Porphyromonas gingivalis genes conferring fitness in a tobacco-rich environment.

Smokers are more likely than non-smokers to harbour Porphyromonas gingivalis, they are more susceptible to destructive periodontal disease and smokers may, ultimately, benefit from tobacco-specific preventive and treatment strategies. A Mariner transposon insertion library for P. gingivalis ATCC 33277 was exploited to define 256 genes as essential for P. gingivalis survival in a tobacco-rich environment. Genes whose products play roles in protein transport and catabolism, nicotinamide processing, protection against oxidative stress, drug resistance, and transcriptional regulation have all been identified as essential for CSE survival. Many of these tobacco-essential genes are also requisite for epithelial colonization and abscess formation, suggestive of a core stress-related P. gingivalis genome. Single-gene deletions in several of the TnSeq-implicated genes led to significantly reduced P. gingivalis fitness upon competition with the parent strain, under conditions of cigarette smoke extract-induced stress (1,000 ng/ml nicotine equivalents). This study identifies, for the first time, a subset of P. gingivalis genes required for surviving the plethora of insults present in cigarette smoke. Such conditionally essential genes may delineate bacterial persistence strategies and represent novel therapeutic foci for the prevention of P. gingivalis infection and related diseases in smokers and in general.

The Porphyromonas gingivalis Hybrid Cluster Protein Hcp Is Required for Growth with Nitrite and Survival with Host Cells.

Although the periodontal pathogen Porphyromonas gingivalis must withstand high levels of nitrosative stress while in the oral cavity, the mechanisms of nitrosative stress defense are not well understood in this organism. Previously we showed that the transcriptional regulator HcpR plays a significant role in defense, and here we further defined its regulon. Our study shows that hcp (PG0893), a putative nitric oxide (NO) reductase, is the only gene significantly upregulated in response to nitrite (NO2) and that this regulation is dependent on HcpR. An isogenic mutant deficient in hcp is not able to grow with 200 µM nitrite, demonstrating that the sensitivity of the HcpR mutant is mediated through Hcp. We further define the molecular mechanisms of HcpR interaction with the hcp promoter through mutational analysis of the inverted repeat present within the promoter. Although other putative nitrosative stress protection mechanisms present on the nrfAH operon are also found in the P. gingivalis genome, we show that their gene products play no role in growth of the bacterium with nitrite. As growth of the hcp-deficient strain was also significantly diminished in the presence of a nitric oxide-producing compound, S-nitrosoglutathione (GSNO), Hcp appears to be the primary means by which P. gingivalis responds to NO2--based stress. Finally, we show that Hcp is required for survival with host cells but that loss of Hcp has no effect on association and entry of P. gingivalis into human oral keratinocytes.

Amixicile, a novel strategy for targeting oral anaerobic pathogens.

The oral microflora is composed of both health-promoting as well as disease-initiating bacteria. Many of the disease-initiating bacteria are anaerobic and include organisms such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. Here we investigated a novel therapeutic, amixicile, that targets pyruvate:ferredoxin oxidoreductase (PFOR), a major metabolic enzyme involved in energy generation through oxidative decarboxylation of pyruvate. PFOR is present in these anaerobic pathogenic bacteria and thus we hypothesized that amixicile would effectively inhibit their growth. In general, PFOR is present in all obligate anaerobic bacteria, while oral commensal aerobes, including aerotolerant ones, such as Streptococcus gordonii, use pyruvate dehydrogenase to decarboxylate pyruvate. Accordingly, we observed that growth of the PFOR-containing anaerobic periodontal pathogens, grown in both monospecies as well as multispecies broth cultures was inhibited in a dose-dependent manner while that of S. gordonii was unaffected. Furthermore, we also show that amixicile is effective against these pathogens grown as monospecies and multispecies biofilms. Finally, amixicile is the first selective therapeutic agent active against bacteria internalized by host cells. Together, the results show that amixicile is an effective inhibitor of oral anaerobic bacteria and as such, is a good candidate for treatment of periodontal diseases.

Metabolic crosstalk regulates Porphyromonas gingivalis colonization and virulence during oral polymicrobial infection.

Many human infections are polymicrobial in origin, and interactions among community inhabitants shape colonization patterns and pathogenic potential 1 . Periodontitis, which is the sixth most prevalent infectious disease worldwide 2 , ensues from the action of dysbiotic polymicrobial communities 3 . The keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii interact to form communities in vitro and exhibit increased fitness in vivo 3,4 . The mechanistic basis of this polymicrobial synergy, however, has not been fully elucidated. Here we show that streptococcal 4-aminobenzoate/para-amino benzoic acid (pABA) is required for maximal accumulation of P. gingivalis in dual-species communities. Metabolomic and proteomic data showed that exogenous pABA is used for folate biosynthesis, and leads to decreased stress and elevated expression of fimbrial adhesins. Moreover, pABA increased the colonization and survival of P. gingivalis in a murine oral infection model. However, pABA also caused a reduction in virulence in vivo and suppressed extracellular polysaccharide production by P. gingivalis. Collectively, these data reveal a multidimensional aspect to P. gingivalis-S. gordonii interactions and establish pABA as a critical cue produced by a partner species that enhances the fitness of P. gingivalis while diminishing its virulence.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments.

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

Scratching the surface - tobacco-induced bacterial biofilms.

Individual environmental factors, such as iron, temperature and oxygen, are known to have a profound effect on bacterial phenotype. Therefore, it is surprising so little known is about the influence of chemically complex cigarette smoke on bacterial physiology. Recent evidence has demonstrated that tobacco smoke and components alter the bacterial surface and promote biofilm formation in several important human pathogens, including Staphylococcus aureus, Streptococcus mutans, Klebsiella pneumonia, Porphyromonas gingivalis and Pseudomonas aeruginosa. The mechanisms underlying this phenomenon and the relevance to increased susceptibility to infectious disease in smokers and to treatment are reviewed.

Altered antigenic profiling and infectivity of Porphyromonas gingivalis in smokers and non-smokers with periodontitis.

BACKGROUND: Cigarette smokers are more susceptible to periodontal diseases and are more likely to be infected with Porphyromonas gingivalis than non-smokers. Furthermore, smoking is known to alter the expression of P. gingivalis surface components and compromise immunoglobulin (Ig)G generation. The aim of this study is to evaluate whether the overall IgG response to P. gingivalis is suppressed in smokers in vivo and whether previously established in vitro tobacco-induced phenotypic P. gingivalis changes would be reflected in vivo. METHODS: The authors examined the humoral response to several P. gingivalis strains as well as specific tobacco-regulated outer membrane proteins (FimA and RagB) by enzyme-linked immunosorbent assay in biochemically validated (salivary cotinine) smokers and non-smokers with chronic periodontitis (CP: n = 13) or aggressive periodontitis (AgP: n = 20). The local and systemic presence of P. gingivalis DNA was also monitored by polymerase chain reaction. RESULTS: Smoking was associated with decreased total IgG responses against clinical (10512, 5607, and 10208C; all P <0.05) but not laboratory (ATCC 33277, W83) P. gingivalis strains. Smoking did not influence IgG produced against specific cell-surface proteins, although a non-significant pattern toward increased total FimA-specific IgG in patients with CP, but not AgP, was observed. Seropositive smokers were more likely to be infected orally and systemically with P. gingivalis (P <0.001), as determined by 16S RNA analysis. CONCLUSION: Smoking alters the humoral response against P. gingivalis and may increase P. gingivalis infectivity, strengthening the evidence that mechanisms of periodontal disease progression in smokers may differ from those of non-smokers with the same disease classification.