A gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions—termed coaggregation—by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD, the expression of which are highly up-regulated in the ΔcarR mutant. Significantly, the native culture medium of ΔkamA or ΔkamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the ΔkamA and ΔcarR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (ΔradD or ΔcarS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites.

Considered a commensal, the Gram-negative anaerobe Fusobacterium nucleatum is a key member of the oral microbiome, due to its wide range of interactions with many oral microbes. While the periodontal pathogenic properties of this organism have widely been examined, its connotation with extra-oral infections including preterm birth and colorectal cancer now becomes apparent. Nonetheless, little is known about the mechanisms of pathogenicity and the associated virulence factors of F. nucleatum, most likely due to limited genetic tools and facile methodology. Here, we describe molecular techniques for the genetic manipulation of F. nucleatum, including marker-less, non-polar gene deletion, complementation, and Tn5 transposon mutagenesis. Further, we provide methodology to assess virulence potential of F. nucleatum, using a mouse model of preterm birth. Basic Protocol 1: Generation of a galK mutant strain Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of F. nucleatum Basic Protocol 4: A mouse model of preterm birth This article is protected by copyright. All rights reserved. 3 recombination event (Han, Ikegami, Chung, Zhang, & Deng, 2007; C. W. Kaplan et al., 2010; Kinder Haake et al., 2006), which potentially have polar effects. Here, we report a recently developed gene deletion method that generates markerless, non-polar, in-frame deletion mutants in F. nucleatum and Tn5 transposon mutagenesis that permits genome-wide screening (C. Wu et al., 2018). Basic Protocols 1 and 2 describe methods for plasmid design, construction, and introduction into F. nucleatum for use in making deletion and complementation strains, respectively. Basic protocol 3 describes a procedure for construction of a Tn5 transposon library. Basic protocol 4 reports a mouse model of infection for which the effect of F. nucleatum on preterm birth can be studied. CAUTION: Fusobacterium nucleatum is a Biosafety Level 2 (BSL-2) pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. STRATEGIC PLANNING Preparation and Growth on Agar or Liquid Medium F. nucleatum strains are grown in tryptic soy broth (TSB) supplemented with 1% Bacto peptone (TSP) plus 0.25% autoclaved cysteine (TSPC) or on TSPC agar plates or on BBL Columbia agar plates with 5% sheep blood (see Regents and Solutions). TSPC plates should be freshly made under sterile conditions before use; plates can be air-dried inside a biosafety cabinet with filtered continuous airflow. Columbia agar plates can be stored at 4°C and should be used within ~2 weeks. When necessary, 50This article is protected by copyright. All rights reserved. 4 µg ml −1 kanamycin, 15 µg ml −1 chloramphenicol, or 5 µg ml −1 thiamphenicol should be added to the medium. Anaerobic ConditionsTSP can be combined with cysteine in aerobic conditions prior to inoculation.Inoculation of bacterial strains from glycerol stocks into media or spread onto a plate can be done in aerobic conditions. Cultured plates should be pl...

F. nucleatum colonizes various human tissues, including oral cavity, placenta, and colon. How this obligate anaerobe withstands oxidative stress in host immune cells has not been described.