Metabolic Pathways for Cytotoxic End Product Formation from Glutamate- and Aspartate-Containing Peptides by Porphyromonas gingivalis

Abstract

Metabolic pathways involved in the formation of cytotoxic end products by Porphyromonas gingivalis were studied. The washed cells of P. gingivalis ATCC 33277 utilized peptides but not single amino acids. Since glutamate and aspartate moieties in the peptides were consumed most intensively, a dipeptide of glutamate or aspartate was then tested as a metabolic substrate of P. gingivalis. P. gingivalis cells metabolized glutamylglutamate to butyrate, propionate, acetate, and ammonia, and they metabolized aspartylaspartate to butyrate, succinate, acetate, and ammonia. Based on the detection of metabolic enzymes in the cell extracts and stoichiometric calculations (carbon recovery and oxidation/reduction ratio) during dipeptide degradation, the following metabolic pathways were proposed. Incorporated glutamylglutamate and aspartylaspartate are hydrolyzed to glutamate and aspartate, respectively, by dipeptidase. Glutamate is deaminated and oxidized to succinyl-coenzyme Porphyromonas gingivalis (formerly Bacteroides gingivalis), a black-pigmented gram-negative anaerobe, is frequently detected in the lesions of several types of periodontitis (37-39, 57), and its isolation frequency increases in active sites of periodontitis (50). These observations suggest the strong relation of this bacterium with periodontal diseases.P. gingivalis has several periodontal pathogenic factors, including membrane-associated proteases, immunoactive cellular compounds, and cytotoxic metabolic end products (19, 31). The main cytotoxic end products, butyrate, propionate, and ammonia, have been found to easily penetrate into periodontal tissue, due to their low molecular weights (59), and subsequently to disturb host cell activity and host defense systems at millimolar concentrations (3, 10-12, 24, 40, 42, 49), the concentration levels found in the P. gingivalis culture supernatant and the gingival crevicular fluid of periodontally diseased subjects (7, 41). Among metabolic end products of P. gingivalis, butyrate is considered the most cytotoxic (42). However, only little information is available about the mechanism of cytotoxic end product formation by P. gingivalis.Several attempts have been made to elucidate the metabolic system of P. gingivalis. Shah and Williams (47) demonstrated that P. gingivalis is capable of degrading aspartate and asparagine to succinate, although P. gingivalis usually produces little succinate (18). Joe et al. (22) reported that this bacterium has glutamate dehydrogenase as an enzyme for glutamate degradation. However, most researchers have concluded that P. gingivalis utilizes mainly peptides instead of single amino acids as sources of energy and cell materials (35,46,48,55,60,62). For example, a chemically defined medium for P. gingivalis must be supplemented with a peptide or a protein such as Trypticase (46, 62) or bovine serum albumin (35). Thus, due to the complicated amino acid composition of peptides or proteins, it had been difficult to determine the amino acid metabolic pathway of P. gingivalis. In additio...

Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves increased consumption of formate and reduced utilization of lactate

“…However, PG0690, which encodes 4-hydroxybutyrate coenzyme A transferase, was slightly downregulated (ratio 0.8, results not shown). This transferase mediates the catabolism of glutamate to butyrate (Takahashi et al, 2000). Other downregulated genes included those mediating the biosynthesis of NAD from aspartate: PG0058, encoding nicotinate (nicotinamide) nucleotide adenylyltransferase (NadD), and PG1576, encoding L-aspartate oxidase (NadB) ( Table 2, Fig.…”

Arginine deiminase inhibits Porphyromonas gingivalis surface attachment

“…P. gingivalis produces proteolytic enzymes that specifically cleave at arginine residues within peptides, as well as enzymes dedicated to releasing an arginine molecule from the carboxy-terminus of a peptide (Chen et al, 1992; Masuda et al, 2001Masuda et al, , 2002. Although the mechanism is unknown, some data indicate preferential uptake and utilization of arginine by P. gingivalis (Masuda et al, 2001(Masuda et al, , 2002Pike et al, 1994;Takahashi et al, 2000). What remains unclear is why arginine is such an important amino acid.…”

“…gingivalis is a strict anaerobe that preferentially utilizes protein or peptide substrates for growth. Although studies have shown that P. gingivalis may utilize free amino acids or dipeptides, the uptake and growth rates on these substrates are limited and highly variable among strains (Seddon et al, 1988;Takahashi et al, 2000;Tang-Larsen et al, 1995). In general, single amino acids do not support planktonic growth; however, arginine appears to be an amino acid of particular importance (Masuda et al, 2001;Seddon et al, 1988;Takahashi et al, 2000;Tang-Larsen et al, 1995).…”

“…Although studies have shown that P. gingivalis may utilize free amino acids or dipeptides, the uptake and growth rates on these substrates are limited and highly variable among strains (Seddon et al, 1988;Takahashi et al, 2000;Tang-Larsen et al, 1995). In general, single amino acids do not support planktonic growth; however, arginine appears to be an amino acid of particular importance (Masuda et al, 2001;Seddon et al, 1988;Takahashi et al, 2000;Tang-Larsen et al, 1995). P. gingivalis produces proteolytic enzymes that specifically cleave at arginine residues within peptides, as well as enzymes dedicated to releasing an arginine molecule from the carboxy-terminus of a peptide (Chen et al, 1992;Masuda et al, 2001Masuda et al, , 2002.…”

Degradation of Incretins and Modulation of Blood Glucose Levels by Periodontopathic Bacterial Dipeptidyl Peptidase 4

“…These peptidases are thought to cover most combinations of P2 and P1 amino acid residues and, therefore, efficiently produce N-terminal dipeptides from polypeptides (21). This dipeptide-liberating potential is of crucial importance for asaccharolytic P. gingivalis, which incorporates amino acids mainly as dipeptides, not single amino acids (22,23), and utilizes them exclusively as carbon and energy sources (24). In addition, from the viewpoint of niche differentiation, dipeptide-incorporating and amino acidutilizing properties are likely to provide a benefit for P. gingivalis during symbiosis in the complex of subgingival microbiota, since, for instance, Prevotella intermedia and Fusobacterium nucleatum incorporate single amino acids (25) and P. intermedia, T. denticola, and Aggregatibacter actinomycetemcomitans are saccharolytic.…”