|Director : Jean-Michel ALONSO (firstname.lastname@example.org)|
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 (bacteremias and meningitis) and particularly the clones involved in epidemics in France and in Africa. Experimental research focuses on the characterization of virulence factors and regulatory genes involved in adhesion and invasion at structural and functional levels, in vivo, and the study of genetic alterations involved in resistance to beta-lactam antibiotics.
1. Surveillance of meningococcal infections by the National Reference Center .
N. meningitidis is a major causative agent of meningitis (30% of bacterial meningitis) and of meningococcemias (septicemias) worldwide. In addition to neurologic sequels, purpura fulminans and fatal septic shock can complicate meningococcal infections. Meningococcal infections are potentially epidemic. Their world incidence varies, according to countries, from 1 to 10 per 100,000 inhabitants, with mortality rates reaching up to 10%. In France, the incidence is increasing since 1995. Nevertheless, the cases remain sporadic and no epidemic has been recorded. The proportion of the various serogroups among the strains recovered from invasive infections is 50% for the prominent serogroup B, followed by an increasing proportion of the serogroup C (38%) and of the serogroup W135 (10%).
2. Experimental research.
Our research activities are based on the careful surveillance of the actual status of meningococcal diseases, considering the evolution of the bacterial molecular determinants as well as the criteria of host susceptibility. They are conducted in collaboration with the correspondents of the Reference Center at the national and international levels. Our main goal is to improve the diagnostic and typing methods and to provide better knowledge on the pathogenesis of these diseases.
2.1. Molecular epidemiology of N.meningitidis.
2.1.1. Molecular diagnosis and typing.
Direct characterization of N.meningitidis from clinical samples (blood, cerebrospinal fluid, etc.) permits its determination by amplification of the regulatory gene crgA, followed by that of the genes siaD, encoding the capsule for serogroups B, C, Y and W135 or the gene mynB encoding the synthesis of capsular polysaccharides of serogroup A. Molecular typing of N.meningitidis is essential for assessing an epidemiological link between cases. Polymorphic chromosomal loci pilA, pilD, regF, igaA and crgA are amplified by PCR and further analyzed by multilocus DNA fingerprinting. Various alleles are then defined that are specific for each clonal group. This technique, fully correlates with the multilocus enzyme electrophoresis, and is completed by the pulsed-field electrophoresis and the multilocus sequence typing to characterize several geographic foci in France those confirm an endemic condition due to heterogeneous genotypes.
2.1.2. Global expansion of N.meningitidis W135 belonging to the ET-37 clonal complex after the Hajj-related epidemic of 2000 and the 2001 epidemic in sub-Saharan Africa.
The molecular diagnosis and typing techniques for the bacterial agents of meningitis have been applied to the surveillance of a particular clone of serogroup W135, first revealed in pilgrims returning from the Hajj in 2000 and then to the etiology of major epidemics un subsaharan africa in 2001. Following our work on the molecular detection and spread of serogroup W135, an international surveillance network is being developed. We have shown that this global expansion (Africa, Middle-East, Indonesia, Europe) is due to different genotypes (some of which are members of the ET-37 complex) expressing sometimes analogous phenotypes. The surveillance of this serogroup by using molecular typing techniques is of outmost importance since no adapted vaccines are yet available.
2.2. The polymorphism of the gene penA and its relationship to reduced susceptibility to penicillin.
N.meningitidis strains with reduced susceptibility to penicillin (MIC≥0.125mg/L) were detected since 1994 in France, reaching 30%. We have studied the polymorphism of the gene penA among clinical strains, including penicillin-susceptible strains (MIC<0.125mg/L) and strains with diminished susceptibility (1 mg/L>MIC≥0.125mg/L), of different geographical origin, antigenic formulas, and from different pathologic conditions. We observed that for different genotypes, according to restriction patterns of the genes pilA, pilD, crgA, regF and iga A, strains with MIC of penicillin <0.125mg/L (penS) harbored identical penA allele, whereas strains with MIC >0.125mg/L (penI) harbored 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. Detecting such gene alterations for penicillin susceptibility testing, can now directly be done without previous culture, whenever PCR diagnosis will be used.
2.3. Molecular genetic of virulence of N.meningitidis.
The meningococcal infectious process first involves bacterial adhesion to epithelial target cells. This is mostly due to the pili. The PilC1 protein plays a major role in the pili assembly and the adhesion to the cell. The gene pilC1 harbors a promoter region necessary for the induction of its expression when the bacteria adhere to the cell. A new transcriptional regulator has been identified, the gene crgA, of the lysR family, whose expression is induced by contact with the target cell along with that of pilC1. The gene crgA regulates several genes involved in adhesion, mostly in switching to intimate adhesion of N.meningitidis to the cell, and in the other aspects of bacteria-cell interaction.
2.4. A mouse model of meningococcemia.
We developed a model of sequential influenza A virus (IAV)-Neisseria meningitidis serogroup C (Nm), infection in BALB/c mice. Mice infected intranasally with a subletal IAV inoculum (260 PFU) were superinfected intranasally, with Nm. Fatal meningococcal pneumonia and bacteremia were observed in mice with previous IAV infection at day 7 but not at day 10. Mice susceptibility to Nm superinfection correlated with the peak of IFNg production in the lungs and the decreasing IAV load. After Nm challenge, IAV-infected as well as uninfected control mice produced both inflammatory cytokines IL-1 and IL-6. However, a production of IL-10 could be detected in susceptible mice superinfected at day 7 after IAV infection, but not in resistant mice (without IAV previous infection).
This model of dual IAV-Nm infection was further used for evaluating the role of bacterial virulence factors involved in the synthesis of the capsule. A capsule-defective mutant was cleared from the lungs, whereas a mutant inactivated for the gene crgA, regulating negatively the pili and the capsule, upon contact with the host cells, retained invasiveness. Therefore, this model of meningococcal disease in adult mice reproduces the pathogenesis of human meningococcemia with fatal sepsis, and is useful for analyzing known or new genes identified from genomic studies.
Photo :Experimental meningococcal bacteremia in the mouse.
Keywords: Experimental meningococcal bacteremia in the mouse
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|Pascale VIENNE, email@example.com||Jean-Michel ALONSO (IP), firstname.lastname@example.org
Muhamed-Kheir TAHA (IP), email@example.com
Mireille LARRIBE (Université Paris 7), firstname.lastname@example.org
|Aude ANTIGNAC,PhD student Paris 7
Ala Eddine DEGHMANE, PhD student Paris 11.
Maria Leticia ZARANTONELLI, post-doc, Argentina
|Magaly DUCOS,Senior Technician,IP.
René PIRES, Senior Technician,IP.
Annie GUIYOULE, Senior Technician, IP.
Dario GIORGINI, Senior Technician,IP.