Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

Background: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. Results: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). Conclusions: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence

The mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG influences various growth characteristics

<p>Abstract</p> <p>Background</p> <p>Pathogenic mycobacteria such as <it>M. tuberculosis</it>, <it>M. bovis </it>or <it>M. leprae </it>are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a <it>M. bovis </it>BCG derivative expressing a MDP1-antisense gene.</p> <p>Results</p> <p>The expression rate of the MDP1 protein in the recombinant <it>M. bovis </it>BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain <it>M. bovis </it>BCG containing the empty vector. In comparison to this reference strain, the recombinant <it>M. bovis </it>BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain.</p> <p>Conclusion</p> <p>The MDP1 protein has a major impact on various growth characteristics of <it>M. bovis </it>BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.</p

Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth

<p>Abstract</p> <p>Background</p> <p>Highly pathogenic mycobacteria like <it>Mycobacterium tuberculosis </it>are characterised by their slow growth and their ability to reside and multiply in the very hostile phagosomal environment and a correlation between the growth rate of mycobacteria and their pathogenicity has been hypothesised. Here, porin genes from <it>M. fortuitum </it>were cloned and characterised to address their impact on the growth rate of fast-growing and pathogenic mycobacteria.</p> <p>Results</p> <p>Two genes encoding porins orthologous to MspA from <it>M. smegmatis, porM1 </it>and <it>porM2</it>, were cloned from <it>M. fortuitum </it>strains, which were originally isolated from human patients. Both porin genes were at least partially able to complement the mutations of a <it>M. smegmatis </it>mutant strain lacking the genes <it>mspA </it>and <it>mspC </it>with respect to the growth rate. <it>PorM1 </it>and <it>porM2 </it>were present in different strains of <it>M. fortuitum </it>including the type strain. Comparative expression analysis of <it>porM </it>genes revealed divergent porin expression among analysed <it>M. fortuitum </it>strains. Repression of the expression of porins by antisense technique decreased the growth rates of different <it>M. fortuitum</it>. The effects of over-expression of <it>porM1 </it>as well as <it>porM2 </it>varied depending on the strain and the concentration of antibiotic added to the medium and indicated that PorM1 and PorM2 enhance the growth of <it>M. fortuitum </it>strains, but also the diffusion of the antibiotic kanamycin into the cells.</p> <p>Conclusion</p> <p>This study demonstrates the important role of porin expression in growth as well as antibiotic susceptibility of the opportunistic bacterium <it>M. fortuitum</it>.</p

Mycobacterium tuberculosis DosR Regulon Gene Rv0079 Encodes a Putative, ‘Dormancy Associated Translation Inhibitor (DATIN)’

Mycobacterium tuberculosis is a major human pathogen that has evolved survival mechanisms to persist in an immune-competent host under a dormant condition. The regulation of M. tuberculosis metabolism during latent infection is not clearly known. The dormancy survival regulon (DosR regulon) is chiefly responsible for encoding dormancy related functions of M. tuberculosis. We describe functional characterization of an important gene of DosR regulon, Rv0079, which appears to be involved in the regulation of translation through the interaction of its product with bacterial ribosomal subunits. The protein encoded by Rv0079, possibly, has an inhibitory role with respect to protein synthesis, as revealed by our experiments. We performed computational modelling and docking simulation studies involving the protein encoded by Rv0079 followed by in vitro translation and growth curve analysis experiments, involving recombinant E. coli and Bacille Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations concerning the interaction of the protein with the ribosomes are supportive of its role in regulation/inhibition of translation. We propose that the protein encoded by locus Rv0079 is a ‘dormancy associated translation inhibitor’ or DATIN

Einfluss virulenz-assoziierter Gene von Mycobacterium bovis BCG auf die Interaktion mit Wirtszellen

1\. Introduction ..................................................................................................................... 1 1.1 Pathogenesis .............................................................................................................. 4 1.2 The central role of alveolar macrophages ................................................................... 6 1.2.1 Cytokines, mediators of the innate and adaptive immune system ....................... 6 1.2.1.1 Interleukin-1 beta (IL-1β) ................................................................................ 8 1.2.1.2 Interferon-gamma (IFN-γ) .............................................................................. 8 1.2.1.3 Tumor necrosis factor-alpha (TNF-α) ............................................................. 8 1.2.1.4 Interleukin-10 (IL-10) ...................................................................................... 9 1.2.1.5 Interleukin-8 (IL-8) ........................................................................................ 10 1.2.2 Nitric oxide (NO) ................................................................................................. 10 1.3 The mycobacterial cell wall ....................................................................................... 11 1.4 Multinucleated (giant) cells and granulomas ............................................................. 12 1.5 The dormancy survival regulon (DosR) ..................................................................... 14 1.6 The conserved hypothetical protein Rv2626c ........................................................... 17 1.7 The mycobacterial DNA-binding protein 1 (MDP1) ................................................... 18 1.8 M. bovis BCG contra M. tuberculosis ........................................................................ 19 1.9 Objectives ................................................................................................................. 20 2\. Material & Methods ....................................................................................................... 21 2.1 Bacterial strains and culture conditions ..................................................................... 22 2.1.1 Growth experiments under neutral and low pH conditions ................................. 22 2.1.2 Cultivation of bacteria under pH stress for protein isolation ............................... 23 2.2 Cloning techniques .................................................................................................... 23 2.2.1 Molecular biology techniques ............................................................................. 23 2.2.2 Mutagenesis of the rv2626c-gene in M. bovis BCG ........................................... 24 2.2.2.1 Construction of the rv2626c over-expression plasmid ................................. 24 2.2.2.2 Site-directed mutagenesis of rv2626c .......................................................... 25 2.2.3 Mutagenesis of the mdp1-gene in M. bovis BCG ............................................... 28 2.2.3.1 Construction of the mdp1 over-expression plasmid ..................................... 28 2.2.3.2 Site-directed mutagenesis of mdp1 ............................................................. 28 2.2.2.3 Construction of the mdp1 antisense plasmid ............................................... 28 2.3 Protein analysis ......................................................................................................... 30 2.3.1 Protein extraction ................................................................................................ 30 2.3.2 Western blot & immunodetection ........................................................................ 30 2.4 Cell culture experiments ............................................................................................ 31 2.4.1 Methods of cell isolation from human-derived blood cells .................................. 31 2.4.1.1 PBMCs Isolation .......................................................................................... 31 2.4.1.2 Monocytes isolation ..................................................................................... 32 2.4.2 Infection experiments ......................................................................................... 32 2.4.2.1 PBMC infection and Cytokine measurement ............................................... 32 2.4.2.2 Monocyte infection and intracellular survival ............................................... 33 2.4.2.3 Monocyte infection and fusion of monocyte-derived macrophages ............. 33 2.4.2.4 J774.A1 mouse macrophages infection and Griess assay .......................... 34 2.4.3 DNA extraction methods ..................................................................................... 35 2.4.3.1 DNA extraction from human-derived blood cells .......................................... 35 2.4.3.2 DNA extraction from mouse tissues ............................................................. 36 2.4.3.3 DNA quantification by real-time-PCR ........................................................... 36 2.5 Animal Experiment .................................................................................................... 37 3\. Results ........................................................................................................................... 38 3.1 Mutagenesis of rv2626c and mdp1 ........................................................................... 39 3.1.1 Mutagenesis of the rv2626c-gene ...................................................................... 39 3.1.1.1 Confirmation of the deletion mutant M. bovis ΔRv2626c::hyg ..................... 39 3.1.1.2 Confirmation of the over-expressing strain M. bovis (pRv2626c) ................ 40 3.2.1 Mutagenesis of the mdp1-gene .......................................................................... 40 3.2.1.1 Deleting the mdp1-gene has lethal consequences for M. bovis BCG .......... 40 3.2.1.2 An over-expression of the mdp1-gene in M. bovis BCG is impossible ........ 40 3.2.1.3 Antisense technique as means of choice to down- regulate MDP1 .............. 42 3.2 Determination of the virulence associated capabilities of rv2626c and mdp1 ........... 43 3.2.1 Expression of rv2626c in exponential and stationary phase cultures ................. 43 3.2.2 Adaption of mutagenised M. bovis BCG strains to low pH ................................. 44 3.2.2.1 Rv2626c influences the adaption of BCG to low pH .................................... 45 3.2.2.2 MDP1 influences the adaption of BCG to low pH ........................................ 49 3.2.3 Rv2626c is expressed under pH stress conditions ............................................. 50 3.2.4 Influence on the intracellular survival in human monocyte-derived macrophages .................................................................................................................................... 52 3.2.5 The influence on the secretion of selected cytokines by human PBMCs ........... 53 3.2.5.1 IL-1β secretion ............................................................................................. 54 3.5.2.2 TNF-α secretion ........................................................................................... 55 3.5.2.3 IFN-γ secretion ............................................................................................. 56 3.5.2.4 IL-10 secretion ............................................................................................. 57 3.5.2.5 IL-8 secretion ............................................................................................... 58 3.2.6 The influence on the NO concentration in J774.A1 cells .................................... 58 3.2.7 Fusion of monocyte-derived macrophages ........................................................ 59 3.2.8 The mutant ΔRv2626c::hyg survives better in Balb/c mice than wild-type BCG 63 4\. Discussion ..................................................................................................................... 66 5\. Summary ....................................................................................................................... 82 6\. Zusammenfassung ....................................................................................................... 83 7\. References .................................................................................................................... 84 8\. Attachment .................................................................................................................... 96 8.1 Abbreviations ............................................................................................................ 96 8.2 Plasmid Maps ............................................................................................................ 97 8.2.1 Plasmid pMV261 ................................................................................................ 97 8.2.2 Plasmid pYUB854 .............................................................................................. 98In 1993, WHO declared tuberculosis a global health emergency. The reasons for this decision are the high infection rate as well as problems associated with the healing of tuberculosis. M. tuberculosis is able to survive in an intact immune system over decades and can be reactivated under certain conditions. The lengthy treatment and the appearance of multidrug resistant and extensively drug-resistant tuberculosis strains complicate the healing process. This makes it even more important to find genes in M. tuberculosis allowing the survival in the human body and to understand their influence on the interaction with the immune cells. Against this background, two genes of M. bovis BCG were investigated whose regulation in M. tuberculosis suggested a potential influence on the course of infection. By site-directed mutagenesis, over-expression or antisense-technique new strains of M. bovis BCG were generated in which either the gene rv2626c was over-expressed or deleted or the gene for the “mycobacterial DNA binding protein 1“ (MDP1) was down- regulated. These strains were used for in vitro experiments in which aspects of the natural infection were simulated. The present work dealt with investigations using human-derived primary cells, a mouse cell line and a mouse model. It was shown that both genes possess virulence-associates capabilities. The secretory protein Rv2626c interferes massively in the cytokine expression of human PBMCs, up-regulating IL-1β, TNF-α, and IL-10 and down-regulating IL-8. It reduces the amount of secreted nitric oxide in a mouse cell line and increases the fusion of human macrophages. On the other hand the deletion of Rv2626c improves the increased survivability in Balb/c mice. The down-regulation of mdp1 is probably responsible for modifications of the mycobacterial cell wall which is accountable for the down-regulation of IL-1β and nitric oxide, the decreased survivability in human macrophages and the increased fusion of human macrophages. Further trials are needed to find out whether Rv2626c and MDP1 are qualified for new therapeutic approaches or not.Tuberkulose wurde 1993 von der WHO zu einem globalen Gesundheitsproblem erklärt. Gründe hierfür sind die hohe Infektionsrate sowie Schwierigkeiten bei der Behandlung von Tuberkulose. M. tuberculosis gelingt es über Jahrzehnte in einem intakten Immunsystem zu überleben und unter bestimmten Bedingungen wieder reaktiviert zu werden. Die langwierige Behandlung und das Auftreten von multiresistenten und extrem resistenten Stämmen erschweren zudem die Bekämpfung der Tuberkulose. Umso wichtiger ist es Gene zu finden die M. tuberculosis das Überleben im menschlichen Körper ermöglichen und zu verstehen, wie sie die Interaktion mit Zellen des Immunsystems beeinflussen. Vor diesem Hintergrund wurden zwei Gene von M. bovis BCG untersucht, deren Regulation in M. tuberculosis einen möglichen Einfluss auf den Infektionsverlauf nahelegten. Mittels gezielter Mutagenese, Überexprimierung oder Antisense Technik wurden neue M. bovis BCG Stämme erzeugt, in denen das Gen rv2626c überexprimiert bzw. deletiert oder im Fall des “mycobacterial DNA binding protein 1“ (MDP1) herabreguliert wurde. Mit diesen Stämmen wurden in vitro Versuche durchgeführt in denen bestimmte Aspekte der natürlichen Infektion simuliert wurden. Die in dieser Arbeit durchgeführten Untersuchungen mit humanen Primärzellen, einer Mauszelllinie und einem Mausmodell haben gezeigt, dass beide Gene über virulenzassoziierte Eigenschaften verfügen. So greift das sekretierte Protein Rv2626c massiv in die Zytokinexpression humaner PBMCs ein, indem es IL-1β, TNF-α, IL-10 hoch- und IL-8 herabreguliert, Stickstoffoxid in einer Mauszelllinie herabreguliert und die Fusion humaner Makrophagen fördert. Auf der anderen Seite führt die Deletion des Gens zu einer verbesserten Überlebensfähigkeit in Balb/c Mäusen. Die Herabregulierung von mdp1 wirkt sich wahrscheinlich auf eine Weise auf die Zellwandstruktur aus, die zu einer Herabregulierung von IL-1β und Stickstoffoxid führt, das intrazelluläre Überleben in humanen Makrophagen vermindert und die Fusion von humanen Makrophagen fördert. Weiterführende Versuche müssen zeigen ob die Rv2626c und MDP1 für die Entwicklung neuer Therapieansätze geeignet sind oder nicht

The role of the mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG in host cell interaction

Background: Mycobacterium tuberculosis differs from most pathogens in its ability to multiply inside monocytes and to persist during long periods of time within granuloma in a status of latency. A class of proteins called mycobacterial histone-like proteins has been associated with regulation of replication and latency, but their precise role in the infection process has yet to be uncovered. Our study aimed at defining the impact of the histone-like protein MDP1 from M. bovis BCG (mycobacterial DNA-binding protein 1, corresponding to Rv2986c from M. tuberculosis) on early steps of infection. Results: Previously, a BCG (Bacillus Calmette Guérin) strain had been generated by antisense-technique exhibiting reduced MDP1 expression. This strain was now used to analyse the impact of reduced amount of MDP1 on the interaction with human blood monocytes, macrophage lines and PBMC (peripheral blood mononuclear cells). MDP1 was revealed to be required for growth at acidic pH and for intracellular replication in human blood monocytes. Down-regulation of MDP1 resulted in reduced secretion of the cytokine IL-1β by infected human PBMC. In addition, a reduction of MDP1 expression had a major impact on the formation of fused multi-nucleated macrophages. In monocyte preparations from human blood as well as in human and mouse macrophage cell lines, both the percentage of multi-nucleated cells and the number of nuclei per cell were much enhanced when the monocytes were infected with BCG expressing less MDP1. Conclusion: MDP1 from M. bovis BCG affects the growth at acidic pH and the intracellular replication in human monocytes. It furthermore affects cytokine secretion by host cells, and the formation of fused multi-nucleated macrophages. Our results suggest an important role of MDP1 in persistent infection

Theoretische Grundlagen für erfolgreiche Klimaanpassungsstrategien

Die Theoriestudie dient als Basis für die wissenschaftlichen Analysen und die Umsetzungsprojekte zur Klimaanpassung in ‚nordwest2050‘ und soll die Entwicklung von erfolgreichen Klimaanpassungsstrategien unterstützen. Die Theoriestudie greift dabei auf bestehende Theorieansätze zurück und präzisiert bzw. passt sie im Hinblick auf die Bedingungen der Klimaanpassung an. Bei der Auswahl und Strukturierung der relevanten Theoriearbeiten werden vier grundlegende Bereiche unterschieden: Zielorientierung, Grundlagentheorien, prozessuale Schlüsselthemen und Forschungsbezugsrahmen. Im Kapitel Zielorientierung wird Resilienz als Leitkonzept für ‚nordwest2050‘ beschrieben. Die Grundlagentheorien greifen systemtheoretische und evolutorische Grundlagen auf. Als prozessuale Schlüsselthemen für die erfolgreiche Gestaltung von Klimaanpassungsstrategien werden die Themen Governance, Richtungsgebung in Innovationsprozessen, kulturelle Kompetenzen und Resilienzlernen beschrieben. Im Forschungsbezugsrahmen erfolgt schließlich eine Zusammenführung der Themen im Hinblick auf ‚nordwest2050‘

The mycobacterial DNA-binding protein 1 (MDP1) from BCG influences various growth characteristics-3

Or with Ampicillin (25 μg ml) to give an initial OD (600) nm of 0.02 to 0.04. The growth of the strains was monitored by determination of the ATP content with a luciferase assay during 22 days. The diagram displays the percent growth of the strains in medium with Ampicillin in relation to the growth in medium without Ampicillin. The columns represent the mean of three independent cultures with the standard deviation. The double asterisks indicate values that varied very significantly (P < 0.01) according to the student's test.<p><b>Copyright information:</b></p><p>Taken from "The mycobacterial DNA-binding protein 1 (MDP1) from BCG influences various growth characteristics"</p><p>http://www.biomedcentral.com/1471-2180/8/91</p><p>BMC Microbiology 2008;8():91-91.</p><p>Published online 10 Jun 2008</p><p>PMCID:PMC2453136.</p><p></p