Recent sequencing studies have suggested that the horizontal gene transfer (HGT) of plasmids, a possible mechanism through which antimicrobial resistance (AMR) genes reach pathogens, is prevalent in the human gut. Due to its prevalence in the community, it is possible to harness HGT as a method of manipulating the gut microbiome by introducing plasmids. However, our knowledge of the conditions under which HGT occurs and the rates of transfer between commensal bacteria and new species is lacking. In order to use HGT to perform targeted manipulations of the gut microbiome, such as reducing the burden of AMR genes in the gut, it is necessary to understand how plasmids are transferred throughout the community. In the Brito Lab, we designed a HGT reporter plasmid that can track transfer events through gene edits created by a dCas9- deaminase encoded in the plasmid. Using this reporter plasmid, I aim to determine the optimal parameters in harnessing HGT for the in vivo delivery of plasmids to the gut microbial community. In my first aim, I will optimize three parameters of conjugation-based plasmid delivery to the gut: donor species, donor dosage or microbiota depletion by pre-treatment with antibiotics, in order to improve plasmid dispersal and persistence in an in vivo mouse model. I will use the HGT reporter plasmid in conjunction with a single-cell sequencing method optimized for bacterial communities to obtain taxonomic data of each cell that contains the plasmid through various timepoints. This will allow me to determine which parameters increase dispersal and persistence of the plasmid. In my second aim, I will compare conjugation to four different artificial transformation methods to determine the most efficient plasmid delivery method. In order to determine transformation efficiency for each method, I will deliver the HGT reporter plasmid to individual colonies of a variety of gut commensal bacteria and use quantitative PCR of the plasmid and antibiotic selection to determine the number of plasmid copies and transformants. Additionally, I will analyze how artificial transformation affects dispersal by creating a mock gut community and use single-cell sequencing to determine which species obtained the plasmid. Through the proposed experiments, I will determine the optimal parameters for plasmid delivery to the gut, a necessary step for harnessing HGT for microbiome engineering. The potential to engineer this community provides an opportunity to interrogate the specific host-microbiome interactions that might underlie microbiome-associated diseases, as well as reducing AMR in the gut. I will also gain valuable experience in microbiome research and single-cell sequencing technologies that will aid me in my goal to become a professor in Puerto Rico and help train the next generation of Puerto Rican scientists.
Horizontal gene transfer (HGT) is one mechanism by which antimicrobial resistance genes and other functional genes are transferred within the gut, though our knowledge of the conditions under which HGT occurs and the rates of transfer between commensal bacteria and new species is lacking. I plan to design and use an HGT reporter plasmid that can track transfer events through gene edits created by a dCas9-deaminase encoded in the plasmid. I will analyze how artificial transformation affects dispersal by creating a mock gut community and use single-cell sequencing to determine which species obtained the plasmid so we can manipulate the gut microbiome, such as to reduce the burden of antimicrobial genes.
