Neisseria gonorrhoeae is one of the two major pathogens involved in the majority of cases of sexually transmitted genital infection. Complement forms an important aspect of the innate immune system that impacts upon gonococcal infection. Prior work in our laboratory has shown that sialylation of gonococcal lipooligosaccharide (LOS) results in complement resistance by binding the host complement regulatory molecule, factor H. The porin molecule also binds factor H. In the first Specific Aim, we will investigate the role of an alternatively-spliced version of factor H, called factor H-like molecule 1 (FHL-1)in binding to gonococci and in regulating complement. Some strains of N. gonorrhoeae process complement (i.e. convert complement component-3 [C3b] to the inactivated form [iC3b]) and bind FHL-1, but not factor H. Cofactor activity of FHL-1 will be assessed using serum containing only FHL-1, but not intact factor H. Because both factor H and FHL-1 bind to cells, we will examine the roles of these two molecules in facilitating gonococcal attachment to immortalized cervical and urethral epithelial cells, in the second Specific Aim, three questions that pertain to LOS sialylation will be addressed. First, the specificity of factor H binding to gonococcal lacto- N-neotetraose (LNT) sialic acid, but not to meningococcai LNT sialic acid, will be examined. Porin (Por) influences binding of fH to gonococcal sialic acid. We will perform allelic exchange of porin molecules between the two species to examine the effect of porin on the sialylated LOS interactions with factor H that differs at baseline in the two neisserial species. Second, we will examine the determinants of the functional specificity of the LOS sialyltransferase (IsO enzyme in meningococci and gonococci by performing allelic exchanges of the Ist genes between the two species. Third, we will also determine why the efficiency of LOS sialylation differs between serum-sensitive (high sialic acid uptake) and "stably" serum-resistant (low sialic acid uptake) gonococci. This will be performed by examining the uptake of 3H-labeled CMP-NANA by isogenic gonococci differing only in their Lst enzymes. The possibility that Por modifies Lst activity will also be examined by performing allelic exchanges of the Por genes between high and low sialic acid incorporators. In the third Specific Aim, we will detail the linkages (amide versus ester) between C4 and LOS, the effects of hexose extension of the LOS on binding of C4 to LOS, and the impact of the bond formed between C4 and LOS on bactericidal killing.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI032725-19
Application #
8239539
Study Section
Special Emphasis Panel (NSS)
Program Officer
Hiltke, Thomas J
Project Start
1993-01-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
19
Fiscal Year
2012
Total Cost
$666,562
Indirect Cost
$261,357
Name
University of Massachusetts Medical School Worcester
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
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Lewis, Lisa A; Carter, Matthew; Ram, Sanjay (2012) The relative roles of factor H binding protein, neisserial surface protein A, and lipooligosaccharide sialylation in regulation of the alternative pathway of complement on meningococci. J Immunol 188:5063-72

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