N. J. Parsons

Morphological, biological and antigenic properties of Neisseria gonorrhoeae adapted to growth in guinea-pig subcutaneous chambers.

Gonococci (strain BS3) passaged three times and harvested directly from plastic chambers implanted subcutaneously in guinea pigs were compared with the parent strain (BS) grown in vitro. The strain grown in vivo produced smaller colonies than that grown in vitro and when examined directly in chamber fluid was sometimes not pilated. It was more resistant to the bactericidal action of human serum and more infective for guinea-pig chambers. In gel diffusion, extracts of the organisms adapted in vivo and cultured once on agar appeared to contain one or two antigens that were different from those in extracts of the in vitro grown organisms; and on polyacrylamide gels, electrophoresis of similar extracts showed one or more protein components for strain BS3 which were not seen for strain BS. Gonococci grown in guinea-pig subcutaneous chambers appear to be suitable for studies on the determinants of gonoccal pathogenicity.

Lactate stimulation of gonococcal metabolism in media containing glucose: mechanism, impact on pathogenicity, and wider implications for other pathogens.

Over the past decade, studies of Neisseria gonorrhoeae have shown that in media containing glucose, lactate stimulates metabolism, and this could affect pathogenicity (7, 19). Recently, the probable mechanism of this stimulation has been identified as one that could apply to other pathogens (68). Earlier studies implicated lactate metabolism in the serum resistance of Haemophilus influenzae (30), and during the past year, the use of signature-tagged mutagenesis and an infant rat model identified a putative (i.e., homologous with the gene in Escherichia coli) lactate permease-deficient mutant of Neisseria meningitidis with diminished virulence (62; C. M. Tang, personal communication). There may be a common role for lactate in the pathogenicity of these and other pathogens. Lactate and glucose are present together in most sites where infection occurs in vivo. This review summarizes the present position on gonococci and its implications regarding other pathogens. Throughout, the phrase “stimulation of metabolism by lactate” refers to this event occurring in a medium containing glucose.

Lipopolysaccharide alteration is associated with induced resistance of Neisseria gonorrhoeae to killing by human serum.

On SDS-PAGE, solubilized and proteinase K treated preparations of Neisseria gonorrhoeae strain BS4 (agar) showed differences in silver stained lipopolysaccharide (LPS) patterns, before and after induction to resistance to serum killing by incubation for 3 h at 37 degrees C with low Mr fractions from lysates of guinea pig red blood cells. Preparations from the original serum susceptible gonococci and LPS purified from such bacteria showed two components, but the preparations from the serum resistant gonococci were deficient in the higher Mr component. Furthermore, on immunoblotting with fresh human serum (FHS), the two LPS components of the susceptible gonococci reacted strongly with IgM. With preparations from the serum resistant gonococci there was no reaction in the area corresponding to the higher Mr component and a weaker reaction with the component of low Mr. Purified LPS from the susceptible gonococci neutralized the bactericidal activity of FHS against N. gonorrhoeae strain BS4 (agar) probably by reacting with the relevant antibody, since heated FHS was no longer bactericidal when mixed with a source of complement (human placental serum) after prior reaction with the LPS. These neutralization tests coupled with the results of immunoblotting strongly suggest that increased serum resistance is due to the lack of the high Mr LPS moiety.

Red blood cells, a source of factors which induce Neisseria gonorrhoeae to resistance to complement-mediated killing by human serum.

Lysates of guinea pig or human red blood cells (RBC) contain far more of the factors that induce resistance in gonococci to complement-mediated killing by fresh human serum that do plasma or serum. As was previously found with serum, most of the resistance-inducing activity of guinea pig RBC lysates was found in ultrafiltrates with molecular weights of less than 5000. In contrast, and as with human serum, most of the resistance-inducing activity of human RBC lysates did not pass ultrafilters which removed molecules of less than 5000 daltons, although some active material of low molecular weight was present.

Induction of phenotypically determined resistance of Neisseria gonorrhoeae to human serum by factors in human serum.

Of 47 human sera tested, 13 converted serum-sensitive gonococci [strain BS4 (agar)] to serum resistance in vitro in 3 h at 37 degrees C, as had previously been demonstrated for most samples of guinea pig serum. The resistance-inducing activity of human serum was lower than that of guinea pig serum but, like the latter, did not operate at 8 degrees C, was greater at pH 6.6 than at pH 7.1, was increased by freezing and thawing, and depended on high and low molecular weight serum fractions; the latter fraction had a molecular weight between 1000 and 5000, and was acid- and heat-labile.

Fractionation of Guinea Pig Serum for an Inducer of Gonococcal Resistance to Killing by Human Serum: Active Fractions Containing Glucopeptides Similar to Those from Human Red Blood Cells

The resistance of gonococci to complement-mediated killing by serum is important in the pathogenesis of gonorrhoea. Most urethal strains lose this resistance on subculture. The host product(s) which induces the resistance in vivo is therefore fundamental to pathogenesis. Human genital secretions and some sera induced gonococci to serum resistance in vitro. Guinea pig serum was more active than human serum and low molecular weight fractions from it conferred resistance to gonococci in 3 h at 37 degrees C. Similar active fractions were obtained from human sera. Now guinea pig serum has been further fractionated for the low molecular weight inducer by membrane filtration, gel filtration on Sephadex G25, high performance liquid chromatography (HPLC) with a Spherisorb ODS reverse phase column, chromatography on Sephadex LH20 and HPLC with a Partisil SCX cation exchange column. The small yield (less than 1 mg from 400 ml serum) of highly active material was contaminated with breakdown products from the Partisil SCX column and a mixture of compounds. However, analysis indicated the presence of one or more small glucopeptides containing cysteine, glutamic acid, aspartic acid, threonine, serine, glycine, alanine, valine and lysine. Similar glucopeptides are liberated from fresh human red blood cells in slightly hypertonic saline and samples of them induced gonococci to serum resistance.

Investigation of the Determinants of the Survival of Neisseria gonorrhoeae within Human Polymorphonuclear Phagocytes

In studies aimed at identifying the determinants responsible for the ability of gonococci to survive and grow within human phagocytes, the reduction of intracellular survival of phagocyte-resistant gonococci by prior treatment of the bacteria with homologous antiserum provided two indirect means of testing for possible determinants. First, surface washes of the resistant gonococci and fractions of these extracts were examined for ability to neutralize the above effect of antisera. Second, antisera raised to purified surface components of the resistant organisms were examined for ability to promote intracellular killing. A combination of these methods indicate that the aggressins were not pili but components of the outer membrane. A surface wash of a phagocyte-resistant, pilated strain, BS4 (agar), neutralized the activity of homologous antisera before and after centrifuging at 20 000 g, but pili separated from the supernatant did not. A corresponding supernatant from the surface wash of a phagocyte susceptible strain, BSSH, had little neutralizing ability. Antisera to pili failed to reduce intracellular survival of resistant gonococci, whereas antisera against outer membrane vesicles did so. Finally, the neutralizing activity of the surface wash supernatant was removed by centrifugation after treatment with wheat germ agglutinin, which precipitates outer membrane vesicles.

A determinant of resistance of Neisseria gonorrhoeae to killing by human phagocytes: an outer membrane lipoprotein of about 20 kDa with a high content of glutamic acid.

A protein of about 20 kDa was extracted by sodium cholate (1%, w/v) from outer membranes of a strain of Neisseria gonorrhoeae, BS4 (agar), which is resistant to killing by human phagocytes. When the protein was purified by repeated fractionation on Sephadex G75, contamination with other outer-membrane proteins and lipopolysaccharide was negligible. The protein contained a full complement of amino acids, with high levels of glutamic acid. Carbohydrate, detected by the anthrone method and by sugar and hexosamine analysis, was present, but at very low levels. There was a significant content of fatty acids (about 5.7% of the protein), indicating a lipoprotein. The 20 kDa lipoprotein: (1) neutralized the ability of antiserum against whole organisms of BS4 (agar) to reduce the resistance of this strain to phagocyte killing; (2) evoked in mice an antiserum which reduced this resistance and immunoblotted only with 20 kDa lipoprotein in the cholate extract of outer membranes; and (3) promoted resistance to intracellular killing of an otherwise phagocyte susceptible gonococcal strain (BSSH). This is strong evidence that it is a determinant of gonococcal resistance to phagocyte killing.

Phenotypic changes in the resistance of Neisseria gonorrhoeae to killing by normal human serum.

Gonococci adapted to growth in chambers implanted subcutaneously into guinea pigs are resistant to killing by human serum. This resistance is lost after a few generations in vitro both in culture medium and in fluid taken from guinea-pig chambers. The rate of loss is too rapid to occur by mutation and selection. Furthermore, the resistance is regained after a few generations when bacteria from the first in vitro culture are inoculated back into guinea-pig chambers in vivo. Hence the loss of serum resistance in vitro and the gain in vivo are probably due to phenotypically controlled events. Such events could be important in the pathogenicity of Neisseria gonorrhoeae.

A serum-sensitive, sialyltransferase-deficient mutant of Neisseria gonorrhoeae defective in conversion to serum resistance by CMP-NANA or blood cell extracts

A stable, sialyltransferase-deficient mutant of Neisseria gonorrhoeae strain F62 totally defective in CMP-NANA-dependent lipopolysaccharide (LPS) sialylation was isolated by insertion mutagenesis with transposon Tn 1545-delta 3 and screened for unlabelled colonies following incubation with CMP-14C-NANA. In contrast to the parental strain which became serum resistant on incubation with CMP-NANA or blood cell extracts, the mutant, JB1, remained serum sensitive. French press extracts of strain F62 catalysed LPS sialylation, but corresponding extracts of the mutant were inactive. Five LPS components were detected by SDS-PAGE in the parental strain. Five components of the same Mr were also found in the mutant. Three identical components were detected by Western blotting using MAb 3F11, which recognizes the Gal beta 1-4GlcNAc groups in the conserved LPS components of F62 which can be sialylated. The mutant, JB1, is therefore deficient in the sialyltransferase that is essential for both LPS sialylation and conversion of serum-sensitive gonococci to serum resistance by either CMP-NANA or blood cell extracts. No evidence was obtained for an LPS sialylation pathway by blood cell extracts that is independent of CMP-NANA.