University of Groningen

Porphyromonas gingivalis, the beast with two headsGabarrini, Giorgio

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Citation for published version (APA):Gabarrini, G. (2018). Porphyromonas gingivalis, the beast with two heads: A bacterial role in the etiology ofrheumatoid arthritis. [Groningen]: University of Groningen.

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Chapter 1

General introduction and scope of

the thesis

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Porphyromonas gingivalis, the beast with two heads According to Greek mythology, Orthrus was a two-headed dog, brother of the infamous Cerberus and guardian of Geryon’s cattle. Not much is told about this fictional monster except for its demise, which occurred at the hands of Heracles, during his tenth labor. Equally unknown to the general public is the story of another, sadly very real, beast with two (more metaphorical) heads: Porphyromonas gingivalis. P. gingivalis (Fig. 1) is a Gram-negative, strictly anaerobic, bacterium belonging to the Bacteroidetes phylum1. Discovered in the second half of the 1900s and initially characterized under the name Bacteroides gingivalis, this bacterium started to garner interest in the following years thanks to its increasingly clear role in the widely spread inflammatory disease periodontitis1-3. It was only recently, however, that the notoriety of P. gingivalis crossed the boundaries of the oral microbiology field and reached the seemingly unrelated field of rheumatology. This shift in focus was due to the alleged involvement of this bacterium in the etiopathogenesis of the autoimmune disease rheumatoid arthritis (RA), its second “head”. More specifically, this modern day Orthrus has been regarded as the major causative agent of periodontitis due to its presence in 85% of the severe cases of this oral disease4. This alone categorizes P. gingivalis as a foremost medical concern and an enormous burden on the global health expenditure5. Periodontitis, with its incidence in the human population of ~11% is one of the most common disorders in the world5-7. The pathogenesis of this disease comprises the triggering of immune responses following microbial infection, leading to the activation of a cascade of cytokines, which will, in turn, cause the destruction of the soft and hard tissue surrounding the tooth7. Erosion of these tissues, called periodontium, leaves the tooth unprotected and may cause the patient to become edentulous. Periodontitis is, in fact, the foremost cause of tooth loss5. This, coupled with the higher incidence of the disease among elderly patients, renders periodontitis one of the main problems in the context of 'healthy ageing', the multi-disciplinary initiative tasked with easing the burdens and improving the quality of life of the ageing population. Perhaps due to its inflammatory nature, periodontitis has been linked to a plethora of diseases, especially autoimmune disorders. The most renowned association involving periodontitis and P. gingivalis is with the autoimmune disease rheumatoid arthritis8-12. RA is an

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ailment of heretofore unknown etiology, whose pathogenesis strongly resembles that of periodontitis5. RA, in fact, results in the destruction of the tissue surrounding the synovial joints, leading to a severe loss in mobility. Contrary to periodontitis, rheumatoid arthritis affects only ~1% of the human population, a percentage that, albeit much lower than the incidence of periodontitis, still translates to a significant number of patients, when compared to more commonly known diseases13. Additionally, due to the higher prevalence of this disorder among the elder population and the cumbersomeness of its symptoms, RA is almost emblematic of the problems faced in the battle for healthy ageing.

Figure 1. Electron micrograph of Porphyromonas gingivalis W83.

Since gaining the first pieces of empirical evidence on the correlation between periodontitis and rheumatoid arthritis, scientists searched for the mechanisms and the lynchpin behind this association. Only recently, though, these inquiries were quelled, thanks to the discovery of the citrullinating enzyme of P. gingivalis10. Citrullinating enzymes, called peptidylarginine deiminases (PADs), are responsible for several important physiological processes in mammals, including inflammatory immune responses and apoptosis, which explains their

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high level of conservation14, 15. Interestingly, these enzymes were never encountered in prokaryotes before the discovery of P. gingivalis’ PAD (PPAD) and PPAD itself was erstwhile thought to be the only instance of prokaryotic PAD16-18, before one of the studies reported in this thesis (Chapter 6). Indeed, PPAD is evolutionarily completely unrelated to mammalian PADs, but it shares with these enzymes the citrullinating function, albeit with different specificities19. As confirmed in this thesis, the PPAD enzyme, whose importance is underlined by the extreme level of conservation within the species18, possesses multiple cellular and extracellular localizations (Chapter 5). Indeed, this protein exists in different forms (Fig. 2)19.

Figure 2. Scheme of PPAD secretion and sorting in P. gingivalis and related visualization of PPAD sorting with Western blot analysis. CTD: C-terminal domain; SP: signal peptide; IM: inner membrane; OM: outer membrane; SEC: SEC pathway; PorSS: Por secretion system.

The first one is a soluble secreted form19-21, a condition that confers the protein a higher degree of 'freedom' and an easier choice of targets, at the cost of a shorter half-life. The second PPAD species is

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anchored to the outer membrane of P. gingivalis thanks to a modification with a specific lipopolysaccharide called A-LPS20, 22, supposedly gaining slightly more protection from the highly proteolytic environment of P. gingivalis but less choice of targets. The last form of PPAD is secreted with the outer membrane vesicles, nanostructures resulting from blebbings of the outer membrane of Gram-negative bacteria23-25. The evolutionary advantage of this pathway for PPAD secretion could lie in the protection of the enzyme from the outer proteolytic environment, in the protection of the bacterial targets of PPAD from non-physiological citrullination, or in the ease of delivery of the enzyme molecules to their external targets. This three-pronged localization highlights the importance of the yet unknown role played by PPAD in the survival of P. gingivalis. The real clinical importance of PPAD, though, lies in its purported involvement in the etiology of rheumatoid arthritis8, 9, 17. Albeit a complete overview of the causes of this disease is still missing, it appears that loss of tolerance toward certain citrullinated peptides may play a highly relevant role16, 26. Indeed, latest etiological models for RA depict PPAD as an alleged causative agent of RA due to its catalytic function, which might lead to the citrullination of certain

host proteins, chiefly fibrinogen and -enolase, culminating in the production of anti-citrullinated protein antibodies (ACPAs) (Fig. 3)10.

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Figure 3. Mechanistic model linking periodontitis and rheumatoid arthritis through the PPAD of P. gingivalis.

These autoantibodies have been found to be extremely specific for RA and a potential cause for this disease27, 28. For this reason, PPAD appears to represent the biomolecular lynchpin of the association between periodontitis and rheumatoid arthritis, the two metaphorical heads of Porphyromonas gingivalis. Scope of this thesis

This thesis comprises several published studies aimed at elucidating the potential role of P. gingivalis, and especially its citrullinating enzyme PPAD, in the etiopathogenesis of the autoimmune disease rheumatoid arthritis. A basic background of this topic is presented in Chapter 1, and a more in-depth introduction is offered in Chapter 2. Particularly, chapter 2 focuses on the roles and the effects of gut and oral bacteria, chiefly P. gingivalis, in the etiology of RA. All the formulated hypotheses regarding the mechanisms of action by which P. gingivalis may result in the onset of rheumatoid arthritis are, in fact, detailed in this section. Such mechanisms can be both PPAD-driven or concern the more immunological side of the threat that is P. gingivalis. Additionally, several potential microbiome-based therapies designed to counter RA are listed. Chapter 3 concludes the introductory part offering a more detailed molecular background for understanding the intricacies of the interplay between periodontitis and rheumatoid arthritis. This chapter focuses on the cellular architecture of P. gingivalis and all the known systems of transportation and secretion of proteins between one subcellular compartment to another or to the extracellular milieu. A specific attention is given to the Por secretion system, the system responsible for the export of PPAD and for its peculiar tripartite sorting, and all the known details of the mechanism behind these multiple localizations. Primarily, though, this chapter offers the predicted subcellular localizations of every protein of the three reference strains of P. gingivalis (W83, ATCC 33277, TDC60) and four clinical strains. This feat, achieved using a pipeline that integrates multiple bioinformatics subcellular localization predictors, is tailored on the bacterium P. gingivalis and renders this study a valuable tool for scientists in the field. Not only protein localizations correlate with

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protein functions, but they can also be used to ease the search of targets of a protein of interest such as PPAD. More attention is drawn to this important P. gingivalis virulence factor in Chapter 418. Indeed, this section investigates an ample panel of P. gingivalis samples derived from periodontitis patients, with or without RA, for the presence of PPAD. This study shows the extreme level of conservation of this gene, having been found in the genome of each P. gingivalis isolate of the panel18, and therefore also hints at a lack of correlation between the nucleotide sequence of PPAD and the RA phenotype of the patient from which the bacterium was isolated. The search for a PPAD feature explaining the difference between periodontitis patients with or without RA is continued in Chapter 5. This section analyzes expression of the PPAD protein in an ample panel of isolates to find differences in the sorting of this virulence factor. Specifically, the clinical isolates in this study were probed with an antibody tailored against PPAD. Due to this, the presence of the PPAD protein in outer membrane vesicles of P. gingivalis, previously hypothesized, was finally biochemically demonstrated in this study, rendering the aforementioned tripartite localization of PPAD official. Additionally, these analyses further proved the presence of this protein in every isolate investigated, consolidating the hypothesis that PPAD is a strictly conserved, and therefore probably a highly important, feature of P. gingivalis. Remarkably, in several isolates analyzed in this study and renamed “PPAD sorting type II” isolates, we discovered an anomaly in the sorting of PPAD, reducing or almost completely halting the attachment of the protein to the outer membrane. More in-depth analyses pinpointed a specific amino acid substitution (Q373K) as the potential cause of this aberrant phenotype, shaping the hypothesis that the replaced amino acid (Gln373) is paramount to the A-LPS modification resulting in PPAD's outer membrane-attachment. The trend of the extreme conservation of PPAD presence as documented in Chapters 4 and 5 is continued and concluded in Chapter 6, in which the almost two decades long dogma of the uniqueness of PPAD among prokaryotes is shattered. This section, in fact, investigates a panel of Porphyromonas species strains, isolated from a variety of animals, for the presence of PPAD homologues. The unprecedented discovery of such homologues in two other Porphyromonas species, Porphyromonas gulae and Porphyromonas loveana, has great implications. Aside from disproving the long-lived hypothesis that P. gingivalis is the only

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prokaryote capable of producing a PAD enzyme, in fact, the results discussed in this section show the high level of conservation of PPAD among the three closely related species within the Porphyromonas genus and the even higher species-specific level of conservation, giving a glimpse of the potential importance of this virulence factor. The biggest implication of the findings in this Chapter, however, lies perhaps in the exciting possibility to use the host of these Porphyromonas species as novel, better, and more apt rheumatoid arthritis models, due to the high level of conservation between PPAD and the discovered PPAD homologues. Exhausted the topic of PPAD conservation, the investigation into the molecular details of PPAD, and especially PPAD sorting, opened in Chapter 5 ends in Chapter 7. Here, PPAD’s purported anchor to the outer membrane, the A-LPS, is thoroughly investigated. The study in this Chapter, proves for PPAD what has been proposed for the proteins subject to secretion by the Por secretion system: the presence, and necessity, of an A-LPS modification in the outer membrane-bound form of the protein. Additionally, this chapter further analyzes the subset of isolates discovered in Chapter 5, the “PPAD sorting type II” isolates that displayed diminished levels or nearly complete absence of the outer membrane-bound form of the protein. The unimpeded production of A-LPS observed in sorting type II isolates and the lack of differences when compared to the A-LPS of sorting type I isolates consolidates the hypothesis formulated in Chapter 5 that the aberrant phenotype of sorting type II isolates is the direct consequence of an amino acid substitution in the PPAD protein. Lastly, Chapter 8 summarizes the results and discoveries detailed in the studies composing the previous chapters and analyzes, in light of these, the future perspectives for the field conjugating periodontitis and rheumatoid arthritis. References 1. Kaczmarek, F. S. & Coykendall, A. L. Production of phenylacetic acid by strains of Bacteroides asaccharolyticus and Bacteroides gingivalis (sp. nov.). J. Clin. Microbiol. 12, 288-290 (1980). 2. van Winkelhoff, A. J., Loos, B. G., van der Reijden, W. A. & van der Velden, U. Porphyromonas gingivalis, Bacteroides forsythus and

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other putative periodontal pathogens in subjects with and without periodontal destruction. J. Clin. Periodontol. 29, 1023-1028 (2002). 3. Bostanci, N. & Belibasakis, G. N. Porphyromonas gingivalis: an invasive and evasive opportunistic oral pathogen. FEMS Microbiol. Lett. 333, 1-9 (2012). 4. Yang, H. W., Huang, Y. F. & Chou, M. Y. Occurrence of Porphyromonas gingivalis and Tannerella forsythensis in periodontally diseased and healthy subjects. J. Periodontol. 75, 1077-1083 (2004). 5. Potempa, J., Mydel, P. & Koziel, J. The case for periodontitis in the pathogenesis of rheumatoid arthritis. Nat. Rev. Rheumatol. (2017). 6. Rylev, M. & Kilian, M. Prevalence and distribution of principal periodontal pathogens worldwide. J. Clin. Periodontol. 35, 346-361 (2008). 7. Darveau, R. P. Periodontitis: a polymicrobial disruption of host homeostasis. Nat. Rev. Microbiol. 8, 481-490 (2010). 8. de Pablo, P., Dietrich, T. & McAlindon, T. E. Association of periodontal disease and tooth loss with rheumatoid arthritis in the US population. J. Rheumatol. 35, 70-76 (2008). 9. Detert, J., Pischon, N., Burmester, G. R. & Buttgereit, F. The association between rheumatoid arthritis and periodontal disease. Arthritis Res. Ther. 12, 218 (2010). 10. de Smit, M. J., Brouwer, E., Vissink, A. & van Winkelhoff, A. J. Rheumatoid arthritis and periodontitis; a possible link via citrullination. Anaerobe 17, 196-200 (2011). 11. de Smit, M. et al. Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical, microbiological and serological study. Arthritis Res. Ther. 14, R222 (2012). 12. de Smit, M. J. et al. Effect of periodontal treatment on rheumatoid arthritis and vice versa. Ned. Tijdschr. Tandheelkd. 119, 191-197 (2012). 13. Silman, A. J. & Pearson, J. E. Epidemiology and genetics of rheumatoid arthritis. Arthritis Res. 4 Suppl 3, S265-72 (2002). 14. Chavanas, S. et al. Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6. Gene 330, 19-27 (2004). 15. Maresz, K. J. et al. Porphyromonas gingivalis facilitates the development and progression of destructive arthritis through its unique bacterial peptidylarginine deiminase (PAD). PLoS Pathog. 9, e1003627 (2013).

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16. Routsias, J. G., Goules, J. D., Goules, A., Charalampakis, G. & Pikazis, D. Autopathogenic correlation of periodontitis and rheumatoid arthritis. Rheumatology (Oxford) 50, 1189-1193 (2011). 17. Quirke, A. M. et al. Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis. Ann. Rheum. Dis. 73, 263-269 (2014). 18. Gabarrini, G. et al. The peptidylarginine deiminase gene is a conserved feature of Porphyromonas gingivalis. Sci. Rep. 5, 13936 (2015). 19. Konig, M. F., Paracha, A. S., Moni, M., Bingham, C. O.,3rd & Andrade, F. Defining the role of Porphyromonas gingivalis peptidylarginine deiminase (PPAD) in rheumatoid arthritis through the study of PPAD biology. Ann. Rheum. Dis. 74, 2054-2061 (2015). 20. Sato, K. et al. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system. FEMS Microbiol. Lett. 338, 68-76 (2013). 21. Glew, M. D. et al. PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis. J. Biol. Chem. 287, 24605-24617 (2012). 22. Shoji, M. et al. Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35. PLoS One 6, e21372 (2011). 23. Veith, P. D. et al. Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors. J. Proteome Res. 13, 2420-2432 (2014). 24. Gui, M. J., Dashper, S. G., Slakeski, N., Chen, Y. Y. & Reynolds, E. C. Spheres of influence: Porphyromonas gingivalis outer membrane vesicles. Mol. Oral Microbiol. 31, 365-378 (2016). 25. Xie, H. Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles. Future Microbiol. 10, 1517-1527 (2015). 26. Mangat, P., Wegner, N., Venables, P. J. & Potempa, J. Bacterial and human peptidylarginine deiminases: targets for inhibiting the autoimmune response in rheumatoid arthritis? Arthritis Res. Ther. 12, 209 (2010). 27. Avouac, J., Gossec, L. & Dougados, M. Diagnostic and predictive value of anti-cyclic citrullinated protein antibodies in rheumatoid arthritis: a systematic literature review. Ann. Rheum. Dis. 65, 845-851 (2006).

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28. Nishimura, K. et al. Meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann. Intern. Med. 146, 797-808 (2007).

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