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Issar Smith

Publications and source records attributed to Issar Smith.

At least 19 recordsLinked to original sources
PubMed2006

Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.

Bacterial Proteins↗
PubMed2006

The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis.

Two-component signal transduction systems (2-CS) play an important role in bacterial pathogenesis. In the work presented here, we have studied the effects of a mutation in the Mycobacterium tuberculosis (Mtb) PhoPR 2-CS on the pathogenicity, physiology and global gene expression of this bacterial pathogen. Disruption of PhoPR causes a marked attenuation of growth in macrophages and mice and prevents growth in low-Mg2+ media. The inability to grow in THP-1 macrophages can be partially overcome by the addition of excess Mg2+ during infection. Global transcription assays demonstrate PhoP is a positive transcriptional regulator of several genes, but do not support the hypothesis that the Mtb PhoPR system is sensing Mg2+ starvation, as is the case with the Salmonella typhimurium PhoPQ 2-CS. The genes that were positively regulated include those found in the pks2 and the msl3 gene clusters that encode enzymes for the biosynthesis of sulphatides and diacyltrehalose and polyacyltrehalose respectively. Complementary biochemical studies, in agreement with recent results from another group, indicate that these complex lipids are also absent from the phoP mutant, and the lack of these components in its cell envelope may indirectly cause the mutant's high-Mg2+ growth requirement. The experiments reported here provide functional evidence for the PhoPR 2-CS involvement in Mtb pathogenesis, and they suggest that a major reason for the attenuation observed in the phoP mutant is the absence of certain complex lipids that are known to be important for virulence.

Animals↗
PubMed2006

Infection of human dendritic cells with a Mycobacterium tuberculosis sigE mutant stimulates production of high levels of interleukin-10 but low levels of CXCL10: impact on the T-cell response.

The Mycobacterium tuberculosis genome encodes 13 sigma factors. We have previously shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and can attenuate the virulence phenotype. In this work, we focused on extracytoplasmic factor sigmaE and studied the effects induced by the deletion of its structural gene (sigE) in the infection of human monocyte-derived dendritic cells (MDDC). We found that the wild-type M. tuberculosis strain (H37Rv), the sigE mutant (ST28), and the complemented strain (ST29) were able to infect dendritic cells (DC) to similar extents, although at 4 days postinfection a reduced ability to grow inside MDDC was observed for the sigE mutant ST28. After mycobacterium capture, the majority of MDDC underwent full maturation and expressed both inflammatory cytokines, such as tumor necrosis factor alpha, and the regulatory cytokines interleukin-12 (IL-12), IL-18, and beta interferon (IFN-beta). Conversely, a higher level of production of IL-10 was observed in ST28-infected MDDC compared to H37Rv- or ST29-infected cell results. However, in spite of the presence of IL-10, supernatants from ST28-infected DC induced IFN-gamma production by T cells similarly to those from H37Rv-infected DC culture. On the other hand, IL-10 impaired CXCL10 production in sigE mutant-infected DC and, indeed, its neutralization restored CXCL10 secretion. In line with these results, supernatants from ST28-infected cells showed a decreased capability to recruit CXCR3+ CD4+ T cells compared to those obtained from H37Rv-infected DC culture. Thus, our findings suggest that the sigE mutant-induced secretion of IL-10 inhibits CXCL10 expression and, in turn, the recruitment of activated-effector cells involved in the formation of granulomas.

Bacterial Proteins↗
PubMed2006

Posttranslational regulation of Mycobacterium tuberculosis extracytoplasmic-function sigma factor sigma L and roles in virulence and in global regulation of gene expression.

In this report, we demonstrate that SigL is posttranslationally regulated by a specific anti-sigma factor, RslA, and contributes to the expression of at least 28 genes. Several of these genes could mediate important cell envelope-related processes. Importantly, a sigL-rslA mutant strain was significantly attenuated in a mouse model of infection.

Animals↗
PubMed2006

Identification of an ABC transporter required for iron acquisition and virulence in Mycobacterium tuberculosis.

Iron availability affects the course of tuberculosis infection, and the ability to acquire this metal is known to be essential for replication of Mycobacterium tuberculosis in human macrophages. M. tuberculosis overcomes iron deficiency by producing siderophores. The relevance of siderophore synthesis for iron acquisition by M. tuberculosis has been demonstrated, but the molecules involved in iron uptake are currently unknown. We have identified two genes (irtA and irtB) encoding an ABC transporter similar to the YbtPQ system involved in iron transport in Yersinia pestis. Inactivation of the irtAB system decreases the ability of M. tuberculosis to survive iron-deficient conditions. IrtA and -B do not participate in siderophore synthesis or secretion but are required for efficient utilization of iron from Fe-carboxymycobactin, as well as replication of M. tuberculosis in human macrophages and in mouse lungs. We postulate that IrtAB is a transporter of Fe-carboxymycobactin. The irtAB genes are located in a chromosomal region previously shown to contain genes regulated by iron and the major iron regulator IdeR. Taken together, our results and previous observations made by other groups regarding two other genes in this region indicate that this gene cluster is dedicated to siderophore synthesis and transport in M. tuberculosis.

ATP-Binding Cassette Transporters↗
PubMed2005

Strain-specific mycobacterial lipids and the stimulation of protective immunity to tuberculosis.

Control of Mycobacterium tuberculosis (M. tuberculosis) infection is dependent on recognition of bacilli by cells of the innate immune system in the lung and the subsequent generation of an acquired effector T lymphocyte response. Lipid moieties of M. tuberculosis are important stimulators of innate immunity mediated predominantly through recognition by Toll-like receptors. In this paper, we will discuss how the lipid composition of different clinical isolates (strains) of M. tuberculosis affect that strain's ability to direct innate immunity, and ultimately influence whether infection is controlled or active disease develops.

Antigen Presentation↗
PubMed2005

Progress and new directions in genetics of tuberculosis: an NHLBI working group report.

Tuberculosis (TB), along with AIDS and malaria, is one of the three major killers among infectious diseases. New approaches to preventing, diagnosing, and curing TB are needed, which depend on a better understanding of Mycobacterium tuberculosis and the host. The National Heart, Lung, and Blood Institute convened a working group to develop recommendations for future TB research, including genetic aspects of the disease. The following areas were identified: (1) animal model research to improve understanding of persistence, reactivation, and granulomatous reactions; (2) preclinical studies aimed at shortening treatment of TB; (3) new resources for manipulating and characterizing the M. tuberculosis genome, proteome chips for more specific diagnoses, and studies of genes that appear to be essential but whose functions are not known; (4) prospective studies associated with clinical trials in populations with or at risk of TB to advance development of diagnostics and prognostics; (5) new quantitative and bioinformatic approaches to study the interaction between M. tuberculosis and the infected host and how this influences the infection process; (6) molecular characterization of M. tuberculosis genome diversity and phylogenetic analysis; (7) coordinated studies of human genome scans; (8) genetic epidemiology studies; (9) activities to foster knowledge dissemination, education, and training; and (10) coordination between the National Institutes of Health, the Gates Foundation, the Global Alliance for Tuberculosis Drug Development, and other organizations.

Animals↗
PubMed2005

Hyperglycosylation of glycopeptidolipid of Mycobacterium smegmatis under nutrient starvation: structural studies.

The presence of a polar species of glycopeptidolipid (GPL) in carbon-starved Mycobacterium smegmatis has been reported previously. In this study, the complete structure of this GPL is established with the help of MALDI-TOF (matrix assisted laser desorption/ionization time of flight) and ESI (electrospray ionization) -MS, (13)C-SEFT (spin echo Fourier transform) -NMR spectroscopy, and HPLC analysis. In the molecule, two units of a 3,4-di-O-methyl derivative of rhamnose are attached to l-alaninol via a 1-->2 linkage. Various methyl derivatives of rhamnose and 6-deoxytalose were synthesized as standards to establish this structure. The accumulation of this polar GPL in M. smegmatis is sigB dependent, as a SigB-overproducing strain of M. smegmatis shows the presence of this spot in the exponential phase, and a sigB-knockout strain of M. smegmatis does not show the presence of any polar GPLs.

Glucose↗
PubMed2005

responses of mycobacterium tuberculosis to growth in the mouse lung.

Using a promoter trap, we have identified 56 Mycobacterium tuberculosis genes preferentially expressed in the mouse lung. Quantitative real-time PCR showed that RNA levels of several genes were higher from bacteria growing in mouse lungs than from broth cultures. These results support the current hypothesis that Mycobacterium tuberculosis utilizes fatty acids as a carbon source in the mouse lung.

Animals↗
PubMed2004

Real-time measurement of in vitro transcription.

We have developed a simple method to measure RNA synthesis in real time. In this technique, transcription reactions are performed in the presence of molecular beacons that possess a 2'-O-methylribonucleotide backbone. These probes become fluorescent as they hybridize to nascent RNA during the course of synthesis. We found that molecular beacons synthesized from natural deoxyribonucleotides were not suitable, because they are copied by RNA polymerases, generating complementary product strands that bind to the molecular beacons, causing a conformational change that results in unwanted fluorescence. However, when the molecular beacons are synthesized from 2'-O-methylribonucleotides, they are not copied and fluorescence is strictly dependent upon transcription of the added template. Utilizing these modified molecular beacons, quantitative comparisons were made of the activity of a variety of RNA polymerases and the effect of an inhibitor of transcription was determined.

Bacteriophage T7↗
PubMed2003

Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients.

Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited. In this study, the levels of selected MTB mRNAs were quantified in vitro in axenic culture, in vivo in the lungs of mice, and in lung specimens obtained from TB patients with active disease. We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL/6 mice as compared with bacteria grown in vitro. Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.

Animals↗
PubMed2003

Mycobacterium tuberculosis gene expression in macrophages.

This review provides a discussion on the current information about the response of Mycobacterium tuberculosis to the environment encountered in the macrophage. We focus on the types of genes shown to be upregulated when the pathogen grows in macrophages and discuss the possible roles of these genes in adaptation to the conditions in the eukaryotic cell, in the context of enhancing the survival of the pathogen during infection.

Bacterial Proteins↗
PubMed2003

Mechanisms of iron regulation in mycobacteria: role in physiology and virulence.

The role of iron in mycobacteria as in other bacteria goes beyond the need for this essential cofactor. Limitation of this metal triggers an extensive response aimed at increasing iron acquisition while coping with iron deficiency. In contrast, iron-rich environments prompt these prokaryotes to induce synthesis of iron storage molecules and to increase mechanisms of protection against iron-mediated oxidative damage. The response to changes in iron availability is strictly regulated in order to maintain sufficient but not excessive and potentially toxic levels of iron in the cell. This response is also linked to other important processes such as protection against oxidative stress and virulence. In bacteria, iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. In mycobacteria, this role is fulfilled by the iron-dependent regulator IdeR. IdeR is an essential protein in Mycobacterium tuberculosis, the causative agent of human tuberculosis. It functions as a repressor of iron acquisition genes, but is also an activator of iron storage genes and a positive regulator of oxidative stress responses.

Feedback, Physiological↗
PubMed2003

Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence.

Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents.

Animals↗
PubMed2002

Role of the extracytoplasmic-function sigma factor sigma(H) in Mycobacterium tuberculosis global gene expression.

Like other bacterial species, Mycobacterium tuberculosis has multiple sigma (sigma) factors encoded in its genome. In previously published work, we and others have shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and, in some cases, cause attenuated virulence phenotypes. In this paper, we characterize a M. tuberculosis mutant lacking the ECF sigma factor sigma(H). This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show decreased ability to grow inside macrophages. Using quantitative reverse transcription-PCR and microarray technology, we have started to define the sigma(H) regulon and its involvement in the global regulation of the response to heat shock and the thiol-specific oxidizing agent diamide. We identified 48 genes whose expression increased after exposure of M. tuberculosis to diamide; out of these, 39 were not induced in the sigH mutant, showing their direct or indirect dependence on sigma(H). Some of these genes encode proteins whose predicted function is related to thiol metabolism, such as thioredoxin, thioredoxin reductase and enzymes involved in cysteine and molybdopterine biosynthesis. Other genes under sigma(H) control encode transcriptional regulators such as sigB, sigE, and sigH itself.

Animals↗
PubMed2002

Phospholipases C are involved in the virulence of Mycobacterium tuberculosis.

Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild-type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the Delta plcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT-PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.

Animals↗