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In previous work, a factor which enhances the ability of cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) to sialylate gonococcal lipopolysaccharide (LPS) was liberated at 4 degrees C in diffusates from high M(r) fractions of blood cell sonicates. The diffusates also contained CMP-NANA and converted serum susceptible gonococci to resistance. The enhancer has now been separated from CMP-NANA and material absorbing at 260 nm by HPLC on mu Bondapak-10 NH2. Resistance inducing activity was found only in fractions containing CMP-NANA and recovery was poor (about 25%). However, addition of enhancer fractions to CMP-NANA substantially increased its resistance inducing activity. Blood cell sonicates dialysed at 18-20 degrees C released enhancer in diffusates. These were ultrafiltered (nominal cut off 3000 Da) and fractionated on Biogel P2 which removed saccharides and most material absorbing at 260 nm. Over 90% of a fraction which was enhancer-active in nanogram quantities was identified by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectometry (GC/MS) as lactic acid. A fraction with similar properties was obtained from a different batch of diffusate by fractionation on Dowex 1. Authentic lithium L-lactate in nanogram quantities enhanced LPS sialyation by CMP-NANA and increased its serum resistance inducing activity. These results have important implications for gonococcal pathogenicity.

Serum resistance of gonococci in most patients is due to sialylation of a Gal beta 1-4GlcNAc group on a conserved 4.5 kDa lipopolysaccharide (LPS) component by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) catalysed by a gonococcal sialyl transferase. This sialylation is enhanced by a low M(r) factor(s) which, like CMP-NANA, is released in diffusates from high M(r) fractions obtained from sonicates dialysed at 4 degrees C. Also, as shown here, this factor(s) is released when the sonicates are dialysed at 18-20 degrees C. The enhancement of sialylation, first demonstrated using enzymes in gonococcal extracts, has been shown to occur in live gonococci and hence probably to have a role in pathogenicity. Gonococci, emerging from lag phase and incubated for 2 h with CMP-14CNANA fixed up to 90% more radiolabel than controls when the second factor(s) was present; their LPS separated by SDS-PAGE contained more radiolabel than control samples and label was not detected in any other component. Fractions with enhancing activity absorbed maximally at about 260 nm but a mixture of UDP-galactose (UDP-Gal), UDP-N-Acetyl galactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc) and UDP-N-Acetyl glucosamine (UDP-GlcNAc) showed no significant enhancing activity. The enhancing action of the low M(r) fractions was unaffected by incubation with beta-galactosidase.

In previous work, a low M(r) component from human blood which converts serum-sensitive gonococci to resistance was shown to be indistinguishable from cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) by seven criteria. However, the presence of CMP-NANA was not proved by physicochemical methods. Purified, high M(r) fractions from human blood cells, which confer serum resistance on gonococci and enhance the transfer of sialyl groups from CMP-NANA to lipopolysaccharide (LPS) by a sialyltransferase in gonococcal extracts, were rechromatographed on DEAE Sepharose CL-6B. Both activities co-eluted from the column but on dialysis were found in the diffusate. After desalting the diffusate with Sephadex G10, the presence of CMP-NANA was proved by mass spectrometry. This confirmed previous work and is the first unequivocal demonstration of CMP-NANA in constituents of human blood cells.

A high relative molecular mass (M(r)) component which confers serum resistance on gonococci has been purified about 300-fold from a dialysed sonicate of human blood cells. Serum resistance conferred by the high M(r) factor (RIF), like that induced by cytidine-5' monophospho-N acetyl neuraminic acid (CMP-NANA), decreased when gonococci were incubated with neuraminidase. Also, the resistance-inducing activities of both high M(r) RIF and CMP-NANA were inhibited by CMP and inactivated at pH 4.0. These activities were not additive but synergistic. Neuraminidase decreased the activity of high M(r) RIF but not CMP-NANA. In tests with 14C CMP-NANA and gonococcal lipopolysaccharide, no sialyltransferase activity was detected, even in highly active samples of high M(r) RIF under conditions in which low activities of rat liver sialyltransferase were readily detected. Conversely, rat liver sialyltransferase was neither active in the RIF assay nor able to enhance the RIF activity of CMP-NANA. Nevertheless, high M(r) RIF greatly enhanced the sialyltransferase activity of a gonococcal extract; this enhancement suggests an explanation for the synergism between CMP-NANA and high M(r) RIF in inducing serum resistance in gonococci.