Abstract
Bacterial resistance to antibiotics poses an immense threat to the public’s health and well-being. Nearly every new antibiotic discovered has been met with resistance, and alternative strategies to combat bacterial infections are necessary. Newly resistant bacterial strains spread when some of the cells survive antibiotic treatment, due to a mutation in their genetic makeup or acceptance of new genetic material. General resistance mechanisms involve decreased permeability into the cell or increased pumping of the antibiotic out of the cell, modification of the antibiotic target, or degradation of the antibiotic, rendering it ineffective. The Centers for Disease Control and Prevention has called for action to attempt to prevent the spread of infection and to improve the collection and distribution of information regarding antibiotic resistance. With the need for a better understanding of bacterial cell processes to implement more creative therapeutic approaches to circumvent resistance, the focus of this thesis is on the development of chemical reporters to study the substrate preferences of bacterial cell wall enzymes that are often the target of antibiotics due to their essentiality for cell survival.
Distinct classes of antibiotics and their targets will be discussed more deeply in Chapter 1, as well as specific methods of resistance that are developed by bacterial cells in the presence of β-lactam antibiotics. The details of the bacterial cell envelope (particularly the peptidoglycan) in Gram-negative, Gram-positive, and mycobacterial classes will be described, as this region is a large antibiotic target since it provides the cells with rigidity and prevents lysis due to external turgor pressure. Specifically, transpeptidase enzymes that crosslink the strands of the peptidoglycan layer in bacterial cells will be described, as well as β-lactam antibiotics, which target these enzymes to prevent cell wall crosslinking and thus result in cell death. To glean insight on the many cell wall processes that occur, significant efforts have been made in the field that rely on chemical reporters for metabolic labeling. Chapter 2 will describe the many recently discovered tools from the field to study metabolic processes of cell wall machinery (including those that label the peptidoglycan, glycans, LPS, teichoic acids, and glycoproteins).
Chapter 3 focuses on novel probes that are used by bacterial cells, exclusively as acyl-acceptor strands in peptidoglycan crosslinking, a process that is necessary for cell wall assembly. The critical nature of the cross-bridge on the PG peptide was demonstrated in live bacterial cells using fluorescently modified stem peptide mimics. The acyl-acceptor probes described provided insight on how chemical remodeling of the stem peptide cross-bridge could modulate cross-linking levels. These specifically acyl-acceptor probes supplemented the acyl-donor probes reported by our group previously, and we envisioned that together, they would provide a versatile platform to interrogate crosslinking in physiologically relevant settings.
Chapters 4 and 5 describe stem peptide probes that use facile chemistry and commercially available reagents to synthesize isosteres for meso-diaminopimelic acid (m-DAP). m-DAP synthesis is challenging due to the presence of internal symmetry and therefore requires the need for orthogonal protecting groups to retain that symmetry. m-DAP is not commercially available, thus limiting the probes available to study bacterial cells that contain m-DAP in their peptidoglycan. To meet this limitation, we developed meso-cystine (m-CYT), a cystine-based m-DAP mimetic that retains the structural features of m-DAP but is one atom longer and is formed by disulfide exchange chemistry, and meso-selenolanthionine (SeLAN), a lanthionine analogue that uses selenoether rather than thioether connectivity. These m-DAP mimetics were incorporated into tripeptides mimicking the natural stem peptide found in bacterial peptidoglycan and modified with fluorescent handles to show their incorporation in live bacterial cells via transpeptidase processing.
In Chapter 6, a novel assay platform (“SaccuFlow”) is discussed that preserves the native structure of bacterial peptidoglycan and is compatible with high-throughput flow cytometry analysis. We show the feasibility of isolating sacculi from Gram-positive, Gram-negative, and mycobacterial organisms and subsequently analyzing it using flow cytometry. This platform is intended to serve to access information about how molecules or proteins interact with peptidoglycan, as typical methods that interrogate these interactions are complicated and qualitative, rather than quantitative. Additionally, we show how this assay can be used in a high-throughput example to identify potential inhibitors of sortase A from Staphylococcus aureus, an enzyme of interest as it relates to covalently attaching virulent proteins on the bacterial cell surface.
Lastly, Chapter 7 covers the use of stem peptide mimetic probes modified with a near-infrared (NIR) fluorophore to visualize gut bacteria in a living organism. We sought to image bacterial cells in an in vivo murine model, as the development of methods to monitor gut microbiota can lead to improved foundational understanding of the biological events, underpinning the interactions between gut commensals and the host. We envisioned that this strategy of labeling bacterial cells may enable the analysis of cell wall turnover, which has implications for bacterial cellular growth and division, in a live animal.
