Joseph A. Mangan

Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages Insights into the Phagosomal Environment

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogens cell envelope.

Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays

Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass alpha-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.

Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide

Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.

Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.

Gene expression during host—pathogen interactions: Approaches to bacterial mRNA extraction and labelling for microarray analysis

Publisher Summary The expression of genes in response to the signals associated with environmental stimuli is a prerequisite for the survival of bacterial pathogens within the host and is, therefore, the underlying basis of infectious disease. With the increasing amount of information about gene complement of bacterial pathogens and the development of functional genomics technologies, such as microarrays, it is now possible to monitor bacterial gene expression at a whole genome level both in vitro and during host-pathogen interactions. Such considerations impact on the choice of an experimental approach to extracting bacterial RNA for microarray analysis from different models of infection, for example, axenic culture, cell monocultures infected with bacteria in vitro, or whole tissues from the infected animal models. This chapter presents the experiences and methods that address both the biological and technical issues involved in extracting biologically “meaningful” RNA from the pathogenic mycobacterium, Mycobacterium tuberculosis , for the purposes of studying whole-genome expression during host-pathogen interactions using microarray analysis.

Glass slide microarrays for bacterial genomes

Abstract The acquisition of complete bacterial genome sequence has coincided with important advances in the development of high-density nucleic acid arrays and hybridization technology. Although ‘chip’ technology holds great promise for the future, at the present time spotted arrays represent an accessible format with sufficient reporter element density to interrogate bacterial genomes by comparative genomics and gene expression profiling. A rapidly growing literature using microarrays in functional genomics studies on bacteria confirms the reality and robust nature of spotted DNA microarrays for small genome organisms and also supports the use of total RNA samples for transcriptome analysis. However, microarray experimental systems are an emerging technology and optimization of nearly every step is an ongoing process. Thus, current methods are subject to development and have limitations and pitfalls. This should not be an argument against their widespread use, since the power of arrays as they are now is of undisputed biological significance and may be regarded as a proven technology. The future challenges for microarrays lies with mathematical and statistical analysis of high-dimensional data sets that are produced even in simple biological experiments.

Extraction of RNA from Intracellular Mycobacterium tuberculosis

Pathogenicity in Mycobacterium tuberculosis may be thought of as a multifactorial process with both pathogen and host-response effector molecules contributing to the process of infection, leading either to immunopathology and disease or control of infection and long-term persistence. Little is known about this at a genetic level, but it is becoming recognized that bacterial virulence constitutes the correct temporal and spatial regulation of many genes that may be necessary for a particular phase in infection in response to specific environmental cues.

The heat shock response of Mycobacterium tuberculosis: linking gene expression, immunology and pathogenesis

The regulation of heat shock protein (HSP) expression is critically important to pathogens such as Mycobacterium tuberculosis and dysregulation of the heat shock response results in increased immune recognition of the bacterium and reduced survival during chronic infection. In this study we use a whole genome spotted microarray to characterize the heat shock response of M. tuberculosis. We also begin a dissection of this important stress response by generating deletion mutants that lack specific transcriptional regulators and examining their transcriptional profiles under different stresses. Understanding the stimuli and mechanisms that govern heat shock in mycobacteria will allow us to relate observed in vivo expression patterns of HSPs to particular stresses and physiological conditions. The mechanisms controlling HSP expression also make attractive drug targets as part of a strategy designed to enhance immune recognition of the bacterium.

Microarrays for Microbes: the BμG@S Approach

The Bacterial Microarray Group at St George’s Hospital Medical School (BμG@S; http://bugs. sghms.ac.uk) has been funded by The Wellcome Trust as part of the Resources for Functional Genomics Initiative. A 5 year programme grant entitled ‘A Multi-Collaborative Microbial Pathogen Microarray Facility’ was awarded to Dr Philip Butcher and Joseph Mangan (St. George’s Hospital Medical School), Professor Brendan Wren (London School of Hygiene and Tropical Medicine), Professor Neil Stoker (Royal Veterinary College, London) and Dr Keith Vass (Beatson Institute/Glasgow University). The aim of this funding is to enable a community of collaborating researchers with a common interest in bacterial pathogenesis to have rapid, flexible and cost-effective access to current microarray technology. Rather than each individual group having to invest in the equipment and the lengthy learning and optimization process involved in microarray set-up, the aim was to centralize the expertise within one group that would then be made freely available to collaborating groups as a functional genomics resource. The project was funded to produce whole-genome DNA microarrays for 12 bacterial pathogens within 2 years and provide training and support in the use of the arrays over a period of 5 years. This article provides an overview of the project organization, the technology and support involved in producing and using the microarrays, future developments and a summary of the progress to date.

The expression profile of Mycobacterium tuberculosis infecting the human monocytic cell line THP-1 using whole genome microarray analysis

The expression profile of Mycobacterium tuberculosis infecting the human monocytic cell line THP-1 using whole genome microarray analysis