No abstract

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.

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.

Summary Porphyromonas gingivalis is a Gram-negative anaerobe and keystone periodontal pathogen. A mariner transposon insertion mutant library has recently been used to define 463 genes as putatively essential for the in vitro growth of P. gingivalis ATCC 33277 in planktonic culture [Library 1, (Klein et al 2012)]. We have independently generated a transposon insertion mutant library [Library 2] for the same P. gingivalis strain and herein compare genes that are putatively essential for in vitro growth in complex media, as defined by both libraries. 281 genes (61%) identified by Library 1 were common to Library 2. Many of these common genes are involved in fundamentally important metabolic pathways, notably pyrimidine cycling as well as lipopolysaccharide, peptidoglycan, pantothenate and coenzyme A biosynthesis and nicotinate and nicotinamide metabolism. Also in common are genes encoding heat shock protein homologues, sigma factors, enzymes with proteolytic activity and the majority of sec-related protein export genes. In addition to facilitating a better understanding of critical physiological processes, transposon sequencing technology has the potential to identify novel strategies for the control of P. gingivalis infections. Those genes defined as essential by two independently generated TnSeq mutant libraries are likely to represent particularly attractive therapeutic targets.

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 to compromise IgG generation. The aim of this study was to evaluate whether 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 We examined the humoral response to several P. gingivalis strains as well as specific tobacco-regulated outer membrane proteins (FimA and RagB) by ELISA in biochemically-validated (salivary cotinine) smokers and non-smokers with chronic (CP, n = 13) or aggressive (AP, n = 20) periodontitis. We also monitored the local and systemic presence of P. gingivalis DNA by PCR. 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 towards increased total FimA-specific IgG in CP subjects, but not AP subjects, 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. Conclusions Smoking alters the humoral response against P. gingivalis, strengthening the evidence that mechanisms of periodontal disease progression in smokers may differ from non-smokers with the same disease classification.