Extraintestinal pathogenic Escherichia coli (ExPEC) are major pathogens in mostly the very young, aged or immunocompromised human population resulting in tens of thousands of deaths each year and billions of dollars in healthcare costs. For example, the predominant ExPEC subtype, E. coli O18:K1, expresses O18 somatic and K1 polysialic acid capsule antigens as two major virulence factors responsible for making these strains the leading causes of neonatal bacterial meningitis. Using microbial genetics and innovative methods of carbohydrate analysis, we defined the complete pathways for sialic acid transport, synthesis, polymerization, catabolism and modification of both monomeric and polysialic acids. The modification mechanism was linked to a novel bacterial virus (CUS-3) carrying the phase-variable acetylase gene catalyzing stochastic O-acetylation of the sialic acid exocyclic chain. We hypothesize that understanding the in vivo functions of these bacterial antigens will facilitate approaches targeting them for new therapeutic development. We have designed three specific aims to test this hypothesis: 1) Determine the contribution of monomeric sialic acid O- acetylation to overall polysialic acid modification by genetically altering the acetyltransferase, NeuD, and acetylesterase, NeuA* (NeuA-star).
This aim focuses on the enzymes involved in the reciprocal addition (NeuD) or subtraction (NeuA*) of O- acetyl esters to or from monomeric sialic acids prior to their incorporation into polysialic acid. 2) Determine the dynamics of capsular polysialic acid modification in vivo using an innovative flow cytometric technique and microscopic methods to distinguish between acetylated and unacetylated phases in different host compartments. 3) Determine the molecular basis for coupling polysaccharide synthesis to export by (i) constructing in frame deletions of all region 1 export genes, (ii) determining whether 3-deoxy-D-manno- octulosonate is required for biosynthesis, (iii) identifying the polymerase domain(s) interacting with the accessory protein, KpsC, (iv) using Quantum-dot technology and K1- specific phage to determine the site of capsule export, and (v) determining the chemical structure of the initiation complex.
Extraintestinal pathogenic Escherichia coli (ExPEC) causes tens of thousands of deaths in the United States each year, and is associated with billions of dollars in healthcare costs. The increasing incidence of antibiotic resistance coupled with the lack of safe and effective vaccines or other drug therapies supports research effort directed toward identification of bacterial targets for new therapeutic development. Our renewal application is focused on the acetylated form of the E. coli K1 polysialic acid capsular polysaccharide, present in all of the most virulent K1 strains, and the fundamental machinery for capsular polysaccharide biosynthesis producing the major virulence factor in the majority of ExPEC strains.
|Steenbergen, Susan M; Vimr, Eric R (2013) Chromatographic analysis of the Escherichia coli polysialic acid capsule. Methods Mol Biol 966:109-20|
|Vimr, Eric R; Steenbergen, Susan M (2009) Early molecular-recognition events in the synthesis and export of group 2 capsular polysaccharides. Microbiology 155:9-15|
|Steenbergen, Susan M; Lee, Young-Choon; Vann, Willie F et al. (2006) Separate pathways for O acetylation of polymeric and monomeric sialic acids and identification of sialyl O-acetyl esterase in Escherichia coli K1. J Bacteriol 188:6195-206|
|Vimr, Eric R; Steenbergen, Susan M (2006) Mobile contingency locus controlling Escherichia coli K1 polysialic acid capsule acetylation. Mol Microbiol 60:828-37|
|Steenbergen, Susan M; Lichtensteiger, Carol A; Caughlan, Ruth et al. (2005) Sialic Acid metabolism and systemic pasteurellosis. Infect Immun 73:1284-94|
|Deszo, Eric L; Steenbergen, Susan M; Freedberg, Daron I et al. (2005) Escherichia coli K1 polysialic acid O-acetyltransferase gene, neuO, and the mechanism of capsule form variation involving a mobile contingency locus. Proc Natl Acad Sci U S A 102:5564-9|
|Steenbergen, Susan M; Vimr, Eric R (2003) Functional relationships of the sialyltransferases involved in expression of the polysialic acid capsules of Escherichia coli K1 and K92 and Neisseria meningitidis groups B or C. J Biol Chem 278:15349-59|
|Kalivoda, Kathryn A; Steenbergen, Susan M; Vimr, Eric R et al. (2003) Regulation of sialic acid catabolism by the DNA binding protein NanR in Escherichia coli. J Bacteriol 185:4806-15|
|Ringenberg, Michael A; Steenbergen, Susan M; Vimr, Eric R (2003) The first committed step in the biosynthesis of sialic acid by Escherichia coli K1 does not involve a phosphorylated N-acetylmannosamine intermediate. Mol Microbiol 50:961-75|
|Gonzalez, Mark D; Lichtensteiger, Carol A; Caughlan, Ruth et al. (2002) Conserved filamentous prophage in Escherichia coli O18:K1:H7 and Yersinia pestis biovar orientalis. J Bacteriol 184:6050-5|
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