In previous years, the report on the scientific activities of the Institut Pasteur was presented according to the different research departments. However, this year, the approach is more thematic. All of the results obtained or work currently being carried out on the campus have been divided into ten themes presenting a fairly accurate picture of our current activities. However, no presumptions are made with respect to the future. It is possible that we will have more themes next year. This report will henceforth reflect the rapid development in the work conducted on the campus. The reports issued by the research departments and units are available on the Institut Pasteur website: http://www.pasteur.fr
Virology and AIDS
Molecules and structures
Molecular biology of the eukaryotic cell
Developmental genetics and biology
Genomes Immunology and immunopathology
Pathogenic bacteria and fungi
Neurobiology and neuropathology
Parasites and parasitology
Microbial physiology and environment
Virology and AIDS
The Institut Pasteur has been studying viruses for a long time. The current developments in this field are directly related to the consequences of infection, with regard to public health, by numerous viruses and the place of viral diseases among emerging diseases.
Human immunodeficiency virus (HIV)
Relationship between the host and HIV
The discovery of chimpanzee viruses very similar to HIV-1, group N, present in the same region of Africa, has increased the likelihood of a zoonosis being the source of HIV infection. The relationship between the host and HIV varies according to the individual. Certain individuals, mainly in Central Africa and Southeast Asia, remain seronegative despite frequent exposure to the virus. These patients are being studied in order to identify the markers which could be associated with natural protection. HIV replication and the permanency of infection in human depend on secreted factors, such as TNF in the thymus and IL-7, which are essential to the survival of infected cells. Placental factors are involved in controlling intra-uterine transmission of HIV-1 from mother to child. The portals for HIV entry into target T cells remain unclear. Viral particles first interact with surface heparan sulphates and nucleolin, then with CD4 and some chemokine receptors. Infection could be delayed by acting on viral attachment. A pseudopeptide can effectively block HIV-1 infection by binding to nucleolin. A cytokine, Midkine, also interacts with surface nucleolin and inhibits the attachment of viral particles. Midkine could be a component of the natural defences against HIV. Chemokines control viral entry into cells. Chemokine SDF-1 which, in vitro, binds and internalises viral co-receptor CXCR4, limits HIV contamination at mucosal level. SDF-1, which is continuously produced by mucosal cells, induces the internalisation of CXCR4 by local lymphocytes and thus minimises viral entry. Moreover, SDF-1 stops lymphocyte efflux from blood vessels, in less than a second, by abruptly increasing the avidity of leukocytes for adhesion molecule VCAM-1.
Immune response and AIDS vaccines
In order for an AIDS vaccine to be feasible, we must first determine whether infected cells can be killed by virus-specific cytotoxic T-cells. These cells recognise small fragments of viral proteins, or epitopes, presented by class I histocompatibility molecules of infected cells. Lines of mice expressing human molecules instead of their own presentation molecules have been produced. The T-cells from these animals are highly effective in recognising human molecules carrying viral epitopes. This approach, which has now been validated for class I molecules, is being extended to class II histocompatibility molecules necessary for maintaining long-term antiviral response. The cells specialised in presenting viral antigens could play an important role in activating cytotoxic T-cells by using viral proteins, thus inducing an immune response in the absence of viral replication. We then need to understand how HIV manages to evade the immune system. The specific activation stimulates viral replication in helper T-cells, giving rise to increased viral production. The propagation of the infection is the result of a difference in kinetics: the virus is produced in less than 24 hours. The viral production site is then 'cleaned up' by cytotoxic lymphocytes. However, since the virus has taken the lead over the immune response, the infection cannot be eradicated. Viral proteins are not therefore endowed with special properties enabling the virus to evade the immune system. Control of viral proliferation by cytotoxic cells is hence the result of mechanisms in greater numbers than had been presumed, particularly since HIV-1 protein Nef decreases the expression of histocompatibility molecules by infected cells, thus protecting the latter against killing. These findings nonetheless enable vaccination studies in subhuman primates to be envisaged. Macaca monkeys infected with SIV, then by the hybrid virus, SHIVsbg, corresponding to an SIV in which the envelope has been replaced by that of HIV-1, represent a good model of human infection. This model is being used to study a vaccine against hybrid envelope proteins. A further vaccinal strategy is based on the use of a live attenuated vaccine consisting of the recombinant measles virus, intended for vaccination against HTLV-1, against the simian immunodeficiency virus (SIV) and, ultimately, against HIV. Chemotherapy meanwhile continues to prevail. The nucleoside analogues used in antiretroviral and anti-HIV treatments in particular have to be modified by the recipient: phosphorylation, yielding a triphosphate derivative, active against viral reverse transcriptase, is catalysed by cell kinases, but constitutes a limiting stage. Attention has turned to the development of drugs derived from these compounds by introducing a borane group, superior substrates compared with non-substituted analogues and partially active on mutant AZT-resistant reverse transcriptases. Their cellular effects are currently being studied. Further current studies address immunological reconstitution in HIV patients receiving triple antiviral therapy, particularly in children receiving combined antiretroviral treatments. These findings are useful in defining immunisation and immunotherapy protocols.
HTLV-1 is a human retrovirus and the etiological agent of tropical spastic paraparesis, a neurological disease similar to multiple sclerosis, and of adult acute T-cell leukaemia, both diseases endemic in the Caribbean region, Central Africa and Japan. No suitable animal model for the study of HTLV-1 infection is currently available. Several attempts have been made to produce a model using transgenic mice. A chimeric virus having acquired tropism for murine cells has been constructed in the hope of obtaining a mouse model of the disease. Molecular epidemiological studies of HTLV-1/2 and STLV-related simian viruses are currently under way. Over 30 new strains of STLV-1, from Africa and Southeast Asia, have been sequenced. A new PTLV-L type virus, which is particularly divergent, has been completely sequenced. A vast epidemiological study of HTLV-2 has been conducted in various populations living in the forested regions of Cameroon.
The variability of HHV8 is also being studied. HHV8 infection is associated with Kaposi's sarcoma and other lymphoproliferative syndromes. New variants of HHV8 and of 2 subtypes have been found in patients of African origin. New isolates, closely related to HHV8, have been identified in gorillas and chimpanzees in Africa. These findings will lead to hypotheses as to the origin, progression and spread of the viruses.
Hepatitis A, B and C viruses
Effective translation of messenger RNA from hepatitis A, B and C viruses requires their physical circularisation by proteins. While this phenomenon is observed in all three viruses, the proteins may differ. Inhibition of RNA circularisation represents a preferred potential antiviral target with regard to the hepatitis C virus. Studies on the hepatitis A virus have led to propose a new model for the disassembly and assembly of capsid proteins. With regard to hepatitis B, a key stage in viral infection is the interaction between viral regulatory protein X and a host cell protein, UV-DDB. This interaction represents a new target for the development of antiviral drugs. Using transgenic mice for HBV surface antigen (HBsAg), an immunotherapy model involving genetic vaccination is now available. It is now possible, using various procedures, to break HBsAg tolerance, induce elimination of the virus from the serum, and control viral gene expression in murine liver. 'Humanised' mice have been vaccinated with the plasmids coding for HBV envelope proteins. A specific response against epitopes identical to those recognised by patient T-cells were obtained. These results validate genetic vaccination as a potential therapeutic weapon for chronic carriers. A phase I clinical trial is to be launched shortly. The viral nucleocapsid has been detected in the serum of patients with chronic hepatitis C virus infection. This discovery may be a significant factor in understanding the pathophysiology of the infection as well as for its diagnosis. Since the only animal model available is the chimpanzee, attention has turned to using a small primate as a substitute animal model. In order to do so, hepatotropic chimeric viruses, very close to the hepatitis C virus in molecular terms and multiplying to high titres in tamarin monkeys, have been constructed.
Influenza and respiratory tract viruses
The flu epidemic of 1999-2000 was particularly severe, with an estimated 8.8 million people infected in France. The percentage of positive specimens for influenza A was nearly twice the percentages obtained between 1996 to 1999. The epidemic was characterised by the almost exclusive circulation of influenza viruses A (H3N2), the characteristics of which were close to the composition of the vaccine. New vaccinal forms are under investigation. Chimerae derived from the Semliki Forest virus, enabling expression of the influenza virus nucleoprotein, have been constructed and injected in the form of naked RNA in mice, inducing protective cell-mediated immunity. This is also the case for a chimera derived from the Mengo virus. The species specificity of influenza viruses is mainly determined by the specificity of the viral haemagglutinin for its receptor, sialic acid derivatives. The determinants involved in the ability of haemagglutinin H3 to agglutinate cock or sheep red blood cells are related to the nature of the bond formed between sialic acid and red blood cell surface components. Inter-species transmission of avian influenza viruses is liable to give rise to new influenza pandemics. Heat sensitivity, due to the proteins PB2 and PA of the polymerase complex, has been found for influenza viruses of avian origin, but not for those of human origin. The sensitivity of the complex to a temperature of 33°C, approaching the temperature of the upper airways in mammals, could play a part in reducing proliferation of avian influenza viruses in man. Calves infected with the bovine respiratory syncytial virus, a model very similar to infection with the human virus in children, have been studied. In Europe, bovine virus isolates are subjected to positive selective pressure on the glycoprotein, particularly in countries practising immunisation. Viruses with substantial changes in the glycoprotein have been collected. This rapid development raises design problems for bovine and human vaccines.
Arbovirus diseases, dengue, West Nile virus and haemorrhagic fevers
The expression of dengue virus proteins in target T-cells has been studied. Envelope glycoprotein E and viral RNA helicase modulate cell death in response to viral infection. Assembly of the viral particle is prevented by inhibiting maturation of viral envelope glycoproteins. Due to the differences in glycosylation, the secretion of viral glycoprotein NS1 is observed in mammals, and not in the mosquito vector. Detection of protein NS1 in the blood of infected subjects has thus become a target for early diagnosis of the disease. Dengue is of minor significance as an "imported" disease in France. However, this is not the case in tropical regions. Omnipresent in the tropics, Aedes aegypti is the dengue virus main vector. Complex interactions link the vector to indigenous species, particularly Aedes albopictus. Studying the genetic structure of the populations shows how upheavals related to man affect the Aedes populations, followed by the epidemiology of dengue. The results for the populations in French Guyana, Madagascar, Reunion, Cambodia, Vietnam and the Ivory Coast are available, and genetic markers applicable to genetic studies on the population of Aedes have been defined. Since its discovery in Uganda in 1937, the West Nile virus has been identified in several countries in Africa, Europe, the Middle East, Asia and, recently, in the United States. Two genetically distinct virus groups are present in Africa, but only one is responsible for epidemics giving rise to encephalitis in humans and horses. The strain introduced into the United States in 1999 is very similar to that present in Israel since 1997. The West Nile virus re-emerged in the Languedoc region of France at the end of August 2000, as an epizootic outbreak in horses, but with no confirmed human cases. A study of avian populations, which act as amplifying hosts to the virus, has been initiated. The Rift valley fever virus is responsible for haemorrhagic fever in human, spontaneous abortion and foetal malformation in cattle. Since September 2000, the virus has spread from the African continent and caused a severe epidemic in Saudi Arabia and Yemen, with numerous human cases. Studies of the virus have shown that a non-structural viral protein (NSs) plays a major role as a virulence factor, by blocking interferon production. A natural mutant with defective NSs is non-virulent.
Rabies virus and lyssaviruses
The vulpine epizootic outbreak is decreasing in Western Europe, replaced by one due to insectivorous bats. Cases of asymptomatic infection have been detected in fruit bats in zoos. The importation of rabid animals, mainly infected by non-rabies lyssaviruses, has been confirmed on a European level. Lyssaviruses are a public health problem since neither rabies vaccines nor immunoglobulins are efficient on them. The protective spectrum of rabies vaccines may be extended so as to cover all the lyssaviruses by immunising with genes coding for chimeric glycoproteins. The efficacy of naked DNA vaccination has been demonstrated in dogs. With regard to vaccine production, the apoptotic bodies of lymphocytes infected with the rabies virus have been shown to act as potent immunogens.
Virological monitoring, necessary to eradicate poliomyelitis, has evidenced the presence of poliovirus strains derived from the vaccine in liquid waste in countries where oral polio vaccines are used. These strains are highly mutated and have acquired a very neurovirulent phenotype. Polioviruses having recombined at preferential sites and combining wild strains with attenuated strains, have been isolated and characterised. A molecular taxonomic study of enteroviruses has been initiated in view of creating a new taxonomic instrument. Although polioviruses generally do not persist in human, occasionally asymptomatic, chronic infection in immunodeficient patients complicates the problem of eradicating poliomyelitis. A study of polioviruses persisting in the gut of certain immunodeficient subjects has been initiated.
Virus and cancer
Intra-epithelial neoplasm of the uterine cervix, associated with productive infection by oncogenic papillomavirus, is fairly frequent in female patients suffering from severe HIV-related immunodeficiency. Triple antiretroviral therapy has a positive effect on cervical disease. The warts and neoplasms induced by papillomavirus in the rabbit have shown the crucial dual role of the histocompatibility + variability of certain viral genome regions, in the course of oncogenic papillomavirusrelated lesions towards regression or malignant transformation. Verruciform epidermodysplasia, associated with a high risk of skin cancer, is characterised by abnormal sensitivity to oncogenic papillomavirus HPV-5. Two chromosomal loci predisposing to the disease have been identified. Patients with psoriasis constitute a reservoir for HPV-5 and the importance of intensive keratinocyte proliferation in unmasking latent HPV-5 infections has been demonstrated. Chronic hepatitis B virus (HBV) carriers present a high risk of developing liver cancer. A transgenic mouse model has shown that regulatory gene X of HBV acts as a cofactor in the induction of hepatic tumours. The mechanisms by which gene X controls the cell cycle are under investigation. In addition, a cell oncogen, a-catenin, is frequently mutated and activated in liver cancer. aNew proteins acting as partners to a-catenin have been identified. A comparative study of chromosomal anomalies in hepatic carcinoma in human and mouse should allow the identification of shared transformation mechanisms and of new markers for early screening of tumours. Tumour suppressor gene inactivation is of vital importance in the development of human neoplasms. This provides the rationale to try and identify the suppressor genes associated with liver cancer and drive the cells to a normal differentiation behaviour.
Viral derivatives and gene therapy
Gene therapy consists in introducing foreign genes into the cells of a selected tissue and obtaining their expression in order to correct a genetic defect. The tools used are frequently derived from viruses which are naturally capable of introducing their genetic material into foreign cells. Modification of viral genomes enables therapeutic DNA to be vectorised. Vectors derived from HIV-1, which are sufficiently effective for considering their use in human clinical practice, have been developed. Animal models are required in order to evaluate strategies for gene therapy in human. Two models are currently being studied. Lysosomal storage diseases are rare, but hazardous in young children, in whom they mainly affect the brain. Strategies to correct the central nervous system deficiency have been developed, using HIV- or parvovirus-derived vectors in mouse models. These strategies are currently being evaluated in large mammals. In contrast, a-thalassaemia is extremely widespread worldwide. Corrective strategies, based on expression of erythropoietin in muscle, have been described. The aim is to obtain re-expression of the foetal haemoglobin chains in response to stimulation by erythropoietin. nternational Network Research Teaching Health
Molecules and structures
The importance of protein studies is growing, with organic chemistry playing an increasing role in the design of biological molecule analogues.
Design, improvement and production of macromolecules of biological and medical significance
The aim is to build new therapeutic proteins and a new generation of analytical or diagnostic tools. Protein affinity, specificity and stability are thus modified, as required, by site-directed mutagenesis, in order to determine the basis for their properties and increase their utility. Optical biosensors, able to assay an antigen continuously, are currently being prepared. Murine and human antibodies are essential reagents in biomedical research. Emphasis is placed on the production and characterisation of the antibodies involved in autoimmune diseases and capable of neutralising micro-organisms or toxins. In addition, antibodies can be selected on random antigens, by means of a kind of antigen combinatorial biochemistry. Special attention is paid to anti-DNA antibodies in the context of systemic lupus. Proteins of medical significance, particularly yellow fever virus envelope protein and glycoprotein E of dengue virus 2, are produced in large quantities by recombinant insect cells. The same method is used to produce the pro-apoptotic peptide of dengue virus 1. West Nile virus glycoprotein is produced for diagnosis, while membrane protein prM and glycoprotein E from the same virus are prepared for vaccination.
Identifying the mechanisms of correct protein refolding has lead to enhance protein production and, above all, understand the protein refolding 'errors', responsible for diseases such as the prion diseases. The refolding intermediates of certain proteins developing in the first few milliseconds following synthesis are therefore being studied. The refolding of proteins exported by bacteria is the key to biotechnological production. Quality control with regard to refolding (detection, repair and degradation of poorly folded proteins) has been analysed on a mutant bacterial envelope protein presenting a refolding defect. This has clarified the role of two other envelope proteins in the mechanism. It is essential to calculate the effect of mutations on the energy of association and refolding of proteins. A calculation method has been validated using antibiotic resistance bacterial protein mutants.
Structure and interactions of macromolecules
This field of research requires sophisticated facilities, the installation of which is a matter of priority for the institute. The structure of numerous macromolecules is currently being studied. The determination of threedimensional structures by X-ray diffraction of macromolecule crystals is gaining in scale. The structural and functional study of prokaryotic nucleoside monophosphate kinases, potential targets for antibacterial therapy, has been continued. Several pathogenic bacterial UMP kinases have been crystallised: the crystallisation studies on Mycobacterium tuberculosis UMP kinase are promising. The enzyme is reported to be a trimer of dimers, in which each subunit is at the same level. This model has also made it possible to define the interaction sites with various ligands. The 3D structure of Escherichia coli CMP kinase, co-crystallised with its ligands and analogues, has enabled the bonds between the protein and its substrates to be demonstrated. Lastly, the inhibitory effect of certain nucleoside analogues on M. tuberculosis TMP kinase, the 3D structure of which has just been resolved, is opening up new prospects for the search for antibacterial agents. Nuclear magnetic resonance (NMR) imaging is used to study structural relationships: the function of biological molecules and the problems of molecular recognition of the DNA-protein, protein-protein or peptide-macromolecule type..The NMR study of the structure of the heme-carrying protein of Serratia marcescens has been completed. The study has demonstrated a new way of refolding for a heme-binding protein. The structure of scorpion venom toxins specific to sodium and potassium channels has made it possible to clarify their action, specific to each species. Other studies are addressing proteinprotein and protein-DNA interactions and have enabled new types of refolding to be demonstrated. Lastly, research into the use of very low volume probes has been continued, namely in order to study microorganisms which only divide slowly.
Molecular combing is a method of aligning DNA molecules, which consists in fixing one extremity of DNA molecules in solution to a treated glass surface. The molecules are then stretched. At the end, the DNA molecules are aligned and regularly stretched as if they had been combed. In addition to its applications in the physical mapping of genomes and the diagnosis of genetic diseases, this relatively simple method is opening up new prospects in oncology. It may be used to detect phenomena occurring during the malignant transformation of a cell: microdeletions, inversions or amplifications of genes. In addition to these applications, molecular combing provides a totally new approach to DNA replication in eukaryotic cells. The identification and molecular definition of an origin of replication remains a fundamental challenge. Molecular combing has made it possible to study the organisation of replication units and determine their distribution in the genome together with their activity during cell division. This method thus enables systematic analysis of the mechanisms controlling replication. A high-resolution physical map of the origins of replication in brewers' yeast is currently being established. A similar study has been initiated in the amphibian Xenopus laevis.
The research activities carried out in organic chemistry are at the interface between chemistry and biology. The 'synthetic vaccines' approach aims to elucidate the immune response against Shigella flexneri by using synthetic complex sugars as antigens. The branching of those sugars is the key element in recognition by antibodies. A new type of immunogen, consisting of a lysine core carrying several copies of tumour antigens Tn and a peptide epitope, has been synthesised and used as an antineoplastic vaccine in clinical research. The synthesis of peptide ligands has made it possible to study the interactions between their receptors and the AIDS virus, and to search for antagonist molecules interfering with the mechanism. Furthermore, nucleoside a-boranophosphate derivatives have been prepared with a view to developing antivirals active against resistant strains of HIV. Various prodrugs have been synthesised to overcome the problem of drug membrane crossing. Similarly, ribavirin analogues against the dengue virus have been prepared. Steps are also being taken to extend the range of nucleic acids able to be replicated in vitro or in vivo. A synthetic combinatorial process has given rise to a collection of nucleoside triphosphates which are undergoing DNA polymerase incorporation tests. The objective is to screen for monomers endowed with unequivocal or equivocal pairing ability and/or replication inhibitors. A second modification concerns the phosphopentose skeleton: mimics of nucleic acids, in which the sugar consists of a six-atom ring, are recognised by DNA polymerases and are able to transfer genetic information in vivo. The enzyme directed evolution group is applying a Darwinian strategy to protein populations in view of isolating specific catalysts for given chemical reactions. The expression of DNA-dependent DNA polymerases has thus been enhanced and in vitro selection of nucleotidyl transferases on the basis of their catalytic activity has been conducted. In the essential field of DNA microarrays, methods for covalently bonding pre-synthesised oligonucleotides, in the 3' and 5' positions, on a silanised glass slide carrier are being developed.Network Research Teaching Health
Molecular biology of the eukaryotic cell
The almost complete sequencing of the human genome by an international consortium represents a major scientific progress. However, this information does not explain how the products of our genes determine our development and the postnatal functioning of our body, or the mechanisms that regulate expression (or transcription) of our genes in time and space in response to internal or external signals. Gene-regulating sequences are recognised by transcription factors, DNA-affine proteins, which account for approximately 6% of our genes. DNA is packaged in chromatin consisting of repetitive units, the nucleosomes, in which it is wound around a histone octamer. Numerous studies have shown that the accessibility of gene transcription factors is restricted by the chromatin structure. Covalent modifications of the histones together with chromatin-remodelling mechanisms should thus modulate the activation or repression of genes. In other words, processes such as transcription and replication are not randomly located in nuclear space, but take place at given topological positions.
Transcription factors and control of gene expression
The structural organisation of a nuclear sub-domain, the perinuclear domain of transcription repression, has been characterised for the first time. The structural integrity of this domain has been shown to be responsible for anchoring and immobilising factors inhibiting transcription, and the location of a gene relative to this type of domain has also been shown to play a part in regulating its expression. Thus, cells use their intranuclear architecture to code for epigenetic information. Several subunits of the chromatin remodelling complexes are currently being studied, one of which is essential in the very early development of the mouse. In its absence, blastocysts are formed, but implantation fails. Heterozygous mice are viable, but develop tumours. This suggests that the gene may, in fact, be a tumour suppressor gene. A certain number of genes involved in hepatic, renal and pancreatic functions are regulated by two homeoproteins, HNF1a and HNF1b. One of the hepatic genes affected by the absence of HNF1a is phenylalanine hydroxylase, the absence of which induces phenylketonuria. Extinction of this gene is associated with the formation of an inactive chromatin structure and the persistence of gene methylation. The presence of HNF1a is essential during development, within a precise time-frame. An inactive chromatin structure, unable to respond to the transcription factor, is formed in the absence of HNF1a. HNF1a mutations in man and mouse are associated with type II familial diabetes mellitus. Deficient mice also show defects in glucose re-absorption by kidneys. The examination of human patients has revealed such defects, hitherto undetected. Specific inactivation of genes in the mouse is a powerful means of studying gene functioning. Out of five inactivated genes, only two induce embryonal lethality. The function of the other genes is important in postnatal life. Analysis of these mice has thus made it possible to increase our understanding of the fine regulation of cell and organ function. This approach is used to define the activity of transcription factors. Thus, protein junD, a subunit of transcription factor AP1, plays an important role in spermatogenesis and in protecting cells against senescence and apoptosis. The protein also contributes to a network of genes controlling the stability of p53, a product of tumour suppressor gene which controls the response of cells to genotoxic agents. In addition, Jun proteins contribute to the expression of cyclin D1, essential in the cell cycle, and frequently over-expressed in tumours. The transcription factors also regulate the expression of genes coded by DNA viruses, such as papillomavirus or hepatitis B virus, and are important in viral propagation and in the progression of viral infection to tumours.
Chromosomal imprinting and inactivation of the X chromosome
Researchers are attempting to elucidate the molecular mechanisms responsible for a chromosomal imprint phenomenon, which controls the asymmetric divisions in the yeast, Schizosaccharomyces pombe. This process enables the yeast to change sexual types, ensuring an equivalent proportion of both sexes in a colony. Previous studies have shown that the process is initiated by single-strand lesions at the sexual locus and involves DNA replication. The singlestrand lesions fulfil all the criteria of a site-specific break: it occurs on only one of the two daughter chromatids, during replication of the discontinuous strand, is maintained through the cell cycle and becomes active during the following replication. Moreover, it has been shown that the break, although site- and strand-specific, is independent of the sequence. However, the mechanisms underlying the formation and maintenance of the single-strand lesion are still unknown. In addition, study of the inactivation of the X chromosome has been continued. The complete sequence of region Xic (X chromosome inactivation centre) in the mouse and its partial sequence in bovines have been determined. Comparative annotation of these sequences together with those in human should enhance targeting of Xist regulating components, the subject of mutation studies aiming to successively replace previously removed components. Significant results have thus been obtained with the Tsix transcript, believed to control expression of Xist (X-inactive specific transcript) in the early embryo. The parental imprinting on this locus has been shown to be ensured by a mechanism other than methylation.
Various approaches have been used to identify new factors involved in RNA metabolism in yeast, S. cerevisiae. A large database focusing on RNA metabolism (maturation, transport and degradation) has been compiled. New proteins have been identified and related to metabolic pathways. In particular, Lsm proteins have been shown to be physically related to both splicing factors and cytoplasmic factors involved in the degradation of messenger RNA. This suggests that the Lsm complex is involved in two distinct metabolic pathways, as confirmed by functional analyses. In addition, a physical link between the constituents of the nucleoplasm and the nuclear pore has been evidenced. A biochemical method of purification by affinity is being used to validate and extend the network of double-hybrid interactions. The constituents of the various complexes are identified by mass spectrometry. Twenty-four proteins were thus analysed within a few months, enabling identification of six new biochemical complexes, from which 34 proteins have been identified. Functional analysis of the complexes is in progress. For example, 12 new proteins of the small ribosomal subunit of mitochondria have been characterised. Similarly, a new complex involved in the maturation of ribosomal RNA 5, 8S and 25S is now being characterised. Coinciding with these approaches, genetic screening methods are being developed in order to identify functional links such as those between mRNA degradation and cell cycle regulation for example.
Genetics and biology of development
Research on development mainly addresses the formation of the muscles and heart, and the differentiation of neurons in mice, with a view to understanding how the cells ultimately contributing to the differentiated tissue are specified and which genetic factors control the cascade of activities of the various families of differentiation genes.
Muscle and heart
Research into muscle formation focuses on the molecular factors of myogenic determination. The genetic locus, Mrf4-Myf5, contains far-acting constituents, one of which has now been divided into sub-constituents, each corresponding to a different specification in muscle mass space. Among the myogenesis signals, the Notch pathway is involved in the formation of certain muscle masses and the Six genes have a probable regulatory role. The complex regulation of the gene coding for muscle determination factor Myf5 is being studied with a view to tracing the cascade of factors controlling myogenesis. Transcription factor Pax3, which is also located upstream in the genetic hierarchy and regulates myogenesis, is also being studied and a zinc-finger transcription factor and a kinase have been isolated. Analysis of the cardiac actin gene has enabled several activatory sequences to be identified, one of which is specific to the myocardium and, to a lesser degree, skeletal muscle. Cardiogenesis is being studied with 'cardiosensor' mice which express a transgene in specific aeras of the heart or precardiac mesoderm. This approach has made it possible to demonstrate for the first time the role of an anterior mesenchymal region in the formation of the heart in the mouse. Cloning the transgene integration site close to a growth factor gene, expressed in cardiac precursor cells, suggests that this factor, FGF, is involved in precursor cell recruitment into the developing cardiac tube. A further experimental approach, retrospective clonal analysis, is providing new insight into the cell populations which contribute to the mycardium and, hence, to the formation of the various regions of the heart.
As regards the nervous system, genetic control of cephalic region formation is being studied in a strain of mice mutant for homeogene Otx2. The segments which should form the proencephalon and mesencephalon are eliminated by the mutation. The transcriptome of wild embryos at the start of gastrulation was compared with that of Otx2-/- embryos. Attempts were made to identify the genes responsible for induction of the anterior neuroectoderm from among over two hundred genes activated, repressed or strongly modulated by Otx2 at this embryonal stage. Two secreted factors, FGF-15 and Dkk-1, are transcribed in the embryonal ectoderm and visceral endoderm, respectively. Lastly, the important role in neurulation of the X-linked gene Nap1/2 has been examined in detail. This gene seems to control the proliferation of neuronal cells through multiple interactions with molecules such as cyclins. In the absence of this gene, precursor neuronal cells begin to proliferate (or proliferate faster), suggesting that the gene may play a part in tumour genesis.
The ectodermmesoderm choice
Genes Msx1 and Msx2, which code for homeodomain transcription factors, are mainly expressed in the induction regions between the ectoderm and mesoderm. Msx1 mutants display craniofacial defects at birth, showing a role for the gene in the induction at these sites. An unexpected phenotype was detected in a mutant this year. It consisted in the defective formation of the posterior commissura of the diencephalon and correlative absence of the sub-commissural organ resulting in hydrocephaly at birth. The architecture of the median line in that region was impaired. The expression profiles for Msx1 and Msx2 are very similar and the activities of the two genes are probably redundant at numerous sites. Moreover, since gene Msx1 is involved in the BMP4 signalling pathway, particularly in the dental mesenchyma, a conditional inactivation programme for the latter factor has been initiated, while a study of the interactions between protein MSX1 and the promoter of BMP4 has been launched. Mice in which the vimentin and desmin genes have been knocked out were analysed in detail, enabling the biological functions exerted by the proteins to be demonstrated. Furthermore, keratin genes K6a and K6b were knocked out, enabling their essential structural role in the formation of the oral mucosa to be demonstrated.
An analytical method, developed in recent years, has made it possible to elucidate the cellular strategies underlying the organisation of the plan for formation of the central nervous system (CNS) and muscle system in the embryo. After an initial phase of cell dispersion at the very beginning of development, clonal separation between the future anterior and posterior segments of the CNS is observed. The behaviour of the cells in these two territories then becomes radically different. The anterior segment is constructed by progressive subdivision of the territory, while the posterior segment is built by successive production of components from a distinctive cell pool. The next stage consists in restricting the dispersion of precursors along the longitudinal axis. This topological restriction precedes, but perhaps also contributes to the advent of the neuromeres, the basic units in CNS development. The same method, adapted to the muscle system, has enabled a second system of stem cells, giving rise to the precursors of the myotomic segments, to be evidenced. Once again, the system constructs the structures in accordance with a temporal mode. The stem cell pools giving rise to the CNS and muscle system occupy distinct territories in the region of the node and primary groove of the gastrula. The unity in the formation of the constituents of the stem and the radical difference in relation to the more anterior segments may represent major invariants in the plan of CNS formation in all vertebrates. The role in establishing the hepatic phenotype of various liver-specific transcription factors has been defined. Research has mainly focused on the study of HNF4a, a key factor in hepatic differentiation. A DNA sequence stimulating gene HNF4 a has been analysed. In addition, gene HNF4 a has two promoters, the use of which varies during the development of the liver. The protein derived from early expression from the distal promoter shows particularly strong transactivation of the genes expressed early in development. In contrast, the protein produced at birth from the proximal promoter mainly transactivates the late expressed genes. Liver cells from transgenic mice with a constitutionally active form of the hepatic growth factor receptor (HGF/SF) have been immortalised. These consist of epithelial-hepatocytic cells and cells with a fibroblast form, known as 'palmates'. The latter are bipotent and may differentiate into hepatocytes or biliary cells. FGF has been identified as the factor inducing differentiation into hepatocytes, and TGFb as that stabilising the palmate phenotype. Once the essential role of transcription factor vHNF1 in the differentiation of the visceral endoderm had been demonstrated, analysis of the function of the protein was initiated using conditional mutagenesis.
Natural mutants of development
The study of natural mouse mutants is being continued. A molecular and genetic analysis of murine polydactyly is currently under way. The mutants present an anterior duplication in the limb bud of the polarising activity zone, which is normally posterior. Characterisation of the genes giving rise to these mutations should help us understand the mechanisms that position the polarising zone. Steps have therefore been taken in order to isolate the mutated gene responsible for murine polydactyly. Phenotypic determination is complex, with a major locus on chromosome 12 and two other independent genes on chromosome 4. The gene responsible for the abnormal digitation mutation, dan, codes for a receptor of lipoprotein. A neurological mutation in the mouse, known as progressive motor neuropathy and which only seems to affect motor nerves, is also being studied. The gene involved has been localised within a 850kb segment of mouse chromosome 13. The conditional lethal mutation, Om, induces embryonal death at the morula-blastocyst stage. A complete physical map of the genetic region containing locus Om has been plotted and three candidate genes are currently being studied.
Technological progress has now made it possible to sequence the genome of numerous species, particularly bacteria, within a few months. Many aspects of biology will be based on the exploitation of genome sequences in the future.
Bacterial genome sequencing is continuing. Spirochetes, slow-growing bacteria with relatively simple genetics, are now being studied. The sequencing of the Leptospira interrogans genome has been practically completed in Shanghai, and a collaborative venture aiming to annotate the genome is under way.The close genetic proximity between Yersinia pestis and a pathogen of markedly lesser virulence, Y. pseudotuberculosis, is to be used in a comparative genomic approach. In order to do so, the sequencing of the genome of Y. pseudotuberculosis has been initiated. In order to have a better understanding of the biology and pathogenic potential of the mycobacteria responsible for leprosy and tuberculosis, a systematic analysis of the relevant genomes has been undertaken. The genomic sequences of Mycobacterium tuberculosis and M. leprae have been determined. A functional and comparative genomic programme has been initiated. Biochips have been used to evidence the presence of a certain number of variable chromosome regions in the tuberculosis bacillus complex. Some of these regions have more than likely contributed to the attenuation of the vaccinal strain, M. bovis BCG Pasteur. Their relationship with virulence and phenotypic differences is currently being explored, using functional genomic analysis. The genomes of Listeria monocytogenes, responsible for very severe food poisoning, and L. innocua, a closely related non-pathogenic species, have been annotated within the context of a European project. Two genomic sequences of Helicobacter pylori are now available. These will enable comparative analysis of clinical strains based on the study of gene distribution and the beginnings of functional analysis of the genome. Furthermore, the complete sequencing and annotation of the virulence plasmid of Shigella flexneri have been conducted. The results made it possible to identify not only the island of pathogenicity coding for type III secreton and the operon coding for the effectors secreted due to bacterial penetration into epithelial cells and during apoptotic death of macrophages, but also fifteen or so genes coding for secreted proteins, the functions of which have yet to be identified. The genome of another enterobacterium, Photorhabdus luminescens, has been almost completely sequenced. This commensal bacterium of an insect-parasitising nematodes is a model for the study of host-parasite interactions, but is also capable of producing numerous insecticides, bactericides and fungicides. Lastly, sequencing of the genome of Streptococcus agalactiae (group B), responsible for neonatal infections representing a major public health problem, is currently under way.
Identifying key genes in bacterial life
Comparative study of two pairs of genomes, each consisting of the genome of a reference bacterium and that of a disease model bacterium, has made it possible to define the key enzymes in microbial life. Two reference micro-organisms are used: Escherichia coli, the oldest model for geneticists, and Bacillus subtilis, a bacterium which yields industrial enzymes. The bacteria have been compared with Photorhabdus luminescens, which kills insects, and Bacillus cereus, a sporulating bacterium involved in numerous diseases, a close relative of which is responsible for anthrax. The study focuses on the identification and function of the pivotal genes in the overall adaptation of the bacterium to its environment. Emphasis is particularly placed on the metabolism of sulphur-containing molecules. Analysis of all the transcription products, associated with analysis of all the bacterial proteins, has made it possible to distinguish major sets regulated in the same manner. Due to the very large quantity of data generated by genome sequencing and the enigmatic nature of many of the genes discovered in the process, these studies are being supplemented by a bioinformatic research programme (http://genolist.pasteur.fr/GenoList/ SubtiList/). Furthermore, a genomic annotation system, Imagene, is being used to compile a general model for the identification of errors in genomic sequences.
Bacterial genomes, taxonomy and diagnosis
The ease with which homologous sequences are found in different micro-organisms has led to major changes in bacterial taxonomy. The sequencing of the gene coding for ribosomal RNA 16S has provided bacterial taxonomy with a firm phylogenetic basis and has enabled species-specific probes to be developed. In situ hybridisation with fluorescence detection has made it possible to detect, identify and count bacteria in just two hours. Molecular typing instruments are being developed with the epidemiological aim of monitoring colibacilli and Shigella. This is the first time that a molecular system has been able to replace a complex serotyping system with almost the same results. This approach has led to the discovery of a new serotype of Shigella. Similar work has been conducted on the flagellum antigens of Salmonella. With regard to Borrelia, all the strains identified in chronic forms of Lyme disease belong to just 10 clones out of the sixty in the phylogenetic tree drawn with the 150 available sequences of a gene coding for a membrane protein. The genotyping of this gene has both therapeutic and preventive implications. The same applies to the precise identification of gram-positive bacterial species, on the basis of gene sodA sequencing. The Listeria laboratory is contributing to a DNA microarray construction project, in view of precisely typing each strain of Listeria monocytogenes and analysing their epidemic potential and pathogenic characteristics. Numerous units and National Reference Centres are engaged in taxonomic studies with the aim of creating or enhancing instruments for the identification and typing of bacteria and fungi.
The genome of Anopheles gambiae
A new large-scale programme concerns the genome of the mosquito Anopheles gambiae, the vector of malaria. The sequence data base currently contains approximately 24,000 identified sequences, 17,500 of which are derived from the same library and have been annotated by the Institut Pasteur. A. gambiae will thus become the second insect, after Drosophila, to have had its genome sequenced. The objective is to compare the genomes of the two species, particularly since marked resemblances with one region of the Drosophila genome have been found. The preliminary results show that, in very similar zones in the two species, there are pairs of orthologous genes which are shared by the two genomes, revealing synteny between neighbouring genes. Thus, although frequent sequence rearrangements must have taken place during the divergent evolution of the two flies (100 million years), some genes seem to have escaped the drift, for reasons which are poorly understood, but which may perhaps be related to a common selection pressure.
Genetic susceptibility to diseases
The genetic control of susceptibility to type 1 diabetes in the mouse is being analysed due to construction of congenic mouse strains. Definition of loci ldd6, ldd19 and ldd20 on chromosome 6 is under way. Current efforts are focused on two of these loci, situated in the best defined regions, in order to 'fish out' the corresponding genes. The search for genes predisposing to human diabetes is continuing and two regions of susceptibility to insulin-dependent diabetes are being investigated through the combined study of French, American and Scandinavian families. The identification of candidate genes is advancing. The gene involved in Wolcott-Rallison syndrome (an autosomal recessive syndrome associating irreversible neonatal insulin-dependent diabetes and bone disease) and in autoimmune diabetes has been identified with factor EIF2AK3 (translation initiation factor 2-a kinase 3). Other models of human diseases are being studied. The gene for transferrin (Tf), the protein responsible for iron transport in the body, is being studied in lines of transgenic mice. Overexpression of Tf in the brain of these animals is accompanied by increased expression of myelin markers and overdeveloped white matter. Moreover, the axons appear healthy and larger. The animals have normal behaviour and motor coordination superior to that of non-transgenic mice. Research relating to melanotransferrin, a protein strongly expressed by melanomas and an early marker of Alzheimer's disease, is focusing on the proteins controlling the specific expression of the gene and, in particular, the proteins binding to double-strand RNA. Lastly, a group of genes coding for three apolipoproteins, A-I, C-III and A-IV, involved in triglyceride and cholesterol transport, lipid metabolism and, as a result, atherosclerosis, is being investigated in genetically modified animals. Over-expression of these genes has been shown to induce hyperlipaemia, but reduce atherogenesis. The mice in question are also of considerable value in screening for new anti-atherogenic drugs. The persistent infection of susceptible mice by Theiler's virus is accompanied by chronic inflammation and demyelinating lesions which mimic those observed in multiple sclerosis. Identification of the resistance genes in this disease model may enable candidate genes to be identified in man. In addition to histocompatibility gene H-2Db, genes located on chromosome 10, on either side of the gene coding for interferon g, contribute to resistance. Another gene on chromosome 14 affects the extent of demyelinisation. A further gene on chromosome 11 controls the severity of the disease in animals with a high viral load. In man, the study of the determinism of sexual differentiation has made it possible to evidence two autosomal genes responsible for testicular dysgenesis and hypospadias, respectively. The investigation for microdeletions on chromosome Y in a large population of European infertile men has not enabled a risk haplotype to be detected. The study has nonetheless led to the discovery of a haplotype of the Y chromosome, which predisposes to the disease in men, 46, XX, together with a haplotype of the Y chromosome, associated with reduced spermatogenesis. Epidemiologists are now looking to identify the genes which intervene in the variability of the response to malarial infection. The study is being conducted in the Senegalese villages of Dielmo and Ndiop, in an area in which malaria is endemic. Numerous epidemiological, clinical and immunological research projects have been implemented in this area over the last 10 years (the Instituts Pasteur in Paris and Dakar). DNA and malarial phenotypes have been collected from 900 people and the genealogical trees of the population have been plotted. Genome screening together with the study of candidate genes have been initiated. In collaboration with several external laboratories, the Institut Pasteur has shown that a human disease, Incontinentia pigmenti, is due to a mutation of gene NEMO, which codes for the central component of a complex of protein kinases, essential to signal transmission. An animal model of the disease and its clinical signs has been obtained by partial or total knockout of the NEMO gene in the mouse.
Immunology and immunopathology
The study of the physiology of the immune system and the involvement of immune system components in disease, immunology and immunopathology are inextricably linked.
Development of the immune system
The cells of the immune system all derive from haematopoietic stem cells. Study of their regenerative potential is important both in basic immunology and in cell therapy. Quantitative study of the haematopoietic precursors, which migrate toward the thymus during foetal life, has shown that between days 12 and 14 of gestation, colonisation of the thymus is very intense. Moreover, while thymus regeneration depends on the number of stem cells injected, the repopulation of secondary lymphoid organs does not. This inevitably involves peripheral lymphocyte proliferation. The T-cells of the intestinal epithelium are by far the most numerous in the organism, but paradoxically, their function and origin have yet to be clearly elucidated: those expressing CD8 derive from thymus CD4- and CD8- precursors, proliferate locally, then migrate to the intestinal mucosa.
Regulatory cells and autoimmunity Network
Research into the T-cells which regulate immune responses and tolerance is a highly active field. A population expressing a gd T-cell receptor, able to produce interleukin 4, has been characterised, and two genetic loci influencing its development have been identified. Another minor T-cell population, CD4, including memory cells, controls the size of the naive T-cell compartment through production of IL-10. In contrast, the counter-selection of autoreactive lymphocytes has been less studied: T-cell tolerance to antibody light chains depends on T-cells CD4+, which negatively regulate kappa-specific cytotoxic T-cells. A model of major importance in understanding tolerance is that known as 'allotypic suppression', Its mechanism has been elucidated. This model also provides greater insight into the sequential interactions between T-cells CD4, CD8 and dendritic cells (DC). The key to the model resides in the interaction of CD4 T-cells with the CD via the CD40 receptor-ligand structure which enables the CD to induce maturation of CD8 into cytotoxic effectors specific for the same antigen. The experimental system has demonstrated, for the first time, that receptor CD40 plays a negative feedback role. Histocompatibility molecule tetramer technology has recently shown that a large fraction of autoreactive T-cells escapes negative selection in the thymus. Homeostatic and/or peripheral deletion mechanisms must thus exist to counter the emergence of autoimmune diseases. NKT-cells follow very specific selection rules. Selection of their receptor in the thymus takes place on a class I molecule, while their peripheral action is at least partially independent of this molecule. With regard to humoral immunity, the selection of B cells during their development depends on the quantity of autoantigens they are exposed to: very small quantities of autoantigens induce positive selection, but also activation of autoreactive B lymphocytes
In addition to cell to cell contacts, the cells of the immune system exchange molecular messengers, the cytokines. The responses resulting from interactions between cytokines and the membrane receptors of the target cells include modifications in cell survival, proliferation and effector function. IL-2 receptors and T-cell receptors are receiving particular attention. When T-cells are stimulated by antigen-presenting cells, an immune synapse is established. The role of the cytoskeleton in this process has been demonstrated. IL-2 binding to its receptor is rapidly followed by internalisation of the complex. A new pathway for the process has been described. Cytokines play a decisive role in the differentiation of stem cells. In vivo, the switch points at which cytokines act have yet to be fully elucidated. An Institut Pasteur group has studied the role of gc-dependent cytokines (the chain common to receptor signal transmission for IL-2, -4, -7, -9 and -15) and their receptors in lymphopoiesis, peripheral lymphoid homeostasis, and immune responses. The group therefore showed, for the first time, that the survival of naive T-cells requires gc-dependent cytokines, in contrast to memory cells. However, naive and memory cells unexpectedly proliferate in vivo in the presence of the antigen and in the absence of cytokines. The question remains as to whether c-dependent cytokines are really essential growth factors for T-cells. In any event, the concept seems to be validated by the correction of the immunodeficiency induced by the AIDS virus. In patients receiving triple therapy, and in whom there is no rise in CD4 T-cells, immunotherapy with IL-2 is able to correct the deficiency without increasing the viral load. In order to elucidate the differentiation programmes of helper T-cells, an original strategy for the identification of the genes involved in the transition from naive T-cells to the Th1, Th2 and memory state has been devised. The strategy, independent of DNA microarray, has been successfully applied to identification of the genes selectively transcribed in Th1 or Th2 cells.
Mechanisms of intracellular signalling
It is essential to have a clear understanding of intracellular signalling i.e. the molecular mechanisms which decode the information received on membrane receptors. The signals induce rapid changes, which include the recruitment and organisation of a considerable number of intracellular signalling components. The cell integrates the signals, the result of which corresponds to the very nature of the response to the antigen. Studies conducted using mice with knock-in ZAP-70 (protein tyrosine kinase essential to T-cell activation) have clarified the control and function of ZAP-70. Transgenic mice with ZAP-70 knock-in have made it possible to study the disturbances in the state of tolerance and demonstrate the existence of compensation mechanisms in the signalling system. In co-stimulation via CD28, in contrast to what is widely believed, the role of CD28 corresponds to direct amplification of receptor signalling in response to the antigen, by increasing the contact between the T-cell and antigen-presenting cell together with the calcium flux. Cyclosporin A, an immunosuppressant used in organ transplantation, inhibits calcium-dependent T-cell activation of transcription factor NF-AT, but also induces a drastic change in gene expression: the deregulation of nuclear transactivators is responsible for expression of around a hundred new proteins. The mechanisms which regulate signal transmission mediated by type I interferon (IFN) receptor and specific expression of certain genes are being studied. The expression, in the same cell, of a cytokine receptor (such as IL-12R), which shares tyrosine kinase Tyk2 with the IFNa/b receptor, induces a decrease in the response to the latter cytokine. This effect, due to competition for interaction with Tyk2, could play a role in Th1/Th2 polarisation. Although the role of IFNa and a in Th1 polarisation has been demonstrated in man, nothing is known about the sensitivity to these cytokines during differentiation: a marked reduction in sensitivity to IFNa/a during Th1/Th2 differentiation has been observed. A new protein which interacts with a domain of Tyk2, known as Pot-1 (partner of Tyk2), has been identified, together with a motif which gives Tyk2 the ability to migrate into the nucleus. At the same time, the study of mice in which the genes coding for two inhibitors of NF-kB, IkBa and IkBe have been knocked out has enabled a drastic phenotype to be observed. The mice die at birth due to a respiratory defect. Apoptosis of B lymphocyte precursors and positive double thymocytes is also observed. The concomitant absence of IkBa and IkBe thus affects lymphocyte precursors very early on. The innate immunity of bacteria is partly based on the responses of the various cellular components of the body, including granulocytes, to lipopolysaccharides (LPS). It has been shown that the response to non-conventional LPS (e.g. derived from Rhizobium) is independent of Toll-like 4 receptor, in contrast with other LPS. Sensitivity to LPS is abolished in NKT-cell-deficient mice and restored by adoptive transfer of normal NKT-cells in the absence of CD1 expression. These results suggest that NKT-cells are components of innate immunity.
Mechanisms of antigen receptor diversification
The diversity of B and T-cell antigen receptors is the basis of adaptative immunity and the focus of active current study. The presence of N diversity regions in the receptors, for which the enzyme Terminal Transferase (TdT) is responsible, was first established for neonatal murine TCR a chain. Two isoforms of the enzyme have been discovered and their enzymatic properties compared. The three-dimensional structure of TdT is currently being analysed, while a further study has shown that genetic rearrangement at the lg loci is preceded by nucleosome positioning and acetylation of H4 histones. The determination of the diversity of antigen receptors has continued with the study of mice lacking certain histocompatibility antigens or TdT, and in mice presenting a single antigenic peptide. The T-cell receptor shows noteworthy diversity in all cases and is simply ten times lower in TdT-knocked-out mice. NKT-cells also show extreme diversity and a polyclonal nature, despite their activated/memory phenotype. In the same context, a 'monoclonal' mouse with an antibody repertoire restricted to rearranged VHT15 gene and with only two genes is being studied in order to elucidate the mechanisms of diversification in individuals either naive or immunised against defined haptens. Another study has determined the proportion of B cells which do not undergo allelic exclusion in the mouse. The study of mice with three chromosomes 12 has enabled the mechanism to be elucidated. Lastly, a new software package for the study of diversity, ISEApeaks®, has been developed.
New vaccinal strategies and anti-tumour studies
New vaccinal strategies based on our knowledge of basic immunology and exploiting progress in genetic engineering have been defined. One strategy is based on the adenyl cyclase toxin from the agent of whooping cough. Detoxified forms of the toxin show strong vaccinal potential in several antiviral or antineoplastic responses. The toxin binds to the CD11b integrin of dendritic cells, an essential stage for biological activity and immune response. Study of a glycoside antigen, Tn, associated with neoplasms of the breast, colon and skin, has led to the development of a totally synthetic antineoplastic vaccine, consisting of a trimer of epitope Tn associated with a T epitope. The resulting molecule has demonstrated great efficacy in inducing the rejection of neoplastic cells in mice. Furthermore, the development of a melanoma induction system in the mouse has made it possible to study the immune response during the successive stages of neoplastic progression. A protocol monitoring the cytotoxic responses specific of tumour antigens and the memory cells, in subjects receiving specific immunotherapy, has been established.
Inflammation and allergy
The role of inflammatory cells, their recruitment and activation, associated with functional disturbances, are being particularly studied in bronchopulmonary hyperreactivity syndrome. In particular, nasal injection of endotoxins induces neutrophil-dependent and TNF-independent bronchoconstriction resistant to corticosteroids. These mechanisms are also thought to be involved in adult respiratory distress syndrome (ARDS). The roles of alveolar macrophages, neutrophils and various cytokines in the induction of these clinical symptoms are currently receiving considerable attention. The role of secreted phospholipase A2 and prostaglandin H2 synthetase, two enzymes the synthesis of which is increased during inflammation, as well as the mechanisms of their secretion are being studied.
The enzymes are expressed by endothelial cells of the human pulmonary microcirculation and alveolar macrophages. Secreted phospholipase A2 plays a major role in the destruction of pulmonary surfactant. The institute has the largest allergy clinic in the Île-de-France region, with over 10,000 consultations per year. Clinical research in allergology is focusing on the oral polarisation towards allergens and the study of autoantibodies directed against the high-affinity receptor for IgE. Basic research is addressing the relationship between mast cells, dendritic cells and T-cells, the immunoregulatory role of mast cells and the molecular mechanisms of mast cell degranulation.
Diseases of immune system cells
Cold agglutinin disease is characterised by the production of autoantibodies against red blood cells, which recognise the glycolipid determinants and induce autoimmune haemolytic anaemia. A transgenic murine model of the disease has been constructed by transfecting the genes for a human antibody. This model thus represents a murine model of the human disease, and also serves as a model for study of the tolerance mechanisms with respect to red blood cell autoantigens. Chronic lymphoid leukaemia (CLL) is being studied in collaboration with the Groupe Français pour l'étude de la LLC. The major objectives of the research are to map the neoplastic identity of the disease, in particular with respect to predisposing genes and progression genes, using DNA microarray technology. Definition of the optimum conditions for vaccination using dendritic cells sensitised by the neoplastic idiotype is being studied.
Venoms, haemostasis and treatments
Venom toxins, through their highly potent action on haemostasis, may contribute to the creation of new drugs. A plasminogen activator, isolated from the venom of a Chinese pit viper, a thrombin inhibitor from the venom of Bothrops jararaca, a very potent platelet activator, convulxin, from the venom of Crotalus durissus terrificus, and anticoagulant phospholipases A2 have therefore been characterised and are proving good candidates for the development of antithrombotic agents. Research is currently under way in order to improve the immunological treatment of snake bite. A serum factor, isolated from the serum of a rattlesnake, protects the snake against the main toxin, crototoxin, and homologous toxins.
Pathogenic bacteria and fungi
One of the remarkable findings in recent years is the correlation between the response to stress and microbial pathogenicity. Studies on pathogenic fungi are increasing in scope.
The Bordetella species induces respiratory infections in man and animals, such as whooping cough, porcine atrophic rhinitis and canine cough. The year 2000 was marked by the discovery of a specific Bordetella toxin receptor on the surface of leukocytes. Moreover, studies have addressed the successive immune responses to B. bronchoseptica infection, the development of B. pertussis isolates following vaccination, the diversity of an adhesin (pertactin) involved in protective immunity, and the duration of the immunity provided by a new acellular whooping cough vaccine in children. Lastly, a murine model of B. pertussis respiratory infection has been standardised.
Transmitted by ticks, the Borrelia infection, Lyme disease, represents the most frequent vector disease, with about 10,000 cases each year in France. Studies are being conducted in the forests of the Île-de-France region in order to determine the density of the tick vectors, a dominant parameter in disease incidence. Atmospheric pressure, the pressure at ground level and the degree of plant cover affect tick density. It is therefore possible to predict the tick activity curve 4 months in advance by monitoring temperatures. This is, of course, essential with regard to prevention.
Cholera is still rife in a large part of the world, and particularly in poor countries. The causal agent of cholera, Vibrio cholerae, has two ecological niches: the sea and man. The objective of cholera vaccination is to eliminate cholera vibrios from the latter. The structure of a V. cholerae antigen, a polysaccharide forming a complex with a protective antibody, has been studied in great detail. The protective efficacy in mice of a chemically defined vaccine, consisting of this antigen coupled with a carrier protein, tetanus toxoid, is leading the way for a vaccine for use in human. Molecular techniques have been developed to identify Vibrio species in the environment which are pathogenic to man. The presence of these species in French coastal waters is promoted by ecological changes in the marine environment namely induced by industry. A study of vibrios in the bay of La Rance is therefore being conducted. These techniques are also used to characterise the pathogenicity factors of Vibrio species in exported seafoods.
Helicobacter pylori is the aetiological agent of chronic gastritis, which may progress towards ulcer and gastric atrophy, a precursor of gastric adenocarcinoma. The research aims to further elucidate the mechanisms that enable H. pylori to survive in, and colonise the gastric environment and generate lesions. The research also aims to elucidate the link with the metabolism of the bacterium. The development of murine models of H. pylori infection has made it possible to study the role of the inflammatory response in these processes. Epidemiological, clinical (resistance to metronidazole) and therapeutic issues are also being addressed. a The same group is also studying the pathogenic potential of the Escherichia coli strains associated with urinary tract infection and diarrhoea, with special emphasis on the adhesion systems expressed by pathogenic strains. Among the different systems, AFA is a prime example of a genetic structure specifying both a protein involved in adhesion and another involved in internalisation of the bacterium. The structure is carried by an island of pathogenicity which must be investigated in order to understand and monitor the evolution of the species. Study of the adhesion factors of E. coli associated with persistent diarrhoea in HIV-seropositive patients has led to the definition of a new adhesion system.
Molecular approaches were hitherto non-existent for the spirochetes of the genus Leptospira. A shuttle vector able to move between Leptospira biflexa and Escherichia coli has been constructed, enabling a genetic approach. An immobile mutant has thus been obtained by inactivating the gene coding for the flagellum. With regard to cell responses, the lipopolysaccharide of L. interrogans activates macrophages via the toll-like receptor, TLR2, unlike the LPS of gram-negative bacteria, which uses receptor TLR4.
Listeria monocytogenes, responsible for severe cases of food poisoning, has become a model system in order to elucidate intracellular parasitism and exploitation of cell functions by pathogens. L. monocytogenes is characterised by its ability to cross barriers which are usually highly impermeable, namely the intestinal, blood-brain and placental barriers. In the infected tissues, Listeria occupies an intracellular position, penetrating and proliferating in numerous cell types. Following lysis of the phagocytosis vacuole, the bacteria, while replicating in the cytosol, move around in it and pass from cell to cell using a highly original propulsion mechanism, generated by continuous polymerisation of cellular actin from one of the poles of the bacteria. The advances in pathogenesis have mainly focused on several aspects of penetration into cells, particularly the role of these events in vivo, the analysis of phagocytosis vacuoles, the study of intra- and inter-cellular movements, the control of virulence gene expression and identification of new virulence genes.
The study of tuberculosis led to the definition of new virulence mechanisms (identification of the virulence genes) in Mycobacterium tuberculosis. A systematic search for virulence genes has been conducted in view of investigating new therapies and new vaccines. BCG-derived recombinant vaccines are being studied. The administration route of these vaccines, the role of glycosylation in the antigenicity of immunodominant molecules, the incidence of forms of the disease resistant to conventional antibiotics, and the mycobacterial flora in drinking water distribution networks are being studied.
The molecular pathogenesis studies on Neisseria have demonstrated the major role of a newly discovered regulation gene, crgA, in the decisive stages of the interaction between N. meningitidis and the target cells of the host. These findings lead the way for a better elucidation of the mechanisms by which invasive infection is triggered, together with the development of new strategies for prevention and treatment.
The research into Salmonella focuses on the mechanisms by which Salmonella persist in the environment, and how they penetrate intestinal epithelial cells, a crucial stage in the emergence of salmonellosis. Bacterial survival is partially ensured by a sigma factor, RpoS, a key regulator in the generalised resistance to various stresses, and which plays an essential role in the regulation of Salmonella virulence genes spv. The genes required for the penetration of Salmonella into intestinal epithelial cells (invasion genes) are grouped on an island of pathogenicity. The expression of those invasion genes is very finely controlled by a cascade of regulators, which are active as a function of the environmental conditions. Two families of chemical compounds (short-chain fatty acids and aromatic acids) inhibit the expression of Salmonella invasion genes in vitro. Sodium caprylate induces a very significant reduction in the bacterial load of infected mice and is highly effective in protecting the animals. Intestinal cells specialised in antigen transport, the M cells of Peyer's patches, are used as the portal of entry by numerous pathogens. The mechanisms for interaction between M cells and the pathogens are being studied with a view to targeting the very first stage of numerous infective processes.
The molecular, cellular and tissue bases of the rupture, invasion and inflammatory destruction of the intestinal barrier by invasive pathogens such as Shigella and Entamoeba histolytica, together with the inductive and effector mechanisms of the protective response against Shigella, continue to be investigated. The data provide the basis for development and clinical studies of candidate vaccines against dysentery. The progress made in the past year dealt with cell signalling events on bacterial invasion of the cell and the elucidation of the mechanisms inducing intestinal inflammation in the course of Shigella infection. A new series of cytosol proteins, homologous with plant resistance proteins, is responsible for the effect of lipopolysaccharide on epithelial cells. The immunogenicity of peptide antigens mimicking the polysaccharide sub-units of the bacterium and constituting a protective antigen has been demonstrated. Phase I and II clinical trials on a S. flexneri SC602 oral live candidate vaccine are pursued in Bangladesh.
One of the objectives of the study of Staphylococcus is to characterise the factors for adhesion to biomaterials and extracellular matrix proteins in coagulase-negative staphylococcal strains responsible for nosocomial infections in the context of joint replacement. A gene coding for an autolysin-binding fibronectin and those involved in the biosynthesis of the biofilm, together with two genes coding for 'serine-aspartate' proteins of the wall, have been isolated from the chromosome of an infectious strain of Staphylococcus caprae and sequenced. Mutagenesis will be carried out in order to evaluate their role in the infection process
Virulence factors of Streptococcus agalactiae, a major aetiological agent in neonatal infections, have been identified. Several genetic tools enabling the construction of mutants have been developed. A murine model simulating the most important consequence of neonatal invasive S. galactiae infection, i.e. meningitis, has been developed. The gene coding for a superoxide dismutase containing manganese, which protects S. agalactiae from oxidative stress, is involved in the pathogenicity of the bacterium. The superoxide dismutase is necessary for maintaining elevated bacteraemia, probably by promoting cerebral invasion and the persistence of bacteria in the spleen and liver of infected mice. The incorporation of D-Ala residues in lipotechoic acids (LTAs) is also an important factor for the virulence of S. Agalactiae.
The genus Yersinia consists of three species pathogenic to man: Y. pseudotuberculosis and Y. enterocolitica, responsible for gastrointestinal disorders, which are widespread in France, and Y. pestis, the aetiological agent of plague, with an endemic-epidemic status in Africa, Asia and America. The Yersinia laboratory is studying the factors responsible for the pathogenicity of Yersinia and the parameters modulating expression of the factors. Special attention is being paid to the grouping of certain genes in islands of pathogenicity.
Bacillus anthracis, an extracellular toxin-secreting bacterium, is the agent of anthrax. During the initial stage of the infection, B. anthracis spores germinate in macrophages. This phenomenon is associated with toxin synthesis. A plasmid germination operon involved in virulence, has been identified. Study of the mode of action of the oedema-inducing and lethal toxins of B. anthracis is crucial. In 1998, two target proteins of the protease activity of the lethal factor were identified. A third has been discovered last year. This constitutes an important avenue for elucidation of the mechanisms of the toxic shock induced by the toxin. Lastly, total protection against anthrax was obtained in animals immunised with one of the components of the toxin associated with inactivated spores. This should represent a significant improvement in the acellular human vaccine, which currently only aims at protection against toxins.
The botulinic neurotoxins secreted by Clostridium botulinum combine with non-toxic proteins to form large complexes. The production of neurotoxins and associated proteins is controlled by a gene named botR, which is homologous with gene tetR of C. tetani. The mode of action of the two genes is being analysed. The way in which the botulin neurotoxin, alone or in the form of a complex, crosses the intestinal barrier is being studied in view of elucidating the factors controlling the toxicity of C. botulinum. A further area of interest corresponds to the binary toxins of Clostridium. Due to the fact that it is associated with heterologous proteins, the iota toxin of C. perfringens is the candidate for internalisation of proteins in cells. The epsilon toxin of C. perfringens, a very potent oedema-inducing toxin responsible for enterotoxaemia, is also being studied using artificial lipid layers and a cell model. Once again, the adaptation to oxidative stress of C. perfringens, the aetiological agent of diseases ranging from simple food poisoning to necrotic enteritis or gangrene, probably represents an important pathogenicity factor for this ubiquitous bacterium. The pathogenic potential of C. difficile, responsible for pseudomembranous colitis and most cases of diarrhoea following antibiotic therapy, is mainly based on its ability to produce two major toxins, ToxA and ToxB, the genetics of which is currently being studied.
Fungi and fungal infections
Two pathogenic yeasts, Cryptococcus neoformans and Candida albicans, responsible for serious infections in immunocompromised patients, are being studied. The year 2000 was devoted to the study of the genes coding for synthesis of the polysaccharide capsule of C. neoformans, an essential component of its virulence, the genetic variability of C. neoformans, the pathophysiology of cryptococcosis through the study of the immune response to C. neoformans infection in man and the mouse and, lastly, the virulence factors of Candida albicans. DNA microarrays have been prepared and have made it possible to analyse part of the transcriptome of Candida albicans, a yeast responsible for the majority of fungal infections in man. The DNA microarrays have already been used to identify new transcriptional targets of the repressors involved in the yeast-hypha transition, which is an important determinant of pathogenicity. New microarrays are being designed for global analysis of the transcriptome of C. albicans. The interactions between Aspergillus fumigatus and man are a topical issue. Aspergillus fumigatus is a saprophytic filamentous thermophilic fungus, the conidia of which are constantly present in the atmosphere and continuously inhaled by man. The conidia may give rise to serious, often fatal, respiratory diseases in immunocompromised patients. With regard to invasive aspergillosis, particular attention is being given to the potential virulence factors of the fungus and the defence mechanisms employed by alveolar macrophages to counter germination of infective conidia. The structural organisation of the cell wall and the enzymes responsible for biosynthesis of wall polysaccharides are the keys to understanding the cellular development of all fungi. Various transcriptional and functional analysis tools have been developed, aiming to facilitate the study, at the genomic level, of the model filamentous fungus, A. nidulans, and the fungi pathogenic to man, A. fumigatus and Candida albicans. With regard to A. nidulans, these methods have been applied to characterising the molecular and biochemical events intervening during the early phase of spore germination. With regard to A. fumigatus, a systematic search for essential genes has been launched with a view to defining original targets for the development of new antifungals drugs.
Neurobiology and neuropathology
The nervous system represents one of the new frontiers of biology. The growing significance of diseases affecting the nervous system has transformed the study of the nervous system into an important discipline at the Institut Pasteur.
Research into the formation of the nervous tissue mainly focused on the membrane proteins that ensure the contacts between cells and their migration in the body. Thus, a chemokine, which binds to receptor CXC-R4, is expressed in the cerebellum and hippocampus, in zones of migration and neurogenesis. Neural stem cells synthesise a membrane neuregulin, which stimulates their division and survival. A soluble receptor of neuregulins transforms the cells into oligodendrocytes, the cells which synthesise myelin. Several molecules that connect stem cells together have been identified. One of the molecules, connexin 45, is expressed in oligodendrocytes at a very early stage of development. Three new connexins of the electrical synapses of the retina have been characterised and form a new branch of the connexin family. A genetic mapping method for neuronal circuits, based on the construction of proteins bound to a fluorescent protein and which cross the synapses, has enabled direct imaging of the neuronal networks. Neurons 'inform' the cells that they innervate via specific structures, the synapses, where neurotransmitters are released. The biology and plasticity of synaptic transmission, particularly inhibitory transmission, are being studied in fish and rat brains. The mechanisms of neurotransmitter release and their modification by learning are being studied. Neurotransmitters act on ion channel receptors, located on the membrane of the neurons situated opposite the endings. Analysis of glycine receptors, chloride channels, aims to elucidate the physiology and pathology of inhibitory synapses. The spontaneous activity of afferent neurons induces fluctuations in neuronal activity. Algorithms based on non-linear dynamics have demonstrated the variations in this activity, consisting of chaotic phases, reinforced by learning, and predictability of neuronal discharges. The excitability of the cells of the hippocampus, a brain region subject to epilepsy and involved in memory, is controlled by specialised inhibitory inter-neurons, the anatomical and functional diversity of which has been studied. Research into the nicotinic receptor of acetylcholine, which intervenes in the neuromuscular junction and the central nicotinic synapse, has been carried out with three objectives: to identify the receptor regions involved in acetylcholine recognition, ion transport and various couplings; to identify the mechanisms controlling receptor distribution; and to study the processes involved, in terms of the central nervous system, in motor control, learning, nicotine dependence and various brain diseases. The potential applications of the research focus on the pharmacology of innervated muscle and central nicotinic receptors, the pathological phenomena associated with skeletal muscle denervation and myasthenia, and the central disorders which result from impairment of, or which affect nicotinic receptors: epilepsy, Alzheimer's disease, Parkinson's disease, ageing and nicotine dependence. Studying the origin of human hereditary neurosensory deficiencies, in addition to its medical interest, constitutes an approach to the sensory organs. The investigation for genes giving rise to isolated deafness has been facilitated by the study of affected consanguineous families, living in geographic isolation. The genes involved in 8 forms of isolated deafness and 5 forms of deafness associated with other symptoms have been identified. The genes responsible for a new form of isolated deafness and Usher syndrome 1C (marked deafness and pigmented retinopathy progressing to blindness) were discovered this year. Significant advances in the understanding of Usher syndrome 1B, another form of isolated deafness, and in Kallmann and Morsier syndrome (which combines deafness, deficiency of the sense of smell and absence of puberty) have been achieved. Unexpectedly, despite the heterogeneity of congenital isolated, half of the forms involve the gene coding for connexin 26. Moreover, a particular mutation of this gene alone accounts for 75% of all the mutations. This deficiency thus represents the most frequently observed form of genetic deafness. Molecular diagnosis can be used in genetic counselling. Research is now turning towards the study of connexin 26 disorders with a view to developing an appropriate therapy.
Numerous nervous system disorders related to viral or microbial infections have been studied in the context of the specific study of these infectious agents. In rabies, the first disease of the nervous system with an infectious aetiology studied by Louis Pasteur, the adhesion molecule, NCAM, is a virus receptor. Neuronal and muscle cells express different isoforms of NCAM, containing variable quantities of sialic acid. The combinations of the various forms of NCAM, responsible for rabies virus penetration into nerve cells, have been characterised. Furthermore, one of the internal proteins of the lyssaviruses has been shown to interact with the light chain of cytoplasmic dynein. This may explain the retrograde axonal transport of lyssaviruses. Recombinant rabies viruses have made it possible to study axonal transport and the trans-synaptic passage of rabies virus, together with the lesions induced by the virus, such as the compromise of the integrity of the blood-brain barrier. In a murine model of rabies encephalitis, the ability of the virus to induce (or not to induce) programmed cell death (apoptosis) determines the course of the infection: the degree of apoptosis in the early phase of infection is considered to determine the spreading of the virus in the central nervous system and the fatal or non-fatal outcome of the infection. Viral pathogenicity is directly related to maintaining the immunological privilege of the central nervous system: by strengthening that privilege, the virus is able to complete its life cycle, protected from an immune response. After inoculation by a bite, the virus is then able to transit through the nervous system to the salivary glands. An animal model of encephalitis and dementia associated with AIDS has been developed in the rat and the neurotoxic properties of HIV protein Nef have been demonstrated in vivo. Functional magnetic resonance imaging of HIV-seropositive patients has shown that they present cognitive deficiencies associated with dysfunction of the frontal cortex. Lastly, in a murine model of paralytic poliomyelitis, caspase-dependent apoptosis accounts for the destruction of motor neurons of the central nervous system. Studies designed to elucidate the neuropathogenesis induced by HTLV-1 have been carried out and include immunovirological studies of lesions in patients with paraparesis, together with the study of HTLV-1 passage through the blood-brain barrier.
Parasites and parasitology
Considerable resources have been dedicated to study the relationship between parasites and hosts, an important stage in identifying the targets on which it may be possible to act. Study of the relationship between the immune system, parasite and infected cells represents a major avenue of parasitological research.
The aetiological agent and its vector
Only mosquitoes of the genus Anopheles enable the development of Plasmodium falciparum. A. gambiae is the main vector in Africa. In order to analyse the adaptation of P. falciparum to Anopheles, the absence of an immune response of the gastrointestinal tract of A. gambiae was studied. The development of P. falciparum in the non-vector mosquito, Aedes aegypti, is blocked at the time of crossing the intestinal epithelium. Understanding the mechanisms by which development is blocked in A. aegypti should make it possible to stop transmission in A. gambiae. As is the case in all insects, A. gambiae has a seryl protease cascade, responsible for the synthesis of melanin subsequent to injury or invasion by a foreign body. In A. gambiae, P. falciparum has subverted this defence reaction and is only very rarely melanised. For genetic reasons, the activation of the enzymatic cascade is being studied in Drosophila, which has the same cascade. A serpin, a natural inhibitor of serine protease, inhibits activation of the cascade and also regulates coagulation of insect haemolymph. It is thus involved in the two defence mechanisms of the insect. The homologue in A. gambiae is being sought in order to determine whether it is responsible for inhibition of the intestinal response. Until recently, only one of the two species of chicken-parasitising Plasmodium, P. gallinaceum, had been discovered. The host-parasite association is a natural system used as a model for the study of transmission to mosquitoes. An isolate from the other species, P. juxtanucleare, was obtained from Vietnam. This will make it possible to elucidate the effects of co-infection of the two species on transmission of the parasite to mosquitoes: co-infections show that the presence of P. juxtanucleare decreases the transmissibility of P. gallinaceum to its vector. This phenomenon seems to be due to cross-fertilisation between the two species, resulting in a non-viable zygote.
Plasmodium and the red blood cell
The parasite penetrates red blood cells following inoculation into the body by a vector insect bite. Enzymatic activities appear to be central to this process. The parasitic seryl protease, which ensures the last stage in maturation of protein MSP1 (major surface protein of merozoites) is essential for parasite penetration in the red blood cell. Maturation is probably ensured by the product of gene sub2, recently characterised in P. falciparum and in rodent-parasitising Plasmodium, P. berghei and P. vivax. During the year 2000, sub2 was shown to be essential indeed to the erythrocytic cycle of Plasmodium. Tools elucidating the enzymatic activity of protein SUB2 have been developed, together with specific inhibitors of SUB2 and another parasitic seryl protease, which also plays a role in parasitic penetration of red blood cells. The ability of peptides to inhibit the enzymatic activities in vitro together with erythrocyte invasion will enable stable pseudopeptides to be designed, possibly with an antiparasitic activity. Cerebral malaria and malaria during pregnancy appear to be related to the presence, on the surface of red blood cells, of parasitic adhesins, Var proteins. Variant expression of these proteins gives rise to antigenic variation and differences in adherence of the infected erythrocytes to the vascular endothelium. The relation between the two phenomena, sometimes accompanied by a variation in parasite tropism, has been confirmed. A parasitic population of defined cytoadherence only expresses one of the 50 var genes in its genome. Among the mechanisms involved in the expression of and variation in var genes, the telomeres appear to play an important role, as do major rearrangements in the family of var genes. Side-by-side positioning of var genes carried by heterologous chromosomes facilitates gene conversion and, at the same time, the diversity of adherence antigens and phenotypes. The elective adherence of a parasite sub-population to chondroitin sulphate (CSA), a receptor present in large quantities in the placenta, is responsible for the sensitivity of women to infection by P. falciparum during their first pregnancy. There is a var gene which codes for a ligand of CSA and this may result in a method for controlling gestational malaria. Knobs, protuberances resulting from changes in the erythrocytic membrane induced by the parasite, may be observed at the sites of adherence to endothelial cells. Several parasitic proteins present in the knobs have been characterised. An in vitro cell adherence study, under flow conditions similar to those prevailing in the blood, has shown that parasites devoid of knobs adhere weakly to endothelial cells. The protein transport and addressing mechanisms ain P. falciparum have yet to be fully elucidated. It seems likely that at least one of the adherence proteins migrates to the surface by an alternative pathway, different to the classic pathway, which may lead to the development of new drugs.
The host response to Plasmodium
The parasite is most sensitive to the host immune system during migration toward the liver. In addition to numerous studies on Plasmodium migration/slipping proteins, studies have been conducted with a view to elucidating the dual immune response during malarial infection. In mice infected by P. yoelii, a very marked increase in hepatic T-cells CD4-CD8-NK is observed, which persists following remission of the disease. These cells exert an inhibitory activity on sporozoite development in the liver. With regard to human malaria vaccines, we have shown that an antigen of the pre-erythrocytic stage induces a protective response in primates infected by P. falciparum. The first clinical trials were initiated in January 2001 on 36 volunteers. An alternative strategy consists in using a fragment of the Plasmodium MSP1 antigen, the three-dimensional structure of which, in the form of a complex with a specific antibody, has been determined. The convincing results obtained in primates have made this antigen a vaccinal candidate for P. falciparum, which should soon be undergoing phase I clinical trials in man. Field studies on the diversity of P. falciparum and its consequences on the immune system have given rise to two noteworthy findings. First, examination of peripheral blood may not yield an accurate reflection of the state of infection. Secondly, the study over time of the antibody response to the highly polymorphic regions of MSP1 shows that the reactivity profile is preserved fairly well and that new antibody specificities are absent, despite accumulation of several Plasmodium variants. This phenomenon would appear to reflect an escape strategy vis-à-vis the immune response. Lastly, a study on the potential contribution of lymphocytes to the disease associated with P. falciparum has shown, in infected African children, that these cells have been activated and produce high levels of INF-a and TNF-a, respectively.
Leishmania, Trypanosoma and Theileria
The blood-sucking insect vectors in which Leishmania accomplish part of their life cycle may feed on man. If the insects are carrying Leishmania, they inoculate invasive parasites into the cutaneous compartment, where the parasites create or remodel an environment conducive to their development, up to the stage of a 'parasite transmissible' to vectors. Original models have been defined in order to characterise the stages underlying the multiple processes triggered by Leishmania in their mammalian hosts, from initial inoculation into the dermis to the stage of transmission to the vector. Research into Trypanosoma cruzi aiming to elucidate the mechanisms paradoxically contributing to parasitic induction of immunocompromise intitially, followed by autoimmunity, is being conducted. The immunosuppressant activity appears to be related to secretion of a proline racemase by the parasite. This enzyme strongly activates naive B lymphocytes in a polyclonal manner. The racemase may represent a new therapeutic target. The analysis of B lymphocytes from resistant or sensitive mice, before and after infection by T. cruzi, has shown that the B repertory in resistant mice is paradoxically composed of multi-reactive anti-'self' antibodies. This could maintain 'physiological' autoreactivity and prevent the development of severe autoreactivity. The strategy used by the bovine parasite, Theileria parva, to transform B lymphocytes is being studied. T. parva targets a phosphatase activity and thus controls induction of host cell transcription factors and apoptosis. Moreover, lymphocytes infected by T. parva have a specific enzymatic activity, essential for transformation, probably through induction of continuous proliferation.
Microbial physiology and environment work
The study of bacterial responses to the environment has been a permanent feature of microbiology at the Institut Pasteur for over fifty years. More in-depth studies are constantly being carried out due to the modern methods of genomics and transcriptome analysis.
Bacterial membranes and protein secretion
Bacterial wall and membrane crossing by molecules, vital to a bacterium, is also of biotechnological interest. The activation of the genes involved in the transport and metabolism of maltose and maltodextrins in Escherichia coli and the secretion of a product of one of these genes, pullulanase, by Klebsiella oxytoca, are the subject of extensive investigation. The former process depends on the transcriptional activator, MalT, which is activated by maltotriose and repressed by three other proteins. The latter process involves the twelve proteins of the membrane complex which carries pullulanase across the membrane. The two systems were selected since homologous systems exist in numerous bacteria. In addition to the genetic approaches, fine structural analyses are being conducted. The ABC transporters of gram-negative bacteria carry proteins without a signal peptide, but with a secretion signal at the end of the protein. The transporters consist of three proteins of the bacterial envelope: protein ABC which supplies energy and two ancillary proteins. Protein ABC forms a complex with the protein to be secreted, the three constituents of the carrier, and protective proteins known as 'chaperones', which vary according to the protein. Hemophores are receiving considerable attention. Hemophores are proteins able to bind to free heme and extract the heme from proteins such as haemoglobin, then deliver it to a specific receptor. Serratia marcescens hemophore HasA is directed to the carrier, thanks to chaperone SecB and remains in a 'secretable' form until its C-terminal end has been synthesised and the secretion signal recognised by the carrier. The special structure of the hemophore demonstrates a new type of binding site for heme and iron. The interaction between the hemophore and its receptor is mediated by contact between the two proteins, without heme intervening. As heme-carrying and non-carrying hemophores bind to the same site on the receptor, with the same affinity, simple exchange between the carrying and non-carrying hemophores bound to the receptor is believed to occur.
Control of bacterial metabolism and virulence
The adaptation of bacterial metabolism to environmental variations is determined by complex genetics, now receiving constant attention. The overall genetic response to environmental variations is being approached through study of the transcriptome of B. subtilis and S. aureus. This strategy has made it possible to identify the sets of genes which respond in a coordinated manner to a given event. Numerous studies addressing control of nitrogen and sugar metabolism and others on resistance to aggressions are being conducted. Heat stress induces expression of proteins known as heat-shock proteins, which ensure protection of the key enzymes of microbial physiology, but are also related to pathogenicity. Control of their expression has been greatly studied in numerous micro-organisms. The heat-shock genes coding for catalytic and regulatory sub-units of the ATP-dependent protease of B. subtilis are placed under the control of repressor CtsR. Protein CtsR and its target sequences are highly conserved in gram-positive bacteria with a low GC content, including certain pathogens (Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis). This suggests a common physiology. In S. pneumoniae, the expression of chaperone proteins GroESL depends both on CtsR and another general regulator, which are the subject of a single regulation system. Neighbouring systems of variable complexity have been identified in various other bacteria. Genetic analysis of bacterial virulence has continued. In S. aureus, a twocomponent system, ArlS/ArlR, intervening in the virulence of this bacterium, has been analysed. Deletion induces a very marked increase in the production of the antibody-binding protein A. A pleiotropic regulator of the expression of virulence factors activates the transcription of at least 15 genes coding for extracellular proteins (phospholipases C, proteases, haemolysin, enterotoxins). Knockout of the gene markedly decreases the haemolytic and cytolytic properties of B. thuringiensis and B. cereus. In mice, the mortality induced by spores from these bacteria is markedly reduced, or even eliminated, with mutant plcR. plcR expression is selfregulated, negatively controlled by a sporulation gene, and also dependent on ABC transporter, involved in the transport of small proteins.
Antibiotic resistance mechanisms and bases
The genetics and biochemistry of bacterial resistance to the main series of antibiotics currently in use or in development (fluoroquinolones and streptogramins) are being studied in major pathogens for man. Study of certain resistance determinants has led to the development of sensitive genotypic methods, enabling concomitant identification of bacteria and associated resistance mechanisms for both gram-positive and gram-negative bacteria. Genetic information transfer from bacteria to mammalian cells is being studied in view of its potential value in gene therapy and with regard to stimulating mucosal immunity. The studies on staphylococci focus on the resistance to streptogramins and related antibiotics. A variant of the gene coding for protein ABC, which confers resistance to streptogramin A, has been recently characterised. The gene is carried by a mobile genetic element. The Pasteur-Necker joint research laboratory for streptococci has studied the spread of antibiotic resistance in cocci. The first strain of streptococci (S. bovis), highly resistant to vancomycin, has been characterised and the role of transposons in resistance spread has been demonstrated. Analysis of streptococcal transposon Tn916 has shown that the transcription of its transfer functions necessitates excision of the component and considerably increases in the presence of tetracycline. These results reveal a further degree of complexity in antibiotic resistance since the latter may also induce the transfer of the resistance gene. Isoniazid is a potent antituberculotic, the toxicity of which derives from its transformation by an enzymatic reaction. Considerable progress has been made in elucidating the mechanism. The pathogenic potential of certain clinical isolates of isoniazidresistant bacteria has been evaluated in an animal model. Furthermore, field evaluation of a molecular detection test for leprosy bacilli resistant to rifampin, has yielded highly satisfactory results.
Micro-organisms and environment
Cyanobacteria, which appeared on our planet some 3 billion years ago, were the first living organisms to use water as a source of reducing power and light as an energy source. These bacteria were responsible for enriching the primitive atmosphere with oxygen and are considered the ancestors of chloroplasts. They also represent excellent bacterial models for the study of cell functions, incorporating morphological and functional changes in response to variations in light or nutritional conditions in the environment. Photoreceptors and their function, together with the functions of the proteins involved in the signal transmission pathways necessary for coordination of the metabolic pathways for carbon and nitrogen, are being studied. The 'Institut Pasteur Collection', of cyanobacteria (http://www.pasteur.fr/recherche/ banques/PCC/), with over 720 strains from 50 different genera derived from various ecosystems, is constantly being enhanced. The collection includes 'spirulins', cyanobacteria widely used in industry, together with pathogenic strains producing hepatotoxins and neurotoxins fatal to animals and man. Studies designed to identify potentially toxic cyanobacteria, particularly those of the genus Microcystis, which invade stagnant water during the summer period, are being conducted both in vitro and in situ. Another Institut Pasteur group is studying nitrogen-fixing bacteria and, in particular, the cell function which enables the bacteria to fix nitrogen in their distinctive environment. The production of a phytohormone, indoleacetic acid, by Azospirillum brasilense, is being studied. This bacterium is associated with cereal crops and fixes nitrogen. Three different synthesis routes are involved in the synthesis of the hormone from tryptophan. The study of Pseudomonas stutzeri, a nitrogen fixer and endophyte of rice, has been continued. The biodegradation of isoalkanes and fuel ethers is relevant to eradicating hydrocarbon pollution. These compounds, among the most recalcitrant of the constituents of gasoline, create problems in the biological restoration of polluted sites. The enzymes and genes of the degradation pathways are being studied in Mycobacterium austroafricanum and Gordonia terrae, two species of mycobacteria. A group of genes responsible for the degradation of ethyl-tert-butyl ether has been cloned and sequenced in G. terrae. This result leads the way for the development of tools (nucleic acid probes, strains carrying the carrier genes) in order to better evaluate the potential for biodegradation and the development of the microbial flora in sites polluted by gasoline.
Microbiological control of insects
A specific receptor for the binary toxin (Bin), produced by Bacillus sphaericus, is present on the intestinal membranes in the larvae of the mosquito, Culex pipiens. The receptor has been cloned and functionally expressed in insect cells. The BinA component of the toxin is able to insert itself into phospholipid membranes and form pores, confirming its role as the active component of the toxin. The component BinB is responsible for binding to the specific receptor. C. pipiens populations are developing resistance to B. sphaericus binary toxin. Several resistance mechanisms are present in geographically neighbouring populations. A number of B. thuringiensis toxins have been studied in order to identify new active toxins. Proteins Cry29A and Cry30A are not active, individually, on the larvae of the mosquitoes, Aedes aegypti, Culex pipiens, and Anopheles stephensi, but appear to act in synergy with other B. medellin toxins on the three species. Protein Cyt2Bc is toxic for the 3 species of mosquitoes and exerts haemolytic activity following activation by trypsin. Three toxins of B. thuringiensis have been shown to be toxic to C. pipiens larvae resistant to B. sphaericus and Bt. israelensis toxins. A recombinant B. sphaericus, producing B. jegathesan protein Cry11Bb and active against Aedes aegypti larvae, has been constructed and placed under the control of the strong promoter sequences of B. sphaericus. With regard to entomopathogenic fungi, the diversity of the species of Zygomycetes attacking insects has been studied in the laboratory and in the field. Molecular analysis of the original strains from Greece pointed to the close relation between Erynia neoaphidis, considered to be an aphid-specific pathogen, and E. dipterigena, considered a specific pathogen for Diptera. In the latter species, molecular typing evidenced geographical infra-specific variability. A prospective mission in West Africa resulted in the entomopathogenic fungi of Guinea being studied for the very first time. The mediumaltitude zones of Fouta Djalon seem particularly favourable to the occurrence of Entomophthorale or Hyphomycetes fungal infections in insect populations of various orders, occasionally in an epizootic form. The genus Zoophthora, infrequent in the tropics, has been observed in particular. Strains have been isolated from various Homoptera for subsequent analysis.