zhou, p. | Search bioRxiv

bioRxiv2024

Development of a Conditional Plasmid for Gene Deletion in Non-Model Fusobacterium nucleatum strains

Fusobacterium nucleatum is an opportunistic pathogen with four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within F. nucleatum. While ATCC 23726 is highly transformable, most other Fusobacterium strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an fdx promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the galK gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species Fusobacterium periodonticum. Incorporating a sacB counterselection marker in this conditional plasmid enabled the deletion of the radD gene in non-model strains. Interestingly, while radD deletion in 23726, 10953, and 21_1A abolished coaggregation with Actinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant F. nucleatum strains, enhancing our understanding of this pathogen. IMPORTANCEFusobacterium nucleatum is implicated in various human diseases, including periodontal disease, preterm birth, and colorectal cancer, often linked to specific strains and reflecting the species genetic and phenotypic diversity. Despite this diversity, most genetic research has centered on the model strain ATCC 23726, potentially missing key aspects of other strains pathogenic potential. This study addresses a critical gap by developing a novel conditional plasmid system that enables gene deletion in genetically recalcitrant strains of F. nucleatum. We successfully deleted genes in the clinical strain CTI-2, the FNA strain 21_1A, and F. periodonticum for the first time. Our findings, particularly the varying behavior of the radD gene production in coaggregation across strains, underscore the complexity of F. nucleatum and the need for broader genetic studies. This work advances our understanding of F. nucleatum virulence at the strain level and provides a valuable tool for future bacterial genetics research.

microbiology↗

bioRxiv2023

Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum

Gene inactivation via creating in-frame deletion mutations in Fusobacterium nucleatum is time-consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPRi system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX, and radD, pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing coaggregation. The system was then extended to probe essential genes bamA and ftsZ, vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ-targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains. IMPORTANCEHow can we effectively investigate the gene functions in Fusobacterium nucleatum, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPRi system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and sgRNA expression is driven by the robust, constitutive rpsJ promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered resistant strains like CTI-2 and Fusobacterium periodontium. With CRISPRis potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.

microbiology↗