UCLA

The emergence of multi-drug resistant bacteria has prompted novel antibiotic development by targeting non-essential pathways, such as virulence factor production and display during cell wall biosynthesis. Within Gram-positive bacteria, sortase transpeptidases covalently attach proteins to the cell wall or assemble pili using class A-F enzymes. Interestingly, class E sortases display proteins via recognition of a non-canonical LAXTG motif. We have determined the first crystal structure of a class E sortase, the 1.93 � resolution structure of SrtE1 from Streptomyces coelicolor. The SrtE1 enzyme possesses structurally distinct β3/β4 and β6/β7 active site loops that contact the LAXTG substrate. Furthermore, molecular dynamics studies have identified a conserved tyrosine residue that likely confers substrate specificity for class E sortases. A second anti-virulence target, the TarA glycosyltransferase (GT), is highly conserved among Gram-positive bacteria and produces surface-anchored wall teichoic acid (WTA) polymers. The WTA biosynthetic mechanism involving TarA and other membrane-associated, enzymes is poorly understood due to a lack of structural characterization. We have determined the 2.0 � resolution crystal structure of the TarA enzyme from Thermobacter italicus, which adopts a structurally novel protein fold, termed class GT-E, and represents the first structurally characterized member of the WecB-TagA-CpsF GT family. Sequence conservation mapping onto experimentally observed TarA oligomer structures has identified putative functional residues and suggests formation of a competent active site through oligomerization, which will guide studies of substrate binding and catalysis. Furthermore, we describe two target-specific, cell-based assays for the discovery of sortase and TarA inhibitors. The first assay monitors sortase-dependent growth inhibition of wild-type and sortase-deficient Actinomyces oris strains in the presence of small molecule inhibitors. A pilot screen of 1280 compounds returned a hit rate of 0.3%, which has prompted large-scale high-throughput screening. The second assay utilizes a TarA-dependent morphological transition of a mutant B. subtilis strain complemented with the TarA enzyme from S. aureus (TarA+) to replace the endogenous enzyme activity. The drastic rod-shape to spherical morphological transition provides a robust HTS platform with a Z-prime score of 0.76. Ultimately, these structural and cell-based studies will promote the development of anti-virulence inhibitors to combat bacterial resistance.