Brigitte Gicquel

Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature‐tagged transposon mutagenesis

Tuberculosis remains the greatest cause of death worldwide due to a single pathogen. In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis, a functional genomic approach was developed. A library of signature‐tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice. From 1927 mutants tested, 16 were attenuated for their virulence. The insertions harboured by the selected mutants were mapped on the M. tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane. Four independent mutations identified a cluster of virulence genes located on a 50 kb chromosomal region. These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens. The interaction of five mutant strains with mouse bone marrow macrophages was investigated. These five mutants were still able to multiply in this cell type. However, in three cases, there was a growth defect in comparison with the wild‐type strain. The other two strains exhibited no clear difference from the virulent strain, MT103, in this model. This study, which is the first global research of virulence factors of M. tuberculosis, opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host.

DC-SIGN Is the Major Mycobacterium tuberculosis Receptor on Human Dendritic Cells

Early interactions between lung dendritic cells (LDCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, are thought to be critical for mounting a protective anti-mycobacterial immune response and for determining the outcome of infection. However, these interactions are poorly understood, at least at the molecular level. Here we show that M. tuberculosis enters human monocyte-derived DCs after binding to the recently identified lectin DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). By contrast, complement receptor (CR)3 and mannose receptor (MR), which are the main M. tuberculosis receptors on macrophages (Mφs), appeared to play a minor role, if any, in mycobacterial binding to DCs. The mycobacteria-specific lipoglycan lipoarabinomannan (LAM) was identified as a key ligand of DC-SIGN. Freshly isolated human LDCs were found to express DC-SIGN, and M. tuberculosis–derived material was detected in CD14−HLA-DR+DC-SIGN+ cells in lymph nodes (LNs) from patients with tuberculosis. Thus, as for human immunodeficiency virus (HIV), which is captured by the same receptor, DC-SIGN–mediated entry of M. tuberculosis in DCs in vivo is likely to influence bacterial persistence and host immunity.

Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome.

Mycobacterial interspersed repetitive units (MIRUs) are 40–100 bp DNA elements often found as tandem repeats and dispersed in intergenic regions of the Mycobacterium tuberculosis complex genomes. The M. tuberculosis H37Rv chromosome contains 41 MIRU loci. After polymerase chain reaction (PCR) and sequence analyses of these loci in 31 M. tuberculosis complex strains, 12 of them were found to display variations in tandem repeat copy numbers and, in most cases, sequence variations between repeat units as well. These features are reminiscent of those of certain human variable minisatellites. Of the 12 variable loci, only one was found to vary among genealogically distant BCG substrains, suggesting that these interspersed bacterial minisatellite‐like structures evolve slowly in mycobacterial populations.

A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10)

The early secreted antigenic target 6 kDa protein (ESAT-6) is a potent T-cell protein antigen synthesized by Mycobacterium tuberculosis. Its corresponding gene (esat-6) is located in RD1, a 10 kb DNA region deleted in the attenuated tuberculosis vaccine strain Mycobacterium bovis BCG. The promoter region of M. tuberculosis esat-6 was cloned and characterized. A new gene, designated lhp and cotranscribed with esat-6, was identified. Moreover, computer searches in the M. tuberculosis genome identified 13 genes related to the lhp/esat-6 operon, defining a novel gene family. The transcription initiation sites of the lhp/esat-6 operon were mapped using M. tuberculosis RNA. The corresponding promoter signals were not recognized in Mycobacterium smegmatis, in which transcription of lhp/esat-6 is initiated at different locations. The M. tuberculosis lhp gene product was identified as CFP-10, a low-molecular-mass protein found in the short-term culture filtrate. These results show that the genes encoding CFP-10 and ESAT-6 are transcribed together in M. tuberculosis and that both code for small exported proteins.

IS6110, an IS-like element of Mycobacterium tuberculosis complex

Mobile genetic elements are useful genetic tools. They have been found in most organisms which have been examined (for recent reviews see 1). /S900 was isolated from M. paratuberculosis (2) and IS6100 from a M. fortuitum strain (our unpublished results). IS elements have been used as taxonomic markers useful for diagnostic purposes, (2, 3). From a IA. tuberculosis cosmid library constructed in pHC79 (4), an IS-like element, 1S6110, was identified as a repeated sequence, by screening the library with labelled M. tuberculosis total DNA. This sequence (1361 nt) possesses characteristics of IS elements, i.e., inverted (28bp with 3 mismatched bp) and direct (3bp) repeats of the target sequence at its extremities. A search in the EMBL data bank has revealed homologies with IS3411, an insertion element from E. coli (5). Cross-hybridization was observed between 1S6110 and a repeated sequence previously isolated from M. tuberculosis (6). IS6110 has been found in M. tuberculosis and M. bovis but not in any of the other mycobacteria tested (our unpublished data). Therefore, IS6110 will be used as probe for the identification of the M. tuberculosis complex. ACKNOWLEDGEMENTS

RAPID DIAGNOSIS OF TUBERCULOSIS BY AMPLIFICATION OF MYCOBACTERIAL DNA IN CLINICAL SAMPLES

A method based on DNA amplification and hybridisation for the rapid detection of Mycobacterium tuberculosis was used to test 35 clinical specimens (sputum, gastric aspirate, abscess aspirate, biopsy sample) from 34 patients in whom tuberculosis was suspected. M tuberculosis was detected in 15 specimens, 2 of which were negative by standard microbiological criteria (microscopy and/or culture). 20 specimens, negative by standard methods, were also negative by the amplification method. M tuberculosis was also detected in peripheral blood samples of 2 of 4 patients with AIDS from whom the organism had been isolated.

An essential role for phoP in Mycobacterium tuberculosis virulence.

Two‐component regulatory proteins function in bacteria as sensory and adaptive factors in response to a wide range of environmental stimuli. Some two‐component systems, such as PhoP/PhoQ, control transcription of key virulence genes essential for survival in host cells in diverse intracellular bacterial pathogens, including Salmonella sp., Shigella sp. and Yersinia sp. In this study, we have disrupted the phoP gene from Mycobacterium tuberculosis, which codes for a putative transcription regulator factor of the two‐component system PhoP/PhoR. The phoP mutant strain exhibited impaired multiplication when cultured in mouse bone marrow‐derived macrophages. However, the mutation did not appear to affect survival of the organisms adversely inside macrophages. The mutant strain was also attenuated in vivo in a mouse infection model, with impaired growth observed in the lungs, livers and spleens. The results suggest that the phoP gene is required for intracellular growth of M. tuberculosis but is not essential for persistence of the bacilli.

Analysis of the Phthiocerol Dimycocerosate Locus ofMycobacterium tuberculosis EVIDENCE THAT THIS LIPID IS INVOLVED IN THE CELL WALL PERMEABILITY BARRIER

Among the few characterized genes that have products involved in the pathogenicity of Mycobacterium tuberculosis, the etiological agent of tuberculosis, are those of the phthiocerol dimycocerosate (DIM) locus. Genes involved in biosynthesis of these compounds are grouped on a 50-kilobase fragment of the chromosome containing 13 genes. Analysis of mRNA produced from this 50-kilobase fragment in the wild type strain showed that this region is subdivided into three transcriptional units. Biochemical characterization of five mutants with transposon insertions in this region demonstrated that (i) the complete DIM molecules are synthesized in the cytoplasm of M. tuberculosis before being translocated into the cell wall; (ii) the genesfadD26 and fadD28 are directly involved in their biosynthesis; and (iii) both the drrC andmmpL7 genes are necessary for the proper localization of DIMs. Insertional mutants unable to synthesize or translocate DIMs exhibit higher cell wall permeability and are more sensitive to detergent than the wild type strain, indicating for the first time that, in addition to being important virulence factors, extractable lipids of M. tuberculosis play a role in the cell envelope architecture and permeability. This function may represent one of the molecular mechanisms by which DIMs are involved in the virulence ofM. tuberculosis.

Detection and identification of mycobacteria by amplification of mycobacterial DNA.

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Tag polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 106 human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.

Mycobacterium abscessus: a new antibiotic nightmare

The intrinsic and acquired resistance of Mycobacterium abscessus to commonly used antibiotics limits the chemotherapeutic options for infections caused by these mycobacteria. Intrinsic resistance is attributed to a combination of the permeability barrier of the complex multilayer cell envelope, drug export systems, antibiotic targets with low affinity and enzymes that neutralize antibiotics in the cytoplasm. To date, acquired resistance has only been observed for aminoglycosides and macrolides, which is conferred by mutations affecting the genes encoding the antibiotic targets (rrs and rrl, respectively). Here we summarize previous and recent findings on the resistance of M. abscessus to antibiotics in light of what has been discovered for other mycobacteria. Since we can now distinguish three groups of strains belonging to M. abscessus (M. abscessus sensu stricto, Mycobacterium massiliense and Mycobacterium bolletii), studies on antibiotic susceptibility and resistance should be considered according to this new classification. This review raises the profile of this important pathogen and highlights the work needed to decipher the molecular events responsible for its extensive chemotherapeutic resistance.