79 Understanding Tailocin Killing Specificity

Faculty Mentor: Talia Karasov (Biological Sciences, University of Utah)

Phage-tail-like bacteriocins, also known as tailocins, are virus-like toxins used by bacteria to compete with other bacteria for resources and space. Tailocins have highly specific killing abilities; they are able to kill genetically closely related bacteria, but not the producing strain or more distant bacterial relatives, and because of this specificity they are promising alternatives to antibiotics. In 2019, the CDC estimated more than 2.8 million antibiotic-resistant infections occur in the U.S. annually, with more than 35,000 deaths. As antibiotic resistance is becoming more prevalent, alternative therapies are becoming increasingly necessary. To ultimately develop tailocins as a clinical option for antibiotics, more research will be needed to understand the underlying mechanisms of tailocin specificity. To identify genes which contribute to a bacteria’s sensitivity to a tailocin, we employed Random Barcode Transposon Sequencing (RB-TnSeq). Tailocin was applied to a saturated mutagenesis library, a library of bacteria which have a barcode inserted in every non-essential gene in the genome thus making the gene non-functional and able to be tracked throughout the experiment. The culture post tailocin application was sequenced for barcode count. Any barcodes which increased in frequency were identified as genes which contributed to a bacteria’s sensitivity to tailocin. 70 genes were identified and 6 of those belonged to the O-Antigen Biosynthesis Gene Cluster (Figure 1). This data indicates that O-Antigen is a target for the tailocin to identify and bind to its target bacteria. Tailocins bind target cells with high specificity by using their tail fibers to interact with target cell lipopolysaccharide (LPS) akin to a lock and key interaction (Figure 2). Additionally, four different length variants of the tail fibers have been identified in wild pathogenic populations of pseudomonas syringae. The bacteria each tailocin variant can kill is predictable based on what tail fiber variant the target bacteria possess, but the specific interactions between tail fibers and O-Antigen have not been well characterized. For the O-Antigen genes significant in the RB-TnSeq results individual knockouts were generated. To understand the trade-off between sensitivity to tailocin and the ability to infect and kill a host both plant and soft agar assays were performed with the O-Antigen mutants (Figure 3). For each O-Antigen mutant plant growth was nearly identical to the control, thus indicating that the bacteria could no longer infect the plant host. Similarly, almost all knockouts were unable to be killed by tailocin. This data indicates that if bacteria lose or mutate their O-Antigen to escape tailocin killing they are unable to effectively infect a host. Similarly, if bacteria retain their O-Antigen and their ability to infect the host, they are still able to be killed by tailocin. This trade-off likely indicates that it would be difficult for bacteria to evolve resistance to a tailocin. Thus, tailocins are a highly promising alternative for antibiotics. In conclusion, tailocins are highly effective weapons used by bacteria in competition with one another. They use their tailfibers to bind to the O-Antigen of target bacteria. Our data indicates that there is not an easy path for bacteria to evolve resistance to tailocins. As such, they are a promising alternative to antibiotics as antibiotic resistance continues to rise.

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Bibliography

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