Towards the discovery of virulence and survival factors in Fusobacterium using host-mimicking DNA methylation to transcend genetic barriers.

Fusobacterium nucleatum (Fn) is an anaerobic, Gram-negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well-established that most Fusobacteriumexhibit genetic recalcitrance, mainly owing to robust restriction modification systems. These systems allow bacteria to distinguish self from non-self DNA via two enzymatic activities: a restriction endonuclease and a modification methyltransferase. Though the association of Fnin diseases is well-established, the majority of our experimental work stems from the strain F. nucleatum 23726 because it is genetically tractable. To enable the study of Fn clinical isolates that are currently genetically intractable, here we show that we are able to fool host restriction modification systems that protect the bacteria against the introduction of foreign DNA by using FnDNA methyltransferases to protect our gene knockout plasmids against nuclease digestion. This genetic breakthrough has enabled the first mutants in F. nucleatum 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cell invasion and bacterial intracellular survival, which is enabling detailed mechanistic studies of these virulence proteins in Fn-driven disease. Our results demonstrate the simplicity and convenience of this host-mimicking approach, which enables circumvention of innate restriction modification barriers - the most common underlying cause of bacterial genetic intractability. We anticipate in future applications our system could be expanded to study novel genetic elements of which are fundamental to the pathophysiology of a wide range of relevant Fusobacterium species.