Identification of Critical Virulence Factors of Fusobacterium nucleatum in Promoting Colorectal Carcinoma

Background: The presence of an oral commensal, Fusobacterium nucleatum, in colorectal cancer(CRC) has been identified as an indicator of poor prognosis and has also been shown to increase gradually from stage I to IV. Several adhesion molecules in F. nucleatum, including RadD, FadA, and Fap2, have been identified as virulence factors in CRC. However, given that F. nucleatum contains over 2,000 genes, it is possible that additional undiscovered pathogenic factors are contributing to F. nucleatum-induced CRC stimulation.

Aims: (1) Identification of potential pathogenic genes involved in CRC progression; (2) Evaluation of biofilm properties of clinical F. nucleatum isolates and their carcinogenicity in CRC cell line.

Methods: (1) A F. nucleatum transposon library was created through EZ-Tn5 transposon mutagenesis. The catP transposon DNA fragment contains a chloramphenicol/thiamphenicol resistant cassette that can be used as a selection marker for the insertion. F. nucleatum mutant colonies were inoculated into 96-well plates and stored at -80°C in 100% glycerol.

(2) Clinical F. nucleatum isolates were successfully identified from saliva samples of 101 oral squamous cell carcinoma (OSCC) patients and 158 non-OSCC patients at a positive rate of 10%. The overnight bacterial culture was adjusted to OD 0.1 and anaerobically incubated at 37°C in 24-well plates for 96 hours. Bacterial biofilm stability was determined by comparing the density of attached biomass before and after washing with 500µl of 1X PBS twice.  1x10⁶ of HCT116, a CRC cell line, were seeded in 6-well plates for 24 hours and a “+” mark was scratched with 200µl pipette tips. Overnight bacterial culture was resuspended in McCoy’s 5a medium (no supplements or antibiotics). HCT116 cells were then infected with MOI 10 of various clinical F. nucleatum isolates. Cell migration rates were calculated based on the distance traveled in 24 and 48 hours.

Results and conclusions: A transposon mutant library of 9,600 mutant colonies of F. nucleatum was established, with a minimum genome coverage of 99% achieved. This robust genetic tool will be used to identify novel virulence genes. Clinical isolates from both OSCC and non-OSCC patients demonstrated varying levels of biofilm stability and CRC cell migration. Further experiments, such as bacterial cell adhesion and invasion, as well as RNA sequencing, will be performed to expand the knowledge towards the development of novel OSCC prevention and treatment strategies.