IADR Abstract Archives
Filifactor Alocis Arginine Deiminase is Involved With Virulence Modulation and Host Cell Citrullination
Objectives: Filifactor alocis, an emerging periodontal pathogen has unique virulence properties. There is a gap however in our understanding of its pathogenic mechanism(s). Previously our proteome analysis of F. alocis during the invasion of epithelial cells showed increased abundance of arginine deiminase (AD). Citrullination, an AD-induced posttranslational modification (PTM) of proteins, can exacerbate the inflammatory response and regulate other host cell signaling pathways that may play a role periodontal disease. The objective of this study is to explore the role of F. alocis AD (FaAD) in virulence and host cell modulation.
Methods: Bioinformatics analysis, molecular modeling and virtual protein interaction studies were performed to evaluate the structural characteristics FaAD. Overlapping extension PCR was used to generate FaAD deletion mutants. The recombinant FaAd protein was purified from E. coli. In vitro studies were conducted using TIGK (telomerase immortalized gingival keratinocytes) cells. Western blot analysis used anti-peptidyl citrulline mouse monoclonal antibody (clone F95) and anti-citrulline antibody (clone C4). Mass spectrometry was used for the confirmation of proteins and PTMs.
Results: FaAD showed molecular (80%) and structural relatedness (70%) with AD from Porphyromonas gingivalis (PgPAD) and other oral bacteria. AD-defective mutants of F. alocis showed reduced invasion of epithelial cells and biofilm forming capacity compared to the wild type. In contrast to cell lysates from the parent strain, anti-citrulline immunoreactive bands were only detected in the FaAD-defective mutant in the presence of the rFaAD protein. Lysates from TIGK cells incubated with the purified rFaAD protein showed anti-peptidyl citrulline immunoreactive bands. The citrullination of these host cell proteins was confirmed using orbitrap mass spectrometry.
Conclusions: Collectively our data suggest that FaAD may play a role in the citrullination of pathogen and host proteins and thus may modulate virulence and host cell functions.
This work was supported by Public Health Services Grants R-56-DE13664, DE019730, DE022508, DE022724 (to H.M.F).
Methods: Bioinformatics analysis, molecular modeling and virtual protein interaction studies were performed to evaluate the structural characteristics FaAD. Overlapping extension PCR was used to generate FaAD deletion mutants. The recombinant FaAd protein was purified from E. coli. In vitro studies were conducted using TIGK (telomerase immortalized gingival keratinocytes) cells. Western blot analysis used anti-peptidyl citrulline mouse monoclonal antibody (clone F95) and anti-citrulline antibody (clone C4). Mass spectrometry was used for the confirmation of proteins and PTMs.
Results: FaAD showed molecular (80%) and structural relatedness (70%) with AD from Porphyromonas gingivalis (PgPAD) and other oral bacteria. AD-defective mutants of F. alocis showed reduced invasion of epithelial cells and biofilm forming capacity compared to the wild type. In contrast to cell lysates from the parent strain, anti-citrulline immunoreactive bands were only detected in the FaAD-defective mutant in the presence of the rFaAD protein. Lysates from TIGK cells incubated with the purified rFaAD protein showed anti-peptidyl citrulline immunoreactive bands. The citrullination of these host cell proteins was confirmed using orbitrap mass spectrometry.
Conclusions: Collectively our data suggest that FaAD may play a role in the citrullination of pathogen and host proteins and thus may modulate virulence and host cell functions.
This work was supported by Public Health Services Grants R-56-DE13664, DE019730, DE022508, DE022724 (to H.M.F).