Conjugation-based genetic manipulation of Fusobacterium animalis

Fusobacterium is an oncobacterium associated with colorectal cancer (CRC) development. Despite that F. animalis (Fa) dominates the CRC niche in humans, studies on the pathogenesis of Fusobacterium have been limited by the paucity of genetically tractable Fa strains. Here, we constructed a conjugative DNA transfer system for genetic manipulation of Fa clinical isolates. A mobilizable shuttle plasmid can be transferred by conjugation from Escherichia coli to various Fa strains, including Fa 7_1, which has been considered genetically intractable using electroporation methods. The conjugation efficiency of Fa 7_1 was further improved by ethanol treatment. Next, we manipulated the E. coli donor strain to express Fusobacterium methyltransferases, which can protect plasmids from degradation by restriction systems in Fa recipient strains. With this donor strain, we could isolate transconjugants of Fa harboring chromosomally integrated plasmid. As a test case, we disrupted megL which is involved in cysteine metabolism and hydrogen sulfide (H₂S) production. The plasmid integrated into the megL locus was stably maintained without antibiotic selective pressure. The megL disruption in Fa 7_1 significantly decreased the level of H₂S production in vitro in the presence of cysteine and resulted in lower cecal H₂S level in a mouse colonization model. The same conjugation-based system was successfully applied to another Fa strain CTI-1, although this strain showed 10-fold lower conjugation efficiency compared to Fa 7_1. Altogether, this work suggests that conjugation is an efficient method for genetic manipulation of Fa strains, allowing us to mechanistically study their pathogenicity in CRC at the molecular level.IMPORTANCEAmong the Fusobacterium species associated with colorectal cancer (CRC), F. animalis (Fa) is the most clinically relevant species showing distinct genetic features. However, due to its genetic intractability, little is known experimentally about the molecular factors of Fa contributing to CRC development. Here, we showed that foreign DNA can be transferred from Escherichia coli to Fa via conjugation. The conjugation system was developed by expressing Fusobacterium-derived methyltransferases in the E. coli donor strain to transfer methylated plasmid to Fa. Finally, the megL gene, encoding an enzyme responsible for hydrogen sulfide (H₂S) production, was successfully disrupted in Fa strains. The effects of the megL disruption in Fa on H₂S production were verified both in vitro and in vivo. This conjugation-based approach would be applied to not only Fa but also a broader range of Fusobacterium species, expanding our understanding of their virulence traits.