Genetic study of virulence determinants (Brigitte Gicquel & Christophe Guilhot)
A thermo-sensitive vector derived from pAL5000 of Mycobacterium fortuitum and containing the counterselective sacB (ts/sacB vector) marker was used to select rare genetic events in mycobacteria, in particular, in species belonging to the M. tuberculosis complex. This vector was used to identify allelic exchange events leading to the inactivation of certain genes, chosen for study based on their similarity to genes known to be involved in virulence or intracellular survival in other pathogens. In collaboration with Carlos Martin and his team (Zaragoza Medical School, Spain), we recently isolated a mutant in which phoP, which encodes a putative regulator, is inactivated by an insertional mutation. This gene, which is close to the putative phoR gene, is essential for the multiplication of M. tuberculosis in cultured mouse macrophages in vitro and in mice, particularly in the lungs and, to a lesser extent, in the spleen and kidneys. The phoP/phoR gene pair is similar to the two-component system phoP/phoQ, which regulates several virulence determinants in Salmonella. In the M. bovis stain with multiple antibiotic resistance that caused an epidemic in Spain, the IS6110 insertion sequence is located several base pairs upstream from phoP. (Perez et al. Molec. Microbiol. in press). This suggests that this gene is regulated differently in this clinical isolate, possibly rendering this strain more virulent and therefore more transmissible than most multiple antibiotic-resistant M. tuberculosis strains.
The te/sacB vector has also been used to build transposition mutant libraries for M. bovis BCG and M. tuberculosis. One library, containing 7000 mutants derived from M. tuberculosis was ordered and used for a PCR search for mutations in candidate genes. A second library containing 4000 mutants was constructed using a tagged transposon. This library was used for STM (signature-tagged transposon mutagenesis) to search directly for mutants with attenuated virulence in mice. About 2000 mutants were screened for the ability to multiply in the lungs of mice. Sixteen attenuated mutants were selected and the corresponding mutations characterised. Four mutations were identified in a 50 kb region of the genome containing 13 genes involved in the synthesis and transport of phthiocerol dimycoserosates. RT-PCR analysis confirmed the results of in silico analysis suggesting that these genes were organised into three operons. One mutation affected the fadD26 gene, located at the start of the phthiocerol synthesis operon, inhibiting the synthesis of these molecules. Two genes, drrC and mmpl7, affect phthiocerol transport. Two different families of transporter are required: ABC transporters and RND permeases, required for the translocation of these molecules across the bacterial envelope. Biochemical analysis, carried out in collaboration with Mamadou Daffé's team at the IPBS at Toulouse, showed that these molecules are synthesised in the cytoplasm before being translocated through the wall. Defects in the synthesis or transport of phthiocerol dimycoserosates affect the permeability and structure of the bacterial envelope. Mutants with such defects are considerably more permeable to chenodeoxycholate (a small, negatively charged, hydrophobic molecule able to diffuse across lipid layers) and are considerably more sensitive to the detergent SDS (Camacho et al., J. Biol; Chem., in press). The role played by phthiocerol dimycoserosates in M. tuberculosis virulence may be associated with the impermeability of the wall to the toxic products produced by the host or the interaction of these molecules with unidentified host targets.
Functional analysis of virulence factors (Jean-Marc Reyrat)
Reporter genes (alkaline phosphatase), tagged insertional mutagenesis and computer tools have been used to identify a number of virulence genes. Several virulence loci are currently being studied, including those encoding Rv1395, a transcriptional activator of the AraC/XylS family and Erp (Rv3810), a protein secreted by M. tuberculosis.
Erp, which is secreted by the bacteria of the tuberculosis complex, was identified by a method involving the use of alkaline phosphatase as a reporter. In M. tuberculosis, inactivation of the erp gene by allelic exchange greatly attenuated virulence both in vitro, in cultured macrophages, and in vivo, in the mouse model of tuberculosis. We recently showed that Erp is ubiquitous in mycobacteria. This secreted protein is composed of three domains. The central domain consists of a tandem repeat of the PGLTS domain. This central domain is the most variable between species: with four repeats in M. leprae and 24 repeats in M. xenopi. (De Mendonça-Lima L. et al. MICROBIOLOGY, in press). Analysis of the genomic region surrounding the erp gene showed that most of the genes in this region are cell wall biosynthesis genes. We are currently studying the role of Erp in development of the structure of the mycobacterial wall and the effect of the number of PGLTS repeats on M. tuberculosis virulence.
Another aspect of the project concerns secretion by myobacteria. A certain number of virulence factors and antigens are present on the surface of the bacillus or in the extracellular environment. We used the Staphylococcus aureus nuclease gene as a reporter gene to show that this protein is secreted into the extracellular environment independently of any potential signal sequence. Cytoplasmic markers showed that this phenomenon was independent of bacterial autolysis. These results suggest the existence of an unidentified secretion pathway independent of the general pathway.
BCG and other vaccines derived from BCG (Nathalie Winter)
Mycobacterium bovis BCG, used as a live attenuated vaccine, confers a certain level of protection against tuberculosis and leprosy. Its persistence in professional antigen-presenting cells, such as macrophages (MP) and dendritic cells (DC), makes it an attractive immunogen for the development of efficient long-term vaccines. The development of genetic tools, in our laboratory in particular, has made it possible to introduce heterologous genes into BCG and to construct recombinant BCG strains (rBCG). We are studying the vaccination potential of rBCG strains expressing the genes of the simian immunodeficiency virus, SIVmac251, in the cynomolgus macaque. Macaques infected with SIVmac251 develop a disease similar to human AIDS, making it possible to evaluate new candidate vaccines against HIV. The nef gene, which is involved in regulation of the viral cycle, and the structural gag (p26) and env genes of SIVmac251 were expressed in BCG. In the mouse model, three strains induced cellular and humoral immune responses after administration by the parenteral or mucosal route.
We studied the immunogenicity of a mixture of these three rBCG strains in the macaque. The animals were inoculated by the intradermal route, the classical pathway for BCG injection in humans, and a booster was given via the mucosal route (oral or rectal). Cellular immune responses induced were assessed in peripheral blood mononuclear cells (PBMC). A cytotoxic T lymphocyte response directed against the Gag and Env antigens was detected in most of the animals vaccinated with the rBCG mixture. This lysis was accompanied by the production of large amounts of IFNg, indicating that a Th1/Tc1 response had been induced. The specific lysis rate also increased after the booster. Although massive proliferation of PBMC against mycobacterial antigens (PPD) was observed, no lymphoproliferation in response to the SIVmac251antigens was observed after the administration of rBCG. After rectal challenge with the SIVmac251 virus, an memory cell response against the Gag antigen was detected in several animals immunised with rBCG. The virus was also detected in all the animals after the challenge. However, one in four of the animals that received the rBCG booster via the rectal route had completely eliminated the virus. These encouraging results indicate that rBCG strains can induce a certain degree of protection against a virulent SIVmac251 challenge. These studies were carried out in collaboration with the team of R. Legrand (CEA, Fontenay-aux Roses) and A. Venet (Faculté de Médecine Paris Sud).
In parallel, we developed new expression vectors with the aim of increasing the stability of rBCG strains. Integrative vectors were used to obtain strains of rBCG with perfect genetic stability in vivo in the mouse model. Consistent with the strong and persistent expression of heterologous genes after immunisation with these integrative strains, very strong and long-lasting cellular and humoral immune responses were observed after immunisation with rBCG strains harbouring these plasmids.
The laboratory is also interested in the mechanisms by which the immune response is induced after administration of the BCG vaccine. Of particular importance is the interaction between BCG and dendritic cells (DC), the only professional antigen-presenting cells capable of activating naive T lymphocytes. We have carried out several studies, notably in vitro, after the development of a technique for obtaining DC from mouse bone marrow precursors. In collaboration with Eric Prina and Jean-Claude Antoine (Immunophysiology and Intracellular Parasitism Unit) the phagocytosis vacuole of BCG in the DC has been characterised to elucidate the mechanisms initially involved in the presentation of mycobacterial antigens to T lymphocytes.
L'Unité est un laboratoire d'accueil du "Marie Curie Host Fellowship" de la Commission Européenne. "Unravellling Mycobacterium pathogenicity. MCFH-1999-00615". http://www.cordis.lu/improving/
L'unité fait partie des réseaux suivants de la commission européenne :
- "A cluster for tuberculosis vaccine development" (N° QLK2-CT-1999-01093). http://www.pasteur.fr/EC_TBvaccine/
- " Mucosal Immunization Cluster Project ", (N° QLK2-CT-1999-00228).
- " New generation genetic markers and techniques for the epidemiology and control of tuberculosis " (N° QLK2-CT-2000-00630).
- " New strategies for treatment and prevention of mycobacterial diseases " (N° QLK2-CT-2000-01761).
- " Development of recombinant BCG multivaccine and complementary diagnostics for predominant parasitic and epizootic disease of ruminants in Latin America ", (N° ICA4-CT-2000-30032 (INCO-DEV).
- " Mucosal immunization- Cluster project", (N° QLK2-CT-1999-00228).
- "Improved diagnosis, drug resistance detection and control of tuberculosis in Latin America".Projet (N°: ICA4-2000-10054).
- "Structural and functional genomics of M. tuberculosis", (N° QLRT-2000-02018).