Biosynthesis and Modification of Helicobacter pylori Lipid A

Hdl Handle:
http://hdl.handle.net/10675.2/319916
Title:
Biosynthesis and Modification of Helicobacter pylori Lipid A
Authors:
Stead, Christopher Michael
Abstract:
The secondary acylation steps of Helicobacter pylori lipid A biosynthesis are poorly understood because H. pylori only has one homolog (Jhp0265) to the Escherichia coli secondary acyl transferases LpxL and LpxM. Jhp0265 was shown to be responsible for the transfer of a secondary C18 acyl chain to the 2′-linked acyl chain of lipid A, making Jhp0265 homologous to LpxL. An activity was also demonstrated for the addition of a secondary acyl chain to the 3′-linked acyl chain of H. pylori lipid A, although the enzyme responsible for the transfer remains unknown. After synthesis, H. pylori lipid A is modified by the action of five enzymes. Mutation of the candidate modification enzyme Jhp0634 demonstrated that the enzyme catalyzes the removal of the 3′-linked acyl chains of H. pylori lipid A, producing a tetra-acylated lipid A species. Continuing with the characterization of H. pylori lipid A modification enzymes, we were also able to demonstrate an activity for a Kdo trimming enzyme in vitro. Requirement for a Kdo hydrolase in vivo was confirmed after the Kdo transferase of H. pylori was shown to be bifunctional despite the presence of only one Kdo sugar in H. pylori lipopolysaccharide. Attempted identification of the Kdo hydrolase revealed that both Hp0579 and Hp0580 were required for the removal of the Kdo sugar, which occurred in the periplasm. A Kdo hydrolase mutant revealed two unexpected phenotypes related to interaction with the innate immune system. The first was an increased sensitivity to cationic antimicrobial peptides, which was explained by a downstream effect on modification to the 4′- phosphate group of lipid A. The second phenotype related to the expression of Oantigen on the bacterial cell surface. The Kdo hydrolase mutants produced a reduced amount of fully extended lipopolysaccharide and conversely, an increased amount of core-lipid A. The type of O-antigen epitope displayed was also affected by a Kdo hydrolase mutation, in a strain specific manner.
Affiliation:
Department of Biochemistry and Molecular Biology
Issue Date:
May-2010
URI:
http://hdl.handle.net/10675.2/319916
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/288329298?accountid=12365
Type:
Dissertation
Language:
en
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorStead, Christopher Michaelen
dc.date.accessioned2014-06-06T16:45:59Z-
dc.date.available2014-06-06T16:45:59Z-
dc.date.issued2010-05-
dc.identifier.urihttp://hdl.handle.net/10675.2/319916-
dc.description.abstractThe secondary acylation steps of Helicobacter pylori lipid A biosynthesis are poorly understood because H. pylori only has one homolog (Jhp0265) to the Escherichia coli secondary acyl transferases LpxL and LpxM. Jhp0265 was shown to be responsible for the transfer of a secondary C18 acyl chain to the 2′-linked acyl chain of lipid A, making Jhp0265 homologous to LpxL. An activity was also demonstrated for the addition of a secondary acyl chain to the 3′-linked acyl chain of H. pylori lipid A, although the enzyme responsible for the transfer remains unknown. After synthesis, H. pylori lipid A is modified by the action of five enzymes. Mutation of the candidate modification enzyme Jhp0634 demonstrated that the enzyme catalyzes the removal of the 3′-linked acyl chains of H. pylori lipid A, producing a tetra-acylated lipid A species. Continuing with the characterization of H. pylori lipid A modification enzymes, we were also able to demonstrate an activity for a Kdo trimming enzyme in vitro. Requirement for a Kdo hydrolase in vivo was confirmed after the Kdo transferase of H. pylori was shown to be bifunctional despite the presence of only one Kdo sugar in H. pylori lipopolysaccharide. Attempted identification of the Kdo hydrolase revealed that both Hp0579 and Hp0580 were required for the removal of the Kdo sugar, which occurred in the periplasm. A Kdo hydrolase mutant revealed two unexpected phenotypes related to interaction with the innate immune system. The first was an increased sensitivity to cationic antimicrobial peptides, which was explained by a downstream effect on modification to the 4′- phosphate group of lipid A. The second phenotype related to the expression of Oantigen on the bacterial cell surface. The Kdo hydrolase mutants produced a reduced amount of fully extended lipopolysaccharide and conversely, an increased amount of core-lipid A. The type of O-antigen epitope displayed was also affected by a Kdo hydrolase mutation, in a strain specific manner.en
dc.language.isoenen
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/288329298?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.-
dc.subjectH. pylorien
dc.subjectlipid Aen
dc.subjectKdoen
dc.subjectAcyltransferaseen
dc.subjectLipopolysaccharideen
dc.subjectouter membraneen
dc.titleBiosynthesis and Modification of Helicobacter pylori Lipid Aen
dc.typeDissertationen
dc.contributor.departmentDepartment of Biochemistry and Molecular Biologyen
dc.description.advisorTrent, Stephenen
dc.description.committeeCaughman, Gretchen; Liu, Kebin; Martin, Scott; Thompson, Stuarten
dc.description.degreeDoctor of Philosophy (Ph.D.)-
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