes. The aim of this work was to evaluate the leukotoxic activity of A. actinomycetemcomitans strains, isolated from individuals with (n = 7) and without (n=1) periodontal disease, and from marmosets (n=8). Two A. actinomycetemcomitans reference strains (ATCC 29523 and FDC Y4) were included in the experiments. By the methodology of in vitro tripan blue exclusion, the strains were classified as high, medium and low producers, respectively. Chemiluminescence was used to evaluate the leukotoxic potential of 2 strains that showed high leukotoxin production by the first method employed, one of them isolated from a patient and the other from a marmoset. Both of them were submited to different growth conditions. This method showed to be more sensitive than the tripan blue one. Leukotoxic activity detection started from 6 hours of growth; the results were similar when the assays were performed either in anaerobic or microaerophilic conditions. Glucose concentration influenced the levels of leukotoxin production: the production was extremely low, high and higher if the growth occured under glucose 0.25%, 0.75 and 1.5%, respectively. When the strains were cultivated in Brain-Heart Infusion Broth, we detected a decrease on leukotoxin production, when comparing to the results obtained if the growth occured in Tryptic Soy Broth or Thioglicolate medium. This results seem to be related to different glucose and protein concentrations. Leukotoxic activity was not affected by sodium bicarbonate l0mM. On the other hand, tripsin and heat shock inhibited leukotoxin production, which suggests that the substance produced has a proteic nature. The diversity of the results obtained regarding leukotoxic activity expression points to the need of new parameters to better evaluate the leukotoxin role in tissue injury.

Supported by CNPq, FAPEMIG, CAPES and PRPq/UFMG.