Production of unmarked mutations in mycobacteria using site-specific recombination

Gene disruption experiments play an important role in the functional characterization of genes in mycobacteria and rely mostly on the use of one or two antibiotic resistance markers. We have developed a system for mycobacteria which features both the advantages of the use of antibiotic resistance markers for gene disruption experiments and the ability to efficiently rescue the marker leaving an unmarked mutation on the chromosome. This new genetic tool relies on the transposon gammadelta site-specific recombination system. A res-OmegaKm-res cassette was used to generate an insertional mutation by allelic exchange both in Mycobacterium smegmatis and Mycobacterium bovis BCG. Upon expression in the mutated strains of tnpR, the transposon gammadelta resolvase gene, res-OmegaKm-res, was excised efficiently leaving behind a single res sequence at the mutated locus. A plasmid was engineered allowing expression of tnpR from an easily curable mycobacterial vector. This system will be useful for simple construction of unmarked mutations or repeated use of the same antibiotic marker to generate multiple mutants.     

Deciphering the genetic bases of the structural diversity of phenolic glycolipids in strains of the Mycobacterium tuberculosis complex

Phenolic glycolipids (PGL) play a major role in the virulence of mycobacteria, notably in strains of the Mycobacterium tuberculosis complex and in Mycobacterium leprae. The structure of the carbohydrate domain of these compounds is highly variable, and the genetic bases for these variations remain unknown. We demonstrated that the monoglycosylated PGL formed by Mycobacterium bovis differs from the triglycosylated PGL synthesized by M. tuberculosis (PGL-tb) because of the following two genetic defects: a frameshift mutation within the gene Rv2958c, encoding a glycosyltransferase involved in the transfer of the second rhamnosyl residue of the PGL-tb, and a deletion of a region that encompasses two genes, which encode a GDP-D-mannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/reductase, required for the formation of activated L-fucose. Expression of these three genes in M. bovis BCG allowed synthesis of PGL-tb in this recombinant strain. Additionally, we showed that all M. bovis, Mycobacterium microti, Mycobacterium pinnipedii, and some Mycobacterium africanum strains harbor the same frameshift mutation in their Rv2958c orthologs. Consistently, the structure of PGLs purified from M. africanum (harboring the Rv2958c mutation) and M. pinnipedii strains revealed that these compounds are monoglycosylated PGL. These findings explain the specificity of PGL-tb production by some strains of the M. tuberculosis complex and have important implications for our understanding of the evolution of this complex.     

A Lipid Profile Typifies the Beijing Strains of Mycobacterium tuberculosis: IDENTIFICATION OF A MUTATION RESPONSIBLE FOR A MODIFICATION OF THE STRUCTURES OF PHTHIOCEROL DIMYCOCEROSATES AND PHENOLIC GLYCOLIPIDS*

The Mycobacterium tuberculosis Beijing strains are a family highly prevalent in Asia and have recently spread worldwide, causing a number of epidemics, suggesting that they express virulence factors not found in other M. tuberculosis strains. Accordingly, we looked for putative characteristic compounds by comparing the lipid profiles of several Beijing and non-Beijing strains. All the Beijing strains analyzed were found to synthesize structural variants of two well known characteristic lipids of the tubercle bacillus, namely phthiocerol dimycocerosates (DIM) and eventually phenolglycolipids (PGL). These variants were not found in non-Beijing M. tuberculosis isolates. Structural elucidation of these variants showed that they consist of phthiotriol and glycosylated phenolphthiotriol dimycocerosates, eventually acylated with 1 mol of palmitic acid, in addition to the conventional acylation of the β-diol by mycocerosic acids. We demonstrated that this unusual lipid profile resulted from a single point mutation in the Rv2952 gene, which encodes the S-adenosylmethionine-dependent methyltransferase participating to the O-methylation of the third hydroxyl of the phthiotriol and phenolphthiotriol in the biosynthetic pathway of DIM and PGL. Consistently, the mutated enzyme exhibited in vitro a much lower O-methyltransferase activity than did the wild-type Rv2952. We finally demonstrated that the structural variants of DIM and PGL fulfill the same function in the cell envelope and virulence than their conventional counterparts.The factors contributing to the development of tuberculosis after infection with the etiologic agent of this disease, Mycobacterium tuberculosis, are not completely understood. It is currently thought that the genetic traits of both host and pathogen influence the outcome of the disease (1, 2). On the pathogen side, molecular epidemiological studies have differentiated clinical isolates of M. tuberculosis into defined genetically related lineages (3). One of the most important worldwide lineages, the “Beijing” lineage, was initially described by van Soolingen et al. (4) who showed that more than 85% of M. tuberculosis isolates from the Beijing region belong to this genetically conserved genotype. The Beijing strains were grouped together on the basis of their highly similar multicopy IS6110 restriction fragment polymorphism patterns, the deletion of spacers 1–34 in the direct repeat region found in all M. tuberculosis strains, and the insertion of IS6110 into the dnaA-dnaN locus (5). This genotype family of M. tuberculosis is highly prevalent in Southeast Asia, where more than 50% of the strains isolated correspond to the Beijing genotype (6). However, these strains have spread worldwide and have been responsible for major outbreaks of tuberculosis. The “W” strain responsible for a multidrug-resistant tuberculosis in the New York region, for example, belongs to this genotype (7, 8). A Beijing strain was introduced into Gran-Canaria Island in 1993 and rapidly spread; this strain accounted for 27.1% of tuberculosis cases on the island in 1996 (9). This spread of Beijing strains from the region of the world in which they are endemic to other areas in which they have become epidemic suggests that these strains may be more virulent than others. Consistent with this hypothesis, several studies have demonstrated that mice infected with Beijing strains die more rapidly than mice infected with other strains (10–13). This hypervirulence phenotype has been associated with a failure of Beijing strains to induce or to maintain an adequate Th1-type immune response (10, 12) and with more severe pneumonia resulting in the death of the infected animals. A recent study suggests that patients infected with Beijing strains are more likely to develop extrathoracic tuberculosis than people infected with strains of other lineages (14). This observation is consistent with the results obtained in a rabbit model with the Beijing strain HN878, which was found more likely to infect the brain and to disseminate (15). A recent study in Vietnam pointed out that the typical lineage of Beijing genotype strains is more frequently found in patients who received a Bacillus Calmette-Guérin vaccination. This suggests that bacteria of this lineage are more capable of circumventing Bacillus Calmette-Guérin-induced immunity (16).In the case of the HN878 strain, the production of a phenolic glycolipid (PGL)³ was shown to be important for the hypervirulent phenotype. The disruption of pks15/1, a PGL biosynthetic gene (17), in strain HN878 increased the release of pro-inflammatory cytokines by infected murine bone marrow macrophages and decreased the virulence of the bacterium in the mouse and rabbit disease models (13, 15). PGL are very unusual molecules with a lipid core and a saccharide domain. The lipid core is formed by a long chain β-diol, naturally occurring as a diester of polymethyl-branched fatty acids and common to phthiocerol dimycocerosates (DIM), another group of lipids found in the cell envelope of M. tuberculosis and a few other slow-growing Mycobacterium species (18) (Fig. 1A). In the major form of PGL from M. tuberculosis (PGL-tb), this lipid core is ω-terminated by an aromatic nucleus glycosylated with a 2,3,4-tri-O-methyl-l-fucopyranosyl-(α1→3)-l-rhamnopyranosyl-(α1→3)-2-O-methyl-l-rhamnopyranosyl-(α1→) (19) (Fig. 1A). This structure is specific to M. tuberculosis and Mycobacterium canettii (18, 20). Only a few M. tuberculosis strains synthesize PGL-tb because of a natural frameshift mutation in the pks15/1 gene (17). Thus, based on the results of Reed et al. (13), it was thought that the production of this glycolipid was the principal cause of hypervirulence in Beijing strains. However, even within the Beijing lineage, only a minority of isolates produce PGL-tb (21, 22). In addition, complementation of the laboratory strain H37Rv with a functional pks15/1 allele, allowing the production of PGL-tb, does not significantly increase the virulence of this strain in the mouse and rabbit models (22). The molecular reasons for the higher virulence of strains of the Beijing lineage therefore remain unclear.Open in a separate windowFIGURE 1.DIM and PGL profile in M. tuberculosis Beijing strains. A, structure of phthiocerol dimycocerosates and related compounds in M. tuberculosis and role of Rv2951c and Rv2952 enzymes in the last steps of DIM and PGL formation. In M. tuberculosis, p, p′ = 3–5; n, n′ = 16–18; m1 = 20–22; m2 = 15–17; R = -CH₂-CH₃ or -CH₃. B, TLC analysis of lipid extracts from bacterial cells of H37Rv, Mt103, HN878, 94-1707, and 1237 strains. Lipid extracts were dissolved in CHCl₃ and run in petroleum ether/diethyl ether (90:10) for DIM analysis. Lipids were visualized by spraying the TLC plate with 10% phosphomolybdic acid in ethanol, followed by heating. The positions of DIM A, DIM B, triglycerides (TAG) are indicated. C and D, TLC analysis of lipid extracts from [¹⁴C]propionate-labeled bacterial cell of H37Rv, H37Rv:pPET1, HN878, and 94-1707. Lipid extracts were dissolved in CHCl₃ and run in petroleum ether/diethyl ether (90:10) for DIM analysis (C) and in CHCl₃/CH₃OH (95:5) for PGL analysis (D). Lipids were visualized with a Typhoon PhosphorImager. Positions of DIM A, DIM B, compounds X, and PGL are indicated.In this study, we identified a mutation specific to the Beijing lineage that affected the activity of an enzyme involved in PGL-tb and DIM biosynthetic pathways. We analyzed the impact of this mutation on the structure of these compounds and explored the potential changes in cell envelope function and virulence induced by this mutation.     

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.     

Identification of genetic loci implicated in the survival of Mycobacterium smegmatis in human mononuclear phagocytes

A luminescence-based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand-mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacteium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.     

Biochemical and Structural Study of the Atypical Acyltransferase Domain from the Mycobacterial Polyketide Synthase Pks13

Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction.     

Fluctuation of a clone 46,XX,i(7q) in bone marrow in a Fanconi anaemia

Summary Fixed metaphase chromosomes of different species and genera of Primates (five species of Macaca genus and Callithrix acchus) have been studied after Alu I restriction enzyme digestion and DA-DAPI counterstaining, in the attempt to determine some qualitative characteristics of their DNAs and specifically of the DNA localized in the heterochromatic components of the karyotypes. The results have been discussed in the light of those already published on humans, confirming the potentiality of this approach in the study of the phyloevolutive relationships in Primates.     

ESX‐1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis

Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT‐6). This small protein, which is secreted by the type VII secretion system ESX‐1 (T7SS/ESX‐1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock‐out or knock‐in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX‐1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.