Children in Low- and Middle-Income Countries (LMICs) are exposed to pathogens and antimicrobial resistance (AMR) genes at high rates due to more exposure to animals and insufficient access to water, sanitation, and hygiene (WASH) resources. This study analyzes the prevalence of AMR gene carriage in young children aged 6, 12, and 18 months living in communities spanning a rural to urban gradient in Northwestern Ecuador. Stool samples were collected from 428 children at ages 6 months (n=327), 12 months (n=373), and 18 months (n=368) in Ecuador. Stool samples were extracted for total nucleic acids and assayed for pathogen and AMR targets via TaqMan Array Cards. This analysis focuses on 15 AMR targets, which include beta lactams, class 1 integrons, fluoroquinolones, and folate pathway inhibitors. There was a very high prevalence of AMR genes, especially SHV (80.7%), sul2 (82.7%), Intl1 (88.9%), qnrB1 (90.0%), sul1 (90.2%), and TEM (98.9%). There was very low prevalence or no detection of KPC (0.0%), NDM (0.1%), and VIM (0.0%). This study demonstrated that there is a high prevalence of diverse AMR genes in children in Ecuador across all community types and ages. This suggests that these children have high exposure rates to AMR genes that could potentially lead to difficulties in determining appropriate antibiotic treatment for future illnesses.

Current methods of cancer immunotherapy, such as CAR T-cell therapy, can treat blood cancers. However, treating solid tumors with T-cells remains a challenge, as the necrotic cores of solid tumors are a toxic environment for human immune cells. Bacteria are inexpensive, easy to genetically modify, and have many species which can colonize tumors. Bacteria, therefore, have potential to be an effective alternative to T-cell based treatments. Our challenge is to engineer E. coli bacteria to secrete immunomodulatory payloads upon colonizing the tumor microenvironment. This could be a useful avenue for immunotherapy, especially if the bacteria could produce multiple cargos with synergistic effects. However, we have limited data on what therapeutics E. coli can secrete, and whether it can secrete multiple therapeutics simultaneously. In the fall, I tested whether known E. coli secretion tags could export immunomodulatory minibinder proteins designed by the Baker lab. These minibinders interact with cytokine receptors on tumor cells and are hypothesized to reduce rates of tumor metastasis, which could make them effective anti-cancer therapeutics. Through western blot analysis, I successfully detected secretion of one of these candidate minibinders. My next step is to test whether it can be secreted together with another designed cytokine, Neo-2/15. I anticipate that combining cargos might lower each individual therapeutic’s secretion, since expressing multiple proteins may increase the cell’s burden past its secretion capabilities. If secretion or expression is observed, I will work on optimizing secretion of each therapeutic. The results of this experiment will broaden our understanding of E. coli’s potential as a delivery mechanism for individual and combined therapeutics, open future avenues to test more human immunomodulatory therapeutics and combinations thereof, and hopefully someday facilitate more effective forms of cancer immunotherapy.

Glycogen Synthase Kinase 3 (GSK3) is a well-studied enzyme that is implicated in many diseases due to its regulatory role in numerous signaling pathways, both known and unknown. The scaffold protein Axin binds to GSK3 and the substrate β-catenin (Bcat), specifying GSK3 so that it primarily acts within the cancer-implicated Wnt signaling pathway. My project seeks to determine if GSK3 is recruited to other potentially unknown signaling pathways by other scaffold proteins that compete with Axin and each other to bind GSK3 at its Axin-binding site. A previous proteomics and computational experiment identified five proteins that potentially interact at this interface of GSK3. Five peptides were designed from the theorized binding sites of the proteins to evaluate whether these proteins compete with Axin. I am using an engineered mammalian cell line to indirectly measure cellular levels of GSK3's substrate, Bcat, and test whether the peptides are capable of displacing Axin from GSK3. When GSK3 is both active and bound to Axin, it causes degradation of Bcat; when GSK3 is inactivated or unbound from Axin, Bcat builds up in the cell. Lithium chloride is a known pathway-independent GSK3 inhibitor that will be used to compare the effect of the peptides on the amount of Bcat, and thus the effect on the amount of Axin-bound GSK3. Displacement of Axin by these peptides indicates that the proteins specify GSK3 for signaling pathways in a similar mechanism to Axin, and that in normal cell states, some equilibrium exists between pools of pathway-recruited GSK3. Understanding the equilibrium between these binders and their associated signal pathways would give insight into how overexpression of one can cause the development of disease states such as cancer.

Technologies like CRISPR-Cas9 have emerged as promising tools for gene regulation and single nucleotide editing. The field has recently developed mRNA responsive base editors that can edit a genomic scratch pad and record mRNA expression and abundance in bacterial cells over time. RNA responsive base editors can let us retroactively study gene expression that can result in phenotypic differences. However, in complex heterogeneous communities, such as biofilms, monitoring the phenotype and expression of individual cells in real time is challenging. Pairing fluorescence signals to levels of mRNA can convey spatial information about how individual cells behave differently in complex communities. Our goal is to achieve mRNA-responsive base editing to generate fluorescent reporter output. To accomplish this, we will utilize the two existing systems, Rptr, which performs mRNA-responsive base editing, and CRISPR activation (CRISPRa), which can activate a fluorescent signal. These two systems will simultaneously perform base editing and CRISPR activation within the same cell. For this purpose, we will prototype orthogonal CRISPR systems that can independently recruit either activators or base editors through RNA hairpins attached to the guide RNA. My work focuses on designing synthetic fluorescence reporters with installed stop codons that can be modified with base editing and then activated with CRISPRa. My reporters will allow for rapid prototyping of mRNA responsive base editing with RNA hairpin recruitment. We can then find our best performing RNA-recruited base editing system to use in a multiplexed effector system. Ultimately, this integrated approach will couple mRNA expression with a fluorescent reporter read out, allowing us to monitor individual bacterial cells within complex populations.