Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Pertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The S1 subunit is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits S2 and S3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Asn 105 in S2 and of Lys 105 in S3 resulted in the more drastic reduction of binding to haptoglobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the S1 subunit led to the production of a toxin analog with reduced ADP-ribosyltransferase-dependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monoclonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the S1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.

Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Early behavioral development of mice is affected by staggerer mutation as soon as postnatal day three.

Staggerer is a neurological mutation of mice that affects the development of the central nervous system and causes abnormal behaviors. The staggerer cerebellum is already abnormal at birth and as the animal grows up there is a progressive loss of granule cells which have all disappeared by day 28. The earliest behavioral disturbance observed is a motor deficiency which occurs between 10 and 15 days-i.e. several days later than the appearance of the cortical abnormalities. To show that staggerer mutant mice also differ from normal mice in behavioral aspects before the age of 10 days, 28 staggerer pups and 246 normal pups aged from 1 to 9 days underwent different motor tests. In addition, the number of ultrasounds emitted during 40 s was recorded, and the animals were weighted every day. Differences between staggerer and normal mice were found as early as 3 days: staggerers were less efficient in motor tasks and they weighed less than normal mice. Staggerers also differed from normal mice in ultrasound production.

Social isolation partially restores reproduction of male staggerer mutant mice.

Social isolation is generally known to promote sexual performances in male mice. We employ here other indices to investigate the positive effects of social isolation on sexual behavior. Mutant staggerer male mice generally do not mate. However some of their reproductive traits can be restored by environmental modifications. We demonstrate that a postpubertal period of social isolation can significantly enhance mutant male sexual behavior. After a period of partial social isolation of one or 2 mo a significantly larger number of mutant males were observed to attempt mounting receptive non mutant C57BL/6 females during a 30 min test encounter. When reproductive success is considered, a larger number of males who were isolated for a period of 2 mo and then allowed to remain with non mutant females were found to sire pups than control (non-isolated) males. The results show that staggerer males isolated for 2 mo can partially overcome the reproductive deficit usually associated with the mutation.

Discrimination of olfactory sexual cues in staggerer mutant male mice.

Generally, staggerer male mice do not express any preference between oestrous and anoestrous female odours in a choice test situation. The staggerer ability to discriminate between these olfactory sexual cues was evaluated in an habituation-dishabituation paradigm. In this situation it was found that the staggerer mice discriminate between these two odours. The lack of sexual odour preference in staggerer male mice is discussed through hormonal and neurological interpretation.

Reactions of staggerer and non-mutant male mice to female urine and vaginal secretion odors.

Previous studies have shown that staggerer male mice do not copulate spontaneously. When meeting unfamiliar non-mutant females either in estrus or in anestrus condition, these neurological mutants behave similarly. One possible explanation is that staggerer males are unable to detect female odors. To test this hypothesis, male reactions to urine and vaginal secretions of females, either in estrous or in anestrous, were studied in a circular device allowing mutant and non-mutant males to move and to explore sources of odors during 20 minutes. Concerning vaginal secretions odors, the duration of time spent in different sectors by mutants was identical for both conditions of female sexual receptivity whereas non-mutant males spent more time on location with vaginal secretions of estrus females. For non-mutant and mutant males as well, duration of time spent in sectors with urine odors was similar for both conditions of female sexual receptivity. We hypothetised a possible deficiency for social odor detection and/or integration due to the staggerer mutation.

Post-weaning social factors promote motor and social behaviors in staggerer mice.

Male staggerer mutant mice generally do not mate. This lack of spontaneous sexual behavior may be partially due to motor disturbances associated with cerebellar abnormalities. Social experience and probable sensory motor stimulation produced by the post-weaning cohabitation with several non-mutant mice improve the adult interactive behaviors of staggerer males when encountering sexually receptive non-mutant females. Tested for their motor capacities, these experienced males perform better when compared to control males who cohabited with only one non-mutant after weaning. These results correspond to the first demonstration of post-weaning social experience effects both on motor control and on social interactions in the same individuals of homozygous staggerer mice.

Male-female interactions in staggerer and non-mutant mice: impairment to react to novelty as a possible explanation of staggerer male social behaviour.

Associated with neurological anomalies, many behavioural deficits are induced by the staggerer mutation. In order to define the consequences of this mutation on the staggerer male social behaviour we realised experimental dyadic encounters with non-mutant and unfamiliar females, either in estrous or in anestrous condition. We compared mutant behaviour to non-mutant male behaviour. Staggerer male behaviour presents the same characteristics during encounters with both types of females. It differs from non-mutant male behaviour in a similar context. Non-mutant males present more interactions (social interest, sniffing and sexual behaviours) with females than staggerer males. Females modify their behaviour as a function of their state of receptivity and according to their partner. Behaviours of staggerer males towards females may be interpreted in terms of general exploratory behaviour.

Effect of post-weaning social experience with normal females on the behaviour of adult male staggerer mice interacting with normal females.

Adult male staggerer mice reared under standard conditions display no sexual behaviour. When maintained for a time with normal female mice however 5% of the mutant males were able to copulate. We hypothesized that the effects of such social experience would be revealed most clearly by changes in the pattern of male behaviour. Two groups of mutant males were subjected to different social experience after weaning. One group was maintained with normal female mice and the other with staggerer mutant females. There were differences between the two groups in the frequency, duration and temporal organization of interactions between mutant males and normal females. Social experience with normal females modified male behaviour, producing greater synchrony with female behaviour and a reduction in stereotypic behaviours such as scratching.

Behavioral differentiation during collective building in wild mice Mus spicilegus.

Although well documented in social insects, the possibility of behavioral differentiation during collective building has been poorly studied in mammals. In this context, the mound-building mouse Mus spicilegus is an interesting model. Under natural conditions, juveniles from different litters gather vegetal material and build a sophisticated structure, the mound, under which the mice will spend winter. The first steps of this complex building process may be elicited under laboratory conditions by offering cotton balls as building material. Spatio-temporal distribution of both animals and cotton balls was automatically recorded by RFID (Radio-Frequency Identification Device) technique. Our results revealed a behavioral differentiation during a collective building task. In a group of six individuals, only two mice (called carriers) transported 80% of the building material whereas the contribution of the remaining mice was weak or even non-existent. The proportion of carriers was constant in all of the six groups studied. This behavioral differentiation was implemented immediately after the building material was made available and remained stable during the 4 days of experiment. The high contribution level of carriers did not result from resource monopolization, nor did it depend on the gender or parental origin of the mice.

Sexual experience and preferences for odors of estrous females in staggerer mutant male mice.

Staggerer mutant male mice generally do not mate and olfactory disabilities may be involved. Choice tests were used to determine the preference of C57BL/6 male mice with the staggerer mutation for urine and vaginal secretions from receptive and unreceptive females. The staggerer mutation does not prevent the olfactory discrimination between vaginal secretions of estrous and anestrous females. Male preferences for odors of receptive females are related to the presence of sexual experience.

[Reduced influence of penile disability on the mating capacity of male staggerer mice]. / Influence réduite d'une particularité pénienne sur la capacité d'accouplement des souris mâles staggerer.

Most staggerer mutant mice do not mate spontaneously. This deficiency may be attributed to a penile disability (during erection, the penis in extension is directed backward). The main characteristics of this phenomenon and its involvement in the reproduction of the staggerer mutant have been considered in our study. Seventy-four percent (n = 66) of staggerer males presented this temporary abnormality at least once. It appeared when the males were 84 +/- 37-d old (M +/- SD). In most animals the penile abnormality was labile and did not exceed 1 wk duration in 48% (n = 32) of the males. Three males mated in spite of presenting this abnormal erection. Moreover, 25% of males (n = 23) did not present this disability; nevertheless, most of them (91%) still did not reproduce. Other mechanisms are certainly responsible for the inefficient mating. In any case, the influence of penile disability on this deficiency appears to be weak.

Social isolation induces preference for odours of oestrous females in sexually naive male staggerer mutant mice.

The staggerer murine mutation induces olfactory deficits. Mutant males do not prefer oestrous odours to anoestrous ones. A period of social isolation after weaning induces such a preference in mutants.

Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.

[Modifications of the olfactory bulb electrocorticogram and trans-glomerular evoked potentials in staggerer mutant mice]. / Modifications de l'électrocorticogramme du bulbe olfactif et des potentiels évoqués transglomérulaires chez la souris mutante cérébelleuse staggerer.

Electrophysiological observations have been made on normal C57-BL/6J and staggerer mutant mice. Morphological observations have given evidence it existed various neurones modifications which affected the olfactory bulb in the mutant mice. Olfactory bulb electrocorticograms (ECoG) of mutant have shown rare bursts of potentials of longer time duration than in normal mice. These bursts were less affected by odor stimulations (ammonia and urine of opposite sexes) than in the normal mice and never varied under urine odor influence in female mutant. Evoked potential induced by the odors had long latency and long duration (up to 50 ms vs 30 ms). In a large amount of them, the late phases and the late oscillatory potentials, which generally followed the evoked potential, were absent. All these results improved the idea that staggerer mutation, which mainly affected the N-CAM gene, not only induced cerebellar diseases, but also functionally affects the olfactory bulb.

The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis.

The S1 subunit (Mr 28,000) of pertussis toxin expresses thiol-dependent enzymatic ADP-ribosyltransferase and NAD-glycohydrolase activities. Site-directed mutagenesis experiments were performed on the codon for Cys-41 of this subunit to investigate the role of this residue in both enzymatic activities. Deletion of Cys-41 caused a decrease in both activities below detectable levels, whereas replacement of this residue by serine, glycine, proline, or asparagine only slightly reduced the activities. The enzymatic activities of these mutants were thiol-independent. The deletion of Ser-40, adjacent to Cys-41, again caused reduction of the enzymatic activities to undetectable levels. Steady-state kinetic experiments showed that the kcat of the mutant protein in which Cys-41 was replaced by glycine was nearly identical to the kcat of the parent version. However, the Km for NAD of the mutant was significantly higher relative to that of the wild type version. These results indicate that the side-chain of Cys-41 is not essential for enzymatic activities and that Cys-41 is not involved in the rate of catalysis but is probably located at or close to the NAD-binding site. The introduction of a negative charge at position 41 through the replacement of Cys-41 by either aspartate or glutamate reduced the enzymatic activities to very low but measurable levels, suggesting a charge-charge repulsive interaction between these residues and possibly one or both of the phosphates of NAD. Cys-41 may therefore be located close to the phosphate subsite of the NAD-binding site.

Tick transmission of Borrelia burgdorferi to inbred strains of mice induces an antibody response to P39 but not to outer surface protein A.

Natural tick transmission of infection by Borrelia burgdorferi induces a very different serum antibody response than needle inoculation of spirochetes. We present data, obtained by using the mouse model, that show that the OspA response was barely detectable, whereas all animals developed significant anti-P39 titers after exposure to B. burgdorferi-infected ticks.

Purification, expansion, and multiple fluorochrome labeling of cord blood hemopoietic precursors: preliminary results.

CD34-positive cells were isolated from a total of 23 cords using CellPro Ceprate columns. AIS MicroCellector flasks, and panning. The cells were (1) expanded in serum-free culture supplemented with a variety of combinations of cytokines and (2) immunophenotyped using multiple fluorochrome labeling. The results indicated that the avidin column produced the highest purity of CD34-positive cells, and that immature blast cells could be expanded in serum-free culture. Preliminary results suggested that the four fluorochrome labeling technique may provide useful information on the lineage commitment of cord blood precursor and blast cells.

Expression, refolding and crystallization of the OpcA invasin from Neisseria meningitidis.

OpcA is an integral outer membrane from the Gram-negative pathogen Neisseria meningitidis that plays a role in adhesion of meningococci to host cells. The protein was overexpressed in Escherichia coli in an insoluble form and a procedure developed for refolding by rapid dilution from denaturant into detergent solution. The refolded material was identical to native OpcA isolated from meningococci, as judged by overall molecular weight, migration on SDS-PAGE and reaction against monoclonal antibodies. Both native and recombinant OpcA crystallized under similar conditions to give an orthorhombic crystal form (P2(1)2(1)2), with unit-cell parameters a = 96.9, b = 46.3, c = 74.0 A. Complete data sets of reflections were collected from native and refolded OpcA to 2.0 A resolution.