PPAD Activity Promotes Outer Membrane Vesicle Biogenesis and Surface Translocation by Porphyromonas gingivalis

Many bacteria switch between a sessile and a motile mode in response to environmental and host-related signals. Porphyromonas gingivalis, an oral anaerobe implicated in the etiology of chronic periodontal disease, has long been described as a non-motile bacterium. Yet, recent studies have shown that under certain conditions, P. gingivalis is capable of surface translocation. Considering these findings, this work aimed to increase our understanding as to how P. gingivalis transitions between sessile growth and surface migration. Here we show that the peptidylarginine deiminase secreted by P. gingivalis (PPAD), an enzyme previously shown to be upregulated during surface translocation and to constrain biofilm formation, promotes surface translocation. In the absence of PPAD, the production of outer membrane vesicles (OMVs) was drastically reduced. In turn, there was a reduction in gingipain-mediated proteolysis and a reduced zone of hydration around the site of inoculation. RNA-Seq and metabolomics analyses also showed that these changes corresponded to a shift in arginine metabolism. Overall, this study provides new evidence for the functional relevance of PPAD and proteases, as well as the importance of PPAD activity in OMV biogenesis and release. Our findings support the model that citrullination is a critical mechanism during lifestyle transition between surface-attached growth and surface translocation by modulating OMV-mediated proteolysis and arginine metabolism. IMPORTANCE Gram-negative bacteria produce nanosized OMVs that are actively released into their surroundings. The oral anaerobe P. gingivalis is prolific in OMV production and many of the proteins packaged in these vesicles are proteolytic or protein modifying enzymes. This includes key virulence determinants, such as the gingipains and PPAD (a unique peptidylarginine deiminase). Here, we show that PPAD activity (citrullination) is involved in OMV biogenesis. The study reveals an unusual mechanism that allows this bacterium to transform its surroundings. Since OMVs are detected in circulation and in systemic tissues, our study also supports the notion that PPAD activity may be a key factor in the correlation between periodontitis and systemic diseases further supporting PPAD as an important therapeutic target.

Cell-surface translocation of annexin A2 contributes to bleomycin-induced pulmonary fibrosis by mediating inflammatory response in mice

Bleomycin, a widely used anti-cancer drug, may give rise to pulmonary fibrosis, a serious side effect which is associated with significant morbidity and mortality. Despite the intensive efforts, the precise pathogenic mechanisms of pulmonary fibrosis still remain to be clarified. Our previous study showed that bleomycin bound directly to annexin A2 (ANXA2, or p36), leading to development of pulmonary fibrosis by impeding transcription factor EB (TFEB)-induced autophagic flux. Here, we demonstrated that ANXA2 also played a critical role in bleomycin-induced inflammation, which represents another major cause of bleomycin-induced pulmonary fibrosis. We found that bleomycin could induce the cell surface translocation of ANXA2 in lung epithelial cells through exosomal secretion, associated with enhanced interaction between ANXA2 and p11. Knockdown of ANXA2 or blocking membrane ANXA2 mitigated bleomycin-induced activation of nuclear factor (NF)-κB pathway and production of pro-inflammatory cytokine IL-6 in lung epithelial cells. ANXA2-deficient (ANXA2−/−) mice treated with bleomycin exhibit reduced pulmonary fibrosis along with decreased cytokine production compared with bleomycin-challenged wild-type mice. Further, the surface ANXA2 inhibitor TM601 could ameliorate fibrotic and inflammatory response in bleomycin-treated mice. Taken together, our results indicated that, in addition to disturbing autophagic flux, ANXA2 can contribute to bleomycin-induced pulmonary fibrosis by mediating inflammatory response.

EPAC1 regulates endothelial Annexin A2 cell surface translocation and plasminogen activation

ABSTRACTAnnexins, a family of highly conserved calcium-and phospholipid-binding proteins, play important roles in a wide range of physiological functions. Among the twelve known annexins in human, Annexin A2 (AnxA2) is one of the most extensively studied and has been implicated in various human diseases. AnxA2 can exist as a monomer or a heterotetrameric complex with S100A10 (P11) and plays a critical role in many cellular processes including exocytosis/endocytosis and membrane organization. At the endothelial cell surface, (AnxA2•P11)2 tetramer, acting as a coreceptor for plasminogen and tissue plasminogen activator (t-PA), accelerates t-PA dependent activation of the fibrinolytic protease, plasmin, the enzyme responsible for thrombus dissolution and degradation of fibrin. This study shows that exchange proteins directly activated by cAMP isoform 1 (EPAC1) interacts with AnxA2 and regulates its biological functions by modulating its membrane translocation in endothelial cells. Using genetic and pharmacological approaches, it is demonstrated that EPAC1, acting through the PLCε-PKC pathway, inhibits AnxA2 surface translocation and plasminogen activation. These results suggest that EPAC1 plays a role in the regulation of fibrinolysis in endothelial cells and may represent a novel therapeutic target for disorders of fibrinolysis.