Eliminating Salmonella with CRISPR-Cas9 and bacterial conjugation
Seung (Joon) Kim, BSc.
May 5, 2021
By composition, we are actually more “bacteria” than we are human by a large margin. The number of bacteria in our gut outnumbers that of our “own” human cells by a factor of 10. This may sound rather like interesting but ultimately irrelevant trivia, but the totality of these bacteria, collectively called the gut microbiome, is intimately linked to our health. Having the wrong kinds or species of bacteria within our microbiome often leads to conditions like diarrhea or inflammation. Furthermore, studies have found that certain species of bacteria are associated with obesity, inflammatory bowel disease, and even colorectal cancer (Cho & Blaser, 2012). These observations point to the fact that a healthy microbiome consists of “good” bacteria or the kind that our immune system can tolerate. Not only that, they compete with “bad” bacteria, which can make us sick, and stop them from expanding. Outbreak of infections with food borne “bad” bacteria like Salmonella is an ongoing public health concern. Take for example some 70 cases in Eastern Canada between late October 2020 and mid-March 2021 with nineteen hospitalized (Canada, 2021). The key obstacle to therapeutically establishing the balance between “good” and “bad” bacteria is to find new ways to selectively modulate the microbiome: keeping the good ones but eliminating the bad ones like Salmonella.
In a collaborative effort by the members of Drs. Gloor, Karas and Edgell lab in the Department of Biochemistry at Western University, a new way of efficiently, selectively eliminating Salmonella has been discovered (Hamilton et al., 2019). The team leveraged the fact that bacteria have a natural system that allows them to share information, in the form of DNA, with each other. This transfer process, called conjugation, uses a certain set of proteins that allow for an arm-like structure to extend from one bacterium to another that enables them to fuse together momentarily. Then, any “DNA-to-be-shared”, called plasmids, can be copied from the donor to the recipient, resulting in the recipient carrying the exact copy of the DNA the donor had.
The researchers engineered a plasmid that encoded CRISPR-Cas9, a “molecular scissor” that can cut specific DNA sequences. If CRISPR-Cas9 inside a bacterium is directed to its own DNA, DNA that is essential for survival, the bacterium will die. The researchers engineered a plasmid that contains CRISPR-Cas9, which once inside Salmonella, will kill the bacteria. They found that E. coli, generally considered “good” bacteria, efficiently transferred the CRISPR-Cas9 plasmid (through conjugation) to Salmonella. Even better, Salmonella that received the plasmid then transferred the same plasmid again to other Salmonella in a vicious self-killing cycle that made the process more efficient. An important element in eliminating Salmonella so efficiently was the fact that the proteins required for conjugation and CRISPR-Cas9 system were contained all in the same plasmid—called a cissystem—which had not been tested before.
The concept of using this cis system combining conjugation with CRISPR-Cas9 has now been validated in a test tube model. The research team will next examine this approach in a living system. The fact that some disease-causing bacteria in a real gut form a protective layer called a biofilm may represent a new challenge. However, the researchers are optimistic that by using donor bacteria that also form biofilms as a Trojan horse, even the tough-to-kill bacteria will be amenable to their strategy. This concept could even be applied to target Fusobacterium infection associated with colorectal carcinoma (Castellarin et al., 2012). Being able to fine-tune the kinds of bacteria that make up our microbiome has important implications, not just for our bowel movement, but also our health.
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Hamilton, T. A., Pellegrino, G. M., Therrien, J. A., Ham, D. T., Bartlett, P. C., Karas, B. J., Gloor, G. B., & Edgell, D. R. (2019). Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing. Nature Communications, 10(1), 4544. https://doi.org/10.1038/s41467-019-12448-3*