Protective role of the PG1036-PG1037-PG1038 operon in oxidative stress in Porphyromonas gingivalis W83.

Leroy G Henry,Lawrence Sandberg,Wilson Aruni,Hansel M Fletcher,Özlem Yilmaz

doi:10.1371/journal.pone.0069645

Abstract

As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2 induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H2O2 and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H2O2-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.

Highlights

Porphyromonas gingivalis, a black-pigmented Gram-negative anaerobic bacterium has been recognized as a major pathogen in adult periodontitis and is associated with other systemic diseases including cardiovascular disease and rheumatoid arthritis [1,2]
Transcriptional profiling of P. gingivalis showed that the PG1036 and PG1038 genes were upregulated in cells exposed to hydrogen peroxide-induced stress [45]
To confirm that the gene encoding PG1037 was part of a three gene operon, total RNA was isolated from the wild-type P. gingivalis W83 grown to mid-log phase [OD600 of 0.7]

Summary

Introduction

Porphyromonas gingivalis, a black-pigmented Gram-negative anaerobic bacterium has been recognized as a major pathogen in adult periodontitis and is associated with other systemic diseases including cardiovascular disease and rheumatoid arthritis [1,2]. Colonization, growth and survival of P. gingivalis in the inflammatory microenvironment of the periodontal pocket are important attributes that are vital for its pathogenesis This is facilitated by many virulence factors, which include adhesion proteins such as hemagglutinins, that can mediate its interaction with host tissues and other commensal bacteria [4]. It has the ability to form adducts (hydrogen-bonded chelate structures) with various cell constituents such as amino acids (e.g. histidine, alanine, glycine, aspartic acid), succinic acid and DNA bases, which act as H2O2 carriers [15]. These characteristics allow H2O2 to act at sites distinct from the site of its production, enhancing its damaging potential. Hydrogen peroxide formation could be one of the main sources of toxicity for anaerobic microorganisms

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Conclusion