Principal Investigator Laura Kiessling
The need for new antibiotics is dire. Pathogenic microbes use a suite of monosaccharide building blocks absent from mammalian cells to build their glycans. Enzymes that process these glycan building blocks and use them represent unmined targets. We are investigating the molecular mechanisms underlying the synthesis of microbial glycans and using this knowledge to seek small molecules that can function as new antibiotic leads. We are especially interested in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), which is responsible for over 1 million fatalities each year. Many of the most successful antibiotics used for the treatment of TB target the cell envelope. Our research aims to understand how different layers of the envelope are constructed, dissect the importance of these layers to bacterial physiology, identify new diagnostics, and new target pathways to identify next-generation antibiotics.
Probing cell wall biosynthesis -- Mycobacteria possess a mycolic acid membrane that is a barrier to antibiotics. The mycolic acids and arabinan, which tethers the mycolic acids to the bacterial surface, are validated targets of clinically useful antibiotics. Still, a molecular level understanding of envelope assembly is lacking. Chemical probes of the assembly of these structures would be valuable. We are developing agents to understand how and when key cell wall polysaccharides, the arabinan and galactan, are synthesized. We are especially interested in how the galactan is synthesized and how its length is controlled. Understanding polysaccharide length and sequence control is intriguing, as the process template independent. We have studied the carbohydrate polymerase GlfT2. We determined that GlfT2 is a processive polymerase with an intrinsic ability to control length and sequence. To understand the role of galactan length in mycobacteria, we generated mutants with truncated galactan. These agents are yielding surprising insights into how chain length impacts bacterial survival and physiology.
UGM inhibitors -- The galactan is essential for mycobacteria, yet no clinically used antibiotics target galactan biosynthesis. We took advantage of this untapped potential by developing potent inhibitors of the enzyme UDP-galactopyranose mutase (UGM also referred to as Glf). UGM catalyzes the interconversion of UDP-galactopyranose and UDP- galactofuranose, reaction required for any organism that generates glycans with galactofuranose. We showed the UGM uses a surprising catalytic mechanism: The enzyme’s flavin cofactor engages in covalent catalysis (Soltero-Higgin). This catalytic mechanism had not been previously observed. In conjunction with our knowledge of UGM catalysis, we employed high throughput screening, X-ray crystallography, and computer-based docking screens to identify UGM inhibitors (Kincaid). These inhibitors can block mycobacterial cell growth. Intriguingly, some analogs are also effective against nematode worms, another class of important human pathogens.
Visualizing Mycobacterial Cell Division -- Watching enzymes in action as bacteria build their cell walls can lead to new strategies to identify and treat them. The field’s ability to monitor bacterial cell growth is generally limited to peptidoglycan assembly, an important part of the cell wall that is found throughout bacteria. We generated a new probe that can be used to follow the assembly of the cell wall of mycobacteria (Hodges, Brown). This probe is processed by an enzyme complex called Ag85, which is present in mycobacteria (but not bacteria like E. coli). Ag85 is essential for building the mycobacterial cell wall, so understanding how and when it is active could lead to new antibiotic strategies. To our knowledge, this probe is the first bacterial imaging probe that enables real-time, continuous monitoring of mycobacteria. Using QTF, we found that Ag85 activity increases prior to cell division. Although Ag85 is abundantly secreted, its activity is localized to the growing cell wall. Because the cell wall of mycobacteria is different than that of other bacteria, this new probe also has the properties needed for a diagnostic agent. We therefore anticipate QTF can be exploited to detect and monitor mycobacteria.