Resistance to human serum of gonococci in urethral exudates is reduced by neuraminidase

Gonococci examined directly from urethral exudates are resistant to killing by human serum, but most strains become susceptible on subculture. Previous work with gonococci grown in vitro indicates that resistance in vivo is due to sialylation of gonococcal lipopolysaccharide (LPS) by a host factor, cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) or a related compound present in urogenital secretions and blood cells including phagocytes, which exude during inflammation. This sialylation inhibits the reaction between bactericidal IgM in serum and its target LPS sites. Here, we confirm the indication by using gonococci grown in vivo. Crucial to the above conclusions was the marked reduction of CMP-NANA-conferred serum resistance when gonococci were treated with neuraminidase to remove sialyl groups from their LPS. We now show that the serum resistance of gonococci in urethral exudates was reduced by treatment with neuraminidase from more than 95% (calculated in relation to controls incubated with heated serum) to 2-11% according to sample and incubation time. Subculture of the gonococci also reduced resistance to 9-11% but resistance was restored to more than 95% by incubation with CMP-NANA. This work is the culmination of an investigation that underlines the need to identify specific host factors and the virulence determinants they induce in vivo in future studies of pathogenicity.     

A surface polysaccharide forms when gonococci are converted to serum resistance by cytidine 5''-monophospho-N-acetyl neuraminic acid

A serum-susceptible, guinea-pig chamber-passaged, laboratory strain (BS4 (agar)) of Neisseria gonorrhoeae was converted to serum resistance by incubation with cytidine 5-monophospho-N-acetyl neuraminic acid (CMP-NANA) and examined by electron microscopy after staining with ruthenium-red. In contrast to serum susceptible gonococci incubated without CMP-NANA, the majority (60-70%) of the serum resistant organisms showed a surface accumulation of polysaccharide. This surface polysaccharide was enhanced on all the resistant gonococci after incubation with fresh human serum. Control susceptible gonococci were devoid of the polysaccharide after incubation with heated human serum. Identical results were obtained with a fresh gonococcal isolate which had lost serum resistance on subculture but which, in common with 3 other isolates, was restored to serum resistance by incubation with CMP-NANA.     

The sialylation of gonococcal lipopolysaccharide by host factors: A major impact on pathogenicity

The resistance of gonococci in most patients to complement mediated killing by human serum is due to sialylation of their lipopolysaccharide (LPS) which prevents bactericidal antibody from reacting with target sites. Two of the host factors responsible are: cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA), a well-known sialylating agent, and another factor which enhances the transfer of sialyl groups from CMP-NANA to LPS catalysed by a gonococcal sialyltransferase. The bacterial determinant of resistance is a conserved LPS component of about 4.5 kDa which is sialylated at a terminal Gal beta 1-4GlcNAc site on its side chain. The sialylated LPS forms a surface coat which is stainable by ruthenium red and connected with previously described 'capsules'. These observations sparked off an explosion of research. Recent publications show that sialylation of LPS by CMP-NANA affects additional important aspects of gonococcal pathogenicity, notably interactions with antibodies and phagocytes, and rendering the gonococcal surface more 'host-like'. Also, the observations have prompted an examination of LPS from some other pathogens for the presence of sialyl groups with positive results for Neisseria meningitidis and Haemophilus influenzae.     

Kinetics of conversion ofNeisseria gonorrhoeae to resistance to complement by cytidine 5′-monophospho-N-acetyl neuraminic acid

Freshly isolated gonococci upon subculture are readily lysed by normal human serum although a few strains remain inherently resistant to the complement activity. The sensitive gonococci can be converted to serum resistance by incubation with a host derived factor referred to as cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NANA). These gonococci resist complement mediated killing due to their sialylation of an epitope structure on a component of lipo-oligosaccharide (LOS). In the present study, the kinetics of conversion to serum resistance by the action of sialyltransferase (STase) inNeisseria gonorrhoeae was followed with very low concentrations of CMP-NANA. This conversion could not be perceived at 2×10^–3 nmol.ml^–1 but was fully attainable from 8×10^–3 to 2×10^–2 nmol.ml^–1 CMP-NANA. When pretreated up to 100 min in presence of the very low concentration of 2×10^–3 nmol.ml^–1, a potentiating effect on the conversion of gonococci by 2×10^–2 nmol.ml^–1 was observed in relation to the time of preincubation. This action was abolished after exposure to a subinhibitory concentration of chloramphenicol (0.5 µg.ml^–1). The gonococci recovered their ability to convert to serum resistance following adequate washing. The potential for increase in STase activity should be of interest for understanding the conversion from a serum sensitive to a serum resistance state.     

Cytidine 5'-monophospho-N-acetylneuraminic acid or a related compound is the low Mr factor from human red blood cells which induces gonococcal resistance to killing by human serum

A low-Mr factor which induces gonococcal resistance to complement-mediated serum killing has been partially purified from lysates of mixed red and buffy coat cells from human blood. The lysates were dialysed against Tris buffer for 24 h at 25 degrees C with the diffusate being continuously recycled through a column of QAE-Sephadex A25. After elution in an NaCl gradient, the active fractions were both desalted and further purified on Sephadex G10. A second fractionation on QAE-Sephadex A25 and desalting with Sephadex G10 preceded further purification by repeated high-pressure liquid chromatography (HPLC) using a DEAE anion exchange column and desalting with Sephadex G10. Less than 500 micrograms of material showing one peak in HPLC was obtained from 1 litre of blood. After NMR had indicated the possible presence of pyrimidine nucleotide, carbohydrate and N-acetyl groups, nanogram quantities of a commercial preparation of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) were shown to induce gonococci to serum resistance. The synthetic CMP-NANA also co-eluted with the preparation from blood cells in HPLC, and the two materials were indistinguishable in their patterns of acid and heat lability. Furthermore, the resistance-inducing activity of both materials was inhibited by cytidine monophosphate, which is known to inhibit sialylation reactions by CMP-NANA. It appears therefore that the resistance-inducing factor is CMP-NANA or a closely related compound. If the factor is CMP-NANA, biological activities indicated that the cell lysate from 1 litre of blood contained about 40 micrograms, and the most purified preparation contained only about 1%. With this minute amount in a mixture, the presence of CMP-NANA or a closely related analogue could not be established unequivocally by NMR.     

Antibodies to N-terminal peptides of gonococcal porin are bactericidal when gonococcal lipopolysaccharide is not sialylated

Six synthetic 25-mer peptides corresponding to certain presumed surface-exposed regions of gonococcal porin protein I (PI) were made from strains FA19 (PIA) and MS11 (PIB). Four peptides were immunogenic in rabbits. Affinity-purified antisera against both PIA and PIB N-terminal peptides were bactericidal for homologous gonococci and many heterologous PI serovars. However, sialylation of gonococcal lipopolysaccharide (LPS) by growth of gonococci in the presence of cytidine monophosphate-neuraminic acid (CMP-NANA) abrogated the bactericidal activity of these antisera. Binding of anti-PI monoclonal antibodies to whole gonococci was reduced two- to fourfold by sialylation of LPS, suggesting that sialylation may inhibit bactericidal activity by masking porin epitopes. However, binding of anti-PII (Opa) monoclonal antibodies was not inhibited, yet complement-mediated killing was inhibited by sialylated LPS. Binding of complement components C3 and C9 was inhibited in the presence of either anti-PI or anti-PII monoclonals when gonococci were grown in the presence of CMP-NANA. Thus sialylation inhibited both anti-PI antibody binding and complement deposition, with a resultant decrease in bactericidal activity.     

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.     

Maintenance of Virulent Gonococci in Laboratory Culture

WE have previously shown¹ that gonococci in urethral exudates are resistant to the bactericidal action of complement plus natural or immune antibodies, yet after subculture the same strains are rapidly killed. Earlier workers² could infect volunteers more readily with gonococci from urethral pus than with the same strains grown in the laboratory. Together, these findings suggest that on subculture gonococci may lose both virulence and resistance to serum killing in parallel. Nevertheless, Kellogg et al.^{3, 4} believe that gonococci can be maintained in a virulent form on culture and that the virulent can be distinguished from avirulent forms by their different colonial morphology on their colonial type medium (CT medium). Freshly isolated gonococci had colony types referred to as 1 and 2; on random subculture different colonies known as types 3 and 4 appeared. Even after repeated subculture one strain, F62, in the type 1 colony form was able to infect volunteers, whereas the so-called avirulent type 4 colonies had lost this ability after some sixty-nine passages in vitro. Nevertheless the dose of type 1 organisms needed to set up infection was about 1.5 × 10¹⁰ microbes⁵. These results need not conflict with our¹ impression that virulence, if correlated with resistance to antibody plus complement, is lost after even one subculture. If one postulates that loss of virulence occurs in two steps type 1 organisms might be phenotypically avirulent while the type 4 colonies might have actually lost the genetic information required for virulence. The vast dose used to challenge volunteers would enable a few gonococci to survive in the host long enough to revert to the phenotypically virulent form.     

Gonococci in vivo and in vitro. Further studies on the host and bacterial determinants of gonococcal resistance to killing by human serum,and by phagocytes

A small M, heat and acid labile, host inducer(s) of gonococcal resistance to complement mediated killing by fresh human serum (-FHS), being purified from red blood cell (RBC) extracts, produced changed in lipopolysaccharide (LPS) structure, surface antigens and proteins; and acquirement of resistance related to loss of a target antigen for bactericidal IgM, possibly LPS components. A 20 kDalt, lipoprotein with a high content of glutamic acid isolated from outer membranes of a gonococcal strain selected in vivo is a determinant of gonococcal resistance to killing by human phagocytes. Somic extracts of gonococci may contain a cytotoxin for human phagocytes.At the 4th International Pathogenic Neisseriae Conference, we reported (Parsons et al. 1985) that conditions in vivo induced phenotypic change leading to gonococcal resistance to complement-mediated killing by human serum; and, also, selected gonococcal types which showed a greater resistance to intracellular killing by human phagocytes than laboratory strains. Furthermore, evidence was presented that not only was resistance to complement mediated killing important in gonococcal pathogenesis, but also resistance to phagocytic defences. This paper describes the continuance of our studies on the determinants of induced serum resistance and of resistance to killing by phagocytes including toxicity to these cells. Each section begins by summarising previous work that was referenced in Parsons et al. (1985).     

Protein changes associated with induced resistance of Neisseria gonorrhoeae to killing by human serum are relatively minor

Serum-susceptible (SS) Neisseria gonorrhoeae were induced to resistance (SR) to complement-mediated killing by fresh human serum (FHS) by a small-Mr factor(s) from guinea-pig blood in 3 h at 37 degrees C, but not in the presence of bacteriostatic concentrations of chloramphenicol or neomycin, indicating that proteins mediated the acquisition of resistance. SDS-PAGE protein profiles of lysates of equal numbers of gonococci showed only two qualitative differences between SR and SS organisms, both in minor components (a protein A of about 205 kDa in the former and not the latter and vice versa for a protein B of about 16 kDa). Many proteins, however, including the three principal outer-membrane proteins, were present in larger amounts in SR gonococci. The lack of major changes in proteins when resistance is acquired was confirmed by immunoblotting the two protein profiles with the IgG of hyper-immune rabbit anti-SR and anti-SS sera, of rabbit anti-SR serum after absorption by SS organisms and of FHS used alone and after absorption with SS organisms. The IgM of FHS, which is responsible for most of the bactericidal activity, showed only faint reactions with a few proteins common to both SS and SR gonococci and no reactions when the FHS was absorbed with SS gonococci. This is in contrast to the strong and different reactions given with lipopolysaccharide (LPS) components of SS and SR organisms, which, prepared from the former organisms, neutralize the bactericidal activity of FHS. Hence, the relatively small protein changes accompanying induction are less likely to be directly responsible for serum resistance than the more profound LPS changes.     

Selection from gonococci grown in vitro of a colony type with some virulence properties of organisms adapted in vivo.

Gonococci from subcutaneously implanted chambers in guinea pigs produced, on agar, more than 95% small colonies showing a "double highlight" (DH) effect in oblique reflected light combined with transmitted light. Laboratory strains of gonococci produced some DH colonies, but other showed a single highlight (SH) or no highlight (NH). Selection of DH colonies and comparison of their organisms with gonococci grown in vivo and with those from SH colonies, showed that the DH character was associated with high infectivity for guinea-pig chambers, resistance to killing by human phagocytes and heavy pilation. Furthermore, DH colonies were found in the first culture of three fresh samples of urethral pus. Thus, the DH colony characteristic may be a more reliable criterion of pathogenicity of gonococcal isolates than systems used previously. There were, however, some differences between the gonococci grown in vivo and the DH colony types. The gonococci grown in vivo and cultured once on solid medium possessed one or two antigens which differed from those of DH (or SH) colonies. They also formed smooth suspensions (which separated slowly) in saline, compared with the rough suspensions (which separated quickly) formed by gonococci from DH (or SH) colonies. Finally, the organisms grown in vivo were resistant to killing by human serum whereas the DH (and SH) colony types were susceptible; the resistance of the organisms grown in vivo was lost during one subculture on agar suggesting that the property is a phenotypic characteristic. Hence, in addition to selecting DH colony types the conditions in vivo produce organisms which differ, probably phenotypically, from cultured organisms.     

The surface properties of Neisseria gonorrhoeae: determinants of susceptibility to antibody complement killing.

Monovalent rabbit antisera were prepared to highly purified gonococcal lipopolysaccharide (LPS), to pili and to two major purified outer envelope proteins. All these antisera were free from significant specific IgM antibody and were standardized to 4 microgram specific IgG antibody per test, permitting accurate comparisons between the different gonococcal surface antigens as triggers of the complement-dependent bactericidal reaction. LPS was the most effective antigen at inducing a bactericidal response to homologous and heterologous gonococci, followed by the two individual outer envelope proteins. Pili were relatively ineffective. Strain P9 gonococci grown in vivo or which possessed a 'capsule' in vitro were more resistant to serum killing than the non-capsulated parent strain. One highly susceptible strain, F62, which was killed by complement in the absence of any LPS antibody, was able to directly activate complement by the alternative pathway.     

The Leeuwenhoek Lecture, 1991. The influence of the host on microbes that cause disease.

Microbial pathogenicity or virulence, the capacity to cause disease, depends on microbial gene products that promote infection and penetration of mucous membranes, multiplication in the tissues, interference with host defence and sickness. Formation of these virulence determinants by microbes is influenced by the environment of the host, which differs from that in laboratory cultures. Studies of microorganisms grown in vivo, and of the host's influence on the production of virulence determinants, are increasing. In most studies, however, the complex conditions in vivo are not dissected to show the influence of particular factors. In future we should define specific host factors that are responsible for producing identified virulence determinants. There are three studies which point the way. Iron limitation in vivo causes production of bacterial siderophores, outer membrane receptors and some toxins. Erythritol, a growth stimulant for brucellae, causes intense placentitis and hence abortion in cattle, sheep and pigs. Cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) sialylates a conserved component of gonococcal lipopolysaccharide (LPS), thereby rendering gonococci in patients resistant to complement-mediated killing by serum. Although the lecture uses bacteria for examples, the principle applies equally to studies of viral and fungal pathogenicity.     

Ruthenium red staining of the neural lamella of the brain of Galleria mellonella

Summary The outer surface of the neural lamella, the connective tissue ensheathing the brain, shows the ability to bind ruthenium red in the wax moth larva. Ruthenium red-positive material is sensitive to neuraminidase, hyaluronidase and to some extent to phospholipase C, what suggests that the negative charge on the external surface of the neural lamella depends on the presence of the anionic groups of sialic and hyaluronic acids and phospholipids.     

The binding of Ruthenium red to tubulin

The inhibition of microtubule assembly by Ruthenium red (Deinum, J., Wallin, M., Kanje, M. and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209-213) could be counteracted by either taxol or dimethyl sulfoxide. Ruthenium red remained bound to the assembled microtubules. Microtubules assembled in the presence of Ruthenium red and taxol showed the typical taxol-dependent stability. The dimethyl sulfoxide-induced microtubules showed normal assembly characteristics, e.g., were GTP dependent, could be disassembled by cold, colchicine and Ca2+ and had no alterations in ultrastructure. The absolute disassembly induced by Ca2+ in the presence of dimethyl sulfoxide and Ruthenium red was dependent on the microtubule protein concentration, but independent in the absence of Ruthenium red. Ruthenium red was strongly bound to purified tubulin also in the presence of 8% (v/v) dimethyl sulfoxide. The dimethyl sulfoxide-induced assembly of purified tubulin in the presence of Ruthenium red was slightly stimulated, although the critical protein concentration was the same. It was found by resonance Raman spectroscopy with a flow technique that Ruthenium red did not bind to a specific calcium binding site on tubulin, although binding to a GTP binding site cannot be excluded. The wavenumbers of the lines in the region 375-500 cm-1 differ from those found for Ruthenium red bound to typical calcium-binding proteins such as calmodulin. Although Ruthenium red binds to serum albumin as well, the spectrum with albumin resembled that of the free dye.     

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry.

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.     

The cohesive properties of variants of Neisseria gonorrhoeae strain P9: specific pilus-mediated and non-specific interactions

The cohesive properties of virulent pilated Neisseria gonorrhoeae strain P9 (P++) have been compared with those of a non-pilated isogenic variant (P-) possessing the same outer membrane components. The binding of P++ gonococci to buccal epithelial cells was dependent on pH, with an optimum at pH 6.5 to 7.0 . This adhesion was markedly inhibited by treatment of the buccal epithelial cells with a neuraminidase/exoglycosidase mixture. In contrast, the binding of P++ gonococci to erythrocytes was unaffected by pH. A possible explanation is that pili bind to a carbohydrate receptor present on buccal epithelial cells but lacking on erythrocytes. The adhesion of P- gonococci to erythrocytes and to buccal epithelial cells was unaffected by pH but enhanced by treatment of the cells with neuaminidase or periodate. Presumably, neuraminic acid residues on host cell surface carbohydrates inhibit adhesion. The finding that P- gonococci bind to amphipathic gels suggests hydrophobic interactions as a possible non-specific mechanism attaching P- gonococci to host cell surfaces.     

Phenotypically determined resistance of Neisseria gonorrhoeae to normal human serum: environmental factors in subcutaneous chambers in guinea pigs.

Some gonococci obtained from human urethral exudate or from subcutaneously implanted chambers in guinea pigs show a resistance to killing by human serum which is lost on sub-culture in vitro after a few generations. The environmental factors which may influence the phenotypic expression of resistance to serum killing were investigated in guinea pig chambers and in chamber fluid in vitro. The redox potential in chambers before and after infection was lower than that of heart blood but conditions were not anaerobic; H2O2 increased the redox potential but did not decrease gonococcal serum resistance. The chambers were slightly alkaline before and after infection. When the concentration of glucose (depleted in infected chambers by the abundant polymorphonuclear cells) was restored to excess, the serum resistance of the gonococci was unaffected. Concentrations of free amino acids in chambers changed little during infection. Gonococci adapted to growth in chambers and subsequently rendered serum-sensitive by growing once on agar reverted to serum-resistance after 0.5 to 1 h incubation in chamber fluid in vitro at 37 degrees C but not at 25 degrees C or 4 degrees C. After 16 to 24 h growth at 37 degrees C, resistance was again lost. The reversion to serum resistance did not occur in a complex laboratory medium. Examination of the chamber fluid after growth of gonococci in vitro showed depletion of lactate, glutamine and proline.     

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.     

Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis

Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells. Ruthenium red staining followed by electron microscopic analysis was used to demonstrate the presence of an external glycoprotein layer surrounding the spore of both Bacillus anthracis and Bacillus subtilis. This layer is less apparent with traditional staining methods used for electron microscopy. Renografin gradients were used to purify B. subtilis spores. These purified spores displayed greatly enhanced staining with ruthenium red, indicating nonspecific binding of renografin, which has a major carbohydrate constituent, methylglucamine. For B. anthracis, staining with ruthenium red was sufficiently intense that it was not significantly enhanced by renografin purification. In addition to demonstrating a previously undiscovered layer surrounding the spores of B. subtilis, the results help explain a long-standing controversy as to ultrastructural differences among these genetically closely related organisms. Ruthenium red staining provides an important addition to the identification of surface glycoproteins in studies to define similarities and differences in the exosporium layers of Bacillus species.