Effect of hemin on the physiology and virulence of Bacteroides gingivalis W50

Abstract

Bacteroides gingivalis W50 was grown in a chemostat under steady-state conditions at pH 7.5 ± 0.2 and a constant growth rate of 6.9 h for periods of up to 6 weeks (146 bacterial generations) in a complex medium. Hemin was capable of limiting the growth of cells up to a concentration of approximately 0.5 ,ug/ml since higher concentrations of hemin did not increase cell yields; cells grew in the absence of exogenously added vitamin Kl. Only a limited number of amino acids was metabolized during growth, but because none of these was totally depleted, the limiting nutrient under hemin excess conditions was probably a peptide. A range of fermentation products was produced under all conditions of growth; higher concentrations of cytotoxic metabolites such as propionate and butyrate were formed under hemin excess conditions, although more ammonia was released under hemin limitation. When viewed by electron microscopy, cells grown under hemin limitation appeared to be either coccobacillary or short rods and possessed few fimbriae per cell, but large numbers of extracellular vesicles could be seen both surrounding the cell surface and free in the environment. In contrast, cells grown under hemin excess conditions were more commonly coccus shaped and were more heavily fimbriated but had fewer extracellular vesicles. Marked differences were found in the susceptibility of mice to infection with cells grown under different concentrations of hemin. Cells transferred to media without any added hemin were avirulent, whereas those grown under conditions of hemin limitation (0.33 and 0.40 ,ug/ml) produced a 20 and 50% mortality in mice, respectively. In contrast cells grown under hemin excess always caused 100% mortality in mice, although this virulence was dose dependent. When virulent, the bacteria caused an extensive, spreading infection with necrosis of the skin and subcutaneous tissues. Collagen disintegration was seen histologically, implying a role for collagenase production in the pathogenicity of these bacteria.

Surface properties and ultrastructure of Porphyromonas gingivalis W50 and pleiotropic mutants

“…Recently colonial variants of a virulent strain of P. gingimlis W50 were isolated from continuous culture (4). Using an animal model to evaluate the virulence of these strains, the brown mutant of strain W50 (W50/BR1) showed reduced virulence compared to the wild strain W50, whereas the beige variant (W50/BE1) was avirulent (4,5).…”

“…Recently colonial variants of a virulent strain of P. gingimlis W50 were isolated from continuous culture (4). Using an animal model to evaluate the virulence of these strains, the brown mutant of strain W50 (W50/BR1) showed reduced virulence compared to the wild strain W50, whereas the beige variant (W50/BE1) was avirulent (4,5). Differences in biochemical properties were recently reported between strain W50, W50/BP1 (a black pigmented variant of W50 which over produced protohaem), and the less pigmented brown and beige pleiotropie mutants (6).…”

Pathogenicity and virulence of black-pigmented Gram-negative anaerobes

“…The addition of haemin supported the development of the spreading fatal disease, while succinate supported development of the less severe, localized disease. McKee et al [69] further substantiated the essential role of haemin in the virulence of P. gingivalis. They found that the virulence of P. gingivalis could be modulated by the level of protohaem added to the culture medium.…”

“…Smalley et al [68] have shown recently that the collagenolytic activity of the strain W50 was considerably localized in an extracellular vesicle fraction, in contrast to strain W50/BE1 which had very little activity in the vesicles. In considering the role of collagenase activity on virulence it is noteworthy that strain W50 grown under haemin excess conditions is more virulent than when grown under haemin limitation [69] and that collagenolytic activity of cells grown under conditions that increase the virulence was one-third of that when grown under haemin limitation [70].…”

“…Indications that this enzyme activity is related to virulence have come from studies showing that the trypsin-like enzymatic activity of strain W50 is more than three-fold higher than that of the avirulent strain W50/BE1 [65][66][67]71]. Further information implicating an association between trypsin activity and virulence of P. gingivalis is provided by the observation that the virulence of strain W50 was markedly reduced during growth under haemin-limitation [69], and this was associated with a 3.5-fold reduction in trypsin-like activity, but not in other enzymes [70]. There were also differences in the distribution of trypsin-like enzyme activity between strains W50 and W50/BE1.…”