Human Practices

We met with Karen Guillemin, UO Professor of Biology, PI of the Guillemin Lab, and world-renowned gut professional. She specializes in studying the gut microbiota linkage to overall human health to better understand both the potential impact of our project as well as the probiotic space in terms of treatment and pathogens.

Dr. Karen Guillemin

We explained our project and asked key questions regarding efficacy, long term effects of pathogen/probiotic treatments, and host consequences.

Other topics discussed were the impacts of an H. pylori treatment, the efficacy of current antibiotics, and most importantly why they fail to meet efficacy parameters in H. pylori vs other pathogens. She explained that H. pylori possesses one of the most unique colonization strategies of any gut microbe–a complex method of gastric pit localization that is heavily influenced by pit architecture. Fascinatingly, different H. pylori populations can have specific preferences on colonization sites, with some preferring the upper stomach, some preferring thinner pits, or even different “depths'' within each pit, meaning we had yet another variable to factor into our design.

Now knowing that we needed to be able to access deeper and more diverse areas of the gastric epithelium than once thought, we needed an increased response to acidic environments than our initial design using arginine deiminase upregulation.

Another key takeaway from our conversation was the efficacy of treatments for common GI pathogens. Given our goal of surpassing antibiotics and becoming a safer and more sustainable alternative, she explained that we would need to solve one of the major challenges facing probiotic development: survival in the stomach. Dr. Guillemin explained that the acid challenge stomach purges an overwhelming majority of bacteria, excluding the hardiest and most adapted organisms, posing a difficult roadblock to our goal of both survival and performance of specific, programmable tasks. Most probiotics work in the intestines, and endure the “acid challenge” for a few hours at most. We are faced with a much more challenging task, long-term survival and growth in the most hostile environment of the human body.

Illustrating the need for improved colonization, we focused on creating a colonization cassette, or set of parts that would improve survivability in incredibly harsh conditions for an extended duration. A deeper literature search (and inspiration from hardy microorganisms’ abilities) yielded two potential candidates: curli, a self assembling, functionalizable polymer mesh that can forms a protective matrix around colonies and GadE, a master regulator of acid resistance metabolic pathways in E. coli. We have more information regarding these in our contribution section.

Our meeting concluded with a more impact driven conversation. We compared the severity of other common pathogens, such as Salmonella Enterica, Salmonella Enterica subsp. Enterica, serovar Typhi, EHEC and ETEC, and Clostridium difficile, considering both their potential morbidity and the lack of effective treatments. While these other bacteria are not as noteworthy for their antibiotic resistance as H. pylori, they still pose significant health risks to humans.

Dr. Guillemin’s advice didn’t deter us from our initial drive of targeting H. pylori, but instead to expand our scope from a targeted therapeutic to a modular platform with a core set of survival adaptations.

In a radical change to our planned design pipeline, we incorporated modularity into every aspect of our project. 1) Our conjugative plasmid, RP4, was broken apart and reassembled with two cloning sites, providing both golden gate and homology-based compatibility, allowing incorporation of new parts into the DNA transfer machinery. See more details on our contribution section Both of our surface expression constructs, Neae and curli have interchangeable regions located on the tips allowing for targetable specificity and functionalization, respectively. To this end, our chemotaxis library generation method uses one-pot, combinatorial assembly. See our contribution section for a more complete explanation of combinatorial assembly.

Thanks to a generous donation of engineered Nissle-1917 strains from the Voigt Lab at MIT (AJT202, AJT206), and associated genome integration plasmids -- our strains contain three landing pads for straightforward genomic integration as well as recombination removal of antibiotic selection markers. Our final project will integrate all the machinery required for colonization into the genome, with no selection markers. Our strain will also be incapable of transmitting plasmid-borne antibiotic resistances because of the modified RP4 transfer machinery. Once the infection is exterminated, our strain can be removed by administration of conventional antibiotics, or through our planned killswitch (also integrated into the genome).