Demonstration of lipooligosaccharide immunotype and capsule as virulence factors for Neisseria meningitidis using an infant mouse intranasal infection model

Using an infant mouse intranasal infection model, we have compared the virulence of 17 epidemiologically related isolates of Neisseria meningitidis associated with an outbreak of meningococcal disease in Gloucestershire, UK, and one germane isolate. The isolates were all of serotype 15 subtype P1:7, 16 and were identical by restriction fragment length polymorphism analysis, but differed in either (i) whether they were isolated from a case or a carrier, (ii) the presence or absence of group B capsule, or (iii) their lipooligosaccharide (LOS) immunotype. The results indicate that capsule is a major virulence determinant and is required for colonization and hence for invasion. In addition, the LOS L3,7,9 immunotype, when compared to the L1,8,10 immunotype, is a secondary virulence factor which enhances colonization of nasal passages and invasion of the blood stream by both case and carrier isolates. Two case isolates which were unusual in possessing the L1,8,10 immunotype, established invasive infection, but this was associated with a switch to the L3,7,9 immunotype. The results confirm that LOS is a virulence factor for N. meningitidis and that immunotype L3,7,9 is associated with invasive disease.     

Tn916-generated, lipooligosaccharide mutants of Neisseria meningitidis and Neisseria gonorrhoeae.

A library of Tn916-generated, tetracycline-resistant (Tc) mutants of the group B Neisseri meningitidis strain NMB was screened by using monoclonal antibodies (MAbs) that recognize structural differences in neisserial lipooligosaccharide (LOS). The LOS of parental strain NMB had a relative molecular mass of 4.5 kDa, reacted with MAbs 3F11 and 6B4 but not with MAb 4C4 or 6E4, and contained a lacto-N-neotetrose unit. Two phenotypically stable mutants, SS3 and R6, altered in LOS, were identified by colony immunoblots, electrophoresis, and Western immunoblots. The LOS of mutant SS3 was 3.4 kDa and reacted with MAbs 4C4 and 6E4 but not MAb 3E11 or 6B4. The LOS of mutant R6 was 3.1 to 3.2 kDa and reacted with MAb 6E4 but not MAb 3F11, 6B4, or 4C4. Thus, the LOSs of the R6 and SS3 mutants were predicted to contain different truncations of the core oligosaccharide. The LOS phenotype of each mutant was linked to Tc(r), as determined by transformation of the parent strain with DNA from the mutant. Southern hybridizations and single-specific-primer PCR revealed in each mutant a single truncated tn916 insertion which had lost genes required for mobilization. Tn916 mutagenesis was used to identify two distinct genetic sites in the meningococcal chromosome involved in biosynthesis of the oligosaccharide chain of LOS and to create genetically defined LOS mutants of N. meningitidis and Neisseria gonorrhoeae.     

Differential role of lipooligosaccharide of Neisseria meningitidis in virulence and inflammatory response during respiratory infection in mice

Meningococcal lipooligosaccharide (LOS) induces a strong proinflammatory response in humans during meningococcal infection. We analyzed the role of LOS in the inflammatory response and virulence during the early infectious process in a mouse model of meningococcal respiratory challenge. An lpxA mutant strain (serogroup B) devoid of LOS (strain Z0204) could not persist in the lungs and did not invade the blood. The persistence in the lungs and invasion of the bloodstream by a rfaD mutant expressing truncated LOS with only lipid A and 3-deoxy-d-manno-2-octulosonic acid molecules (strain Z0401) was intermediate between those of the wild-type and Z0204 strains. Both LOS mutants induced acute pneumonia with the presence of infiltrating polymorphonuclear leukocytes in lungs. Although tumor necrosis factor alpha production was reduced in mice infected with the mutant of devoid LOS, both LOS mutants induced production of other proinflammatory cytokines, such as interleukin-1beta (IL-1beta), IL-6, and the murine IL-8 homolog KC. Together, these results suggest that meningococcal LOS plays a role during the early infectious and invasive process, and they further confirm that other, nonlipopolysaccharide components of Neisseria meningitidis may significantly contribute to the inflammatory reaction of the host.     

Immunotype epitopes of Neisseria meningitidis lipooligosaccharide types 1 through 8.

Lipooligosaccharides (LOS) of the eight immunotypes found in serogroup B Neisseria meningitidis were purified from their prototype strains grown in tryptic soy broth. Rabbit antisera to these LOS were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining revealed that most of the LOS antigens contained two major components; the larger components had apparent molecular weights (Mrs) in the range of 4,800 +/- 300, and the smaller components had an apparent Mr of 4,300. Immunoblot analysis showed that the larger major component of an LOS, in general, was much more immunogenic because the rabbits produced antibodies exclusively or primarily to this component even though the LOS immunogen contained both large and small major components. Antibodies to the smaller 4,300-Mr components were infrequently observed but, when present, were cross-reactive with the same-size components of all heterologous LOS. Hence, the immunotype epitopes reside in the larger major components of all immunotypes except type 5, in which a smaller major component having an apparent Mr of 4,400 carries the epitope. Rabbit antisera to types 1, 5, and 6 were immunotype specific. Antisera to other types had cross-reactivities with some heterologous LOS, and the larger components, but not the 4,300-Mr components, of the LOS were primarily responsible for the cross-reactivities. This finding suggests that the larger components of cross-reactive LOS have a similar structure in addition to their type-specific sugar moieties. The LOS of N. meningitidis M986, a strain used for the production of a serotype 2a vaccine, was found to contain the immunotype 7 epitope.     

Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB.

Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Failure of adherence to buccal cells and surface hydrophobicity as virulence markers in Neisseria meningitidis

Surface hydrophobicity and adherence to buccal epithelial cells were studied in 33 carrier and 34 invasive Neisseria meningitidis strains. It was found that hydrophobicity is statistically similar in both groups (P = 0.0507) although it could be considered that carrier strains are slightly more hydrophobic than invasive ones. Adherence was similar in both groups although more homogeneous in the carrier strains. No correlation could be demonstrated between these two properties nor can they be considered relevant as markers of the carrier or invasive status of this bacterium, indicating that at least in N. meningitidis they are not good properties to discriminate virulent strains.     

The msbB mutant of Neisseria meningitidis strain NMB has a defect in lipooligosaccharide assembly and transport to the outer membrane

A deletion-insertion mutation in msbB, a gene that encodes a lipid A acyltransferase, was introduced into encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of the same strain. These mutants were designated NMBA11K3 and NMBA11K3cap-, respectively. Neither lipooligosaccharide (LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both were easily extracted from NMBA11K3cap-. Immunoelectron microscopy using monoclonal antibody (MAb) 6B4, which recognizes the terminal Galbeta1-4GlcNAc of LOS, demonstrated that NMB, NMBcap-, and NMBA11K3cap- expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3. However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Mass-spectrometric analyses demonstrated that the relative amounts of the lipid A-specific C12:0 3-OH and C14:0 3-OH present in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25- and 23-fold, respectively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substantially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences the assembly of the lipid A and consequently the incorporation of porin in the outer membrane.     

Increased virulence of Neisseria meningitidis after in vitro iron-limited growth at low pH.

At low pH (6.6) and under conditions of iron limitation, Neisseria meningitidis group B (strain SD1C) exhibited an atypical outer membrane protein profile and an increased relative virulence for the mouse. Cells grown in a buffered medium were effectively deprived of iron by the addition of ethylenediamine-diorthohydroxyphenylacetate. The pH of the medium selected for characteristic colonial morphologies: type M3 predominated at pH 6.6, and type M5 predominated at pH 7.7. A mixed population of M1, M3, and M5 colonies was observed at pH 7.2. Isolated outer membrane proteins were analyzed by sodium dodecyl 99 99 sulfate-polyacrylamide gel electrophoresis, and surface exposed proteins were labeled by the [125I]lactoperoxidase method and subsequently identified by autoradiography. Cells grown at pH 6.6 elaborated a major outer membrane protein (protein III; molecular weight, 69,000), which was also present in the outer membrane of iron-limited cells grown at pH 7.2. At pH 7.2 in an iron-sufficient medium, protein III was present only in small quantities in sodium dodecyl sulfate-polyacrylamide gel was present only in small quantities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A study of the relative virulence (50% lethal dose) of the meningococcus for C57/BL mice revealed that iron-limited cells grown at low pH had an increased relative virulence 1,200-fold (50% lethal dose, 4.0 CFU) greater than that of cells grown in the same medium but at pH 7.2 and with sufficient iron. These studies indicate that pH and iron can be important factors in the determination of meningococcal virulence.     

Affinity-purified human immunoglobulin G that binds a lacto-N-neotetraose-dependent lipooligosaccharide structure is bactericidal for serogroup B Neisseria meningitidis

Despite technological advances, no vaccine to prevent serogroup B meningococcal disease is available. The failure to develop a vaccine has shifted the focus to an alternative outer membrane structure, lipooligosaccharide (LOS), because disseminated disease induces bactericidal immunoglobulin G (IgG) that binds LOS. The purpose of this study was to identify the LOS structure(s) that induces human bactericidal IgG by purification and characterization of these antibodies. Human LOS IgG antibodies were affinity purified by passage of intravenous immunoglobulin through purified, type-specific LOS having a known structure coupled to epoxy-activated Sepharose 6B. Pathogenic group B strains representing the major LOS serotypes were used to examine the binding and bactericidal activities of four LOS-specific IgG preparations. All four LOS-specific IgG preparations bound to strains expressing homologous, as well as heterologous, LOS serotypes as determined by flow cytometry and an enzyme-linked immunosorbent assay. With human complement, IgG that was purified with L7 LOS was bactericidal for strains expressing L3,7 and L2,4 LOS, serotypes expressed by the majority of disease-associated group B and C meningococci. In conclusion, we purified human LOS-specific IgG that binds meningococci across LOS glycose-specific serotypes. An antigen that is dependent on the glycose lacto-N-neotetraose induces IgG in humans that is bactericidal for L2, L3, L4, and L7 strains. A vaccine containing this antigen would have the potential to protect against the vast majority of group B meningococcal strains.     

Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway

Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants of Neisseria meningitidis that contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS alpha or beta chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an approximately 10-fold (P < 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2 from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2 linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.     

Neisseria meningitidis RTX proteins are not required for virulence in infant rats

RTX cytotoxins play an important role in virulence of numerous gram-negative pathogens. Unexpectedly, however, we show here that the RTX proteins of Neisseria meningitidis are dispensable for virulence in the infant rat model of infection.     

Organism resembling Neisseria gonorrhoeae and Neisseria meningitidis.

A problem isolate resembling Neisseria gonorrhoeae and Neisseria meningitidis is reported. Growth and biochemical characteristics indicated the organism to be N. meningitidis, whereas serological characteristics indicated it to be N. gonorrhoeae. This vaginal isolate may be a genetically transformed gonococcus with the ability to utilize maltose. Conversely, it may be a meningococcus which has acquired antigenic determinants of N. gonorrhoeae.     

Lipooligosaccharide structure contributes to multiple steps in the virulence of Neisseria meningitidis

Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galbeta-GlcNAc-Galbeta-Glcbeta-Hep2 (GlcNAc, Glcalpha) 3-deoxy-D-manno-2-octulosonic acid (KDO2)-lipid A; 1,4' bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.     

Relationship of Serogroups of Neisseria meningitidis II. R Variants of Neisseria meningitidis

Meningococci isolated in primary cultures from nasopharyngeal carriers occasionally consisted of mixtures of smooth (S) and rough (R) strains. The R strains were separated from the S strains and their morphological and serological characteristics were studied. Some of these R strains reverted spontaneously to S strains which subsequently produced group-specific polysaccharide. Several R strains, grown in the presence of deoxyribonucleic acid from either an R strain of known parentage or an S strain, formed recombinants with serological group specificity.     

Molecular analysis of lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A HindIII gene bank of Neisseria gonorrhoeae MUG116 was constructed in the cosmid vector pHC79. A cosmid (pSY81) was isolated that was able to convert N. gonorrhoeae FA5100 to reactivity with monoclonal antibody (MAb) 2-1-L8. Several MAb-reactive transformants were isolated and characterized with respect to lipooligosaccharide (LOS) production as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their ability to react with two other LOS-specific MAbs (3F11 and O6B4), and Southern blot analysis. Escherichia coli containing the clone had altered lipopolysaccharide expression as determined by electrophoretic analysis; however, no reactivity was seen with gonococcus-specific MAbs. The introduction of pSY81 into FA5100 had a pleiomorphic effect, giving rise to transformants having the full parental phenotype or transformants lacking reactivity to a combination of LOS-specific MAbs. Southern blot analysis indicated that the LOS biosynthetic mutation in FA5100 was not due to chromosomal rearrangement or large deletions.     

Urethritis caused by Neisseria meningitidis.

A glucose-negative group B strain of Neisseria meningitidis isolated from a meningitis case is described. A brief review of Neisseria identification procedures is also presented.     

Morphological differences in Neisseria meningitidis pili.

Disease and carrier isolates of Neisseria meningitidis were examined for their ability to adhere to human buccal epithelial cells and human cell lines and to hemagglutinate human erythrocytes, properties thought to be associated with the presence of pili. Seventy percent (7 of 10) of carrier isolates were found to be highly adherent to human buccal epithelial cells and to agglutinate human A, B, O, Rh-, and Rh+ erythrocytes. In contrast, 60% of the disease isolates adhered poorly to human buccal epithelial cells and 80% failed to agglutinate human erythrocytes. No adherence of either disease or carrier isolates was observed when several human cell lines were tested. When the meningococcal strains were examined by electron microscopy, 7 of 10 disease isolates were found to possess large bundles of aggregated pili (alpha-type pili), while 7 of 10 carrier isolates were found to have numerous unaggregated pili (beta-type pili). A monoclonal antibody against meningococcal pili and one against gonococcal pili reacted with 6 of 10 piliated carrier isolates and 4 of 10 piliated disease isolates. These results suggest that meningococci, like gonococci, possess different types of pili which differ in morphological, antigenic, and binding properties. In addition, antigenic and morphological differences between pili from carrier and disease isolates were observed as well as differences in adherence and hemagglutinating properties.     

Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes

Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.