Although Neisseria gonorrhoeae (Ng) and Neisseria meningitidis (Nm) cause very different diseases, they share the ability to covalently add sialic acid to their lipooligosaccharide (LOS). Sialylation of LOS leads to decreased association with host cells, and to resistance to phagocytic killing and serum killing. Therefore, sialylation is considered a major virulence factor. However, Ng and Nm are found within host cells during disease. In addition, they are not exposed to serum components during initial infection or carriage. Thus, there are times or conditions when the neisseria either needn't be sialylated (because it would be a waste of energy) or may not want to be sialylated (because they want to enter host cells). Thus, there is an apparent paradox. One way to explain this is to suggest that LOS sialylation is regulated by environmental signals in vivo. Dr. Rest has observed that Ng express more constitutive stase than Nm, and that the two organisms have very different DNA sequences upstream of their respective stase (lst) genes, which leads to differential expression at the transcriptional level. Also, Ng and Nm express up to 10 times or more stase activity when grown in broth than when grown on plates. Stase expression is also regulated by the absence of iron or the presence of nitrite. He would like to further define the regulation of stase expression in Ng and Nm, in vitro and in vivo. Toward this goal, he proposes the following specific aims: 1. Investigate the expression of stase by Ng and Nm in vitro. A. Perform mutational and deletional analyses. B. Investigate expression in various Ng and Nm regulatory mutants, including PilA/PilB, Fur, FNR, NarL/NarQ, BasS/BasR, DegS-DegU and IHF. C. Determine if the newly defined 105 bp repeat is involved in stase expression. D. Determine the role of the above systems in regulation of stase expression by environmental signals. 2. Investigate the expression of stase by Ng and Nm in vivo. A. Determine stase expressed by Ng or Nm in contact with human epithelial or endothelial cells, neutrophils or serum. Investigate the role of extracellular vs intracellular residence. B. Use the regulatory mutants mentioned in Aim 1 as tools to investigate the mechanisms of in vivo lst regulation. 3. Determine whether stase is expressed on the outer or inner surface of the outer membrane of Ng and Nm. 4. Investigate whether LOS sialylation modulates serum resistance and association with host cells by DGI (disseminated gonococcal infection; stable serum resistant) strains.
