Bacterial Cell Surfaces: Exploiting Metabolic Pathways for Fundamental Understanding of Antibiotic Resistance and Growth

Abstract The worldwide problem of antibiotic resistance has become one of the most serious health threats of this century and will undoubtedly become top clinical priority. In the U.S. alone, antibiotic-resistant bacteria cause at least 2 million infections and 23,000 deaths a year resulting in a $55-70 billion per year economic impact. The rise in the number of resistant bacteria is mainly attributed to the improper and overuse of antibiotics, and the ability of these bacteria to evolve elaborate genetic systems to counteract pharmaceuticals. As the number of efficacious antibiotics continues to rapidly dwindle without replenishment, the possibility of entering a post-antibiotic era can become a reality. To combat this ever-growing threat, the Centers of Disease Control and Prevention (CDC) stress four core actions; (1) preventing infections from occurring and spreading, (2) tracking resistant bacteria, (3) improving the use of antibiotics, and (4) promoting the development of new antibiotics and new diagnostic tests for resistant bacteria. With those actions in mind, our goal is to rededicate efforts towards understanding fundamental bacterial physiology and pathology, with a special focus on mechanisms of cell wall growth and drug resistance. A clearer understanding of the molecular events underpinning antibiotic resistance phenotypes can be instrumental in guiding the design of next generation diagnostics, antibiotics, and therapeutics.The bacterial cell envelope is a vital extracellular component of prokaryotic cells, providing structural support and osmotic stability. Some of the most potent antibiotics in use today are molecules that inhibit the assembly of the bacterial cell wall. Despite the tremendous clinical importance of bacterial cell wall targeted antibiotics, it is surprising that several key aspects of cell wall biosynthesis and processing remain poorly characterized. A deeper understanding of the catalysis of cell wall associated enzymes will undoubtedly bestow researchers with the complemented ability to identify new antibiotic targets and antibiotics. Chapter 2 will discuss the technologies that have been developed to date to understand cell wall coordination. These molecular probes are designed for the understanding of enzymatic mechanisms and dynamics of biosynthesis pathways.The work discussed in Chapter 3 describes the initial efforts to combat antibiotic resistance, with the specific goal of developing a novel bacterial diagnostic assay. Diverse fluorescent D-amino acids were synthesized and incubated with different bacterial species. These D-amino acids containing C-terminus variations were shown to be incorporated into bacterial peptidoglycan with subtle differences within and between bacterial species. We show that the enzymes responsible for incorporation (transpeptidases) have remarkable flexibility in accepting unnatural D-amino acid derivatives. The incorporation profile has the potential to form the basis of a novel bacterial detection method.We next exploited the incorporation of unnatural D-amino acids for decoration of bacterial cell surfaces with tetrazine ligation handles, as discussed in Chapter 4. Such an approach can provide an alternative method of installing molecules of interest to the exterior of the cell. Peptidoglycan labeling of live bacteria through this ligation approach paves the way for future in vivo studies due to its non-toxic effects and proven biocompatibility, as mentioned in Chapter 5. In this section, we present the first evidence that bacteria remodel their PG with exogenous D-amino acids in a live host animal. These results suggest that extracellular D-amino acids may provide pathogens with a mode of late-stage in vivo cell-surface remodeling.The work discussed in Chapter 6 and 7, as opposed to previous methods, starts to explore and exploit the intracellular pathway of peptidoglycan biosynthesis. We developed a novel strategy aimed at hijacking peptidoglycan biosynthetic machinery to install specific reporter handles that track changes in cell wall composition. Described is a panel of synthetic cell wall precursor analogs that mimic substrates for vancomycin resistant-linked enzymes. Reporter handles were included at strategic points within these molecules to generate resistance-specific output signals. Monitoring cell wall alterations during drug evasion with temporal resolution revealed insight into adaptation dynamics and kinetics. In Chapter 8, we demonstrated that cell wall analogs can be unparalleled chemical probes in revealing key features of the cell wall crosslinking in live bacteria. We assessed the proficiency of two vital enzymes (D,D- and L,D-transpeptidases) with the goal of unraveling the interplay between these two modes of crosslinking. We showed how subtle structural modifications to the primary sequence of peptidoglycan can control crosslinking efficiency. Such probes may guide drug regimen and establish new drug targets.