Structural, Biochemical, and Cellular Studies of TarA, the Novel Wall Teichoic Acid Glycosyltransferase, for the Discovery of Gram‐positive Bacterial Inhibitors

Antibiotic‐resistant bacteria, such as methicillin‐resistant Staphylococcus aureus (MRSA), are rapidly countering treatment options, demonstrating a considerable need to explore novel targets for drug development. The wall teichoic acid (WTA) biosynthetic enzymes are promising anti‐virulence targets that produce and display WTA on the Gram‐positive cell surface. Accounting for >50% of the cell wall content, WTA functions in regulation of cell morphology and cell division, as well as cell adhesion and pathogenicity. In fact, deletion of the WTA glycosyltransferase, TarA, renders S. aureus avirulent, and MRSA strains lacking WTA polymers become sensitized to β‐lactam antibiotics. This study focuses on TarA, the novel, membrane‐associated glycosyltransferase that constructs the highly conserved WTA linkage unit in Gram‐positive bacteria. We first identified the membrane‐anchoring feature of TarA, which when truncated, shifts partitioning into the soluble fraction of E. coli cell lysate. Limited proteolysis of the S. aureus TarA (SaTarA) extracytoplasmic domain (ECD) reveals a protease‐susceptible loop connecting two stable polypeptides; cleavage is delayed when incubated with substrate mimics, suggesting that the loop may play a role in substrate binding. A thermophilic homolog of the TarA ECD is more resistant to proteolytic cleavage, suggesting improved stability. In fact, the thermophilic TarA ECD‐ligand complex crystallizes under a range of conditions, which has produced a 3.1 angstrom resolution native diffraction dataset. Optimization of heavy metal derivative crystals is underway to complete de novo phasing and structure determination. To demonstrate that WTA linkage unit assembly is a highly conserved process among Gram‐positive bacteria, we disrupted WTA production through knockout of the endogenous TarA enzyme within B. subtilis ( tarA‐ ), which produces a drastic shift from rod‐shaped to spherical morphology; a similar strain produced by Brown et al. revealed a similar morphological shift that correlated with a lack of WTA on the cell surface. Interestingly, subsequent complementation of the tarA‐ strain with the SaTarA enzyme re‐establishes the wild type B. subtilis morphology, indicating that WTA production is restored. The morphological dependency of B. subtilis on SaTarA activity will be exploited to identify important functional residues through mutation. Confocal microscopy studies of B. subtilis expressing mCherry‐tagged, full‐length and truncation constructs of SaTarA are underway to track cellular localization and determine the effect of membrane localization on enzyme activity. In addition, the SaTarA‐complemented tarA‐ strain is well‐suited for high throughput screening (HTS); by monitoring the drastic morphological change of B. subtilis upon inactivating the TarA enzyme, we have established a robust, cell‐based HTS assay with a Z′ score of 0.76 to pursue pilot screening. Ultimately, this work facilitates the discovery and design of novel inhibitors towards Gram‐positive bacteria to address the urgent need for novel treatments to combat antibiotic resistance.