Characterization of the Porphyromonas gingivalis protein PG1881 and its roles in outer membrane vesicle biogenesis and biofilm formation

© 2016 Dr. May Ju GuiPorphyromonas gingivalis is a non-motile, Gram-negative coccobacillus, oral anaerobe that is implicated in chronic periodontitis, a polymicrobial inflammatory disease resulting in the destruction of the supporting tissues of the teeth. P. gingivalis OMVs are an important vehicle for secretion of virulence factors, nutrient acquisition, biofilm development and pathogenesis. PG1881 was first identified to be preferentially sorted and enriched to the OMV membrane, and thus became the focus of this study. The purpose of this thesis was to characterize PG1881 protein and its potential roles in P. gingivalis virulence particularly in OMV biogenesis and biofilm formation. This study determined that pg1881 was transcribed as part of a four gene operon with pg1878-pg1880 in P. gingivalis strain W50. PG1881 was bioinformatically identified as a Type V pilin capable of forming filament-like structures and is exclusively conserved in limited families of the Bacteroidales order. PG1881 shares a similar overall structure to FimA and Mfa1 and possesses the essential conserved motifs in the N- and C-terminal regions. Similar to FimA and Mfa1, PG1881 undergoes a step-wise maturation process including maturation by proteolytic cleavage by the gingipains. PG1881 polymerisation is mediated by non-covalent interactions likely to be utilized via a donor stand mechanism. Phenotypic characterization of a P. gingivalis W50 mutant strain, ΔPG1881 lacking PG1881 revealed no significant difference in ΔPG1881 growth, whole cell morphology, SDS sensitivity, autoaggregation activity and cellular arginine-specific proteinase activity as compared to the parent strain W50. Although proteomic analyses of ΔPG1881 whole cells revealed minimal altered protein expression, ΔPG1881 displayed a significant decrease in OMV production as compared to W50. ΔPG1881 OMVs showed a significant decrease in the abundance of proteinases (including RgpA), consistent with the significant reduction in ΔPG1881 OMV arginine-specific proteinase activity in comparison to W50 OMVs. ΔPG1881 OMVs were also more enriched in TonB-dependent receptors. ΔPG1881 OMVs contained greater amount of nucleic acids on the surface and in the lumen than W50 OMVs. These results suggest that PG1881 may play a role in the vesiculation mechanism(s) of P. gingivalis given that OMVs originate from the bulging of bacterial OM, thus substantiating the correlation between pilins and vesiculation. PG1881 protein expression was increased in acidic pH conditions suggesting a role of PG1881 in P. gingivalis adaptability in response to pH changes. Using a flow cell model, PG1881 was shown to play a suppressive regulatory role in mono-species biofilm formation. PG1881 expression was also crucial for mutualistic biofilm formation with T. denticola as determined using a static biofilm model. These data suggest that PG1881 may play a role in response to environmental cues and for polymicrobial interaction with other oral bacteria such as T. denticola. Collectively, this study has identified a novel Type V pilin which has been designated as filament-forming protein 1, Ffp1 that affects P. gingivalis vesiculation and biofilm formation and extends the current knowledge concerning P. gingivalis pili. Further investigation may lead to greater insights in host-microbe interactions and the pathogenicity of P. gingvalis in periodontitis disease progression