Dario Giorgini

Meningococcal Meningitis: Unprecedented Incidence of Serogroup X—Related Cases in 2006 in Niger

BACKGROUND In Niger, epidemic meningococcal meningitis is primarily caused by Neisseria meningitidis (Nm) serogroup A. However, since 2002, Nm serogroup W135 has been considered to be a major threat that has not yet been realized, and an unprecedented incidence of Nm serogroup X (NmX) meningitis was observed in 2006. METHODS Meningitis surveillance in Niger is performed on the basis of reporting of clinically suspected cases. Cerebrospinal fluid specimens are sent to the reference laboratory in Niamey, Niger. Culture, latex agglutination, and polymerase chain reaction are used whenever appropriate. Since 2004, after the addition of a polymerase chain reaction-based nonculture assay that was developed to genogroup isolates of NmX, polymerase chain reaction testing allows for the identification of Nm serogroup A, Nm serogroup B, Nm serogroup C, NmX, Nm serogroup Y, and Nm serogroup W135. RESULTS From January to June 2006, a total of 4185 cases of meningitis were reported, and 2905 cerebrospinal fluid specimens were laboratory tested. NmX meningitis represented 51% of 1139 confirmed cases of meningococcal meningitis, but in southwestern Niger, it represented 90%. In the agglomeration of Niamey, the reported cumulative incidence of meningitis was 73 cases per 100,000 population and the cumulative incidence of confirmed NmX meningitis was 27.5 cases per 100,000 population (74.6 cases per 100,000 population in children aged 5-9 years). NmX isolates had the same phenotype (X : NT : P1.5), and all belonged to the same sequence type (ST-181) as the NmX isolates that were circulating in Niamey in the 1990s. Nm serogroup W135 represented only 2.1% of identified meningococci. CONCLUSIONS This is, to our knowledge, the first report of such a high incidence of NmX meningitis, although an unusually high incidence of NmX meningitis was also observed in the 1990s in Niamey. The increasing incidence of NmX meningitis is worrisome, because no vaccine has been developed against this serogroup. Countries in the African meningitis belt must prepare to face this potential new challenge.

Down-regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein

The initial attachment of Neisseria meningitidis to the target cell surface appears to be largely pilus depend‐ent in capsulated bacteria. Intimate adhesion subsequently occurs to permit colonization. We recently reported that insertional inactivation of the crgA gene, which encodes a transcriptional regulator belonging to the LysR family, decreased meningococcal adhesion to epithelial cells and abolished intimate adhesion. In this report, we analyse expression of the pilE and sia genes, which are involved in the biosynthesis of pili and capsule respectively, during bacteria–host cell interactions. Western blotting, transcriptional fusion and reverse transcriptase polymerase chain reaction (RT‐PCR) analysis showed that the expression of these genes was downregulated during intimate adhesion. DNA‐binding assays, footprinting and RT‐PCR analysis indicated that this downregulation was directly mediated by the CrgA protein. The pilE and sia promoters were found to have a CrgA binding motif in common. These results strongly suggest that N. meningitidis displays an adaptive response upon cell contact. CrgA may play a central regulatory role in meningococcal adhesion, particularly in switching from initial to intimate adhesion by downregulating the bacterial surface structures that hinder this adhesion.

The Role of Particular Strains of Neisseria meningitidis in Meningococcal Arthritis, Pericarditis, and Pneumonia

The clinical presentations of meningococcal diseases other than meningitis or meningococcemia may lead to erroneous diagnosis. Although several reports have described unusual meningococcal diseases, the Neisseria meningitidis strains involved in these forms have been poorly characterized. In this study, meningococcal arthritis and pericarditis were confirmed by isolation of N. meningitidis and/or detection of meningococcal DNA in synovial or pericardial fluid, respectively, and meningococcal pneumonia was detected by isolation of N. meningitidis from blood. From 1999 through 2002, meningococcal disease was bacteriologically confirmed in 26 cases of arthritis, 6 cases of pericarditis, and 33 cases of pneumonia by the National Reference Center for the Meningococci in Paris. We found a statistically significant association between strains of serogroup W135, mostly of the clonal complex ET-37, and arthritis. Pneumonia was most frequently diagnosed in patients aged >70 years, and 54.5% of the strains belonged to serogroup W135, although these strains had heterogeneous phenotypes. Bacteremia is a key step in the pathophysiology of meningococcal disease and precedes any form of invasive infection.

Intimate adhesion of Neisseria meningitidis to human epithelial cells is under the control of the crgA gene, a novel LysR-type transcriptional regulator

PilC1, a pilus‐associated protein in Neisseria meningitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact‐regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR‐type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria–cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.

Pilus‐mediated adhesion of Neisseria meningitidis: the essential role of cell contact‐dependent transcriptional upregulation of the PilC1 protein

Pilus‐mediated adherence makes an essential contribution to the pathogenesis of Neisseria meningitidis by allowing the initial localized adherence. Pili are assembled from a protein subunit called pilin. Two proteins, PilC1 and PilC2, are also key elements in the formation of pili as the production of at least one PilC protein is required for pilus assembly. In addition, PilC1 but not PilC2 modulates adhesiveness, most probably by being the adhesin. Recently, both genes have been demonstrated to be controlled by different promoters, pilC2 is expressed from a single transcription starting point (TSP), whereas pilC1 has three TSPs. One of these, PC1.1, corresponds to the unique TSP of pilC2, and two others, PC1.2 and PC1.3, are located in a region upstream of pilC1 but not pilC2. This suggests that both genes may be under the control of separate regulatory pathways. In this work, by engineering pilC1–lacZ and pilC2–lacZ transcriptional fusions, we provide evidence that expression of pilC1, but not that of pilC2, is transiently induced by bacterial cell contact. This induction required viable cells, did not need the presence of pili and relied on the expression of pilC1 from PC1.3. Destruction of this TSP by site‐directed mutagenesis did not significantly diminish the piliation level or the basal expression of PilC1, but led to the loss of cell contact‐dependent upregulation of pilC1 and to a dramatic decrease in bacterial adhesiveness. Taken together, these data demonstrate that cell contact‐dependent upregulation of the transcription of pilC1 at PC1.3 is essential for meningococcal pilus‐mediated adhesion.

Continuing Diversification of Neisseria meningitidis W135 as a Primary Cause of Meningococcal Disease after Emergence of the Serogroup in 2000

ABSTRACT The occurrence of a clonal outbreak of serogroup W135 (of the electrophoretic type 37 [ET-37] clonal complex) meningococcal disease among Hajj pilgrims in 2000 has led to enhanced surveillance of the evolution of this particular serogroup, formerly considered rare, in invasive infections. Since the first case of meningococcal disease due to a serogroup W135 strain was detected in France in 1994, all isolates were characterized phenotypically. We further used phenotypic and genotypic approaches to type the 101 serogroup W135 strains isolated from patients with invasive meningococcal diseases in France in 2001 and 2002. Overall, 55% of these isolates had Hajj strain-related phenotypes (60 and 52% in 2001 and 2002, respectively), although only 45% belonged to the ET-37 clonal complex. Moreover, pulsed-field gel electrophoresis of the ET-37 clonal complex isolates showed that only 32% of the serogroup W135 isolates were indistinguishable from the 2000 Hajj-related strain. Our results suggest the continuous emergence of new genetic lineages of serogroup W135 independently of the 2000 global outbreak.

Transgenic Mice Expressing Human Transferrin as a Model for Meningococcal Infection

ABSTRACT The pathogenesis of meningococcal disease is poorly understood due to the lack of a relevant animal model. Moreover, the use of animal models is not optimal as most meningococcal virulence determinants recognize receptors that are specifically expressed in human tissues. One major element of the host specificity is the system of meningococcal iron uptake by transferrin-binding proteins that bind specifically human transferrin but not murine transferrin. We developed a new mouse model for experimental meningococcal infection using transgenic mice expressing human transferrin. Intraperitoneal challenge of transgenic mice induced bacteremia for at least 48 h with an early stage of multiplication, whereas the initial inoculum was rapidly cleared from blood in wild-type mice. Inflammation in the subarachnoidal space with a high influx of polymorphonuclear cells was observed only in transgenic mice. Meningococcal mutants that were unable to use transferrin as a source of iron were rapidly cleared from both wild-type and transgenic mice. Thus, transgenic mice expressing human transferrin may represent an important advance as a new mouse model for in vivo studies of meningococcal virulence and immunogenicity factors.

Microevolution through DNA exchange among strains of Neisseria meningitidis isolated during an outbreak in the Czech Republic

Neisseria meningitidis is a highly variable bacterium. Indeed, N. meningitidis is naturally competent for transformation, and horizontal DNA exchange between strains may lead to mosaic genetic loci in N. meningitidis. We studied such an exchange in nature during an epidemic provoked by N. meningitidis. This epidemic started in the Czech Republic in 1993 and the original epidemic clone was shown to have the antigenic formula (serogroup:serotype:serosubtype) C:2a:P1.2,5. We analysed 145 meningococcal strains isolated in the Czech Republic between 1993 and 1997 using serological and genetic typing methods (multilocus enzyme electrophoresis and polymorphism of pilA and pilD genes). This analysis showed that genetic exchange between epidemic and endemic strains had occurred. Exchanges involved mostly surface-exposed structures such as the capsule, giving rise to new meningococcal variants. The expansion of these variants should be kept under close surveillance.

Hyperinvasive genotypes of Neisseria meningitidis in France

Clinical isolates of Neisseria meningitidis from cases of meningococcal disease, collected between January 2000 and December 2004, were identified and typed at the French National Reference Centre. A representative subset of 546 isolates from among 2882 isolates was further genotyped by multilocus sequence typing to determine their genetic lineages (clonal complexes) and the degree of diversification among different clonal complexes. Representative isolates of the main clonal complexes were tested for their virulence in mice and for proapoptotic effects on human epithelial cells. High genetic diversity in some genetic lineages (ST-32 and ST-41/44) was correlated with heterogeneity in virulence in mice and proapoptotic effects on human epithelial cells. In contrast, the homogeneous genetic structure of isolates of the ST-11 clonal complex, regardless of their serogroup, correlated positively with a fatal outcome of the infection, increased virulence in mice and increased proapoptotic effects on human epithelial cells.

THE PILA REGULATORY GENE MODULATES THE PILUS-MEDIATED ADHESION OF NEISSERIA MENINGITIDIS BY CONTROLLING THE TRANSCRIPTION OF PILC1

Adherence to eukaryotic cells is essential in the pathogenesis of Neisseria meningitidis. Pilus‐mediated adhesion has been shown to play an essential role in this process. Pilin, the pilus major subunit, and two pilus associated proteins, PilC1 and PilC2, are key components in meningococcal adhesiveness. Phase and/or antigenic variation of these molecules are the only identified means by which N. meningitidis modulates pilus‐mediated adhesion. PilA/PilB is a pleiotropic regulatory system first characterized in Neisseria gonorrhoeae where it controls pilin gene transcription. Similar alleles are found in N. meningitidis. To address the role of this regulatory pathway in N. meningitidis, we engineered a meningococcal pilA mutant strain and analysed the consequences of this mutation on pilus‐mediated adhesion using epithelial Hec‐1‐B cells. This mutation resulted in a threefold reduction in adhesiveness. As no change in the amount of pilin nor in pilin gene mRNA was detected, we compared the expression of the pilC genes in both pilA and parental strains. Two transcriptional fusions pilC1–lacZ and pilC2–lacZ were constructed. A threefold reduction in β‐galactosidase activity was observed in the pilA mutant strain harbouring the pilC1–lacZ fusion. No effect of the pilA mutation on β‐galactosidase activity was observed in the strain carrying the pilC2–lacZ fusion. Gel retardation experiments confirmed that the PilA protein binds to the promoter region of pilC1 but not of pilC2. Taken together, these data demonstrate that PilA modulates meningococcal adhesiveness via the transcription of pilC1. Thus, in addition to phase variation, a more co‐ordinate and responsive system may allow a fine adaptation of adhesiveness of meningococci to various environmental signals.