Bacteria utilize a wide array of metabolic pathways to conserve energy from various substrates, powering many physiological processes including bacterial adhesion, motility, biofilm formation, polymicrobial interaction, and virulence. The Gram-negative oral anaerobe, Fusobacterium nucleatum, is an emergent pathogen implicated in a multitude of extra-oral disorders including colorectal cancer (CRC), breast cancer, adverse pregnancy outcomes, cardiovascular disease, and rheumatoid arthritis. F. nucleatum owes its ability to spread from the oral cavity and colonize a range of extra-oral tissues in large part to the expression of several outer membrane proteins (OMPs) that act as molecular adhesins. However, the mechanisms by which F. nucleatum metabolically adapts to changing extra-oral environments to maintain its virulence potential remain poorly understood. Informed by our Tn5 transposon mutagenesis screens to identify additional F. nucleatum virulence factors involved in biofilm formation and polymicrobial interaction, we hereby describe in this dissertation the metabolic role of a conserved prokaryotic respiratory enzyme, the Rhodobacter nitrogen-fixation (Rnf) complex, and its impact on fusobacterial virulence.
The Rnf complex is a well-studied, six-subunit, ferredoxin:NAD+ oxidoreductase that promotes microbial fitness via conserving energy from several metabolic niches, including those relying on autotrophic, heterotrophic, and/or syntrophic metabolism. Here, we show that genetic disruption of the fusobacterial Rnf complex, via in-frame deletion of subunit-encoding genes, causes pleotropic defects in polymicrobial interaction, biofilm formation, ATP biosynthesis, toxic hydrogen sulfide production, bacterial cell growth, and morphology. Targeted metabolomic screening demonstrates loss of Rnf causes global deficiencies in amino acid fermentation, negatively impacting fusobacterial virulence in a mouse model of preterm birth. This study establishes the fusobacterial Rnf enzyme as a metabolic conduit of F. nucleatum pathophysiologywithin and outside of the oral cavity.
F. nucleatum is an oncobacterium that promotes carcinogenesis of colorectal cancer, and the amyloid-forming adhesin FadA is integral to this process. Previous studies suggest that the highly conserved Rhodobacter nitrogen-fixation (Rnf) complex modulates the virulence potential of this pathobiont via metabolic signaling; however, a mechanism for this modulation remains unknown. Here, we show that genetic disruption of the Rnf complex, via rnfC deletion, significantlyreduced the transcript level of fadA, relative to the wildtype. This was accompanied by near complete abolition of the precursor form of FadA (pFadA) and reduced surface assembly of FadA at the mature cell pole. Noticeably, rnfC deletion caused a severe defect in osmotic stressinduced amyloid formation that was rescued by ectopic expression of rnfC. Gene deletion analysis identified three response regulators – CarR, ArlR, and S1 – that modulate expression of pFadA, without affecting its transcript level, suggesting that these response regulators control expression of factors that process FadA. Consistently, deletion of rnfC, arlR, and s1 significantly reduced expression of the signal peptidase-encoding gene lepB, and CRISPR-induced depletion of lepB nearly abolished FadA expression. Importantly, while rnfC deletion did not affect the ability of mutant cells to adhere to cancer cells HCT116, rnfC deficiency significantly diminished fusobacterial invasion of HCT116. Consistent with the role of FadA in tumor formation, the rnfC mutant was markedly defective in promotion of spheroid tumors. Evidently, the Rnf complex modulates expression of FadA and formation of FadA-associated amyloids and tumors via regulation of LepB by multiple response regulators.
