0.1mM replicate2
Fusobacterium nucleatum is an important oral pathobiont that contributes to periodontal disease and has been linked to multiple systemic disorders, including colorectal cancer. FomA, the major outer membrane protein of F. nucleatum, has long been recognized as both an adhesin and a porin; however, its physiological role remains unclear. Unexpectedly, despite previous reports that fomA is dispensable, we were unable to generate a deletion mutant and found that CRISPRi-mediated depletion of FomA caused growth arrest and cell death. These findings reveal that FomA is essential for fusobacterial viability and suggest an unrecognized function beyond its established role in adhesion. To gain insight into the molecular basis of FomA essentiality, we performed comparative transcriptomic analyses following CRISPRi-mediated repression of fomA. An ATCC 23726 strain carrying a CRISPRi plasmid targeting fomA was grown overnight in TSPC medium and subsequently diluted 1:20 into fresh medium containing 0, 0.1, or 0.5 mM theophylline to achieve different levels of FomA depletion. After 8 h of growth, cells were harvested and total RNA was isolated using TRIzol reagent followed by purification with the Direct-zol RNA MiniPrep Kit (Zymo Research).RNA samples were submitted to the Cancer Genomics Center at The University of Texas Health Science Center at Houston (CPRITRP240610) for quality control and sequencing. RNA integrity was assessed using the Agilent RNA 6000 Pico Kit (#5067-1513) on the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), and samples with RNA integrity numbers (RIN) greater than 7 were used for library construction.RNA-seq libraries were prepared from rRNA-depleted RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L, New England Biolabs) in combination with NEBNext Multiplex Oligos for Illumina (E6609S, New England Biolabs), following the manufacturers instructions. Library quality was evaluated using the Agilent High Sensitivity DNA Kit (#5067-4626) on the Bioanalyzer 2100, and concentrations were quantified via qPCR using the Collibri Library Quantification Kit (#A38524500, Thermo Fisher Scientific). ). The libraries were pooled evenly and went for the paired-end 150-cycle sequencing on an Illumina NovaSeq X plus System (Illumina, Inc., USA).
SUB16246374
WTfomA0.1 A2