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Substitutions in the Proximity of the Active Site of Peptidylarginine Deiminase from P. gingivalis ‐ Is There a Correlation with Enzyme Activity?
Author(s) -
Bereta Grzegorz,
Gawron Katarzyna,
ŁazarzBartyzel Katarzyna,
Łazarz Mieczysław,
Stożek Karol,
Naskalska Antonina,
Burmistrz Michał,
Pyrć Krzysztof,
ChomyszynGajewska Maria,
Potempa Jan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.925.8
Subject(s) - genbank , porphyromonas gingivalis , biology , microbiology and biotechnology , genomic dna , gene , genetics , bacteria
Background Periodontitis (PD) is a chronic inflammatory disease leading to tooth loss, which affects over 30 % of adults worldwide. A major causative agent of the disease is an anaerobic bacterium, Porphyromonas gingivalis (Pg). Recently, considerable interest has been focused on peptidylarginine deiminase (PPAD) from Pg, an enzyme which catalyzes conversion of arginine to citrulline. Our recent results imply PPAD activity as a critical factor in gingival cell infection and prostaglandin E 2 induction. Those findings encouraged us to analyze polymorphisms of the PPAD gene and enzyme activity among Pg clinical strains. Objective Analysis of PPAD gene polymorphisms and enzyme activity in clinical isolates of Pg from patients with chronic periodontal disease (CPD), reference strains (RS) and healthy volunteers (HV). Material and methods Gingival crevicular fluid was collected from 9 CPD and 4 HV, seeded on blood agar and grown under anaerobic conditions. Pg was confirmed by 16S rRNA and PPAD PCR, followed by genomic DNA purification. Next, PPAD coding sequences were cloned into a pTZ57R vector, amplified and sequenced. The sequences from CPD and HV were aligned with the PPAD sequences deposited in the GenBank database, i.e. 2 of RS (ATCC33277, W83), 4 strains of PD patients (F0568, F0569, F0570, SJD2), and 5 other strains (F0185, F0566, JCVI SC001, W50, W4087). Further, PPAD activity from CPD, RS and HV was analyzed using colorimetric method. Results In total, mutations were identified in 50 nucleotide positions in the PPAD sequence from all clinical isolates and those deposited in GenBank. One sequence was identical to the ATCC33277, but varied from the W83 strain, while 4 sequences were identical to the W83 strain. One isolate harboured 7 nucleotide substitutions, which resulted in a change of 3 amino acid residues in the proximity of the PPAD active site. This isolate displayed significantly increased PPAD activity in comparison to W83 strain. The identical nucleotide substitutions were noted in PPAD of strains from patients with PD found in USA and China. Conclusions Our analyses shown variations in nucleotide and amino acid sequence of PPAD among clinical and reference strains. The presence of specific, identical amino acid substitutions in clinical isolates from Poland, USA and China in comparison to the RS along to the variation of enzyme activity may suggest an impact of PPAD on the virulence of Pg. This observation deserves further elucidation of functional and clinical significance of this mutation in the pathogenesis of PD. Support or Funding Information National Science Centre, Poland (2012/07/B/NZ6/03524, K.G.), Foundation for Polish Science (TEAM, DPS/424‐329/10, J.P.)