Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

HcpR of Porphyromonas gingivalis is required for growth under nitrosative stress and survival within host cells.

Although the Gram-negative, anaerobic periodontopathogen Porphyromonas gingivalis must withstand nitrosative stress, which is particularly high in the oral cavity, the mechanisms allowing for protection against such stress are not known in this organism. In this study, microarray analysis of P. gingivalis transcriptional response to nitrite and nitric oxide showed drastic upregulation of the PG0893 gene coding for hybrid cluster protein (Hcp), which is a putative hydroxylamine reductase. Although regulation of hcp has been shown to be OxyR dependent in Escherichia coli, here we show that in P. gingivalis its expression is dependent on the Fnr-like regulator designated HcpR. Growth of the isogenic mutant V2807, containing an ermF-ermAM insertion within the hcpR (PG1053) gene, was significantly reduced in the presence of nitrite (P < 0.002) and nitric oxide-generating nitrosoglutathione (GSNO) (P < 0.001), compared to that of the wild-type W83 strain. Furthermore, the upregulation of PG0893 (hcp) was abrogated in V2807 exposed to nitrosative stress. In addition, recombinant HcpR bound DNA containing the hcp promoter sequence, and the binding was hemin dependent. Finally, V2807 was not able to survive with host cells, demonstrating that HcpR plays an important role in P. gingivalis virulence. This work gives insight into the molecular mechanisms of protection against nitrosative stress in P. gingivalis and shows that the regulatory mechanisms differ from those in E. coli.

The capsule of Porphyromonas gingivalis leads to a reduction in the host inflammatory response, evasion of phagocytosis, and increase in virulence.

Periodontal disease is a chronic oral inflammatory disease that is triggered by bacteria such as Porphyromonas gingivalis. P. gingivalis strains exhibit great heterogeneity, with some strains being encapsulated while others are nonencapsulated. Although the encapsulated strains have been shown to be more virulent in a mouse abscess model, so far the role of the capsule in P. gingivalis interactions with host cells is not well understood and its role in virulence has not been defined. Here, we investigated the contribution of the capsule to triggering a host response following microbial infection, as well as its protective role following bacterial internalization by host phagocytic cells with subsequent killing, using the encapsulated P. gingivalis strain W50 and its isogenic nonencapsulated mutant, PgC. Our study shows significant time-dependent upregulation of the expression of various groups of genes in macrophages challenged with both the encapsulated and nonencapsulated P. gingivalis strains. However, cells infected with the nonencapsulated strain showed significantly higher upregulation of 9 and 29 genes at 1 h and 8 h postinfection, respectively, than cells infected with the encapsulated strain. Among the genes highly upregulated by the nonencapsulated PgC strain were ones coding for cytokines and chemokines. Maturation markers were induced at a 2-fold higher rate in dendritic cells challenged with the nonencapsulated strain for 4 h than in dendritic cells challenged with the encapsulated strain. The rates of phagocytosis of the nonencapsulated P. gingivalis strain by both macrophages and dendritic cells were 4.5-fold and 7-fold higher, respectively, than the rates of phagocytosis of the encapsulated strain. On the contrary, the survival of the nonencapsulated P. gingivalis strain was drastically reduced compared to the survival of the encapsulated strain. Finally, the encapsulated strain exhibited greater virulence in a mouse abscess model. Our results indicate that the P. gingivalis capsule plays an important role in aiding evasion of host immune system activation, promoting survival of the bacterium within host cells, and increasing virulence. As such, it is a major virulence determinant of P. gingivalis.

AdpC is a Prevotella intermedia 17 leucine-rich repeat internalin-like protein.

The oral bacterium Prevotella intermedia attaches to and invades gingival epithelial cells, fibroblasts, and endothelial cells. Several genes encoding proteins that mediate both the adhesion and invasion processes are carried on the genome of this bacterium. Here, we characterized one such protein, AdpC, belonging to the leucine-rich repeat (LRR) protein family. Bioinformatics analysis revealed that this protein shares similarity with the Treponema pallidum LRR (LRR(TP)) family of proteins and contains six LRRs. Despite the absence of a signal peptide, this protein is localized on the bacterial outer membrane, indicating that it is transported through an atypical secretion mechanism. The recombinant form of this protein (rAdpC) was shown to bind fibrinogen. In addition, the heterologous host strain Escherichia coli BL21 expressing rAdpC (V2846) invaded fibroblast NIH 3T3 cells at a 40-fold-higher frequency than control E. coli BL21 cells expressing a sham P. intermedia 17 protein. Although similar results were obtained by using human umbilical vein endothelial cells (HUVECs), only a 3-fold-increased invasion of V2846 into oral epithelial HN4 cells was observed. Thus, AdpC-mediated invasion is cell specific. This work demonstrated that AdpC is an important invasin protein of P. intermedia 17.

Porphyromonas gingivalis ferrous iron transporter FeoB1 influences sensitivity to oxidative stress.

Porphyromonas gingivalis FeoB1 is a ferrous iron transporter. Analysis of parental and feoB1-deficient strains of the periodontal pathogen revealed that the feoB1-deficient mutant strain had an increased ability to survive oxidative stress. Specifically, survival of the mutant strain was increased 33% with exposure to peroxide and 5% with exposure to atmospheric oxygen compared to the parental strain. Interestingly, the ability to survive intracellularly also increased fivefold in the case of the feoB1-deficient mutant. Our data suggest that although the FeoB1 protein is required for ferrous iron acquisition in P. gingivalis, it also has an adverse effect on survival of the bacterium under oxidative stress conditions. Finally, we show that feoB1 expression is not iron dependent and is dramatically reduced in the presence of host cells, consistent with the observed deleterious role it plays in bacterial survival.

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

Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in the presence of low concentrations of oxygen. Microarray analysis revealed alteration in gene expression in the presence of 6 % oxygen. During the exponential growth phase, 96 genes were upregulated and 79 genes were downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative stress protection were upregulated, including alkyl hydroperoxide reductase (ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant changes in gene expression of proteins that mediate oxidative metabolism, such as cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The expression of genes encoding formate uptake transporter (PG0209) and formate tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that formate metabolism plays a major role under aerobic conditions. The concomitant reduction of expression of a gene encoding the lactate transporter PG1340 suggests decreased utilization of this nutrient. The concentrations of both formate and lactate were assessed in culture supernatants and cells, and they were in agreement with the results obtained at the transcriptional level. Also, genes encoding gingipain protease secretion/maturation regulator (porR) and protease transporter (porT) had reduced expression in the presence of oxygen, which also correlated with reduced protease activities under aerobic conditions. In addition, metal transport was affected, and while iron-uptake genes such as the genes encoding the haemin uptake locus (hmu) were downregulated, expression of manganese transporter genes, such as feoB2, was elevated in the presence of oxygen. Finally, genes encoding putative regulatory proteins such as extracellular function (ECF) sigma factors as well as small proteins had elevated expression levels in the presence of oxygen. As P. gingivalis is distantly related to the well-studied model organism Escherichia coli, results from our work may provide further understanding of oxygen metabolism and protection in other related bacteria belonging to the phylum Bacteroidetes.