Otto Solberg

Endotoxin liberation from neisseria meningitidis correlates to their ability to induce procoagulant and fibrinolytic factors in human monocytes

Endotoxin released from different strains of Neisseria meningitidis were studied for their ability to induce procoagulant (tissue factor, TF), fibrinolytic (plasminogen activator, PA) and antifibrinolytic (plasminogen activator inhibitor 2, PAI-2) factors in human monocytes. Two meningococcal strains that liberate endotoxin (E+; 270+ and 840+) and 2 non-liberating (E-; 270- and 840-) strains were used. The endotoxin activity in culture filtrates of these strains was monitored with the Limulus amoebocyte lysate (LAL) test. There was a marked difference between E+ and E- strains in their ability to liberate endotoxin. Suspensions of whole bacteria of all 4 strains induced a significant (14-19-fold) increase in monocyte TF expression when present in concentrations > 10(5) CFU/ml. At lower concentrations (10(4) CFU/ml), E+ strains were clearly more potent stimulators of TF synthesis than E- strains. Culture filtrates of E+ strains were up to 10(4)-fold more potent in inducing TF synthesis than filtrates from E- strains. This marked difference in inducing potency between E+ and E- strains was also observed when monocyte PAI-2 synthesis was examined. The PA expression, on the other hand, was suppressed when monocytes were incubated in the presence of culture filtrates, especially filtrates from the E+ strains. The increased procoagulant and antifibrinolytic activity, together with reduced profibrinolytic activity of monocytes, was closely correlated to the amount of endotoxin measured in the culture filtrates. These changes may contribute substantially to the coagulopathic state seen during systemic meningococcal disease.

Endotoxin liberation associated with growth, encapsulation and virulence of Neisseria meningitidis.

Endotoxin liberation, encapsulation and growth was studied in 123 isolates of Neisseria meningitidis. Free endotoxin appeared in culture filtrates during exponential growth. Meningococci with free endotoxin titre greater than or equal to 10(2) (E+) showed a higher mean number of viable bacterial counts (CFU/ml) during growth than isolates with titre less than 10(2) (E-), p less than 0.001. Differences in endotoxin liberation was, however, far more pronounced than what was indicated by growth differences alone. E+ property and increased growth rate was significantly more often found among encapsulated than non-encapsulated meningococci. Within the serogroup B isolates, there tended to be a higher mean number of CFU/ml during growth in E+ than E- meningococci, particularly among carrier isolates studied separately (p = 0.009). Case isolates of B meningococci, which had generally a higher amount of capsular material and a higher proportion of E+ strains, had also higher mean CFU/ml than B carrier isolates. This indicates that the endotoxin liberation and growth may be correlated to presence and amount of capsular polysaccharide. Endotoxin liberation, presence of capsular polysaccharide and growth ability are 3 factors which are likely to coincide in meningococci. This combination of properties may be of importance for the development of meningococcal disease.

Endotoxin liberation studied by biological and chemical methods

Release of endotoxin (or lipopolysaccharides, LPS) from four meningococcal strains was studied with a chemical and a biological technique. Two strains were endotoxin‐liberating (E+; 270E+ and 840E+) and two had no or low endotoxin release E‐; 270E‐ and 840E‐). LPS was quantitated by gas chromatography (GC) of LPS‐specific hydroxy fatty acids, in parallel with assay of endotoxin by Limulus Amebocyte Lysate (LAL), in cell suspensions of equal O.D. and in filtered samples. The GC and LAL methods showed a reasonably good agreement in the determination of LPS in filtrates, which had distinctly higher levels (approx. 10–100 times) for the E+ strains than the E‐ strains, in accordance with earlier LAL studies. This difference was not due to overproduction of LPS in the E+ strains, since all four strains had the same level of LPS (by GC) in cell suspensions of equal O.D. Here the agreement between the GC and LAL methods was substantially less, with lower values by LAL for the two E‐ strains. The chemical composition of purified LPS was determined by methanolysis and GC for the four strains and for two additional strains 247 and 714 with a high degree of genetic similarity with strains 270E‐ and 840E‐, respectively. Amounts of unphosphorylated L‐glycero‐D‐manno heptose and 2‐keto‐3‐deoxyoctonic acid were the same in all 6 LPS. Otherwise distinct differences were found between LPS of the 6 strains. LPS of the two E+ strains formed one group with about 2.4 mol of galactose (gal), 1.4 mol of glucose (glc) and 2.8 mol of glucosamine (glcN) in the carbohydrate chain. Another group, LPS of all the E‐ strains except 270E‐, had 1.1 mol of gal, 2.8 mol of glc and 1.3 mol of glcN in the LPS chain. LPS 270E‐ also had 1.3 mol of glcN but deviated strongly form all other LPS by a complete lack of gal and glc. On the basis of genetic evidence strain 270E‐ is regarded as a “rough” LPS mutant of strain 247. The atypical chemistry of LPS 270E‐ may explain an observed hydrophobicity of this LPS, and it may be related to the previously described sulfonamide sensitivity. Whether the chemical difference observed for LPS of the E+ and E‐ strains is a mere coincidence remains to be elucidated by detailed studies of more strains of known tendency of endotoxin liberation.

Endotoxin Release from Invasive Meningococci Related to Sulfonamide Resistance, Serogroup and Serotype

The relationship between endotoxin liberation, sulfonamide resistance, serogroups and serotypes was studied in 28 Neisseria meningitidis strains isolated from patients with meningococcal disease. Sulfonamide resistance was present in 15/28 strains. 22 strains belonged to serogroup B, and 5 to group C; 1 strain was non-groupable. Free endotoxin activity in growing cultures of meningococci with endotoxin titre of greater than or equal to 10(2) was found in 27/28 strains. A high endotoxin activity was present in both sulfonamide-sensitive and -resistant invasive meningococci. A high endotoxin release with titre greater than or equal to 10(3) seemed to be more associated with serogroup C than B, and more to the serotypes 2 and 15/16 than to the non-typable strains.

Multilocus genotypes of two strains of Neisseria meningitidis and their presumed variants obtained upon subcultivation

Loss of sulfonamide resistance and endotoxin liberation have been described in two strains of Neisseria meningitidis serogroup B, upon subcultivation every 1 to 2 months over an 18‐month period. Subsequently, the two laboratory variants, designated 270E‐ and 840E‐, were also found to differ from the parent strains, 270E+ and 840E+, in serotype, outer membrane protein pattern, and virulence in mice. We report here the multilocus genotypes determined by enzyme electrophoresis, of the four isolates 270E+, 270E‐, 840E+, and 840E‐, and demonstrate that 270E‐ and 840E‐ strains could not have originated from subcultivation of 270E+ and 840E+, respectively, but that a mix‐up of strains has occurred.