Genetic Manipulation and Virulence Assessment of Fusobacterium nucleatum

Abstract

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...

The Fused Methionine Sulfoxide Reductase MsrAB Promotes Oxidative Stress Defense and Bacterial Virulence in Fusobacterium nucleatum

“…Lack of knowledge about gene regulation may reflect the inherent difficulty of culturing fastidious anaerobes and the hitherto underdeveloped genetic tractability of fusobacteria. Recent advancements in bacterial genomics and genetic manipulations of this organism, however, have begun to facilitate fruitful molecular studies ( 36 – 38 ). We were then able to undertake a genetic investigation of adaptive response in fusobacteria.…”

“…Bacterial strains and plasmids used in this study are listed in Table S5 in the supplemental material. F. nucleatum was grown in tryptic soy broth supplemented with 1% Bacto peptone plus 0.25% freshly made cysteine (TSPC) or on TSPC agar plates with 1% vitamin K1-hemin solution or BBL Columbia agar with 5% sheep blood in an anaerobic chamber (5% CO 2 , 2.5% H 2 , and 92.5% N 2 ), as previously described ( 38 ). When necessary, chloramphenicol (15 μg/mL) or thiamphenicol (5 μg/mL) was added.…”

Genetic and molecular determinants of polymicrobial interactions in Fusobacterium nucleatum

“…Here, we wished to tackle the central problem of how Fusobacteria achieve this capacity of serving as a "polymicrobial niche." To foster an unbiased and systematic mechanistic study of this problem, we recently made strides in developing several essential genetic tools for F. nucleatum (21,27), and we began to dissect some of the key molecular steps in interspecies interactions and biofilm development. Here, we describe the results of a systematic genetic investigation of one of these steps, the fusobacterial coaggregation with streptococci, which uncovered a hitherto unknown genetic network that promotes fusobacterial interactions with other oral bacteria and its metabolic regulation by a physiologically intriguing pathway for fusobacterial communication with the host that may also be connected to the important process of nutrient acquisition by the microbe.…”

“…Since the deletion of carR/carS affects the expression of radD, lysine metabolic genes, and many other genes, we were motivated to determine if CarR/S are associated with fusobacterial virulence. To do so, we chose to utilize the aforementioned mouse model of preterm birth for F. nucleatum that was utilized in previously reported studies of fusobacterial pathogenesis (3,15,27). Accordingly, a group of five pregnant CF-1 mice was injected via the tail vein with ∼5.0 × 10 7 colony forming units (CFU) of individual bacterial strains on day 16/17 of gestation, and the number of live and stillborn pups was recorded for the next 7 d. Remarkably, the nonpolar, in-frame deletion mutant ΔcarR exhibited a virulence attenuation phenotype as compared to the parental strain, which produced only 6% pup survival at the end point (Fig.…”

Colorectal cancer: the facts in the case of the microbiota

“…Beyond these four adhesins, ten additional type Va SS autotransporters are encoded by Fn ATCC 23726 (91,92). Given that ATCC 23726 is one of the rare genetically tractable Fn strains (93,94), there is the opportunity to better understand the roles of these undercharacterized Fn autotransporters in CRC virulence.…”