Cerebrospinal meningitis is a feared disease that can cause the death of a previously healthy individual within hours. Paradoxically, the causative agent, Neisseria meningitidis, is a common inhabitant of the human nasopharynx, and as such, may be considered a normal, commensal organism. Only in a small proportion of colonized people do the bacteria invade the bloodstream, from where they can cross the blood–brain barrier to cause meningitis. Furthermore, most meningococcal disease is caused by bacteria belonging to only a few of the phylogenetic groups among the large number that constitute the population structure of this genetically variable organism. However, the genetic basis for the differences in pathogenic potential remains elusive. By performing whole genome comparisons of a large collection of meningococcal isolates of defined pathogenic potential we brought to light a meningococcal prophage present in disease-causing bacteria. The phage, of the filamentous family, excises from the chromosome and is secreted from the bacteria via the type IV pilin secretin. Therefore, this element, by spreading among the population, may promote the development of new epidemic clones of N. meningitidis that are capable of breaking the normal commensal relationship with humans and causing invasive disease.

Metagenomic approaches applied to viruses have highlighted their prevalence in almost all microbial ecosystems investigated. In all ecosystems, notably those associated with humans or animals, the viral fraction is dominated by bacteriophages. Whether they contribute to dysbiosis, i.e., the departure from microbiota composition in symbiosis at equilibrium and entry into a state favoring human or animal disease is unknown at present. This review summarizes what has been learnt on phages associated with human and animal microbiota, and focuses on examples illustrating the several ways by which phages may contribute to a shift to pathogenesis, either by modifying population equilibrium, by horizontal transfer, or by modulating immunity.

We have investigated genetic differences between the closely related pathogenic Neisseria species, Neisseria meningitidis and Neisseria gonorrhoeae, as a novel approach to the elucidation of the genetic basis for their different pathogenicities. N. meningitidis is a major cause of cerebrospinal meningitis, whereas N. gonorrhoeae is the agent of gonorrhoea. The technique of representational difference analysis was adapted to the search for genes present in the meningococcus but absent from the gonococcus. The libraries achieved are comprehensive and specific in that they contain sequences corresponding to the presently identified meningococcus-specific genes (capsule, frp, rotamase, and opc) but lack genes more or less homologous between the two species, e.g., ppk and pilCI. Of 35 randomly chosen clones specific to N. meningitidis, DNA sequence analysis has confirmed that the large majority have no homology with published neisserial sequences. Mapping of the cloned DNA fragments onto the chromosome of N. meningitidis strain Z2491 has revealed a nonrandom distribution of meningococcus-specific sequences. Most of the genetic differences between the meningococcus and gonococcus appear to be clustered in three distinct regions, one of which (region 1) contains the capsulerelated genes. Region 3 was found only in strains of serogroup A, whereas region 2 is present in a variety of meningococci belonging to different serogroups. At a time when bacterial genomes are being sequenced, we believe that this technique is a powerful tool for a rapid and directed analysis of the genetic basis of inter-or intraspecific phenotypic variations.The study of bacterial pathogenicity has greatly benefited from tools such as transposon mutagenesis, which have made possible the identification of virulence genes. However, there exist many bacteria for which mutagenesis is inefficient. One way to study virulence in such organisms is to take advantage of naturally occurring differences in pathogenicity between variants of the same species or between closely related species. In such a case, the differences in DNA sequence, including genes responsible for the differential pathogenicities, may be isolated from the generally similar genetic background by a subtractive technique. We set out to define genes or loci that may be responsible for the pathogenesis of meningococcal meningitis by comparing the chromosome of the meningococcus [Neisseria meningitidis (Nm)] with that of the gonococcus [Neisseria gonorrhoeae (Ng)]. These two human pathogens are very closely related, but cause notably different diseases. While Ng is generally responsible for localized inflammation of the urogenital tract, the meningococcus is the cause of lifethreatening disease, meningitis, that follows penetration of the blood-brain barrier and colonization of the meningeal membranes by the bacteria.In contrast to the great differences in pathogenic potential of the gonococcus and the meningococcus, the organisms are closely related at the level of genetic organiza...

Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109 -11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37°C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA؉ phenotype even in the presence of a functional periplasmic DsbA.

Phylogenetic relationships, virulence factors, alone and in specific combinations, and virulence in a rat meningitis model were examined among 132 isolates of Escherichia coli neonatal meningitis from France and North America. Isolates belonging to phylogenetic groups A (n=11), D (n=20), and B2 (n=99) had similar high prevalence rates of the siderophores aerobactin and yersiniabactin and the K1 capsule (>/=70%) yet induced different level of experimental bacteremia. Ectochromosomal DNA-like domains involved in blood-brain barrier passage (PAI III(536) [sfa/foc and iroN; 34%]; GimA [ibeA and ptnC; 38%]; PAI II(J96) [hly, cnf1, and hra; 10%]) were restricted to B2 isolates. Among group B2 isolates, representatives of the O45:K1 clonal group (n=30), which lacked these domains, were as able as the archetypal O18:K1 strain C5 to cause meningitis. Molecular epidemiology combined with experimental virulence assays demonstrate that known virulence factors are insufficient to fully explain the pathophysiology of ECNM and to allow for rational search for new virulence factors.

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis.Neisseria meningitidis colonizes the nasopharynx, from which it can seed the bloodstream before crossing the blood-brain barrier (BBB) to cause meningitis. In contrast, Neisseria gonorrhoeae colonizes and invades the epithelium of the genitourinary tract, where it can cause a localized inflammation; bacteremia, though frequent, is asymptomatic and dissemination is rare. Thus, both species are capable of crossing a cellular barrier at their port-of-entry but they differ in their abilities to subsequently disseminate in the blood. The ability to induce intense and prolonged bacteremia is one of the prerequisites for a bacterial pathogen to cross the BBB. In contrast, the details of specific interactions with the cellular components of the BBB remain unclear. Therefore, in order to understand the mechanisms that allow N. meningitidis to cross the BBB, it will be necessary to identify the genes that are involved in bloodstream dissemination and/or specific interaction with the cellular components of the BBB. Such genes might be present in both N. meningitidis and N. gonorrhoeae but differ subtly in sequence or regulation, or they might be present in only one of the two species.Results from in vitro models have shown that most of the mechanisms mediating cellular interactions are common to both N. meningitidis and N. gonorrhoeae. On the other hand, several determinants have been identified that are specific to N. meningitidis: the polysaccharide capsule (8), the enzyme rotamase (26), the RTX toxin-like Frp proteins (29, 30), and a glutathione peroxidase (20). Of these, the capsule locus is ...

Specific virulence factors associated with the pathogenesis of Escherichia coli strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified. However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM. To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique. The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from E. coli strain C5, isolated from a case of neonatal meningitis. Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM. We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains. Of these clones, 44 were clustered in six distinct regions on the chromosome. The sfa and ibe10 genes were located in regions 2 and 6, respectively. A group of genes (cnf1, hra, hly, and prs) known to be present in a pathogenicity island of the uropathogenic strain E. coli J96 colocalized with region 6. The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitisassociated strains belonging to the same B2 group. Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains. Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of E. coli to invade the meninges of neonates.Escherichia coli is responsible for a third of the cases of neonatal meningitis (NM), with an incidence of 0.1 per 1,000 live births (8). Case fatality rates are still very high and range from 25 to 40%. Furthermore, the occurrence of long-term neurologic sequelae in nonfatal cases is 33 to 50% of neonates with E. coli meningitis (8, 9, 32). Understanding the pathogenesis of this disease and characterizing these pathogenic strains are prerequisites to the development of new treatments.Few specific pathogenic determinants have been described for E. coli strains causing NM (ECNM). Both expression of the K1 capsular polysaccharide (17) and production of aerobactin (21) are believed to be important for bloodstream dissemination. On the other hand, S fimbrial adhesin (sfa) (11,17,23) and Ibe10 protein (13), involved in the adhesion and invasion of brain microvascular endothelial cells, likely promote the crossing of the blood-brain barrier.Phylogenetic approaches have helped to characterize the pathogenic strains. The E. coli species has been divided into four main phylogenetic groups designated A, B1, B2, and D (12, 28). Previous studies have shown that ECNM has a clonal structure (3,27) and that strains mostly belong to the B2 group (5). Considering that only 38% of ECNM have both sfa and ibe10, it is likely that other determinants remain to be identified (5). In favor of this hypothesis is the fact that 10 pa...

The occurrence of antigenic shift during meningococcal infection has been investigated by comparison of paired isolates obtained from the blood, cerebrospinal fluid or nasopharynx of patients. Isolates from any individual produced identical DNA 'fingerprints' and showed stability in expression of both class 2 outer membrane protein and an antigen common to pathogenic Neisseria, confirming their origin as a single strain. One of the four strains examined produced variants which differed in the molecular mass of their class 5 outer membrane proteins. Three of the strains produced pili containing the epitope recognized by monoclonal antibody SM1 and two of these gave rise to variants which expressed pili of differing subunit molecular masses. The two variants of the remaining strain produced pilins lacking the common epitope detected by antibody SM1 but radioimmune precipitation with polyclonal anti-pilus antiserum revealed that variation in the molecular mass of the pilin expressed also occurred with this second class of pili. Antigenic variation in expression of both class 5 outer membrane proteins and pili therefore appears to be a common occurrence during meningococcal infection.