Daniel Chew Bench D 23 September 2016 
Higher order taxa
Bacteria (Kingdom) – Bacteroidetes (Phylum) – Bacteroidetes (Class) – Bacteroidales (Order) – Porphyromonadaceae (Family) – Porphyromonas (Genus)
Porphyromonas gingivalis type strain ATCC 33277
Description and significance
Porphyromonas gingivalis is a Gram-negative, obligate anaerobe which plays a major etiological role in the initiation and progression of clinically severe periodontal disease. As an opportunistic pathogen, it would coexist within the host in harmony, until a shift in ecological balance within the environment would allow it to cause disease in the host (12). P. gingivalis can be isolated and grown, albeit infrequently, with anaerobic culturing techniques using blood agar and incubation at 37°C (3) and has been described as non-motile, rod-shaped and will form black colonies on blood agar (10).
It shares, with other “red-complex” bacteria, Tannerella forsythia and Treponema denticola, the ability to resist innate host responses through inhibition or evasion (6). This ability increases the virulence capabilities of P. gingivalis, in conjunction with other virulence factors such as the presence of fimbriae, which allow it to attach to surfaces within the oral cavity for initial microbial colonisation in pathogenesis of periodontal disease (4). P. gingivalis has also been shown to instigate pathogenesis in hosts, even at low abundance levels, through inhibition of chemokine induction and subversion of the complement system, which would allow normally harmless microbiota to become pathogenic. Therefore, this would have significant implications in regions of similar immune defences, where the maintenance of homeostasis is sought after in the face of a large microbial presence (9).
In 2008, the genome of the type strain of P. gingivalis, ATCC 33277 was completely sequenced. The bacterium contains a single circular chromosome of 2,354,886 bp in size. The genome sequence had an average GC content of 48.4% with 2090 protein coding sequences, 4 RNA operons and 53 tRNA genes that was specific for all 20 amino acids (15).
Cell structure and metabolism
P. gingivalis is a rod-shaped, Gram-negative bacterium that possesses fimbriae and other bacterial surface proteins on its cellular surface. These proteins are indispensable in the adherence to other bacterial species during the secondary or late colonisation phases (15). A study has also shown that P. gingivalis is not capable of independent biofilm formation, thus it will require other species of bacteria, like Streptococcus gordonii, to achieve primary attachment to the host cell surfaces, before a substantial biofilm can be formed (5). Furthermore, after these biofilms has been formed, synergistic interactions between Fusobacterium nucleatum and P. gingivalis have been observed, with F. nucleatum possibly possessing a mechanism to protect P. gingivalis from oxidative stress in a non-strictly anaerobic environment (14).
As an asaccharolytic bacterium, P. gingivalis must rely on other sources of energy generation, such as the fermentation of amino acids. This is done through the highly-dependent enzymatic action of proteases released by the bacterium, which are also known as its gingipains (10). Extracellular proteins are first degraded to oligopeptides by gingipains R (Rgp) and K (Kgp), which are cysteine endopeptidases. The oligopeptides are further processed by exopeptidases, which include dipeptidyl peptidases (DPPs), tripeptidyl peptidases and acylpeptidyl oligopeptidases (AOPs), and produce di- and tri-peptides. These processed peptides are then suitable for uptake by the sodium ion-driven serine/threonine transporter into the interior of the bacterial cell. Once inside the cytoplasm, the peptides are utilised in the metabolic pathways as energy and carbon sources (16).
P. gingivalis has an absolute requirement for iron in its growth and proliferation within a host (12). However, unlike other Gram-negative bacterial species, where siderophores are used for the sequestering and transport of iron, gingipains are used to achieve that goal instead. Studies on Kgp and an arginine-specific gingipain R1 (HRgpA) have shown that they are responsible for the agglutination of red blood cells and degradation of haemoglobin, allowing for heme acquisition by P. gingivalis. Active transport by heme/haemoglobin receptors would bring the heme into the cytoplasm where it is utilised for growth. Surplus heme would be stored on the cellular surface of P. gingivalis, in the form of µ-oxo dimers of heme, which gives the bacterium the characteristic black pigmentation (17).
Ecology & Pathology
P. gingivalis is an obligate anaerobe, with its major habitat being the subgingival sulcus of the human oral cavity (10). As a late coloniser of the oral cavity, its colonisation is assisted, rather than inhibited, by the salivary flow, as a vector for transmission. Salivary pellicle-coated surfaces within the oral cavity provide anchors at which P. gingivalis fimbriae would attach to. Additionally, primary colonisers like facultative anaerobic streptococci would provide additional anchoring points and initial protection from the aerobic environment through reduction of oxidative tension. These mechanisms would allow for the establishment of a foothold within the human oral cavity by P. gingivalis (8).
In order to be a successful pathogen, P. gingivalis has a myriad of virulence factors at its disposal to colonise and cause disease in the human host. Such virulence factors include fimbriae, capsules, lipopolysaccharides (LPS) and gingipains (10). It was reported that the encapsulation of the bacterial cells may lead to higher resistance to host immune responses and allow for high adhesion to gingival epithelial cells by working hand-in-hand with the fimbriae. Indeed, it has been suggested that the more virulent strains are often encapsulated, in comparison to the less virulent strains, which are often non-encapsulated (19). Interestingly, LPS of P. gingivalis has not been documented to be as readily identified by the host immune system, as compared to other Gram-negative bacteria like Escherichia coli. It may be possible that the release of LPS does not have a direct effect on stimulating inflammatory responses, but rather it inhibits the secretion of the chemokine, interleukin-8 (IL-8), which is responsible for the activation of neutrophils, eosinophils and basophils. Thus, chemokine activation is suppressed, leading to a higher survival and proliferation rate of the other periodontal bacteria, which would result in pathogenesis of periodontal disease (10).
Perhaps the most interesting of the virulence factors are the gingipains of P. gingivalis. These gingipains have a myriad of functions, ranging from the cleavage of amino acids to provide nutrients for growth (10) and being involved in mixed-species biofilm formation (2) to the subversion and evasion of host immune responses. Degradation of fibrinogen and host heme proteins by the gingipains would lead to increased bleeding while inhibiting the coagulation of blood in the region, which increases the availability of iron and heme for growth of periodontal bacteria. At the same time, the gingipains constitute a defensive capability of P. gingivalis by actively degrading host antibacterial peptides, interrupting communication between immune receptors such as CD5a and toll-like receptors (TLRs) and imposing a regulation on the inflammatory response by controlling the secretion of various chemokines. As such, the gingipains afford an increased virulence of P. gingivalis by increasing the survivability of it and the surrounding microbiota during harsh host immune responses and generating a large nutritional availability for the whole microbiome to grow (10).
Other than the oral cavity, P. gingivalis has also been documented to be in the vagina and associated with the pathogenesis of bacterial vaginosis (1). Alongside other ‘red-complex’ bacteria, P. gingivalis has also been implicated in the pathogenesis of some systemic diseases, such as atherosclerotic cardiovascular disorder, decreased kidney function and rheumatoid arthritis (16).
Using a mice model, a study has found that P. gingivalis can cause cardiac rupture after myocardial infraction through the action of its gingipains. Invasion and subsequent infection of the ischemic myocardium stimulated the expression of p18 Bax, which was activated by the gingipains and promoted apoptosis of the cardiomyocytes. The release of LPS by P. gingivalis during infection increased the oxidative stress in the infracted myocardium through the activation of matrix metalloproteinase-9 (MMP-9), thus leading to the promotion of rupture in the left ventricle (18).
In another study involving mice models, it was found that P. gingivalis, in association with F. nucleatum, by causing chronic oral infections, can promote the formation of oral squamous cell carcinoma (OSCC). It was suggested experimentally that the exposure of oral epithelial cells to P. gingivalis/F. nucleatum will trigger the activation of the signalling pathway in TLRs, leading to the stimulation of interleukin-6 (IL-6) production, which would activate STAT3, which is prominent transcription factor of many genes and plays a key role in the tumour-promoting pathways of oncogenesis. STAT3 would go on to activate other important effectors of tumorigenesis, such as cyclin D1 and MMP-9, leading to the onset of OSCC (7).
The gingipains of P. gingivalis has been implicated as key virulence factors in the pathogenesis of periodontal disease. As such, the inhibition of these proteinases could have desirable therapeutic effects. In a recent study, a research team has concluded that prenyl flavonoids, which were extracted from the Epimedium species of plants, inhibited the gingipains, leading to a suppression of the growth and biofilm formation by P. gingivalis. Thus, if the mechanisms of inhibition could be improved and understood further, this could have widespread implications and advantages for the development of a synthetic prenyl flavonoid as a therapeutic agent against P. gingivalis (11).
Bacterial colonisation has led to many post-operative complications of dentoalveolar surgery. This is mostly due to periodontal bacteria like P. gingivalis adhering and forming biofilms on the sutures used during the surgical procedures, resulting in the sutures becoming a makeshift portal of entry into the surgical wounds. A recent study looked into the effectiveness of coating surgical sutures with a novel quaternary ammonium compound (QAC), K21, on preventing P. gingivalis colonisation. This was done through the measurement of zone of inhibition on a P. gingivalis culture plate. The results showed that the growth of P. gingivalis was inhibited by K21 at concentrations from 5% to 25% (13). This study is important as it shows the antimicrobial capability of a compound, without the use of antibiotics, whose widespread use has led to the frightening increase in number of antibiotic resistant strains of bacteria.
1) Africa, C.W., Nel, J. and Stemmet, M., 2014. Anaerobes and bacterial vaginosis in pregnancy: virulence factors contributing to vaginal colonisation. International journal of environmental research and public health, 11(7), pp.6979-7000.
2) Bao, K., Belibasakis, G.N., Thurnheer, T., Aduse-Opoku, J., Curtis, M.A. and Bostanci, N., 2014. Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation. BMC microbiology, 14(1), p.258.
3) Boutaga, K., van Winkelhoff, A.J., Vandenbroucke-Grauls, C.M. and Savelkoul, P.H., 2003. Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples. Journal of clinical microbiology, 41(11), pp.4950-4954.
4) Condorelli, F., Scalia, G., Calì, G., Rossetti, B., Nicoletti, G. and Bue, A.M.L., 1998. Isolation of Porphyromonas gingivalisand Detection of Immunoglobulin A Specific to Fimbrial Antigen in Gingival Crevicular Fluid. Journal of clinical microbiology, 36(8), pp.2322-2325.
5) Cook, G.S., Costerton, J.W. and Lamont, R.J., 1998. Biofilm formation by Porphyromonas gingivalis and Streptococcus gordonii. Journal of periodontal research, 33(3), pp.323-327.
6) Curtis, M.A., Zenobia, C. and Darveau, R.P., 2011. The relationship of the oral microbiotia to periodontal health and disease. Cell host & microbe, 10(4), pp.302-306.
7) Gallimidi, A.B., Fischman, S., Revach, B., Bulvik, R., Maliutina, A., Rubinstein, A.M., Nussbaum, G. and Elkin, M., 2015. Periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum promote tumor progression in an oral-specific chemical carcinogenesis model. Oncotarget, 6(26), pp.22613-22623.
8) Hajishengallis, G., 2009. Porphyromonas gingivalis–host interactions: open war or intelligent guerilla tactics?. Microbes and Infection, 11(6), pp.637-645.
9) Hajishengallis, G., Liang, S., Payne, M.A., Hashim, A., Jotwani, R., Eskan, M.A., McIntosh, M.L., Alsam, A., Kirkwood, K.L., Lambris, J.D. and Darveau, R.P., 2011. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell host & microbe, 10(5), pp.497-506.
10) How, K.Y., Song, K.P. and Chan, K.G., 2016. Porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. Frontiers in microbiology, 7.
11) Kariu, T., Nakao, R., Ikeda, T., Nakashima, K., Potempa, J. and Imamura, T., 2016. Inhibition of gingipains and Porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids. Journal of periodontal research.
12) Lamont, R.J. and Jenkinson, H.F., 1998. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiology and Molecular Biology Reviews, 62(4), pp.1244-1263.
13) Meghil, M.M., Rueggeberg, F., El-Awady, A., Miles, B., Tay, F., Pashley, D. and Cutler, C.W., 2015. Novel coating of surgical suture confers antimicrobial activity against Porphyromonas gingivalis and Enterococcus faecalis. Journal of periodontology, 86(6), pp.788-794.
14) Mohammed, M.M.A., Nerland, A.H., Al-Haroni, M. and Bakken, V., 2013. Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K. Journal of oral microbiology, 5.
15) Naito, M., Hirakawa, H., Yamashita, A., Ohara, N., Shoji, M., Yukitake, H., Nakayama, K., Toh, H., Yoshimura, F., Kuhara, S. and Hattori, M., 2008. Determination of the genome sequence of Porphyromonas gingivalis strain ATCC 33277 and genomic comparison with strain W83 revealed extensive genome rearrangements in P. gingivalis. DNA research, 15(4), pp.215-225.
16) Nemoto, T.K. and Ohara-Nemoto, Y., 2016. Exopeptidases and gingipains in Porphyromonas gingivalis as prerequisites for its amino acid metabolism. Japanese Dental Science Review, 52(1), pp.22-29.
17) Olczak, T., Simpson, W., Liu, X. and Genco, C.A., 2005. Iron and heme utilization in Porphyromonas gingivalis. FEMS microbiology reviews, 29(1), pp.119-144.
18) Shiheido, Y., Maejima, Y., Suzuki, J.I., Aoyama, N., Kaneko, M., Watanabe, R., Sakamaki, Y., Wakayama, K., Ikeda, Y., Akazawa, H. and Ichinose, S., 2016. Porphyromonas gingivalis, a periodontal pathogen, enhances myocardial vulnerability, thereby promoting post-infarct cardiac rupture. Journal of molecular and cellular cardiology.
19) Singh, A., Wyant, T., Anaya-Bergman, C., Aduse-Opoku, J., Brunner, J., Laine, M.L., Curtis, M.A. and Lewis, J.P., 2011. The capsule of Porphyromonas gingivalis leads to a reduction in the host inflammatory response, evasion of phagocytosis, and increase in virulence. Infection and immunity, 79(11), pp.4533-4542. (16)
This page is written by Daniel Zi En Chew for the MICR3004 course, Semester 2, 2016