Nitric Oxide Stress Resistance in Porphyromonas gingivalis Is Mediated by a Putative Hydroxylamine Reductase

Abstract

bPorphyromonas gingivalis, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants P. gingivalis FLL455 (PG0893::ermF) and FLL456 (PG2213::ermF) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain. P. gingivalis FLL455 was more sensitive to NO than the wild type. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain. P. gingivalis FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were upregulated 1.4-and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function. PG1181, predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance. Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that is a primary etiologic agent of periodontal disease. This infection-induced disease is characterized by inflammation, which can result in host-mediated destruction of tooth-supporting tissues and structures (18). Nitric oxide (NO) is a key component of the host immune response. Oral neutrophils can constitutively secrete low concentrations of NO via the involvement of multiple NO synthases. However, with activation, these neutrophils have increased secretion of NO with antimicrobial effects. As in other host-pathogen interactions (22), P. gingivalis has been shown to trigger the production of NO in immune and nonimmune host cells by activating the expression of inducible nitric oxide synthases (9, 54) and can survive in NO concentrations ranging from 4.9 M to 19.2 M (47). Elevated NO concentrations are reported to cause vasodilatation and a decrease in platelet aggregation, which may contribute to gingival bleeding (18), and to have cytotoxic effects on surrounding host tissue that can lead to alveolar bone loss (7). NO is present in the saliva and gingival fluid of periodontitis patients (7,18,57). Further, saliva NO concentrations have been shown to increase with the severity of periodontitis (47).P. gingivalis, as an asaccharolytic microorganism, metabolizes nitrogenous compounds as a source of energy and generates a micr...

HcpR of Porphyromonas gingivalis Is Required for Growth under Nitrosative Stress and Survival within Host Cells

“…Thus, it can survive the innate immune response, which is especially elevated in inflamed periodontal pockets. Our data verify the results recently reported by Boutrin et al (7), who have shown that P. gingivalis can grow in the presence of low nitric oxide concentrations.…”

“…The upregulation of hcp was quantified using qRT-PCR to be more than 150-fold higher upon exposure to nitrosative stress. This result is much higher than that observed in a previous study that used NONOate as a nitric oxide donor (7). Such significant upregulation of hcp is in agreement with previous reports demonstrating drastic upregulation of this gene in other anaerobic and facultatively anaerobic bacteria exposed to nitrite (26,46,48,56).…”

“…Such results indicate that the gene product plays an indispensable role in the protection of P. gingivalis against nitrosative stress. Indeed, previous studies have shown that hcp is required for bacterial growth with nitric oxide (7). The upregulation of hcp was quantified using qRT-PCR to be more than 150-fold higher upon exposure to nitrosative stress.…”

“…Indeed, recent studies have shown that Hcp plays a role in the protection of a variety of bacteria from nitrosative stress as well as from nitrosative stress-based killing by macrophages (48). Hcp is also encoded on the genome of P. gingivalis by PG0893 and protects the bacterium from exposure to nitric oxide (7). However, the biological function and the regulatory mechanisms governing expression of this protein remain unknown.…”

13. Reactivity of iron-sulfur clusters with nitric oxide

“…The so called hybrid cluster protein (Hcp) is found in many anaerobic or facultatively anaerobic bacteria, where it functions in response to nitrosative stress [217][218][219][220]. Hcp shares structural homology with anaerobic carbon monoxide dehydrogenases and contain two types of iron sulfur clusters; the first occurs as either a [2Fe-2S] or [4Fe-4S] cluster, while the second, socalled "hybrid cluster", is a unique cluster type, the precise form of which is dependent on the oxidation state.…”