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 the study of genetic alterations involved in resistance to α -lactam antibiotics.

1. Molecular pathogenesis of meningococcal infections. Neisseria meningitidis (Nm) is a commensal bacterium of the human nasopharynx. Invasive infection occurs when bacteria, first adhering to the respiratory epithelium, invade the bloodstream and then cross the blood brain barrier, the synovium or pericardium to provoke meningitis, arthritis or pericarditis, respectively. Adhesion of Nm to epithelial and endothelial cells is a crucial step in meningococcal infection that provokes a complex bacterium-cell cross talk. Nm-cell interaction begins with initial (localized) adhesion and then progresses toward intimate adhesion. PilC1, located on pili is the major adhesin and the expression pilC1 is transiently induced following initial adhesion. This induction depends on a transcription start point localized within the contact regulatory element of Neisseria (CREN) in the promoter region of pilC1. Another gene, crgA, coding CrgA, a new bacterial LysR-like transcriptional regulator, is also induced in a CREN dependent manner. The expression of bacterial surface structures involved in adhesion, such as pili and capsule, is modulated by CrgA, which is involved in a co-ordinate regulation of the genes pilC1, pilE, sia and crgA to permit the shift from initial to intimate adhesion.

1.2. A mouse model of meningococcemia. Based on epidemiological studies on the association between influenza outbreaks and meningococcal diseases, we have developed a model of sequential influenza A virus (IAV)-Nm infection in mice. Previous IAV infection induces a transient phase of susceptibility of mice to challenges with Nm leading to fatal meningococcal pneumonia and bacteremia. Sequential histopathological examination shows localized lesions of the bronchial epithelium in which Nm adher to and cross the epithelial barrier. An intense pneumonia occurs with infiltrating neutrophils and the phagocytosis of Nm. A perivascular infiltration of leukocytes and of meningococci is associated with an inflammation of the adjacent endothelium, and the subsequent bacteremia. This experimental model was used for evaluating the role of some major bacterial virulence factors. A capsule-defective mutant was cleared from the lungs, whereas a crgA mutant retained invasiveness. Other experiments show that a penI isogenic derivative (altered penA gene) of the penS wild-type Nm strain was impaired in its in vivo survival, suggesting that the structural alterations of the peptidoglycan, associated with the modifications of the PBP2, described below, could result in impaired virulence of strains with diminished susceptibility to penicillin. 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.

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

2. Surveillance of meningococcal infections by the National Reference Center (NRCM). Meningococcal diseases occur as sporadic cases with an annual incidence below 1 per 100,000 inhabitants in France. The NRCM receives an average of 1,000 strains of Nm per year from approximately 700 collaborating laboratories in France, for confirmation and typing. Approximately half of the strains are from invasive infections, mainly meningococcemia or meningitis. Strains of serogroup B are prominent (more of 50% of the strains) with a relatively stable incidence at 270± 28 cases per year. An increasing incidence of meningococcal diseases, mainly associated with an increasing incidence of serogroup C, was observed since 2001. However, periodic fluctuation in the incidence of serogroup C has yet been observed. A peak of incidence of serogroup C was yet observed in 1992 with 191 cases (40.2%), followed by a continuous decrease until 1995 with 56 cases (15%) and then a continuous increase reaching 235 cases (38.2%) in 2002, but a decrease to 31% is observed in 2003.The incidence of serogroup W135, first detected in 1994, reached 9.8% in 2002, but decreased to 5,9% in 2003. Serogroup Y is stably found in 2-3% of the invasive infections, but mainly in immunocompromised patients and in the elderly. Case fatality rates vary between 8 to 10% per year. They are mostly associated with sepsis and purpura fulminans.

3. Molecular diagnosis and epidemiology of N. meningitidis.

Direct identification and characterization of N. meningitidis from clinical samples without culture, can be achieved by amplification of the highly conserved regulatory gene crgA, followed by that of the alleles of siaD, involved in the biosynthesis of the capsule for serogroups B, C, Y and W135 or mynB involved in the biosynthesis of the capsule of serogroup A. This molecular diagnosis of meningococcal disease is now recognized by the ministry of health as a one of the criteria for the reporting of meningococcal disease (circulaire DGS/SD5C/2002/400). Molecular typing of Nm is essential for assessing an epidemiological link between cases. Polymorphic chromosomal loci pilA, pilD, regF, iga and crgA are amplified by PCR and further analyzed by multilocus DNA fingerprinting which fully correlates with the multilocus enzyme electrophoresis and is completed by the multilocus sequence typing and the pulsed-field electrophoresis.

4. The surveillance of the Nm W135 (ET-37) epidemic clone, in the "African meningitis belt". Confirmation of its role in the 2001 and 2002 outbreaks.

We were requested in April 2001 by the West African Health Organization to investigate large meningitis outbreaks in Burkina Faso and Niger, while the vaccination campaign with A&C vaccine seemed ineffective. Ninety-eight samples from meningitis patients visited in 16 districts from Bobo Dioulasso to Niamey were tested by PCR for the presence of Nm, or Streptococcus pneumoniae or Haemophilus influenzae DNA. Sixty five percent were positive for Nm, 7% for S. pneumoniae and 2% for H. influenzae type B. Half of the Nm-positive samples corresponded to serogroup W135 and half to serogroup A. The characterization of 12 strains A:4:P1-9 confirmed the involvement of serogroup W135 of to the clonal complex ET-37 for the first time in epidemic conditions in Africa. The emergence of serogroup W135 (ET-37) as new "epidemic clone", escaping the available A&C vaccination, in Africa was confirmed by a new outbreak in Burkina Faso in 2002 where more than 84% of the isolates were of serogroup W135. This study demonstrated the usefulness of the molecular diagnosis for a rapid and reliable surveillance of meningitis in epidemic conditions and for better selection of vaccine strategies. An active laboratory surveillance of meningitis cases during the inter-epidemic period as well as longitudinal epidemic laboratory investigations have been implemented through a collaborative project initiated by our unit, the "Meningitis in Sahel project", in Burkina Faso at the Centre Muraz of Bobo Dioulasso and in Niger at the CERMES in Niamey.

5. The polymorphism of the gene penA and its relationship to reduced susceptibility to penicillin. The current incidence of N. meningitidis strains with reduced susceptibility to penicillin G (MIC?0.125mg/L) reaches approximately 30%. We have studied the polymorphism of the gene penA, encoding the penicillin-binding protein 2 (PBP2), among clinical isolates, including penicillin-susceptible strains (MIC<0.125mg/L) and strains with diminished susceptibility (1 mg/L>MIC?0.125mg/L). Strains with MIC of penicillin <0.125mg/L (penS) harbored identical or highly related penA alleles, whereas strains with MIC >0.125mg/L (penI) harbored altered sequences of penA but the sequence encoding the catalytic site on PBP2 was highly conserved. 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. By comparatively testing the affinity of purified recombinant PBP2 from either penS or penI strains, we have found that PBP2 from penI has lower affinity than that of penS, suggesting conformational changes in the penI PBP2 in the vicinity of the the catalytic site on PBP2.

5.1. Analyzing the structure of N. meningitidis peptidoglycan and its modifications in penI strains. Since PBPs are involved in the synthesis of peptidoglycan, we have analyzed the detailed structure of peptidoglycan 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 peptidoglycan represented by an augmentation in muropeptides sharing a particular pentapeptide (GlcNac and MurNac). These structural alterations of peptidoglycan 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 (see above 1.2).

Keywords: Neisseria meningitidis, Molecular pathogenesis, pathophysiology, molecular epidemiology, Reference Laboratory

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