Antibacterial Action of Polyphosphate on Porphyromonas gingivalis

Abstract

Polyphosphate [poly(P)] has antibacterial activity against various Gram-positive bacteria. In contrast, Gram-negative bacteria are generally resistant to poly(P). Here, we describe the antibacterial characterization of poly(P) against a Gram-negative periodontopathogen, Porphyromonas gingivalis. The MICs of pyrophosphate (Na 4 P 2 O 7 ) and all poly(P) (Na n ؉ 2 P n O 3n ؉ 1 ; n ‫؍‬ 3 to 75) tested for the bacterium by the agar dilution method were 0.24% and 0.06%, respectively. Orthophosphate (Na 2 HPO 4 ) failed to inhibit bacterial growth. Poly-P75 was chosen for further study. In liquid medium, 0.03% poly-P75 was bactericidal against P. gingivalis irrespective of the growth phase and inoculum size, ranging from 10 5 to10 9 cells/ml. UV-visible spectra of the pigments from P. gingivalis grown on blood agar with or without poly-P75 showed that poly-P75 reduced the formation of -oxo bisheme by the bacterium. Poly-P75 increased hemin accumulation on the P. gingivalis surface and decreased energy-driven uptake of hemin by the bacterium. The expression of the genes encoding hemagglutinins, gingipains, hemin uptake loci, chromosome replication, and energy production was downregulated, while that of the genes related to iron storage and oxidative stress was upregulated by poly-P75. The transmission electron microscope showed morphologically atypical cells with electron-dense granules and condensed nucleoid in the cytoplasm. Collectively, poly(P) is bactericidal against P. gingivalis, in which hemin/heme utilization is disturbed and oxidative stress is increased by poly(P).Inorganic polyphosphate [poly(P)] is a ubiquitous compound found in bacteria, fungi, algae, plants, and animals. The poly(P) found in the organisms is a chain of a few or many hundreds of phosphate (P i ) residues linked by high-energy phosphoanhydride. It performs varied functions in bacteria: it can serve as an ATP source and substitute, it is a strong chelator of metal ions and thus can regulate the levels of the ions in the cells, it is a channel for DNA entry, and it is a regulator that contributes to bacterial resistance and survival under stress and stringent conditions (18). Therefore, intracellular poly(P) is considered a virulence factor of microorganisms.In contrast, exogenous poly(P) has attracted considerable attention as an antimicrobial agent, since it can prevent spoilage of food (29, 32), and it is listed as a GRAS (generally recognized as safe) food additive by the FDA. Poly(P) inhibits the growth of various Gram-positive bacteria, such as Staphylococcus aureus (14,17,22,35,52), Listeria monocytogenes (37, 52), Sarcina lutea (35), Bacillus cereus, and Lactobacillus, and of fungi, such as Aspergillus flavus (17,28). Concerning oral bacteria, mutans streptococci were first found to be inhibited by condensed phosphate, resulting in a decrease of plaque formation and dental caries (5, 39). The ability of poly(P) to chelate divalent cations is regarded as relevant to the antibacterial effects of poly(P), contributing to cell division i...

In Vitro Effects of Polyphosphate against Prevotella intermedia in Planktonic Phase and Biofilm

“…Recently, however, polyP was demonstrated to be bactericidal against Porphyromonas gingivalis, a black-pigmented anaerobic Gram-negative rod associated with periodontal disease (31). The MICs of polyP with different linear phosphorus chain lengths (3 to 75) for the bacterium were 0.06%, which is much lower than the MICs previously reported for Gram-positive bacteria (31).…”

“…The MICs of polyP with different linear phosphorus chain lengths (3 to 75) for the bacterium were 0.06%, which is much lower than the MICs previously reported for Gram-positive bacteria (31). Nota-bly, there is ample evidence to show that the mode of bactericidal action of polyP against Gram-negative bacteria is different from that against Gram-positive bacteria: antibacterial activity of polyP against P. gingivalis was independent of the bacterial growth phase, and no P. gingivalis cells with obviously aseptate and elongated morphology were observed when the bacterial cells were exposed to polyP (31).…”

“…Black pigment on the cell surface of the species is believed to result from accumulation of hemin (46). The redox potential of hemin, required as a prosthetic group of cytochrome b, allows it to mediate electron transfer with generation of cellular energy (31,46). These bacteria, by virtue of their hemolytic activity, are capable of liberating the hemoglobin or hemin from erythrocytes, thereby acquiring the essential nutrient for their metabolism (44,47,48).…”

“…polyP with chain length 3 (polyP3; Na 5 P 3 O 10 ), which is listed as GRAS (polyP), was purchased from Sigma Chemical Co. polyP3 was dissolved in distilled water to 10% (wt/vol), sterilized using a 0.22-m filter, and stored at Ϫ20°C until use. The MIC of polyP3 was determined by the agar dilution method as described previously (31). Briefly, various concentrations of polyP3 were prepared and added to brucella blood agar supplemented with 5 g/ml of hemin and 1 g/ml of vitamin K 1 .…”

OxyR Activation in Porphyromonas gingivalis in Response to a Hemin-Limited Environment

“…These differentially expressed genes or proteins were linked to bacterial invasion, iron transport, and an oxidative-stress response. A recent study showed that inorganic polyphosphates (potential antibacterial agents) inhibited energydriven uptake of hemin by P. gingivalis and repressed the expression of oxidative stress-induced proteins, including superoxide dismutase (SOD) (15), which provides indirect evidence linking hemin to SOD expression. To better understand the response of P. gingivalis to hemin-limited conditions and the association between iron/hemin concentrations and oxidative stress, we investigated the expression and activity of OxyR in P. gingivalis under hemin-limited as well as excess-hemin conditions.…”