Given the dearth of new antibacterial drugs and the facile evolution of microbial resistance, some fear that we have entered the “post-antibiotic era.” Our research focuses on both the development and application of new technologies to explore the mechanisms of bacterial growth and pathogenesis and the identification of potential therapeutic agents. One area of particular interest is the generation of tools for the study of two-component signal transduction systems (TCSs), which are commonly used by bacteria to couple environmental stimuli to adaptive responses through gene regulation. They contribute to many critical bacterial functions, including virulence, resistance mechanisms, and survival, making the TCS proteins potential drug targets. The high degree of homology in the ATP-binding site of histidine kinases, a critical player in TCSs, suggests that appropriately designed compounds could serve as probes for the global profiling of TCS activities. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P−N bond. We anticipated that use of a γ-thiophosphorylated ATP analogue, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, BODIPY-FL-ATPγS, labels active HK proteins and is competitive for the ATP binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification.
A second focus of the Carlson group is on understanding the enzymes involved in the biosynthesis of peptidoglycan (PG), a complex polymeric structure that is a major component of the bacterial cell wall and is essential for survival. PG is a common target for antibiotic therapy, but its structure and assembly are only partially understood. PG synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we generated fluorescent derivatives of the β-lactam-containing antibiotic cephalosporin C to enable selective examination of a subset of PBPs. These probes facilitated specific in vivo imaging of active PBPs in both Bacillus subtilis and Streptococcus pneumonia and revealed that even PBPs that are located at a particular site (e.g., septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Presently, we are working to generate probes with other selectivity profiles. Together, the described studies will lead to a more comprehensive understanding of bacterial growth and pathogenesis and may provide novel therapeutic targets and lead structures.
Prof. Raquel Lieberman (404-385-3663)