Unit: Neisseria

Director: Jean-Michel ALONSO

Main research topics of the Neisseria unit, National Reference Center for the Meningococci, are in the field of the molecular epidemiology and pathogenesis of infections due to Neisseria meningitidis, including the molecular typing of clinical isolates from invasive infections. Experimental research focuses on the characterization of virulence factors and protective antigens through the development of cellular and murine models.

1. Global epidemiology. In industrialized countries, meningococcal diseases occur as sporadic cases due to diverse genotypes, mainly serogroup B, C and W135 isolates. In this context, host immunological defects or immunosuppressive conditions are suspected in any invasive infection, whereas the simple contamination of the normal host usually leads to asymptomatic carriage and naturally acquired immunity. In the "subSaharan African meningitis belt" (from Senegal to Ethiopia), meningococcal meningitis epidemics occur periodically that are due to homogenous genotypes, mainly strains of serogroups A and W135. In these cases, the virulence of the epidemic clone determines the transmissibility and severity of the disease among immunologically naive populations.

1.1. Surveillance of meningococcal infections by the National Reference Centre for Meningococci (CNRM). The CNRM expertises an average of 1,000 strains of N. meningitidis per year. Half of the strains are from invasive infections (meningitis, meningococcemia). Sporadic cases occur with an annual incidence below 1 per 100,000 inhabitants. Serogroup B is the most frequent (>55%). An increasing incidence of serogroup C was observed in 2002, reaching 38.2%, followed by a decrease to 31% in 2003 that was confirmed in 2004 (30.85%). The incidence of serogroup W135 reached 9.8% in 2002, then decreased to 5,9% in 2003 and to 4% in 2004. Serogroup Y as a stable incidence at 2-4%, and is mainly isolated from immunocompromised hosts.

1.2. Molecular diagnosis and typing of N. meningitidis. The identification and genogrouping of N. meningitidis can be achieved by the amplification of the crgA gene, followed by that of siaD, encoding the biosynthesis of the capsule of serogroups B, C, Y and W135, or mynB encoding the capsule of serogroup A. This technique is now recommended as a criterion for the obligatory declaration of an invasive meningococcal infection (circulaire DGS/SD5C/2002/400). Various techniques characterizing various polymorphic chromosomal loci are further used, such as the "multilocus DNA fingerprinting", the "multilocus sequence typing" and the pulsed field gel electrophoresis that permit to identify epidemiological links between N. meningitidis strains.

1.2.1. Surveillance of epidemics in the "African meningitis belt". The emergence of the serogroup W135 (ET-37/ST11) as a new epidemic clone, escaping A&C vaccination, in Africa was detected in 2001 in Burkina Faso and Niger by our laboratory, and further confirmed in 2002 in Burkina Faso where more than 84% of the isolates were of serogroup W135. A meningitis biological surveillance system has been set up for the surveillance of all meningitis cases during epidemics, in order to select the most appropriate vaccine. This has been organized with the collaboration of the Centre Muraz of Bobo Dioulasso in Burkina Faso and the CERMES of Niamey in Niger . The use of direct multiplex PCR diagnostic methods now permits to obtain rapid and reliable etiological diagnosis of any case of acute bacterial meningitis, either due to the meningococcus, the pneumococcus or Haemophilus, even in remote areas.

2. Pathophysiology and molecular pathogenesis of invasive meningococcal infections. N. meningitidis is a commensal bacterium of the human rhinopharynx, but it is able to cause an invasive infection (septicemia, meningitis). Some meningococcal virulence factors are responsible for the invasion of blood vessels, as well as the subarachnoïdal or synovial or pericardic fluids. However, the infection of these normally sterile profound anatomical sites does not participate to the cycle of transmission of N. meningitidis from infected to recipient host, since host to host transmission occurs via respiratory dropplets. The genetic variability of the meningococci, due to frequent DNA horizontal exchanges between strains, contributes to continuously generate new variants with modified virulence and/or transmissibility properties.

2.1. An experimental model of meningococcemia. Based upon epidemiological studies showing a correlation between the incidence of influenza and meningococcal outbreaks, we have developed a mouse model of sequential infection with the influenza A virus (IAV) and N. meningitidis. IAV primary infection induces a transitory phase of susceptibility of mice to the superinfection by N. meningitidis, inducing a pneumonia and a subsequent bacteremia. This mouse model of invasive meningococcal infection mimicks the main steps of the disease in human and permits to analyze the functions of known or genome-derived new genes.

2.2. Modulation of virulence and transmissibility meningococcal determinants. Two major virulence factors are involved in the infectious and invasive process, the pili and the capsule. Pili are surface structures of the bacterium, responsible for attachment to host cells. The capsule protects the bacteria against phagocytosis and facilitates their dissemination via blood. The pili and the capsule also play a role in the transmissibility. Indeed, these surface structures are necessary for colonization and extracellular survival of N. meningitidis, but they create a steric impairment during the cellular invasion process and the trans-epithelial and trans-endothelial translocation. The persistence of these structures could therefore facilitate the detachment of the bacteria and their transmission to another host. The modulation of these structures through their transcriptional regulation is hence an essential mechanism in the virulence/transmissibility of N. meningitidis. We have shown, by using the mouse model described above, that a capsule mutant is rapidly cleared from the lungs, whereas a mutant with an inactivated transcriptional regulator crgA has enhanced virulence.

2.3. Genetic variability in N. meningitidis. DNA horizontal transfers, by transformation and recombination, in N. meningitidis influence the evolution of virulence/transmissibility in invasive strains, by generating two types of variants. Short-term variants, with identical genotype to the parent strain, correspond to strains sharing one mutation on one antigenic epitope. This provides a temporary selective advantage to the bacterial population, to circumvent host immunity. Long-term variants, by contrast, represent the emergence and expansion of a new epidemic clone. This may be the result of the replacement of a bacterial population from a particular serogroup by another population of a different serogroup; in this case both populations are often genetically distinct. Or it may result from a change in the serogroup (capsule switching) when the epidemic strain undergoes the replacement of its siaD gene, encoding the capsule, by transformation and recombination. This genetic conversion of one single gene, encoding the biosynthesis of the capsule, occurs without changing the genotype of the strain within the same clonal complex. We are currently evaluating the differences of strains of different clonal complexes and the role of the capsule switching in virulence through the mouse model.

2.4. Impact of meningococcal genetic variability on vaccine strategies. Current meningococcal vaccines are based on capsular antigens that determine four out of the five major serogroups involved in invasive infections worldwide (serogroups A, C, W135, and Y, but not serogroup B). The emergence of antigenic variants within a serogroup allows bacterial escape to vaccine-induced immunity. The risk for vaccine escape was illustrated by the emergence of serogroup W135 among Haj pilgrims vaccinated against serogroups A and C, in 2000, and in Burkina Faso and Niger in 2001, also after vaccination with A and C meningococcal vaccine. There is a high risk of escaping to either natural or vaccine-induced immunity in bacteria which are spontaneously competent for transformation, such as the meningococci, and this requires a constant and careful surveillance of invasive strains, by using modern tools of molecular epidemiology to detect any antigenic variation.

2.5. The polymorphism of the penA gene and its relationship to susceptibility to penicillin. The incidence of N. meningitidis strains with reduced susceptibility to penicillin G reaches 30%. Strains with MIC of penicillin <0.125mg/L (penS) harbor identical penA alleles, whereas strains with MIC >0.125mg/L (penI) harbor altered sequences of penA. Transformation of penS strains into penI was obtained in vitro or by mixed culture of both genotypes, suggesting a direct correlation between alterations of penA and the expression of diminished susceptibility to penicillin.

2.5.1. The structure of N. meningitidis peptidoglycane and its modifications in penI strains. Since PBPs are involved in the synthesis of peptidoglycane, we have analyzed the detailed structure of peptidoglycane by mass-spectrometry and chromatography and characterized 28 different muropeptide species. PenI clinical isolates as well as isogenic penA mutants from a penS strain have altered structure of their peptidoglycane, represented by an augmentation in muropeptides sharing a particular pentapeptide (GlcNac and MurNac). These structural alterations of peptidoglycane are directly linked to the structural changes of PBP2 in penI strains, suggesting that N. meningitidis isolates with diminished susceptibility to penicillin G have important alterations of their cell wall. Moreover, these alterations may affect the virulence of such strains, as suggested by virulence studies in the mouse model.

2.5.2. Intracellular detection of N. meningitidis peptidoglycane by Nod1. Muropeptides sharing a GlcNac-MurNac-tripeptide chain of the peptidoglycane of Gram-negative bacteria, including N.meningitidis, are detected by the intracellular molecule Nod1 activating the transcription of molecules involved in innate immunity in epithelial cells via NFΚ B. This unique muropeptide may be a new "pathogen-associated recognition pattern" (PAMP) and a new putative target molecule for anti-bacterial agents.

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