A central step in the pathogenesis of bacterial meningitis caused by Neisseria meningitidis (the meningococcus) is the interaction of the bacteria with cells of the blood-brain barrier. In the present study, we analysed the invasive potential of two strains representing hypervirulent meningococcal lineages of the ET-5 and ET-37 complex in human brain-derived endothelial cells (HBEMCs). In contrast to previous observations made with epithelial cells and human umbilical vein-derived endothelial cells (HUVECs), significant internalization of encapsulated meningococci by HBMECs was observed. However, this uptake was found only for the ET-5 complex isolate MC 58, and not for an ET-37 complex strain. Furthermore, the uptake of meningococci by HBMECs depended on the presence of human serum, whereas serum of bovine origin did not promote the internalization of meningococci in HBMECs. By mutagenesis experiments, we demonstrate that internalization depended on the expression of the opc gene, which is present in meningococci of the ET-5 complex, but absent in ET-37 complex meningococci. Chromatographic separation of human serum proteins revealed fibronectin as the uptake-promoting serum factor, which binds to HBMECs via alpha 5 beta 1 integrin receptors. These data provide evidence for unique molecular mechanisms of the interaction of meningococci with endothelial cells of the blood-brain barrier and contribute to our understanding of the pathogenesis of meningitis caused by meningococci of different clonal lineages.

Cell surface‐located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface‐located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild‐type bacteria to a capsule‐negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule‐negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element‐based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.

The stx-flanking regions of 49 Shiga toxin-producing Escherichia coli strains and nine Shigella dysenteriae serotype 1 strains containing either stx, stx 1 , stx 2 , or stx 2 variant genes, were examined. We analyzed these regions by PCR using a set of primers with one primer specific for the respective stx gene and a second primer complementary to sequences of Stx phages H-19B and 933W. We further characterized the amplification products by restriction endonuclease digestion and nucleotide sequencing. PCR products of stx 1 -containing E. coli strains of serogroups O157, O26, and 0103 showed the same lengths and similar restriction patterns. However, we failed to amplify the 3 stx-flanking region in stx 1 -harboring E. coli O111:H ؊ strains. Stx2-producing E. coli strains revealed amplification products of different lengths and restriction patterns, suggesting greater heterogeneity than in stx 1 -positive strains. We also obtained specific PCR products for two Stx2c-producing and seven Stx2f-producing E. coli strains when they were subjected to PCR analysis. In nine S. dysenteriae type 1 strains, H-19B-and 933W-specific primers amplified only the 3 stx-flanking region. The results of our study demonstrate that the stx genes of all strains investigated are continuous with phage sequences. Whereas almost all strains except E. coli O111:H ؊ strains were associated with a S-like gene, association with Q could not be demonstrated in nine S. dysenteriae type 1 strains and three E. coli strains. Furthermore, we showed that the organization of the stx-flanking regions is similar in all strains investigated, whereas fine-structure analysis showed subtle differences among the sequences examined. Our results support the hypothesis that stx genes in E. coli and S. dysenteriae are generally phage-borne.Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause a broad spectrum of human diseases ranging from watery diarrhea to hemorrhagic colitis and the hemolytic-uremic syndrome (5). Shiga toxins have been generally accepted to be the main agent in the development of these diseases (5, 16).Two Stx subgroups have been described in E. coli: Stx1 and Stx2. The structural genes for Stx1 and Stx2 are encoded in the genome of temperate, lambdoid bacteriophages in E. coli O157 and O26 strains (9,(28)(29)(30). Such Stx-encoding phages of E. coli O157 and O26 strains were reported to have similar morphologies with regular hexagonal heads and short tails (20). E. coli O157 phages are similar in genome size and have highly related restriction fragment length polymorphism (RFLP) patterns (17). stx RFLP pattern analysis of O157 and non-O157 STEC strains suggested a more heterogeneous structure of phage DNA flanking the stx genes in E. coli O157:H7 than in non-O157 STEC strains (23). Recently, the whole genome of the Stx2-encoding phage 933W and a 17.3-kb fragment of the Stx1-encoding phage H-19B were determined by nucleotide sequencing (15,19).Sequence analysis of a part of the Stx1-encoding phage H-19B has shown that the stxA 1 and s...

In this work we analyzed the roles of meningococcal lipooligosaccharide (LOS) and capsule expression in the interaction of Neisseria meningitidis with human dendritic cells (DC). Infection of DC with serogroup B wild-type meningococci induced a strong burst of the proinflammatory cytokines and chemokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. In contrast, a serogroup B mutant strain lacking LOS expression barely led to cytokine induction, demonstrating that meningococcal LOS is the main mediator of the proinflammatory response in human DC. Sialylation of meningococcal LOS did not influence cytokine secretion by DC. However, we found the phagocytosis of N. meningitidis by human DC to be inhibited by LOS sialylation. In addition, the expression of the meningococcal serogroup A, B, and C capsules dramatically reduced DC adherence of N. meningitidis and phagocytosis to some extent. Hence, LOS sialylation and capsule expression are independent mechanisms protecting N. meningitidis from the phagocytic activity of human DC.

Infection with Neisseria meningitidis serogroup B is responsible for fatal septicemia and meningococcal meningitis. The severity of disease directly correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣), interleukin-1 (IL-1), IL-6, and IL-8. However, the source of these cytokines has not been clearly defined yet. Since bacterial infection involves the activation of dendritic cells (DCs), we analyzed the interaction of N. meningitidis with monocyte-derived DCs. Using N. meningitidis serogroup B wild-type and unencapsulated bacteria, we found that capsule expression significantly impaired neisserial adherence to DCs. In addition, phagocytic killing of the bacteria in the phagosome is reduced by at least 10-to 100-fold. However, all strains induced strong secretion of proinflammatory cytokines TNF-␣, IL-6, and IL-8 by DCs (at least 1,000-fold at 20 h postinfection [p.i.]), with significantly increased cytokine levels being measurable by as early as 6 h p.i. Levels of IL-1␤, in contrast, were increased only 200-to 400-fold at 20 h p.i. with barely measurable induction at 6 h p.i. Moreover, comparable amounts of cytokines were induced by bacterium-free supernatants of Neisseria cultures containing neisserial lipooligosaccharide as the main factor. Our data suggest that activated DCs may be a significant source of high levels of proinflammatory cytokines in neisserial infection and thereby may contribute to the pathology of meningococcal disease.