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.

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.

Prevalence of diabetes, obesity, hypertension and hyperlipidemia in the central area of Argentina.

OBJECTIVES: There is little information available about the prevalence of chronic metabolic diseases in many Latin American countries. Between 1995 and 1998, studies on the prevalence of obesity, hypertension, hyperlipidemia and diabetes were carried out in four cities located in central Argentina: Dean Funes, Oncativo, Pehuajo and Venado Tuerto. The data provided by these surveys are reanalysed here in order to determine prevalence of obesity, hypertension, hyperlipidemia and diabetes using new epidemiological criteria. METHODS: Representative samples of the population, based on a multistage probabilistic sampling design, were taken from each of the four cities. The sample size was calculated to obtain a precision of 4% for the prevalence assessment. The subjects included were aged 20 years and over. Standardization of the prevalence rates used the entire study sample as the reference population. RESULTS: Age-standardised prevalence rates for the cities ranged between 22.4% and 30.8% for obesity, 27.9% and 43.6% for hypertension, 24.2% and 36.4% for hyperlipidemia, and 6.5% and 7.7% for diabetes mellitus. All these prevalences increased with age. 58.1% of the obese subjects and 51.2% of the diabetic subjects had hypertension, while 43.2% of the obese subjects and 52.8% of the diabetic subjects had hyperlipidemia. CONCLUSIONS: While the prevalence of diabetes mellitus was between 6% and 8%, the prevalence of obesity was close to 26% and hypertension and hyperlipidemia affected one third of the population. These data can be considered as indicative of the prevalences of these four diseases in the population aged 20 years and over, in the central region of Argentina.

[Rapid epidemiological characterization of Neisseria meningitidis using polymerization chain reaction from biological samplings]. / Caractérisation épidémiologique rapide de Neisseria meningitidis par réaction de polymérisation en chaîne à partir des prélèvements biologiques.

OBJECTIVES: Due to the spread of the meningococcal infections, a good epidemiological surveillance is needed. Prophylactic measures should be undertaken because of the high transmissibility of these bacteria. One problem which hinders the epidemiological characterization is that the responsible strain should be isolated. The aim of this work is to develop a rapid and non culture typing method of Neisseria meningitidis. METHODS: Six cerebrospinal fluids were obtained from 5 different patients with meningococcal meningitis. A specific locus, pil A, for N. meningitidis was amplified by polymerization chain reaction (PCR). The polymorphism of this locus was then analyzed by digesting the PCR products with one of three different restriction enzymes. RESULTS: The polymorphism of this locus allowed us to establish the clonal relationships between the meningococcal strains involved in the infection. Three CSF corresponded to epidemiological strains. CONCLUSION: This typing method allows a rapid and less expensive epidemiological characterization of meningococcal infections. Moreover, it is a non culture typing method.

Neisseria meningitidis serogroup W135 in Portugal: presence of the ST-11/ET-37 clonal complex.

Invasive serogroup W135 Neisseria meningitidis strains were isolated in Portugal between 1995 and 2002. Nine of 12 isolates showed "Hajj-2000"-associated phenotypes and belonged to the ST-11/ET-37 clonal complex. Pulsed-field gel electrophoresis clustered the ST-11/ET-37 isolates together with the "Hajj-2000" strain although the profiles were distinguishable.

Molecular typing of Neisseria meningitidis serogroup A using the polymerase chain reaction and restriction endonuclease pattern analysis.

A new molecular typing method for identification and characterization of Neisseria meningitidis is reported. Chromosomal DNA from 20 well-documented meningococcal strains of serogroup A originating from France, Central African Republic, Sudan and Burkina Faso were amplified using the polymerase chain reaction. Primers designed in this study were located in the pilA/pilB locus which has been shown to be conserved in the genus Neisseria. The amplified fragments were subjected to restriction endonuclease analysis using three different enzymes, and the restriction endonuclease patterns obtained were compared. Clonal isolates clustered together in distinct restriction endonuclease patterns which are described in this study and coincided with electrotypes as determined by multi-locus enzyme electrophoresis. This DNA-based typing system for meningococci may be useful for epidemiological studies.

Phosphorylation and functional analysis of PilA, a protein involved in the transcriptional regulation of the pilin gene in Neisseria gonorrhoeae.

The transcriptional regulation of the pilE gene, coding for the pilin in Neisseria gonorrhoeae, by PilA/PilB proteins is quite complex. Sequence analysis of PilA suggested that it has multiple domains. PilA appears to have in its N-terminal half a DNA-binding site followed by a region showing sequence similarity with other bacterial transcriptional regulators. In its C-terminal half, PilA has extensive homology with the 54 kDa protein of the eukaryotic signal-recognition particle which is involved in protein secretion. A transcriptional fusion between the promoter of pilE and the lacZ gene was constructed and integrated into the gonococcal chromosome. We show that transcription of the pilE-lacZ fusion is affected in pilA mutants in the absence of any possible interference with pilin secretion. Moreover, pilE transcription depends on a -24/-12-type promoter which could be a member of a family of promoters recognized by the alternative sigma subunit, RpoN, of the RNA polymerase. We also show that PilA binds specifically to the promoter region of pilE and that it is phosphorylated in a manner dependent on acidic residues Glu-59, Asp-149 and Asp-186. The functional organization of PilA suggests that it may be an unusual transcriptional regulator different from other RpoN-dependent activators.

[Localization of mitochondrial cytochrome b using specific antibodies]. / Localisation mitochondriale du cytochrmoe b à l'aide d'anticorps spécifiques.

Specific antibody has been obtained against cytochrome b (pig heart mitochondria). It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome b site of the inner mitochondrial membrane. It has no effect on the isolated submitochondrial particles which are inside-out inner membrane vescicles free of any outer membrane or outside-out inner membrane. These findings indicate a probably not transmembranous topologic localization of cytochrome b; this component of the respiratory chain seems located near the outer side of the inner mitochondrial membrane.

[In vitro effect of morphine on oxidative phosphorylation in mitochondria on non-neural cells]. / Action de la morphine in vitro sur les oxydations phosphorylantes dans les mitochondries de cellules non nerveuses.

In vitro assays carried on rat liver mitochondria show that morphine is a strong inhibitor of oxidative phosphorylations. Thus the neurotropic drug has also a more general effect on non-nervous cells, effect masked till now by the more impressive effects on the nervous system.

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 beta-galactosidase activity was observed in the pilA mutant strain harbouring the pilC1-lacZ fusion. No effect of the pilA mutation on beta-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.

[Mitochondrial localization of cytochrome c1 with specific antibodies]. / Localisation mitochondriale du cytochrome c1 à l'aide d'anticorps spécifiques.

A specific antibody against cytochrome c1 (pig heart mitochondria) has been obtained. It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome c1 site of inner mitochondrial membrane ; but it has no effect on the isolated submitochondrial particles (inside-out inner mitochondrial membrane vesicles free of any outer membrane or outside-out inner membrane). Thus the topologic position of cytochrome c1 in the inner mitochondrial membrane is asymetrically lcoated on the outer side of the inner mitochondrial membrane. These results agree with our previous researches on ATP-ase and cytochromes b, c and a, indicating the location on the inner side for the first one, transmembranous for the last one, on the outer side for the others respiratory chain components. Thus the electron transport from cytochrome b to a takes place in the outer region of inner mitochondrial membrane and the transmembranous location of cytochrome-oxidase facilitates the transfer of the electrons to oxygen.

Genetic heterogeneity of strains of Neisseria meningitidis belonging to serotype 22 isolated in the Czech Republic.

Strains of Neisseria meningitidis of serogroup B isolated in the Czech Republic frequently belong to serotype 22. We analyzed the genetic relationships among strains of this serotype by using the multilocus enzyme electrophoresis technique and the polymorphism of the pilA gene. Our results indicate that these strains correspond to a highly heterogeneous population rather than to the expansion of a single clone.

Genetic analysis of a meningococcal population based on polymorphism of the pilA-pilB locus: a molecular approach for meningococcal epidemiology.

The genetic relationships between 88 meningococcal strains were analyzed by using the polymorphism of the pilA gene and the multilocus enzyme electrophoresis. While a good agreement was observed, correlation with antigenic formula (serogroup, serotype, and serosubtype) was incomplete. The inadequacy of serological classification alone in outbreak surveillance may be overcome by DNA-based approaches.

Role of pilA, an essential regulatory gene of Neisseria gonorrhoeae, in the stress response.

Sequence analysis has shown that PilA, a transcriptional regulator of pilin gene expression in Neisseria gonorrhoeae, has extensive homology with the 54-kDa protein of the signal recognition particle of eukaryotes and its receptor, as well as with two proteins of Escherichia coli, FtsY and Ffh, which have been proposed to be a part of a signal recognition particle-like apparatus. We tested the putative role of PilA in protein export in N. gonorrhoeae and did not find any effect. However, we did observe induction of a heat shock response and a previously described slow-growth phenotype when PilA function was impaired. We also examined the interference of pilA expression in E. coli with the function of the products of ftsY and ffh and observed an accumulation of pre-beta-lactamase. We argue against a direct role for PilA in protein export in gonococci and propose instead that PilA is involved in the modulation of cell growth rate in response to different environmental conditions.

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.

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 menin- gitidis, 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.