DNA Repair of 8-Oxo-7,8-Dihydroguanine Lesions in Porphyromonas gingivalis

Abstract

The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stressinduced DNA damage was investigated. A 3.1-kb fragment containing the uvrB gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black pigmentation and beta-hemolysis to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.

VimA-Dependent Modulation of Acetyl Coenzyme A Levels and Lipid A Biosynthesis Can Alter Virulence in Porphyromonas gingivalis

“…Purification of the His-tagged VimA chimera protein was carried out using nickel-nitrilotriacetic acid magnetic agarose beads (Invitrogen, Carlsbad, CA) as previously reported (21). The protein pull-down assay was performed using a Dynabeads His-tag isolation and pull-down kit (Invitrogen, Carlsbad, CA).…”

“…An LCQ Deca XP Plus system (Thermo Scientific) with nanoelectrospray technology (New Objective) was used to analyze the peptides extracted from each gel piece (21). The four-part protocol used for the mass spectrometry (MS) and MS/MS analyses included one full MS analysis (from 450 to 1,750 m/z), followed by three MS/MS events using data-dependent acquisition, where the most intense ion from a given full MS scan was subjected to collision-induced dissociation, followed by the second and third most intense ions.…”

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

“…UV stress causes DNA mutations similar to the ones found in NO-and H 2 O 2 -induced stress and has been widely used for DNA repair mechanism studies. Unlike other bacteria, P. gingivalis has been shown to use a yet-unknown repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions caused by NO-and H 2 O 2 -induced stress (11,29,53). To investigate the potential effect of PG0893 and PG2213 in the cascade of events for NO detoxification, cell repair (including DNA repair) and survival, we evaluated the sensitivity of the P. gingivalis FLL455 (PG0893:: ermF) and FLL456 (PG2213::ermF) mutants to UV stress compared to the wild-type strain W83.…”

Oxidative Stress Resistance in Porphyromonas Gingivalis

“…Oxidative damage produced by intracellular ROS results in base modifications, single- and double-strand breaks and the formation of apurinic/apyrimidinic lesions, many of which are toxic and/or mutagenic [3]. The most notable is the highly mutagenic guanine residue 7,8-dihydro-8-oxoguanine, which is by far the most common DNA lesion formed as a result of oxidative stress [4]. When the genome of P. gingivalis was surveyed [5,101], essential components that are involved in base excision repair (BER) [6] and nucleotide excision repair [7] were observed, and investigation of these components revealed that the mechanism P. gingivalis uses to repair DNA damage in comparison to Escherichia coli [8] and other well-studied organisms [3,9] is undoubtedly unique.…”