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.

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.Abbreviations: ECF, extracellular function; formyl-THF, 10-formyl-tetrahydrofolate; GNAT, Gcn5-related N-acetyltransferase; JCVI, J. Craig Venter Institute; RNAP, RNA polymerase; TIGR, The Institute for Genomic Research.The microarray data discussed in this paper have been deposited in the Gene Expression Omnibus (GEO) repository available at the National Center for Biotechnology Information (NCBI) under the accession number GSE17960.Four supplementary figures, showing aerobic growth of P. gingivalis W83, a genomic view of the regulated genes, selected pathways affected by the presence of oxygen and the lactate utilization locus, and two supplementary tables, listing genes upregulated and downregulated in bacteria grown in the presence of oxygen, are available with the online version of this paper. INTRODUCTIONPorphyromonas gingivalis is a Gram-negative bacterium that plays a major role in the developme...

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron-dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron- and hemin-dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole-genome microarrays of P. gingivalis genes were used to compare the levels of gene expression under iron-replete and iron-depleted conditions as well as under hemin-replete and hemin-depleted conditions. Under iron-depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron-depleted conditions to those from hemin-depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron-dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron-regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron-stressed bacterium than the iron-replete P. gingivalis cells. The major iron-regulated proteins encoded by PG1019-20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole-genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P. gingivalis.

Summary Little is known about the regulatory mechanisms that allow Porphyromonas gingivalis to survive in the oral cavity. Here we characterize the sigma (σ) factor SigH, one of six extracytoplasmic function (ECF) σ factors encoded in the P. gingivalis genome. Our results indicate that sigH expression is upregulated by exposure to molecular oxygen, suggesting that sigH plays a role in adaptation of P. gingivalis to oxygen. Furthermore, several genes involved in oxidative stress protection, such as sod, trx, tpx, ftn, feoB2 and the hemin uptake hmu locus, are downregulated in a mutant deficient in SigH designated as V2948. ECF σ consensus sequences were identified upstream of the transcriptional start sites of these genes, consistent with the SigH‐dependent regulation of these genes. Growth of V2948 was inhibited in the presence of 6% oxygen when compared with the wild‐type W83 strain, whereas in anaerobic conditions both strains were able to grow. In addition, reduced growth of V2948 was observed in the presence of peroxide and the thiol‐oxidizing reagent diamide when compared with the W83 strain. The SigH‐deficient strain V2948 also exhibited reduced hemin uptake, consistent with the observed reduced expression of genes involved in hemin uptake. Finally, survival of V2948 was reduced in the presence of host cells compared with the wild‐type W83 strain. Collectively, our studies demonstrate that SigH is a positive regulator of gene expression required for survival of the bacterium in the presence of oxygen and oxidative stress, hemin uptake and virulence.

ABSTRACTAlthough the Gram-negative, anaerobic periodontopathogenPorphyromonas gingivalismust 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 ofP. gingivalistranscriptional 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 ofhcphas been shown to be OxyR dependent inEscherichia coli, here we show that inP. gingivalisits expression is dependent on the Fnr-like regulator designated HcpR. Growth of the isogenic mutant V2807, containing anermF-ermAMinsertion within thehcpR(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 thehcppromoter sequence, and the binding was hemin dependent. Finally, V2807 was not able to survive with host cells, demonstrating that HcpR plays an important role inP. gingivalisvirulence. This work gives insight into the molecular mechanisms of protection against nitrosative stress inP. gingivalisand shows that the regulatory mechanisms differ from those inE. coli.