A putative TetR regulator is involved in nitric oxide stress resistance in Porphyromonas gingivalis.

To survive in the periodontal pocket, Porphyromonas gingivalis, the main causative agent of periodontal disease, must overcome oxidative and nitric oxide (NO) stress. Previously, we reported that, in the presence of NO comparable to stress conditions, the transcriptome of P. gingivalis was differentially expressed, and genes belonging to the PG1178-81 cluster were significantly upregulated. To further evaluate their role(s) in NO stress resistance, these genes were inactivated by allelic exchange mutagenesis. Isogenic mutants P. gingivalis FLL460 (ΔPG1181::ermF) and FLL461 (ΔPG1178-81::ermF) were black-pigmented, with gingipain and hemolytic activities comparable to that of the wild-type strain. Whereas the recovery of these isogenic mutants from NO stress was comparable to the wild-type, there was increased sensitivity to hydrogen peroxide-induced stress. RNA-Seq analysis under conditions of NO stress showed that approximately 5 and 8% of the genome was modulated in P. gingivalis FLL460 and FLL461, respectively. The PG1178-81 gene cluster was shown to be part of the same transcriptional unit and is inducible in response to NO stress. In the presence of NO, PG1181, a putative transcriptional regulator, was shown to bind to its own promoter region and that of several other NO responsive genes including PG0214 an extracytoplasmic function σ factor, PG0893 and PG1236. Taken together, the data suggest that PG1181 is a NO responsive transcriptional regulator that may play an important role in the NO stress resistance regulatory network in P. gingivalis.

Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis.

PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared with the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from Escherichia coli with the PG0162 promoter as template. As an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared with the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than two-fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis.

Metabolome variations in the Porphyromonas gingivalis vimA mutant during hydrogen peroxide-induced oxidative stress.

The adaptability and survival of Porphyromonas gingivalis in the oxidative microenvironment of the periodontal pocket are indispensable for survival and virulence, and are modulated by multiple systems. Among the various genes involved in P. gingivalis oxidative stress resistance, vimA gene is a part of the 6.15-kb locus. To elucidate the role of a P. gingivalis vimA-defective mutant in oxidative stress resistance, we used a global approach to assess the transcriptional profile, to study the unique metabolome variations affecting survival and virulence in an environment typical of the periodontal pocket. A multilayered protection strategy against oxidative stress was noted in P. gingivalis FLL92 with upregulation of detoxifying genes. The duration of oxidative stress was shown to differentially modulate transcription with 94 (87%) genes upregulated twofold during 10 min and 55 (83.3%) in 15 min. Most of the upregulated genes (55%), fell in the hypothetical/unknown/unassigned functional class. Metabolome variation showed reduction in fumarate and formaldehyde, hence resorting to alternative energy generation and maintenance of a reduced metabolic state. There was upregulation of transposases, genes encoding for the metal ion binding protein transport and secretion system.

Filifactor alocis: The Newly Discovered Kid on the Block with Special Talents.

Infection-induced periodontal disease has been primarily focused on a small group of periodontal pathogens. A paradigm shift, based on data emerging from the oral microbiome project, now suggests the involvement of as-yet-unculturable and fastidious organisms. Collectively, these studies have demonstrated that there are changes in the periodontal status associated with shifts in the composition of the bacterial community in the periodontal pocket. In addition, it is likely that the emerging new pathogens may play a more significant role in the disease. One of the organisms previously unrecognized is Filifactor alocis. While this Gram-positive anaerobic rod has been identified in peri-implantitis, in endodontic infections, and in patients with localized aggressive periodontitis, its presence is now observed at significantly higher levels in patients with adult periodontitis or refractory periodontitis. Its colonization properties and its potential virulence attributes support the proposal that F. alocis should be included as a diagnostic indicator of periodontal disease. Moreover, these emerging characteristics would be consistent with the polymicrobial synergy and dysbiosis (PSD) periodontal pathogenesis model. Here, unique characteristics of F. alocis are discussed. F. alocis has specific factors that can modulate multiple changes in the microbial community and host cell proteome. It is likely that such variations at the molecular level are responsible for the functional changes required to mediate the pathogenic process.