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.

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-foldhigher 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.

Angiotensinogen is the substrate for the entire RAS cascade and polymorphisms leading to its overexpression are linked to hypertension. SNPs in the promoter of the hAGT gene are associated with hypertension. Importantly, these SNPs can further modulate the gene of interest in various physiological/environmental settings like the high-sodium diet. In this regard, the human angiotensinogen (hAGT) gene has polymorphisms in its 2.5Kb promoter that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A is associated with human hypertension whereas Hap -6G/-217G reduces cardiovascular risk. We have engineered transgenic (TG) mice with these haplotypes (Hap -6A: -6A/-217A and Hap -6G: -6G/-217G) so as to examine the transcriptional regulation of the hAGT in an in vivo setting. This study is designed to study the effects of a high-sodium diet on the transcriptional milieu of renal tissues with consequential effects on the hAGT expression in our two haplotypes. Male TG mice were placed on 4% Na + diet for a period of 8 weeks. High-salt diet induces mineralocorticoid receptor (MR) and SGK-kinase expression in both haplotypes, equally. MR has been shown to bind to GRE elements in the hAGT gene. Importantly, MR-binding (ChIP assay) and hAGT induction are significantly (p&lt;0.05) greater in the -6A haplotype males as compared to -6G males. High-salt also increased the expression of transcriptional regulators including CEBPβ and HNF4 (p&lt;0.05) that are independent of haplotype. Complementary ChIP assay confirmed enhanced transcription factor (TF) binding to the chromatin of male -6A TG mice as compared to their -6G counterparts after high-salt diet treatment. Thus, we show here an effect of high-salt on cellular transcriptional apparatus that is haplotype-independent. However, increased TF affinity of the chromatin in -6A TG mice leads to higher salt-induced AGT levels in this haplotype than -6G. These observations could partly account for increased salt-sensitivity of some adult males that, in turn, is governed by the “risk” haplotype. Identifying these individuals with the -6A haplotype will help guide therapeutic lifestyle changes in patients with essential hypertension.