Discovery and Characterization of Novel Thanatin Orthologs Against Escherichia coli LptA and Pseudomonas aeruginosa LptH

Multidrug resistance (MDR) in bacteria is ever growing and complicates treatment of infections, especially in patients who are critically ill and immunocompromised. Treatment often utilizes a regiment of small molecule drugs, however resistance against them develops after prolonged usage. An alternative class of molecules, antimicrobial peptides (AMPs), have remained of interest due to its vast potential of becoming pharmacological agents. AMPs, or host defense peptides, are naturally expressed in many organisms, including microbes, plants, and humans. AMPs are expressed to control the population of bacteria, fungi, and viruses as a defense mechanism. Mining host genomes for AMPs will prove to be a valuable source of novel alternative drug molecules. Characterization of AMPs will lead to be a better understanding of their mechanism of action and allow for applications to novel targets. Here in this dissertation, we apply these methods to thanatin, an AMP identified from the spined soldier bug (Podisus maculiventris) that was reported to regulate the gut microbiome population by targeting Gram-positive bacteria, Gram-negative bacteria, and fungi.First, we mined genomic databases to discover novel thanatin orthologs. We generated these orthologs and characterized their binding against Escherichia coli LptA, a known target of thanatin, via bio-layer interferometry (BLI) and their antimicrobial activity against several E. coli strains via minimum inhibition concentration (MIC) assays. We found a subset of thanatin sequences that target E. coli better than P. maculiventris thanatin, as shown with increased binding affinity, cell permeability, and overall potency. We crystallized and determined the structures of Chinavia ubica thanatin and Murgantia histrionica thanatin, the two most improved thanatin orthologs, in complex with E. coli LptA to better understand the interaction. We performed mutagenesis studies to show that thanatin residues A10 and M21 interacts with the hydrophobic core of LptA and improves binding and synergistically improves cell permeability to increase antimicrobial activity against E. coli. We redesigned M. histrionica thanatin to truncate the sequence and remove the need for a disulfide bond. Our stapled peptide retained binding affinity to LptA, however potency was hampered. Despite seeing no improvement in antimicrobial activity, we present a novel scaffold for the next generation of thanatin-based AMPs. Next, we characterized thanatin against Pseudomonas aeruginosa, a known but weaker target of thanatin. We confirmed binding of P. maculiventris thanatin to LptH, the P. aeruginosa homolog of E. coli LptA, via BLI and isothermal titration calorimetry (ITC) and showed inhibition of P. aeruginosa strain RP73 via MIC assays. We used homology modeling and an E. coli model system to identify the resistance factor of thanatin to be LptH Y51 at the predicted binding interface. We attempted to overcome the hinderance of LptH Y51 by modeling thanatin to accommodate it. Our designs cooperated in the E. coli model system, however they did not translate to improve binding with LptH. Interestingly, we discovered that thanatin Y10 is essential to binding LptH. We applied the small library of thanatin orthologs to LptH and P. aeruginosa and did not discover any sequences with improved binding or antimicrobial activity. Our small library screening highlighted the necessity of thanatin Y10 and the resistance factor LptH Y51 again. We investigated the role of improved potency of thanatin with P. aeruginosa through c-amidation. Our E. coli model system shows a key rescued interaction between the c-amidated terminus of thanatin and LptA R76Q that mimics LptH. We crystallized and determined the structure of c-amidated truncated thanatin and LptA R76Q to gain insight on the interaction. However, we did not observe the hypothesized rescued interaction. When translating our findings to LptH, we did not observe improved binding due to c-amidation via BLI, but we did via ITC. Conflicting data about how thanatin interacts with LptH could be clarified with a high-resolution protein:peptide complex structure, however attempts to experimentally obtain one has been difficult. Overall, we provide some insight on the mechanism of how thanatin targets LptH in P. aeruginosa, but further studies will be needed to fully elucidate its mechanism of action. Collectively, this dissertation provides an example of how natural sources can be mined to uncover novel AMPs to target bacteria with MDR on the rise. We present various insights gained on the mechanism of action of thanatin by characterizing thanatin and its novel orthologs against E. coli LptA and P. aeruginosa LptH. The characterization of thanatin will allow for improved AMPs to be designed in the next generation of thanatin peptides to target pathogens.