Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems

Abstract

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.

Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states

“…Furthermore, the CRISPR-Cas and associated CAS proteins system represents a unique system that provides prokaryotic cells, as P . gingivalis , adaption and protection from host defenses [54, 55]. Down-regulation of the genes PGN_1924-Cas2, PGN_1925-Cas1 may suggest a decrease in the defensive capability of P .…”

Genetic exchange and reassignment in Porphyromonas gingivalis

“…They provide adaptive immunity against foreign elements such as bacteriophages/viruses, plasmids and transposons. At least 95% of clinical strains from P. gingivalis harbours CRISPR arrays [41]. The presence of transposases and CRISPR-associated sequences in P. gingivalis [11] suggested that some parts of the P. gingivalis genome are mobile so that rearrangement or horizontal gene transfer from one strain to another can take place [20].…”

“…The presence of transposases and CRISPR-associated sequences in P. gingivalis [11] suggested that some parts of the P. gingivalis genome are mobile so that rearrangement or horizontal gene transfer from one strain to another can take place [20]. In the P. gingivalis W83 genome, all four CRISPR regions were transcribed and one of them was active against dsDNA in vivo [41]. Probably the CRISPR-Cas system has developed as a protective system that verifies and selects DNA entering bacterial cells, helping them to regulate horizontal gene transfer [41].…”

“…In the P. gingivalis W83 genome, all four CRISPR regions were transcribed and one of them was active against dsDNA in vivo [41]. Probably the CRISPR-Cas system has developed as a protective system that verifies and selects DNA entering bacterial cells, helping them to regulate horizontal gene transfer [41]. …”

Porphyromonas gingivalis and its CRISPR-Cas system

“…Clustered regularly interspaced short palindromic repeats CRISPRs belong to a family of DNA sequences characterized by short direct repeats (DRs) that are separated by spacers [1]. Almost half (45%) of the species in Bacteria and the majority (80%) in Archaea contain CRISPR-Cas systems [2]. Through an adaptation process, the system is able to store memories of previous exposure to foreign DNA in the spacer sequences across generations of progenies [3][4][5].…”