Galactose Impacts the Size and Intracellular Composition of the Asaccharolytic Oral Pathobiont Porphyromonas gingivalis

Abstract

The asaccharolytic anaerobe Porphyromonas gingivalis metabolizes proteins it encounters in the periodontal pocket, including host-derived glycoproteins such as mucins and immunoglobulins. Often, these proteins are protected by a diverse array of carbohydrates tethered to the polypeptide chain via glycolytic bonds, and P. gingivalis produces enzymes capable of liberating these carbohydrates, exposing the proteinaceous core. In this study, we investigated the effect of individual monosaccharides, including galactose, l-fucose, mannose, and glucose, on the growth and physiology of P. gingivalis Of the carbohydrates tested, only galactose noticeably altered the density of the bacterial culture, and we observed that cultures grown with galactose reached significantly higher densities during stationary phase. Importantly, electron micrographs and plating of P. gingivalis in stationary phase demonstrated that the presence of galactose did not increase cell numbers; instead, the higher densities resulted from the expansion of individual cells which contained large intracellular granules. Initial attempts to characterize these granules revealed only a subtle increase in soluble carbohydrates, suggesting they are likely not composed of stored carbohydrate. Also, an analysis of major surface polysaccharides via an enzyme-linked immunosorbent assay (ELISA) did not reveal significant differences between cells grown with or without galactose. Finally, an initial investigation of the transcriptional changes elicited by galactose in late exponential phase suggested that genes important for cell shape and for the general stress response may play roles in this phenomenon. Overall, galactose, a monosaccharide commonly present on the surfaces of host proteins, substantially alters the physiology of P. gingivalis via the production of large, currently undefined, intracellular granules.IMPORTANCE Environmental perturbations are central to the ability of pathobionts, such as Porphyromonas gingivalis, to promote the development of diseased sites. In the case of periodontal disease, increased local pH, a shift to anaerobic surroundings, and the accumulation of Gram-negative anaerobes at the expense of Gram-positive cocci are known ecological fluctuations prominently associated with progression toward disease. Importantly, in contrast, the alterations to subgingival food webs in disease sites remain poorly characterized. We hypothesized that given the dramatic shift in community structure during disease, it is possible that free carbohydrates, which would typically be readily metabolized by Gram-positive cocci after cleavage from glycoproteins, may increase in concentration locally and thereby affect the physiological state of the subgingival microbiota. In this study, we explored the impact of free monosaccharides on P. gingivalis to gain deeper insight into the effect of dysbiotic conditions on the growth and physiology of this periodontal pathogen.

Keywords: microbial physiology; stress response.

FIG 1

Growth curves of P. gingivalis W83 (A) and 381 (B) on TSBHK alone or TSBHK supplemented with 0.1% of glucose (glc), mannose (man), l-fucose (l-fuc), or galactose (gal). The data are representative of at least three replications showing similar results.

FIG 2

Growth curves of P. gingivalis W83 (A) and 381 (B) on TSBHK alone or TSBHK supplemented with 0.5%, 0.1%, 0.05%, or 0.01% galactose (gal). The data are representative of at least three replications showing similar results.

FIG 3

The presence of galactose alters the size of P. gingivalis W83 cells. Cultures of P. gingivalis W83 were grown in TSBHK alone (A and C) or containing 0.1% galactose (B and D). Samples were prepared and viewed using scanning electron microscopy as described in Materials and Methods.

FIG 4

Cells of P. gingivalis W83 grown in the presence of galactose contain large intracellular inclusions. Cultures of P. gingivalis W83 were grown in TSBHK alone (A) or containing 0.1% galactose (B to D), and samples were stained with ruthenium red and viewed using scanning electron microscopy as described in Materials and Methods. We consistently observed large rod-shaped cells in cultures grown with galactose compared to the control cultures. Furthermore, the larger cells were packed with intracellular inclusions (presently undefined). We did not observe consistent differences in ruthenium red staining between cells grown with or without galactose, which suggests the cultures produce similar levels of surface polysaccharides.

FIG 5

The presentation of major surface polysaccharides by P. gingivalis W83 is not altered by growth in the presence of galactose. Cultures of P. gingivalis W83 were grown in TSBHK alone or TSBHK supplemented with 0.5%, 0.1%, 0.05%, or 0.01% galactose (gal) for 24 h. Before generating autoclaved extracts, a fixed volume of the cultures was removed (A) or the cultures were normalized to an OD₆₀₀ of 1.0 (B), and ELISAs were performed to detect capsular polysaccharides (A and B). The extracts generated using a fixed volume of culture as in panel A were used in subsequent ELISAs to detect the O-LPS (C) and A-LPS (D).