Broad Institute Inhibition of Glycoprotein Biosynthesis in Gram-Negative Pathogens Inhibitor Probe Project
N- and O-linked glycoproteins including the modified sugar building block di-N-acetylbacillosamine (diNAcBac) feature on the cell surface of many Gram-negative pathogens. Despite the strong association between these cell surface glycoconjugates and virulence, there are currently no small molecule inhibitors for acutely perturbing glycoprotein biosynthesis in microbial pathogens. These studies more ..
Barbara Imperiali,MIT,Cambridge, MA,imper@MIT.EDU,617.253.1838
N- and O-linked glycoproteins including the modified sugar building block di-N-acetylbacillosamine (diNAcBac) feature on the cell surface of many Gram-negative pathogens. Despite the strong association between these cell surface glycoconjugates and virulence, there are currently no small molecule inhibitors for acutely perturbing glycoprotein biosynthesis in microbial pathogens. These studies seek to establish a "first in class" probe set to target prokaryotic glycoprotein synthesis. A high-throughput screening strategy is implemented to identify potent and selective inhibitors of PglD, an acetyl-CoA-dependent acetyl transferase that catalyzes the final step in the conversion of UDP-GlcNAc into UDP-diNAcBac in the N-linked protein glycosylation pathway of Campylobacter jejuni. Since UDP-diNAcBac is an essential intermediate in the pathway that ultimately affords bacterial cell-surface N-linked glycoproteins, small-molecule inhibitors would represent valuable chemical tools for elucidating the fundamental roles of highly modified saccharides in microbial pathogenesis and potential leads in the development of novel therapeutic targets.
PglD, glycoproteins, anti-bacterial
Cell surface glycoconjugates, from many medically relevant Gram-negative bacterial pathogens, have been characterized in molecular detail and found to be important for host-dependent virulence and pathogenicity. Recently, major progress in the complete genome sequencing of microbial pathogens, together with the application of bioanalytical tools and bioinformatics has brought into focus the prevalence of unusual, highly modified and prokaryote-specific saccharides as constituents of the glycan structures that decorate bacterial glycoproteins. Of particular relevance here is di-Nacetylbacillosamine (diNAcBac), which is a member of a class of highly modified sugars known as the 2,4-diacetamido-2,4,6-trideoxyhexoses (DATDHs). DATDHs feature in the N- and O-linked glycoproteins and lipopolysaccharides of bacterial glycoconjugates of many microorganisms, including the human pathogens N. meningitidis, N. gonorrhoeae, Pseuodomonas aeruginosa, Vibrio cholera, A. baumanii, and C. jejuni. Recently, studies to investigate the functional consequences of perturbing glycoprotein biosynthesis in selected pathogens have established direct connections between glycoprotein biosynthesis and virulence in targeted hosts. In particular, significant developments in understanding the importance of N- and O-linked glycoproteins have come from studies on C. jejuni, N. gonorrhoeae and N. meningitides. These pathogens biosynthesize highly modified saccharides that are integrated into virulence-associated O- and/or N-linked glycoproteins. Both C. jejuni and C. coli are among the most frequently isolated causes of human acute bacterial enteritis worldwide. C. jejuni is also the infectious agent most often associated with Guillain-Barre syndrome, which is an acute demyelinating polyneuropathy characterized by an immunologic attack upon peripheral nerve myelin. Both O- and N-linked glycoproteins are found in C. jejuni and this was the first pathogen identified to include a general N-linked protein glycosylation system (termed the "pgl pathway").
Evidence for the connection between N-linked glycoprotein biosynthesis and pathogenicity in C. jejuni comes from complementary in vitro and in vivo studies that explored the effect of perturbing glycoconjugate biosynthesis. The phenotypes for genetically impaired N-linked glycosylation in C. jejuni are a reduction in cell adhesion resulting in the inability to interact with epithelial cells in vitro, and reduced epithelial cell adhesion and invasion and subsequent colonization in vivo in chick and mouse animal models. In particular, mutation of key enzymes in the N-linked glycosylation pathway, including the early UDP-diNAcBac biosynthesis enzymes, significantly diminished the ability of C. jejuni to adhere to and invade INT407 cells in vitro and to colonize the intestinal tracts of mice in vivo, providing direct evidence for the relationship between C. jejuni infection and protein glycosylation. In addition to phenotypic studies, high-resolution magic angle spinning NMR (HR-MAS-NMR) analysis revealed that mutagenesis of the genes in the pgl gene cluster including those encoding the early enzymes (PglF, PglE and PglD) involved in the conversion of UDP-GlcNAc into UDP-diNAcBac, caused a complete suppression of the biosynthesis of N-linked glycoproteins in the microbe. Additionally, it was found that mutation of the pglE and pglD genes resulted in a loss of C. jejuni colonization in the baby chick model of animal infectivity. In separate studies, a C. jejuni pglE (aminotransferase) knockout mutant was found to be impaired for flagella-mediated motility, intestinal epithelial cell invasion and persistence in the chicken intestine, again establishing involvement of UDP-diNAcBac biosynthesis in host colonization and virulence. Recent studies also reveal that C. jejuni N-glycosylated proteins interact with the human macrophage-type lectin (MGL) via the terminal N-GalNAc of the N-linked glycan mediating adherence to host cells and suppressing IL-6 production.