ABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Nitric Oxide/pharmacology , Porphyromonas gingivalis/drug effects , Porphyromonas gingivalis/genetics , Stress, Physiological , Adhesins, Bacterial/metabolism , Alleles , Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , Gingipain Cysteine Endopeptidases , Hydrogen Peroxide/pharmacology , Multigene Family , Mutagenesis , Mutation , Oxidative Stress , Porphyromonas gingivalis/metabolism , Promoter Regions, Genetic , Regulatory Elements, Transcriptional , Sequence Analysis, RNA , Sigma FactorABSTRACT
The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2h and 12h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml(-1) bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2h after treatment but viability decreased up to 8h. Even very low concentrations of bvPLA2 (10(-12) mg ml(-1)) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2h bacterial cultures but bacteriostatic to 12h ones. Minimum bactericidal concentrations were 10(-5)-10(-6) mg ml(-1). The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.
