This competing continuation application proposes to continue studies of how lipooligosaccharide (LOS) mimicry of human glycosphingolipids (GSL) enable Neisseria gonorrhoeae to infect both men and women. LOS are involved in attachment to and invasion of epithelial cells and in evasion of clearance mechanisms. One highly conserved LOS has the same lacto-N-neotetrose (LacNAc beta1- >3Lac) glycose moiety as paragloboside-series GSL. Other glycose structures are shared with those of ganglio- (GalMAc beta1->3Gal-R), globo- (Gal alpha1->Lac-R or P series), muco- (Gal beta1->Lac-R) and lacto-series (Lac-R) GSL. The internal Gal residue of lacto-N-neotetrose is a """"""""toggle switch""""""""; i.e., the glycose that is linked to it determines the structure of the mature LOS. A second Gal linked either alpha or beta stops further chain elongation. The terminal lacto-N-neotetrose Gal is a second biosynthetic decision point; it may be unsubstituted, adorned with sialic acid, or substituted with beta-galactosamine. Gonococci sialate paraglobosyl LOS during infection by """"""""borrowing"""""""" sialic acid (NANA) of human origin. Sialylation protects gonococci from lysis by complement, retards their killing by polymorphonuclear leukocytes (PMNs) and enhances their ability to invade female genital epithelial cells. Addition of a terminal GalNAc to paraglobosyl LOS provides a binding site for lytic IgM molecules in human sera. Gonococci shed during gonorrhea make even larger LOS. These high Mr LOS terminate with the same GSL-like glycose sequences. The investigator now wants to know how these biosynthetic """"""""choices"""""""" affect the organism's ability to infect the cervix. He will use mass spectrophotometric techniques to determine the structure of the high Mr LOS made by MS11[mk]C at the onset of urethral leukorrhoea. He particularly wants to know whether these LOS have two GSL-like antennae, as this would provide closely apposed terminal sugar ligands that could cross-link epithelial receptors. Gonococci within PMNs are sialylated; cervical epithelial cells make a number of glycosyl transferases that could also alter LOS during invasion. He will compare the structures of LOS on gonococci within cervical cells with those on bacteria shed from the male urethra. He will use a library of monoclonal antibodies that bind each LOS structure, laser desorption mass spectrometry, and female genital epithelial cells (HEC-1-B) in tissue culture.
His final aim i s to use isogenic mutants that lack the ability to add Gal to the internal Gal of lacto-M-neotetrose and that cannot attach NANA or GalNac to the lacto-N-neotetrose terminal Gal, to find out how these LOS structures facilitate entry into epithelial cells. He will also assess the mutants' abilities to stimulate HEC-1-B cells to make IL-6, IL-8, IL- 10 and sialyl transferase. These studies may make it possible to devise ways to prevent LOS-dependent gonococcal infection.
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