Neil G. Stoker

Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement

Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed: a marked deletion of the plcABC cluster, which encodes three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyADelta plcABCDelta. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PAC:I restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PAC:I site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.

Deletion of two-component regulatory systems increases the virulence of Mycobacterium tuberculosis.

ABSTRACT Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors. We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain. The most hypervirulent mutant (devRΔ) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that of the host.

Characterization of Auxotrophic Mutants of Mycobacterium tuberculosis and Their Potential as Vaccine Candidates

ABSTRACT Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and themetB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpDmutants were killed during this time. In SCID mice, both H37Rv and themetB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. TheproC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and themetB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proCand trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either theproC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.

The common aromatic amino acid biosynthesis pathway is essential in Mycobacterium tuberculosis.

Attempts to construct Mycobacterium tuberculosis strains with a defect in the common aromatic amino acid biosynthesis pathway were made. In other bacteria the genes of this pathway (aro) can be disrupted in the presence of suitable media supplements. The genomic organization of the aro genes in M. tuberculosis reveals that there is one operon (aroCKBQ) and three isolated aro genes (aroE, aroG and aroA). The aroK gene was chosen as a target for disruption; this encodes shikimate kinase, which catalyses the fifth step in chorismate biosynthesis. Attempts to replace the wild-type aroK gene with a disrupted allele (aroKDelta::hyg) by a two-step homologous recombination procedure were unsuccessful in a wild-type strain. When a second functional copy of aroK was integrated into the chromosome, it was possible to isolate a strain carrying the disrupted gene. Excision of the L5-integrated copy of aroK by the L5 excisionase could be not be achieved in the strain carrying the disrupted copy, but was possible in a strain carrying a wild-type copy. These results demonstrate that the chorismate pathway is essential for the viability of M. tuberculosis.

Probing Host Pathogen Cross-Talk by Transcriptional Profiling of Both Mycobacterium tuberculosis and Infected Human Dendritic Cells and Macrophages.

Background Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells. Methodology/Principal Findings In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. Conclusions/Significance This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.

A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis

The Mycobacterium tuberculosis TetR‐type regulator Rv3574 has been implicated in pathogenesis as it is induced in vivo, and genome‐wide essentiality studies show it is required for infection. As the gene is highly conserved in the mycobacteria, we deleted the Rv3574 orthologue in Mycobacterium smegmatis (MSMEG_6042) and used real‐time quantitative polymerase chain reaction and microarray analyses to show that it represses the transcription both of itself and of a large number of genes involved in lipid metabolism. We identified a conserved motif within its own promoter (TnnAACnnGTTnnA) and showed that it binds as a dimer to 29 bp probes containing the motif. We found 16 and 31 other instances of the motif in intergenic regions of M. tuberculosis and M. smegmatis respectively. Combining the results of the microarray studies with the motif analyses, we predict that Rv3574 directly controls the expression of 83 genes in M. smegmatis, and 74 in M. tuberculosis. Many of these genes are known to be induced by growth on cholesterol in rhodococci, and palmitate in M. tuberculosis. We conclude that this regulator, designated elsewhere as kstR, controls the expression of genes used for utilizing diverse lipids as energy sources, possibly imported through the mce4 system.

Electroporation of Mycobacteria

High-efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall but is compounded by the fact that most molecular techniques have been developed for distantly related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified, and DNA transformation of many mycobacterial species has now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow-grower and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterium tuberculosis protocols

Plasmid Vectors Nicola Casali and Sabine Ehrt Isolation of DNA from Mycobacterium tuberculosis Paul D. van Helden, Thomas C. Victor, Robin M. Warren, and Eileen G. van Helden Extraction of RNA from Intracellular Mycobacterium tuberculosis: Methods, Considerations, and Applications Irene M. Monahan, Joseph A. Mangan, and Philip D. Butcher Transposon Mutagenesis in Mycobacteria Using Conditionally Replicating Mycobacteriophages Stoyan S. Bardarov, Svetoslav S. Bardarov, Jr., and William R. Jacobs, Jr Gene Replacement and Transposon Delivery Using the Negative Selection Marker sacB Mary Jackson, Luis Reinaldo Camacho, Brigitte Gicquel, and Christophe Guilhot Gene Replacement Using Pretreated DNA Bhavna G. Gordhan and Tanya Parish Gene Replacement in Mycobacterium tuberculosis and Mycobacterium bovis BCG Using rpsL+ as a Dominant Negative Selectable Marker Peter Sander, Burkhard Springer, and Erik C. Bottger Transcription Start-Site Mapping Farahnaz Movahedzadeh, Jorge A. Gonzalez-Y-Merchand, and Robert A. Cox Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells Rory P. Cooney, Natalie J. Garton, and Michael R. Barer Phage Replication Technology for Diagnosis and Drug Susceptibility Testing Ruth McNerney Detection of Mutations in Mycobacterium tuberculosis by a Dot Blot Hybridization Strategy Thomas C. Victor and Paul D. van Helden Restriction Fragment Length Polymorphism Typing of Mycobacteria Dick van Soolingen, Petra E. W. de Haas, and Kristin Kremer Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis Ida Rosenkrands and Peter Andersen Analysis of the Capsule of Mycobacterium tuberculosis Mamadou Daffe and Marie-Antoinette Laneelle Analysis of the Lipids of Mycobacterium tuberculosis Richard A. Slaydenand Clifton E. Barry, 3rd In Vitro Model of Hypoxically Induced Nonreplicating Persistence of Mycobacterium tuberculosis Lawrence G. Wayne Macrophage Virulence Assays Pauline T. Lukey and Elizabeth U. Hooker Analysis of Mycobacterium-Infected Macrophages by Immunoelectron Microscopy and Cell Fractionation Wandy Beatty and David G. Russell Real-Time PCR Using Molecular Beacons: A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis Riccardo Manganelli, Sanjay Tyagi, and Issar Smith Electronic Access to Mycobacterium tuberculosis Sequence Data Julian Parkhill Proteomics Joanna C. Betts and Marjorie A. Smith Functional Genomics of Mycobacterium tuberculosis Using DNA Microarrays Michael Wilson, Martin Voskuil, Dirk Schnappinger, and Gary K. Schoolnik Storage of Mycobacterial Strains Kristin Kremer, Tridia van der Laan, and Dick van Soolingen Safety in the Laboratory Heidi Alderton and Debbie Smith

Enhanced gene replacement in mycobacteria

Allelic replacement will be a vital tool for understanding gene function in mycobacteria. Disruption of the chromosomal hisD gene of Mycobacterium smegmatis by standard gene replacement methods was surprisingly difficult, with most products being caused by illegitimate recombination (IR) events. A recombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M. smegmatis. Treatment of competent cells with UV, hydrogen peroxide or mitomycin C did not improve the frequency of HR; however, treatment of the DNA with alkali or UV enhanced recombination frequency, while boiling did not. Applying these observations to allele replacement, UV and alkali treatment of transforming DNA increased HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA improved the level of HR and abolished IR. In Mycobacterium intracellulare the use of alkali-denatured DNA increased the numbers of recombinants obtained with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inactivation of a putative haemolysin gene, tlyA, was achieved using both UV-irradiated DNA and ss phagemid DNA. Significantly, IR, which has been reported to be a problem in this species, was not observed. Thus, four genes in three species were successfully knocked-out using non-replicating DNA pretreated with alkali, UV or in an ss form. The use of these methods to enhance HR will greatly facilitate experiments to inactivate other genes in these important species.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium–host interaction

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL−10 production via interaction with the mannose receptor or DC‐SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose‐dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC‐SIGN‐transfected Raji cells, but no differences in IL‐10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose‐capped LAM does not dominate the Mycobacterium–host interaction.