Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated Peptides

Larsen, Daniel Nyberg; Mikkelsen, Christian Engelbrecht; Kierkegaard, Mads; Bereta, Grzegorz; Nowakowska, Zuzanna; Kaczmarek, Jakub Zbigniew; Potempa, Jan; Højrup, Peter

doi:10.1074/mcp.ra119.001700

Cited by 21 publications

(35 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recent publications have shown that proteins carried on OMVs produced by P. gingivalis strain W83 can be citrullinated by PPAD and this included protein PG1881 (20). The data presented in this study demonstrate that when P. gingivalis cannot synthesize SLs, it was proficient in OMV biogenesis, yet the PG1881 protein is absent (or at low abundance) in the OMVs.…”

Section: Discussionmentioning

confidence: 49%

Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis

et al. 2021

View full text Add to dashboard Cite

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells, and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyper-inflammatory immune response from THP-1 macrophage-like cells with elevated TNF- α, IL-1β, and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF by SL-null P. gingivalis. No significant differences in gingipain activity were observed in our infection models and both strains produced OMVs of similar size. Using comparative 2-dimensional gel electrophoresis we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.

show abstract

Section: Discussionmentioning

confidence: 49%

Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis

et al. 2021

View full text Add to dashboard Cite

show abstract

“…gingivalis OMVs can be citrullinated (21). The data presented in this study show that when strain 381 cannot citrullinate peptides and/or proteins (Δppad mutant), the strain is deficient in OMV and biopearling biogenesis (Fig.…”

Section: Discussionmentioning

confidence: 72%

“…7b). Taken together, these findings show that 381Δppad, which is unable to citrullinate substrates and its own proteins, demonstrates reduced proteolytic activity, which in turn makes (11,21). Given our findings that 381Δppad is deficient in the rate of proteolysis and forms a limited zone of hydration during the initial stage of surface translocation, we hypothesized that the secretion of T9SS cargo proteins via OMVs is impacted in the absence of PPAD.…”

Section: Downloaded Frommentioning

confidence: 68%

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

et al. 2021

View full text Add to dashboard Cite

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.

show abstract

“…It was indicated in our previous study that the activity of citrullinating bacterial virulence factor-PPAD-had a certain importance during the formation of dual-species bacterial-fungal biofilm [16]. The modifications that PPAD introduces to diverse molecules play a role not only in relation to the functionality of bacterial proteins, and modulation of biofilm development, but also in the interactions with host proteins and systems [25,[66][67][68]. Under the conditions used in this study citrullination of such C. albicans proteins as glucan 1,3-beta-glucosidase Bgl2, elongation factor 1-alpha 1, Tdh3, heat shock proteins Ssa1 and Ssa2, yeast-form wall protein 1, plasma membrane ATPase 1, agglutinin-like sequence protein 3, 1,3-beta-glucanosyltransferase Pga4 and pH-responsive protein 1 was newly identified, whereas Eno1, Mp65, Adh1 and Eng1 were previously identified to be citrullinated [16].…”

Section: Discussionmentioning

confidence: 99%

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen

Karkowska‐Kuleta

Surowiec

Gogól

et al. 2020

IJMS

Self Cite

View full text Add to dashboard Cite

Microorganisms that create mixed-species biofilms in the human oral cavity include, among others, the opportunistic fungus Candida albicans and the key bacterial pathogen in periodontitis, Porphyromonas gingivalis. Both species use arsenals of virulence factors to invade the host organism and evade its immune system including peptidylarginine deiminase that citrullinates microbial and host proteins, altering their function. We assessed the effects of this modification on the interactions between the C. albicans cell surface and human plasminogen and kininogen, key components of plasma proteolytic cascades related to the maintenance of hemostasis and innate immunity. Mass spectrometry was used to identify protein citrullination, and microplate tests to quantify the binding of modified plasminogen and kininogen to C. albicans cells. Competitive radioreceptor assays tested the affinity of citrullinated kinins to their specific cellular receptors. The citrullination of surface-exposed fungal proteins reduced the level of unmodified plasminogen binding but did not affect unmodified kininogen binding. However, the modification of human proteins did not disrupt their adsorption to the unmodified fungal cells. In contrast, the citrullination of kinins exerted a significant impact on their interactions with cellular receptors reducing their affinity and thus affecting the role of kinin peptides in the development of inflammation.

show abstract

Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated Peptides

Cited by 21 publications

References 60 publications

Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis

Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis

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

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen

Contact Info

Product

Resources

About