Characterization of specific inhibitors of Porphyromonas gingivalis gingipains based on their cognate propeptides
AuthorMohammad Mahmud, Abu Sayeed
AffiliationMelbourne Dental School
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-09-09.
© 2019 Abu Sayeed Mohammad Mahmud
Porphyromonas gingivalis is a pathogenic bacterium that has a significant role in the progression of chronic periodontal disease. The cell-surface proteases RgpA, RgpB and Kgp collectively known as the gingipains, are major virulence factors of P. gingivalis. Furthermore, P. gingivalis is unable to metabolize sugars for energy production and relies on the gingipains to degrade host proteins to produce small peptides which it uses for nutrition. Thus, strategies that inhibit gingipain function may be of use in reducing the incidence and severity of periodontitis. The Arg-gingipains, RgpA and RgpB and Lys-gingipain Kgp are secreted from P. gingivalis as inactive prodomain-bearing precursors. The amino-terminal propeptides render the proteases inactive until they are cleaved away from the protease at the cell surface. Previously it was demonstrated that when added exogenously recombinant forms of the propeptides of RgpA and RgpB were able to effectively inhibit purified mature high molecular weight RgpA (HRgpA) and RgpB whereas the recombinant Kgp propeptide was a relatively poor inhibitor of Kgp. This present study aimed to examine the inhibitory effect of recombinant gingipain propeptides on the activities of gingipains of P. gingivalis strains isolated from global locales. Furthermore, the effect of the recombinant gingipain propeptides on P. gingivalis growth in biofilm, a clinically relevant state was also examined. The previously developed gingipain protease assays were improved by the addition of optimized concentrations of glycylglycine and cysteine to increase the sensitivity. The assays were then applied to measure the Arg- and Lys-gingipain activities of whole cells and vesicle free culture supernatants (VFSN) of 14 strains of P. gingivalis. It was found that P. gingivalis clinical isolates exhibited different rates of gingipain activities with Vmax of whole cell Arg-gingipains varying 5.8-fold and whole cell Lys-gingipain Vmax varying 2.1-fold across the strains. The P. gingivalis strains also showed more than 100-fold variance in Arg-gingipain activity in the VFSN and more than 350-fold variance in Lys-gingipain activity in the VFSN. Thus, the global P. gingivalis isolates manifest varied Arg- and Lys-gingipain activities. Recombinant RgpA, RgpB, and Kgp propeptides were produced in Escherichia coli (rRgpA-PP, rRgpB-PP, rKgpS16-PP) and added to the gingipain activity assays to determine the inhibitory effects. Using rRgpA-PP, the 50% inhibitory concentration (IC50) for Rgp activity inhibition across the strains ranged from 66 to 227 nM at 95% confidence interval (CI) of 61 to 234 nM. The rRgpA-PP at 670 nM inhibited 87 to 98% Arg- gingipain activity of the P. gingivalis strains except for P. gingivalis 381 that was inhibited 77%. The range of IC50 values of rRgpB-PP against whole cell Arg-gingipain activity were higher than those of rRgpA-PP, with IC50 from 0.69 to 1.8 µM at 95% CI of 0.62 to 1.9 µM, a 5.9 to 11-fold difference depending on the strain. The rRgpB-PP applied at 4.8 µM inhibited 87 to 99% of Arg-gingipain activity of the P. gingivalis, except for strain 381, which was inhibited 76%. Thus, both rRgpA-PP and rRgpB-PP are effective at inhibiting Arg-gingipain activities of the P. gingivalis isolates, but rRgpA-PP is a more effective inhibitor than rRgpB-PP. The inhibitory potential of rKgpS16-PP against whole-cell Kgp activity was minimal, reaching at most 17% against strain W50 when applied at 40 µM. Both rRgpA-PP and rRgpB-PP exhibited a non-competitive type of whole-cell Arg-gingipain inhibition. It was also determined that both rRgpA-PP and rRgpB-PP stored in solution at room temperature for 16 months retained 69% and 64% of inhibitory activity respectively. To discriminate the activities of rRgpA-PP and rRgpB-PP toward the whole cell RgpA or RgpB, rgpA gene mutants of P. gingivalis designated W501 (strain W50 parent) and ECR833 (strain ATCC 33277 parent) and the rgpB gene mutants W50B (W50) and ECR834 (ATCC 33277) were used. Similar Vmax were calculated for all mutants, which were approximately 50% of the activity of parent strains. Propeptide-mediated protease inhibition assay showed that rRgpA-PP and rRgpB-PP could each inhibit both RgpA and RgpB on whole cells. However, rRgpB-PP was 7.8 to 12.5-fold less effective than rRgpA-PP at inhibition of whole-cell Arg-gingipain activity. A method for production of a functional 6 x histidine-tagged RgpA proteinase domain, rRgpAH, was successfully developed and the protein isolated. The kinetics of purified rRgpAH and RgpB were compared. The calculated Km of RgpB for benzoyl-arginine 4-nitroanilide (BApNA) substrate was ~1.4-fold lower than the rRgpAH Km for BApNA, whereas the Vmax of rRgpAH was 2.9-fold higher than the RgpB Vmax. The calculated Ki for RgpA-PP against 5 nM each of rRgpAH and RgpB were similar at 13 nM and 15 nM respectively. The rRgpB-PP Ki at 22 nM and 29 nM against rRgpAH and RgpB respectively was significantly higher (p<0.0001) than the rRgpA-PP Ki. Although rKgpS16-PP was a weak inhibitor of whole-cell Kgp 25% inhibition of 10 nM of purified rKgp with a Ki of 37.5 µM was determined. Biofilm forming capacity of the different P. gingivalis clinical strains was compared following growth in microtiter plates, then measuring biofilm by staining the biomass with crystal violet. It was found that each strain of P. gingivalis produced a different amount of monotypic biomass that could be crystal violet stained. Non-capsular strains 381 and ATCC 33277 were found to produce thick and strongly adherent biofilm biomass. Strains 381, ATCC 33277, 13-1, 15-9, and 7BTORR were used for the biofilm inhibition model, in which increasing concentrations of propeptide were added to the initial bacteria inoculum when seeding the wells for biofilm production. In a dose-response manner, each propeptide inhibited biofilm formation by P. gingivalis with IC50 values for both rRgpA-PP and rRgpB-PP in the low micromolar range, with 95% CI of 1.2-5.2 µM. Most significantly, at the same concentrations, the propeptides also disrupted established biofilm. The rRgpA-PP and rRgpB-PP at 8 and 14 µM, respectively inhibited 99% of biofilm biomass production by P. gingivalis strains ATCC 33277, 381, and 13-1 and 94% of biofilm biomass production by strains 15-9 and 7B TORR. To determine the importance of Arg- and Lys-gingipains in the biofilm formation of P. gingivalis, mutants ECR833 (RgpA null), ECR834 (RgpB null), ECR835 (RgpA/B null) and ABK (RgpA/B and Kgp null) P. gingivalis ATCC 33277 mutants were compared. ECR835 (RgpA/B null) formed significantly higher biofilm biomass than ECR834 (RgpB null) and parent strain ATCC 33277 (P <0.05). The P. gingivalis ABK mutant formed a poorly adherent biofilm with less biomass than the parent ATCC 33277 (P<0.0001). Thus, the absence of RgpA and RgpB resulted in increased biofilm formation by P. gingivalis ATCC 33277, but this effect reversed if Kgp was also absent. The IC50 of rRgpA-PP and rRgpB-PP against P. gingivalis RgpA and RgpB and RgpA/B mutants were found to be higher than the parent strain (P<0.05). However, the IC50 of rRgpA and rRgpB propeptide against the ABK mutant was approximately 6.4 and 8-fold respectively lower than the parent strain. Biofilm forming capabilities of other oral pathogens, Treponema denticola, and Tannerella forsythia, alone and in combination with P. gingivalis ATCC 33277 was assessed. The P. gingivalis ATCC 33277, T. denticola, and T. forsythia cohort formed substantially more biofilm with approximately 39% more biomass than the cumulative biomass of the individual biofilms (P <0.0001). The IC50 against the polymicrobial biofilm for rRgpA-PP was 4.8 ± 0.6 µM and 4.2 ± 0.2 µM for rRgpB-PP. As monospecies, it was found that both T. denticola and T. forsythia formed less biofilm in the in vitro model than P. gingivalis. Nether rRgpA-PP nor rRgpB-PP inhibited biofilm formation by T. denticola. However, unexpectedly, the propeptides did inhibit T. forsythia biofilm formation by 55%. To determine if the propeptides could inhibit biofilm formation by other species rRgpA-PP and rRgpB-PP were applied against Streptococcus sanguinis, E. coli, Chryseobacterium indologenes, and Fusobacterium nucleatum. F. nucleatum biofilm formation was completely inhibited by rRgpA-PP at 12 µM and by rRgpB-PP at 14 µM. However, neither propeptide had any inhibitory effect on E. coli, S. sanguinis or C. indologenes biofilm indicating that the effect of the propeptides is species specific. Analysis of the planktonic phase of the in vitro biofilm model revealed that the growth of P. gingivalis, F. nucleatum, and T. forsythia was inhibited by propeptides, which explained the reduction of biofilm biomass. The spreading of the planktonic phase from the assay wells onto agar plates proved that the propeptide antimicrobial activity was bactericidal. Thus, this study suggests that rRgpA-PP and rRgpB-PP are proteins that have selective antimicrobial activity. The rKgp16S-PP exerted no antimicrobial effect. Porphyromonas gulae is closely related to P. gingivalis, produces Arg-and Lys-gingipains, and is implicated in the etiology of periodontal diseases in companion animals. The kinetics of P. gulae whole-cell and VFSN Arg- and Lys-gingipains were found to be similar to the P. gingivalis 84-3 strain. It was found that rRgpA-PP and rRgpB-PP that were designed using P. gingivalis strain W50 sequences were more effective at inhibiting P. gulae gingipain activity and growth than they were at inhibiting P. gingivalis Arg-gingipain activity and growth. However, rKgp16S-PP did not inhibit P. gulae Lys-gingipain activity. In conclusion, recombinant Arg-gingipain protease propeptides rRgpA-PP and rRgpB-PP are effective at inhibiting Arg-gingipain activities of global P. gingivalis isolates; however, the recombinant Lys-gingipain propeptide was not an effective inhibitor of P. gingivalis Lys-gingipains. Antimicrobial activity of rRgpA-PP and rRgpB-PP was displayed against P. gingivalis and other species with inhibition of planktonic and biofilm growth, but this was not ubiquitous, indicating the mechanism of propeptide antimicrobial action is species specific. The recombinant Rgp propeptides were shown to be stable to room temperature storage. Together the data show that recombinant Rgp propeptides may have the potential for development as inhibitors that impact initiation or progression of periodontitis.
KeywordsP. gingivalis; gingipain; propeptides; inhibition, kinetics; biofilm; P. gulae; antimicrobials; proteases; periodontitis; mutants; IC50
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