Establishment and improvement of genetic manipulation tools for Fusobacterium nucleatum

Abstract

An imbalance in oral microbial homeostasis is significantly associated with the onset and progression of several systemic diseases. Fusobacterium nucleatum, a ubiquitous periodontitis-causing bacterium in the oral cavity, is frequently detected in focal sites and contributes to the pathogenesis of many extraoral diseases, including cancers, cardiovascular diseases, and adverse pregnancy outcomes (APOs). F. nucleatum is one of the few oral anaerobes that can be cultured purely in vitro and is a 'model species' for studying the impact of oral health on systemic health. The establishment and development of genetic manipulation tools for F. nucleatum and the construction of pathogenic gene-disrupted strains are important strategies for studying the pathogenicity of F. nucleatum. Here, we review the establishment and development of the genetic manipulation systems for F. nucleatum and summarize the characteristics of various genetic manipulation tools, such as suicide plasmid-based systems for gene inactivation, replicable plasmid-based systems controlling gene expression, and transposon-based random mutagenesis systems. Notably, we summarize and analyze their applications in the study of the pathogenic mechanisms of F. nucleatum. We hope to provide reference information and ideas for future research on genetic manipulation tools and the pathogenic mechanisms of F. nucleatum and other Fusobacterium species.

Keywords: Crispr interference; Fusobacterium nucleatum; Gene inactivation; Genetic manipulation tool; Natural competence; Restriction-modification system.

Graphical abstract

Fig. 1

Schematic representation of constructing and modifying transformation plasmids of F. nucleatum [57,58]. oriFn, F. nucleatum putative chromosomal origin of replication; ermF-ermAM, the clindamycin-resistance cassette; catP, the chloramphenicol-resistance cassette; oriEc, E. coli putative chromosomal origin of replication.

Fig. 2

Schematic representation of the gene inactivation strategies based on a suicide plasmid for F. nucleatum. (a) Single homologous recombination [58]. (b) Double-crossover homologous recombination [66]. (c) Markerless in-frame gene disruption via double-crossover homologous recombination using 2-deoxy-d-galactose (2-DG) [60], theophylline [71], anhydrotetracycline (ATc)[73], or sucrose resistance [74], as the selection marker, respectively. WT, the wild-type strain of F. nucleatum; catP, the chloramphenicol-resistance cassette; galK, the galactokinase-encoding gene; hicA, the HicA expression cassette; mazF, the MazF toxin expression cassette; sacB, the levansucrase expression cassette; ΔgalK, the F. nucleatum-derived markerless in-frame galK-disrupted mutant.

Fig. 3

Schematic representation of a CRISPR interference (CRISPRi) system for gene inactivation that applies to F. nucleatum based on a replicable plasmid [85]. oriFn, F. nucleatum putative chromosomal origin of replication; dCas9, the nuclease-inactive Streptococcus pyogenes Cas9 protein-encoding gene; sgRNA, single guide RNA; RNAPRNA polymerase.

Fig. 4

Schematic diagram of the use of a transposon mutagenesis system to discover specific functional genes of F. nucleatum [91,96]. ITR, inverted terminal repeat; catP, the chloramphenicol-resistance cassette.