Investigation into Enhancement of Lasso Peptide Antimicrobial Activity

Abstract

Since the development of the first antibiotics, the emergence of drug resistance in bacteria has been a pressing issue. In addition to acquiring antibiotic resistance mechanisms, several bacteria are also naturally resistant to various classes of antibiotics, further limiting the pool of antibiotics that are available to effectively treat infections. One group of bacteria with such clinical significance is the Burkholderia cepacia complex (Bcc). These bacteria tend to infect immunocompromised individuals and cystic fibrosis (CF) patients, causing severe symptoms such as pneumonia and sepsis that can turn fatal. Bcc bacteria are naturally resistant to several clinically approved antibiotics such as ampicillin and erythromycin, motivating the need for new antibiotics to be developed. Another group of clinically significant bacteria are the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). This group of bacteria causes nosocomial infections and are known for high levels of multidrug resistance, motivating the development of new antibiotic classes. Lasso peptides are natural products that are promising potential drugs with their lariat structure providing both thermal and proteolytic stability. Several have antimicrobial activity and work by targeting RNA polymerase (RNAP), an essential enzyme that is conserved amongst diverse bacteria but distinct from human RNAP. To take advantage of the antibiotic potential of lasso peptides, I have studied how their antimicrobial activity can be enhanced. Chapter 2 focuses on ubonodin, a lasso peptide with antimicrobial activity against the Bcc. I screen a library of >90,000 ubonodin variants for RNAP activity, leading to key insights on the structure-activity relationship of ubonodin. I also identify variants with enhanced activity compared to wild-type ubonodin against clinical isolates of the Bcc. In Chapter 3, I focus on enhancing the antimicrobial activity of the lasso peptides citrocin and microcin J25 by improving their uptake into susceptible bacteria. I conjugate a cell-penetrating peptide (CPP) to these lasso peptides and find that the conjugated compounds have a broader spectrum of activity than their lasso peptide counterparts, with potent activity against several laboratory strains and multi-drug resistant strains of the ESKAPE pathogens.