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Biomedical subjects

Eric J Rubin

Publications and source records attributed to Eric J Rubin.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of mycobacterial virulence genes through genetic interaction mapping.

We have previously shown that approximately 5% of the genes encoded by the genome of Mycobacterium tuberculosis are specifically required for the growth or survival of this bacterium during infection. This corresponds to hundreds of genes, most of which have no identifiable function. As a unique approach to characterize these genes, we developed a method to rapidly delineate functional pathways by identifying mutations that modify each other's phenotype, i.e., "genetic interactions". Using this method, we have defined a complex set of interactions between virulence genes in this pathogen, and find that the products of unlinked genes associate to form multisubunit transporters that are required for bacterial survival in the host. These findings implicate a previously undescribed family of transport systems in the pathogenesis of tuberculosis, and identify genes that are likely to function in the metabolism of their substrates. This method can be readily applied to other organisms at either the single pathway level, as described here, or at the system level to define quantitative genetic interaction networks.

Animals↗
PubMed2006

Chemical biology and bacteria: not simply a matter of life or death.

Chemical biological approaches to understanding bacteria have largely been confined to screening for antibiotics. More complex phenotypes, such as virulence, have largely been studied using bacterial genetics. However, it has recently become clear that these two methods are complementary and that combining chemical biologic and genetic approaches to studying bacteria brings new power to old problems.

Bacteria↗
PubMed2006

Detecting genetic variability among different Mycobacterium tuberculosis strains using DNA microarrays technology.

Recent advances in functional and comparative genomics have improved our understanding of genetic diversity among the Mycobacterium tuberculosis complex. In this study, we investigated the genetic polymorphism of M. tuberculosis using whole-genome microarray analysis. Amplified fragments of 15 M. tuberculosis strains (from two different geographical origins) and the reference strain H37Rv were produced by random amplification of polymorphic DNA (RAPD) using three different primers. The RAPD products were labeled with fluorescent dyes (Cy3 and Cy5) and hybridized to a TB DNA microarray representing nearly all open reading frames (ORFs) of H37Rv. The final results were analyzed using bioinformatic tools. Some genetic variability was found among the 16 M. tuberculosis strains. The majority of the highly polymorphic DNA sequences were observed in ORFs representing non-essential genes of the bacterium. The future use of comparative genomics based on DNA microarray technology should prove a powerful tool for understanding phenotypic variability among M. tuberculosis isolates of similar genetic composition. It is also a promising approach to provide important insights into evolution, virulence and pathogenesis of M. tuberculosis.

DNA, Bacterial↗
PubMed2006

Scaling down: a PCR-based method to efficiently screen for desired knockouts in a high density Mycobacterium tuberculosis picked mutant library.

Transposon mutagenesis produces random mycobacterial mutants at high frequency. Because they are random, however, it is difficult to isolate mutations in particular target genes. Here we describe the use of an arrayed library of Mycobacterium tuberculosis together with a PCR screening strategy to rapidly identify strains with defined insertion mutations. This method is useful for many genetic applications.

DNA Transposable Elements↗
PubMed2006

Dividing oceans into pools: strategies for the global analysis of bacterial genes.

In bacterial pathogenesis, it is often easy to accept the results of large-scale screens without independent verification of the results. How can one critically read this literature? Here we review issues inherent in genome-wide screens in bacteria, focusing on experiments that attempt to comprehensively identify genes required for bacterial growth under specific conditions. Our analysis suggests that the methodologies employed undoubtedly shape the results. It is clear, however, that the question is not which method is better but which provides the data most suited to a given question.

Bacteria↗
PubMed2005

A new site-specific integration system for mycobacteria.

Site-specific integration into the mycobacterial chromosome can produce stable transformants useful for understanding pathogenesis. However, gene expression can be problematic at certain sites of integration. We have used the Streptomyces phiC31 integration system to integrate vector DNA into Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis through site-specific recombination. A single dominant insertion site was found in M. smegmatis, as previously reported. Three different insertion sites were found in M. bovis BCG. In M. smegmatis, integrated vectors appear to be far more stable than episomal plasmids during unselected passage in vitro, although excision products are detectable. Plasmids based on the phiC31 integration system could make useful tools for the study of mycobacterial genetics.

BCG Vaccine↗
PubMed2005

Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection.

Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.

Animals↗
PubMed2005

Genome-wide RNAi screen for host factors required for intracellular bacterial infection.

Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.

Animals↗
PubMed2005

Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages.

Macrophages are central to host defense against microbes, but intracellular pathogens have evolved to evade their antimicrobial functions. Mycobacterium tuberculosis (MTB) has successfully exploited macrophages as its primary niche in vivo, but the bacterial genome-wide requirements that promote its intracellular survival remain undefined. Here we comprehensively identify the MTB genes required for survival by screening for transposon mutants that fail to grow within primary macrophages. We identify mutants showing decreased growth in macrophage environments that model stages of the host immune response. By systematically analyzing several biologically relevant data sets, we have been able to identify putative pathways that could not be predicted by genome organization alone. In one example, phosphate transport, requiring physically unlinked genes, was found to be critical for MTB growth in macrophages and important for establishing persistent infection in lungs. Remarkably, the majority of MTB genes found by this analysis to be required for survival are constitutively expressed rather than regulated by macrophages, revealing the host-adapted lifestyle of an evolutionarily selected intracellular pathogen.

Adaptation, Physiological↗
PubMed2005

New genetic approaches shed light on TB virulence.

Although tuberculosis has been studied for more than a century, insights into the molecular mechanisms by which it causes disease remain fairly limited. The current genetic boom in this system promises to reveal new virulence mechanisms, making this an exciting time to be studying this disease. Long considered a technical "poor relation", tuberculosis research has developed into a source for creative techniques and ideas. In the midst of this development, it is important to keep in mind the limitations of each new approach that is employed to study this organism. This review examines the genetic approaches that are currently being used to study tuberculosis, with an emphasis on new developments that promise to improve our understanding of the pathogenic mechanisms of Mycobacterium tuberculosis.

Animals↗
PubMed2005

Differential antibiotic susceptibilities of starved Mycobacterium tuberculosis isolates.

We compared the efficacies of 17 agents against rapidly growing and starved Mycobacterium tuberculosis H37Rv. Few compounds have significant activity at attainable concentrations. However, two phenothiazine compounds at similar concentrations were bactericidal for starved and growing cells. These drugs appear to target a process important in both replicating and nonreplicating bacteria.

Anti-Bacterial Agents↗
PubMed2004

Mycobacterium tuberculosis inhibits macrophage responses to IFN-gamma through myeloid differentiation factor 88-dependent and -independent mechanisms.

Mycobacterium tuberculosis overcomes macrophage bactericidal activities and persists intracellularly. One mechanism by which M. tuberculosis avoids macrophage killing might be through inhibition of IFN-gamma-mediated signaling. In this study we provide evidence that at least two distinct components of M. tuberculosis, the 19-kDa lipoprotein and cell wall peptidoglycan (contained in the mycolylarabinogalactan peptidoglycan (mAGP) complex), inhibit macrophage responses to IFN-gamma at a transcriptional level. Moreover, these components engage distinct proximal signaling pathways to inhibit responses to IFN-gamma: the 19-kDa lipoprotein inhibits IFN-gamma signaling in a Toll-like receptor (TLR)2-dependent and myeloid differentiation factor 88-dependent fashion whereas mAGP inhibits independently of TLR2, TLR4, and myeloid differentiation factor 88. In addition to inhibiting the induction of specific IFN-gamma responsive genes, the 19-kDa lipoprotein and mAGP inhibit the ability of IFN-gamma to activate murine macrophages to kill virulent M. tuberculosis without inhibiting production of NO. These results imply that inhibition of macrophage responses to IFN-gamma may contribute to the inability of an apparently effective immune response to eradicate M. tuberculosis.

Acylation↗
PubMed2004

The folate pathway is a target for resistance to the drug para-aminosalicylic acid (PAS) in mycobacteria.

The increasing rate of multidrug-resistant tuberculosis has led to more use of second-line antibiotics such as para-aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS-resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS-resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.

Aminosalicylic Acid↗
PubMed2003

Genetic requirements for mycobacterial survival during infection.

Despite the importance of tuberculosis as a public health problem, we know relatively little about the molecular mechanisms used by the causative organism, Mycobacterium tuberculosis, to persist in the host. To define these mechanisms, we have mutated virtually every nonessential gene of M. tuberculosis and determined the effect disrupting each gene on the growth rate of this pathogen during infection. A total of 194 genes that are specifically required for mycobacterial growth in vivo were identified. The behavior of these mutants provides a detailed view of the changing environment that the bacterium encounters as infection proceeds. A surprisingly large fraction of these genes are unique to mycobacteria and closely related species, indicating that many of the strategies used by this unusual group of organisms are fundamentally different from other pathogens

Animals↗
PubMed2003

Genes required for mycobacterial growth defined by high density mutagenesis.

Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

DNA Transposable Elements↗
PubMed2003

Bacterial genomics and vaccine design.

Complete genome sequences are available for an increasing number of pathogenic bacteria. These new data are beginning to make an impact on the understanding of bacterial evolution and virulence. Thus far, however, vaccine development has had little benefit from genomics. Here we discuss how genomic sequence is being used in ways that could help identify useful bacterial antigens or create attenuated live vaccines.

Bacterial Proteins↗