Sarah Vanderlinde Bench B 31/08/2016 
Higher order taxa
Bacteria; FCB group (Fibrobactere-Chlorobi-Bacteroidetes superphylum); Bacteroidetes; Bacteroidetes; Bacteroidales; Porphyromonadaceae; Porphyromonas
Porphyromonas gingivalis, Type Strain W83
Description and significance
Porphyromonas gingivalis is a gram negative, rod shaped bacterium typically found in the subgingival niche of people suffering from periodontal disease . H. Werner first isolated strain W83 in the 1950s (Bonn, Germany) from an unknown oral disease sample . Oral samples can be cultured on horse blood agar at 37°C under anaerobic conditions to form black-pigmented colonies after 6-10 days . P. gingivalis is a prominent and typically commensal member of the oral microbiome however under certain conditions can for a complex with other oral pathogens to produce periodontal lesions . This complex, termed the “red complex” includes P. gingivalis, Treponema denticola, and Tannerella forsythia and has been associated with severe periodontal disease . Periodontitis is a polymicrobial disease characterized by initial inflammation of gingival tissue which if left untreated, can lead to the eventual destruction of tooth-supporting structures and loss of teeth . The World Health Association have stated that 10-15% of the global population suffer from periodontitis, making the study and potential targeting of its causative bacteria an essential field of biomedical research .
P. gingivalis strain W83 has a circular genome, 2,343,479 bp in length with an average GC content of 48.3% . It contains 1,990 identified open reading frames (ORFs) (85% of the genome), 54% of which have biological roles. The P. gingivalis genome has four ribosomal operons (5S-23S-tRNAAla-tRNAIle-16S), two structural RNA genes and 53 tRNA genes with specificity for all 20 amino acids . Approximately 6% of the genome consists of repetitive elements including DNA repeats and transposable elements. Identified DNA repeats consist of uninterrupted direct repeats and clustered regularly interspaced short palindromic repeats (CRISPRs). Strain W83 does not appear to contain other classes of repetitive elements such as ERIC and REP . Major genes of interest include rgp and kgp which encode for the gingipain virulence proteins unique to P. gingivalis and the fimA gene which transcribes to the major fimbriae virulence protein .
Cell structure and metabolism
As P. gingivalis is an asaccharolytic, non-motile anaerobe that resides in the small crevice between tooth and gum, it is highly dependent on proteases to obtain nutrients for growth. The majority of this organism’s protealytic activity is performed by gingipains, a group of cysteine endoproteases unique to P. gingivalis that have both metabolic and virulent capacities . There are three members in the gingipain family: RgpA, RgpB and Kgp, which are encoded for by three genes, rgpA, rgpB and kgp respectively. Both RgpA and RgpB hydrolyse Arg-Xaa whereas Kgp cleaves Lys-Xaa peptide bonds . Gingipains are essential for iron and haem acquisition through haemoglobin degradation, which causes P. gingivalis to form black colonies on blood agar after 6-10 days of incubation . They also degrade human albumin serum to provide an abundant source of nitrogen and carbon .
The P. gingivali cell wall is gram negative and contains multiple layers of lysine-rich peptidoglycan. P. gingivalis, like many gram negative bacteria, utilizes fimbriae in cell to cell adhesion. This bacterium possesses three distinct types of fimbriae termed long, short and accessory components. Of these, the long fimbriae (FimA) and short fimbriae (Mfa1), also known as major and minor fimbriae are mainly involved in cell-to-cell contact for microcolony and biofilm formation . Bioflim formation is an essential virulence factor in P. gingivalis pathology as it allows for metabolic interaction with other oral microbes. For example, when P. gingivalis forms a biofilm with T. denticola, a relationship characteristic of periodontitis, 34 metabolic genes undergo modified regulation . These changes result in T. denticola stimulating P. gingivalis glycine production via the upregulation of proteases and increasing the synthesis and transport of thiamine .
P. gingivalis is an obligate anaerobe typically found in the subgingival sulcus of the human oral cavity. It is able to survive in the deep periodontal pocket due to its ability to ferment amino acids for energy production . P. gingivalis is a secondary coloniser in dental plaque formation, often adhering to primary colonisers such as streptococci and actinomyces . The transition from gram positive primary colonisers to mainly gram negative secondary colonisers is characteristic of the shift in disease state from gingivitis to periodontitis . Secondary colony formation is typically dependent on the host immune response adapting to biofilm species composition. In the presence of primary colonisers the host initiates a neutrophil-mediated immune response which causes soft tissue inflammation. As biofilm growth continues, the host immune response adapts to include lymphocytes and plasma cells which further increase inflammation and allow for secondary colony formation. As well as in the oral cavity, P. gingivalis has been found in the upper gastrointestinal tract, respiratory tract, colon and women with bacterial vaginosis .
Research has shown that only a small number of bacteria found in the subgingival niche contribute to periodontitis, of which P. gingivalis is the major etiological factor. Datta et al. (2008) identified P. gingivalis in 85.75% of subgingival plaque samples from patients with chronic periodontitis . Host cell invasion is mediated by fimbriae, which are able to bind to salivary enzymes, extracellular matrix proteins, commensal bacteria and the epithelial cell alpha5beta1-integrin . Adhesion to epithelial cells allows for capture of P. gingivalis by pseudopodia and uptake into phagosomes. Here the bacteria can activate cellular autophagy to create a replicative niche and suppress apoptosis . The replicative vacuole provides P. gingivalis with host proteins via autophagy that are essential for its survival . P. gingivalis contributes to periodontal pathogenicity via the production of pro-inflammatory cytokines, such as IL-1β and IL-6, by peripheral CD4+ T helper cells. This is predominantly through the lipopolysaccharide (LPS) of P. gingivalis, which is a major virulence factor in periodontal disease. Biofilm formation and bacterial dipeptidyl peptidase IV (DPPIV) also aid in pathogenicity due to their roles in sustaining growth throughout infection .
Recent studies have shown an etiological link between periodontitis and rheumatoid arthritis . Rheumatoid arthritis occurs as a result of the production of autoantibodies against citrullinated proteins. Citrullination is a post-translational modification of arginine residues that is mediated by the family of peptidylarginine deiminases (PADs) . P. gingivalis has been identified as a possible source of citrullinated proteins in its human host. It is the only bacterium to date that has been found to express a functional bacterial PAD, termed PPAD . Protein citrullination by PPAD uses a different mode of proteolytic processing to human PADs and therefore has the potential to generate epitopes to which immunologic tolerance does not exist . Not only can these epitopes cause an immune response at the site of P. gingivalis infection, but they can also spread to joints causing the inflammation associated with rheumatoid arthritis .
Application to biotechnology
As P. gingivalis is a human pathogen, the main focus of biotechnological research has been in developing a successful vaccine against the bacterium. In most cases, the first step towards developing a successful vaccine is to identify possible gene vaccine candidates. Ross et al. (2001) did this by using shotgun sequencing and bioinformatics to identify 120 possible gene targets. These selected genes were then cloned for expression in Escherichia coli and screened with P. gingivalis antisera before being purified and tested in a mouse model . Two of the identified recombinant proteins, PG32 and PG33 showed significant protection in the mouse model . Further research into vaccine development has found strong evidence for the use of virulence proteins RgpA and RgpB as vaccine antigens. Mice injected by Gene Gun with plasmid DNA carrying rgpA showed production of high levels of serum antibodies as well as diminished protealytic activity of RgpA and RgpB .
Due to its etiological link with rheumatoid arthritis, P. gingivalis may provide a possible therapeutic target to reduce joint inflammation. The bacterial deaminase (PPAD) associated with production of autoreactive epitopes is located on the cell surface, making it a convenient drug target . Inactivation of this protein could not only provide treatment for the otherwise incurable rheumatoid arthritis, but also reduce the inflammatory immune response associated with clinical periodontitis .
Research is currently being conducted into the symbiotic relationship between bacteria that contribute to periodontal disease, in particular P. gingivalis and Aggregatibacter actinomycetemcomitans . Previous studies have found that neither of these bacteria is able to survive and cause disease without the other, therefore making their relationship of great interest. Studies are also being conducted into the structure and production of fimbriae as a potential target to prevent tooth adhesion and biofilm formation . Recent clinical and epidemiological studies have shown that P. gingivalis may facilitate the development and progression of collagen induced arthritis  . Research is being conducted into therapeutic targets for the citrullinating peptidylarginine deiminase enzyme, uniquely expressed by P. gingivalis. As this enzyme has the ability to convert arginine residues in proteins to citrulline and create autoreactive epitopes, removing or inactivating it may be a possible treatment for rheumatoid arthritis .
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This page is written by Sarah Vanderlinde for the MICR3004 course, Semester 2, 2016