Joseph A. Mangan

Bio: Joseph A. Mangan is an academic researcher from St George's Hospital. The author has contributed to research in topics: Mycobacterium tuberculosis & Gene. The author has an hindex of 15, co-authored 20 publications receiving 2851 citations.

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

Abstract: 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 pathogen's cell envelope.

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

Abstract: 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.

Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity.

Abstract: Campylobacter jejuni is the leading cause of bacterial food-borne diarrhoeal disease throughout the world, and yet is still a poorly understood pathogen. Whole genome microarray comparisons of 11 C. jejuni strains of diverse origin identified genes in up to 30 NCTC 11168 loci ranging from 0.7 to 18.7 kb that are either absent or highly divergent in these isolates. Many of these regions are associated with the biosynthesis of surface structures including flagella, lipo-oligosaccharide, and the newly identified capsule. Other strain-variable genes of known function include those responsible for iron acquisition, DNA restriction/modification, and sialylation. In fact, at least 21% of genes in the sequenced strain appear dispensable as they are absent or highly divergent in one or more of the isolates tested, thus defining 1300 C. jejuni core genes. Such core genes contribute mainly to metabolic, biosynthetic, cellular, and regulatory processes, but many virulence determinants are also conserved. Comparison of the capsule biosynthesis locus revealed conservation of all the genes in this region in strains with the same Penner serotype as strain NCTC 11168. By contrast, between 5 and 17 NCTC 11168 genes in this region are either absent or highly divergent in strains of a different serotype from the sequenced strain, providing further evidence that the capsule accounts for Penner serotype specificity. These studies reveal extensive genetic diversity among C. jejuni strains and pave the way toward identifying correlates of pathogenicity and developing improved epidemiological tools for this problematic pathogen.

Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide.

Abstract: 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.

The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences

Abstract: Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium—properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world1. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain–Barre syndrome2. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.

Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens

Abstract: This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity. Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense. In combination, the contribution of these two enzymes appears to be greater than previously appreciated. The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI. Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role. In diseases such as tuberculosis, a molecular understanding of host-pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates. Genetic and biochemical approaches have identified candidates for RNI-resistance genes in Mycobacterium tuberculosis and other pathogens.

Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling.

Abstract: The search for new TB drugs that rapidly and effectively sterilize the tissues and are thus able to shorten the duration of chemotherapy from the current 6 months has been hampered by a lack of understanding of the metabolism of the bacterium when in a 'persistent' or latent form. Little is known about the condition in which the bacilli survive, although laboratory models have shown that Mycobacterium tuberculosis can exist in a non-growing, drug-resistant state that may mimic persistence in vivo. Using nutrient starvation, we have established a model in which M. tuberculosis arrests growth, decreases its respiration rate and is resistant to isoniazid, rifampicin and metronidazole. We have used microarray and proteome analysis to investigate the response of M. tuberculosis to nutrient starvation. Proteome analysis of 6-week-starved cultures revealed the induction of several proteins. Microarray analysis enabled us to monitor gene expression during adaptation to nutrient starvation and confirmed the changes seen at the protein level. This has provided evidence for slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis and cell division in addition to induction of the stringent response and several other genes that may play a role in maintaining long-term survival within the host. Thus, we have generated a model with which we can search for agents active against persistent M. tuberculosis and revealed a number of potential targets expressed under these conditions.

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

Abstract: 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 pathogen's cell envelope.

The spectrum of latent tuberculosis: rethinking the biology and intervention strategies

Abstract: Immunological tests provide evidence of latent tuberculosis in one third of the global population, which corresponds to more than two billion individuals. Latent tuberculosis is defined by the absence of clinical symptoms but carries a risk of subsequent progression to clinical disease, particularly in the context of co-infection with HIV. In this Review we discuss the biology of latent tuberculosis as part of a broad range of responses that occur following infection with Mycobacterium tuberculosis, which result in the formation of physiologically distinct granulomatous lesions that provide microenvironments with differential ability to support or suppress the persistence of viable bacteria. We then show how this model can be used to develop a rational programme to discover effective drugs for the eradication of M. tuberculosis infection.