Genetic Manipulation of <i>Porphyromonas gingivalis</i>

Bélanger, Myriam; Rodrigues, Paulo Henrique Mazza; Progulske‐Fox, Ann

doi:10.1002/9780471729259.mc13c02s05

Cited by 40 publications

(30 citation statements)

References 56 publications

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“…P. gingivalis PPAD (UniProt database [UP] access code Q9RQJ2 or GenBank entry WP_005873463.1 for NCBI gene tag PG_1424) was obtained through small-scale homologous overexpression as a secreted protein from plasmid-transformed P. gingivalis W83 PPAD-deletion mutant strain Δppad . Briefly, plasmid pT-COW, which confers resistance against tetracycline 36 , was used as expression vector, and plasmid derivatives encoding the wild type (wt) and a total of 18 PPAD point mutants (W 127 A, D 130 A, D 130 N, R 152 A, R 154 A, R 154 E, T 180 A, G 182 A, H 236 A, H 236 N, D 238 A, D 238 N, C 239 A, C 239 E, C 239 S, N 297 A, C 351 S, and C 351 A; see Table 1 ) were generated. For this, the wt gene sequence plus 1081 upstream base pairs and 267 downstream base pairs was amplified from P. gingivalis W83 genomic DNA with primers pTCowPPADf and pTCowPPADr (see Table 1 ), which contained recognition sequences for restriction endonucleases Nhe I and Sph I, respectively.…”

Section: Methodsmentioning

confidence: 99%

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase

Goulas

Mizgalska

Garcia-Ferrer

et al. 2015

Sci Rep

108

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Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer’s disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a “Michaelis loop” that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants.

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Section: Methodsmentioning

confidence: 99%

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase

Goulas

Mizgalska

Garcia-Ferrer

et al. 2015

Sci Rep

108

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“…E. coli strain S17-1 was used as a donor strain to introduce the pT-COW plasmid and its derivatives into P. gingivalis by conjugation (42). Plasmids carrying spacer sequences (Table 2) were transformed into chemically competent E. coli S17-1 cells.…”

Section: Methodsmentioning

confidence: 99%

Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems

et al. 2015

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The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3= end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3= terminus by the appropriate PAM element. IMPORTANCEThe CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial communities and, consequently, the process of disease development and progression.

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“…P. gingivalis strain 381 was obtained from F. Macrina (Virginia Commonwealth University) and grown as previously described (21). Genomic DNA was obtained using the Wizard gDNA purification kit (Promega) and processed to generate shotgun and 3-kb paired-end libraries, which were sequenced using the 454 Life Sciences GS-20 instrument (22) (Roche).…”

Section: Genome Announcementmentioning

confidence: 99%