Sialic acid is a widely distributed microbial virulence factor that is expressed on the surface of Neisseria meningitidis, Streptococcus agalactiae, Escherichia coli K1 and N. gonorrhoeae as capsular polysaccharide and/or terminal N-acetylneuraminic acid (NANA) residues attached to lipooligosaccharides. Sialic acids are expressed to circumvent human host defenses and to regulate contact-dependent interactions of the organisms with host cells. Despite the important role of sialic acids in microbial pathogenesis, little is known about the genetic regulation of expression of these molecules in humans. As a paradigm for this process, the investigator seeks to identify how synthesis of the sialic acid capsules of groups B, C, Y and W-135 N. meningitidis are regulated during human infection. The investigator proposes that transcriptional control of two operons involved in capsule biosynthesis and transport is a major regulatory mechanism of the sialic acid capsules of N. meningitidis, and that environmental conditions in the human host and/or human cell factors influence the regulation. In two specific aims, the investigator proposes to explore the genetic basis for expression and transcriptional regulation of sialic acid by meningococci.
In Aim I, the investigator will identify the promoters and regulatory elements of the 134bp intergenic region linking the divergent operons involved in CMP-NANA/sialic acid capsule biosynthesis and capsule membrane transport. This will be accomplished by transcriptional reporter gene constructs, and orchestrated mutagenesis of the intergenic region. Tn916 mutagenesis, in vitro mutagenesis and use of the Hermes plasmid, or E. coli complementation will then be used to identify transcriptional factors which regulate the biosynthesis and transport operons.
In Aim II, the investigator proposes to investigate the expression, regulation and role in pathogenesis of capsule genes during specific events (nasopharyngeal colonization, bloodstream invasion) observed for meningococci during infection. The investigator will first study the regulation of capsule genes during different in vitro environmental growth conditions that mimic those the meningococcus would likely encounter during infection or colonization (pH, iron limitation, Ca++, temperature, and anaerobiosis). The investigator will then expand these studies to human nasopharyngeal organ cultures and human serum. Direct assessment of capsular polysaccharide by ELISA and immunomicroscopic techniques, genetically-defined isogenic mutants, transcriptional reporter gene constructs, and quantitation of capsule gene mRNA will be used. These studies have direct application to the development of new meningococcal vaccine strategies and should provide important information about the regulation of sialic acid in other important bacterial and biologic systems.
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