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Adel M Talaat

Publications and source records attributed to Adel M Talaat.

11 recordsLinked to original sources
PubMed2006

CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator.

Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S(2)N) Cu(I) complex. The 2.6-A crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65' and His61' in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.

Amino Acid Sequence↗
PubMed2006

Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants.

Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G+C content (50.5 to 60.5%) lower than the average G+C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johne's and Crohn's diseases.

Animals↗
PubMed2006

Whole-genome plasticity among Mycobacterium avium subspecies: insights from comparative genomic hybridizations.

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is also implicated in cases of Crohn's disease in humans. Another closely related strain, M. avium subsp. avium, is a health problem for immunocompromised patients. To understand the molecular pathogenesis of M. avium subspecies, we analyzed the genome contents of isolates collected from humans and domesticated or wildlife animals. Comparative genomic hybridizations indicated distinct lineages for each subspecies where the closest genomic relatedness existed between M. avium subsp. paratuberculosis isolates collected from human and clinical cow samples. Genomic islands (n = 24) comprising 846 kb were present in the reference M. avium subsp. avium strain but absent from 95% of M. avium subsp. paratuberculosis isolates. Additional analysis identified a group of 18 M. avium subsp. paratuberculosis-associated islands comprising 240 kb that were absent from most of the M. avium subsp. avium isolates. Sequence analysis of DNA regions flanking the genomic islands identified three large inversions in addition to several small inversions that could play a role in regulation of gene expression. Analysis of genes encoded in the genomic islands reveals factors that are probably important for various mechanisms of virulence. Overall, M. avium subsp. avium isolates displayed a higher level of genomic diversity than M. avium subsp. paratuberculosis isolates. Among M. avium subsp. paratuberculosis isolates, those from wildlife animals displayed the highest level of genomic rearrangements that were not observed in other isolates. The presented findings will affect the future design of diagnostics and vaccines for Johne's and Crohn's diseases and provide a model for genomic analysis of closely related bacteria.

Animals↗
PubMed2004

The temporal expression profile of Mycobacterium tuberculosis infection in mice.

Infection with Mycobacterium tuberculosis causes the illness tuberculosis with an annual mortality of approximately 2 million. Understanding the nature of the host-pathogen interactions at different stages of tuberculosis is central to new strategies for developing chemotherapies and vaccines. Toward this end, we adapted microarray technology to analyze the change in gene expression profiles of M. tuberculosis during infection in mice. This protocol provides the transcription profile of genes expressed during the course of early tuberculosis in immune-competent (BALB/c) and severe combined immune-deficient (SCID) hosts in comparison with growth in medium. The microarray analysis revealed clusters of genes that changed their transcription levels exclusively in the lungs of BALB/c, SCID mice, or medium over time. We identified a set of genes (n = 67) activated only in BALB/c and not in SCID mice at 21 days after infection, a key point in the progression of tuberculosis. A subset of the lung-activated genes was previously identified as induced during mycobacterial survival in a macrophage cell line. Another group of in vivo-expressed genes may also define a previously unreported genomic island. In addition, our analysis suggests the similarity between mycobacterial transcriptional machinery during growth in SCID and in broth, which questions the validity of using the SCID model for assessing mycobacterial virulence. The in vivo expression-profiling technology presented should be applicable to any microbial model of infection.

Animals↗
PubMed2004

Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis.

Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.

Animals↗
PubMed2002

Genomic DNA standards for gene expression profiling in Mycobacterium tuberculosis.

A fundamental problem in DNA microarray analysis is the lack of a common standard to compare the expression levels of different samples. Several normalization protocols have been proposed to overcome variables inherent in this technology. As yet, there are no satisfactory methods to exchange gene expression data among different research groups or to compare gene expression values under different stimulus-response profiles. We have tested a normalization procedure based on comparing gene expression levels to the signals generated from hybridizing genomic DNA (genomic normalization). This procedure was applied to DNA microarrays of Mycobacterium tuberculosis using RNA extracted from cultures growing to the logarithmic and stationary phases. The applied normalization procedure generated reproducible measurements of expression level for 98% of the putative mycobacterial ORFs, among which 5.2% were significantly changed comparing the logarithmic to stationary growth phase. Additionally, analysis of expression levels of a subset of genes by real time PCR technology revealed an agreement in expression of 90% of the examined genes when genomic DNA normalization was applied instead of 29-68% agreement when RNA normalization was used to measure the expression levels in the same set of RNA samples. Further examination of microarray expression levels displayed clusters of genes differentially expressed between the logarithmic, early stationary and late stationary growth phases. We conclude that genomic DNA standards offer advantages over conventional RNA normalization procedures and can be adapted for the investigation of microbial genomes.

DNA, Bacterial↗
PubMed2002

DNA microarray analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete.

DNA microarrays were used to survey the adaptive genetic responses of Borrelia burgdorferi (Bb) B31, the Lyme disease spirochete, when grown under conditions analogous to those found in unfed ticks (UTs), fed ticks (FTs), or during mammalian host adaptation (Bb in dialysis membrane chambers implanted in rats). Microarrays contained 95.4% of the predicted B31 genes, 150 (8.6%) of which were differentially regulated (changes of > or = 1.8-fold) among the three growth conditions. A substantial proportion (46%) of the differentially regulated genes encoded proteins with predicted export signals (29% from predicted lipoproteins), emphasizing the importance to Bb of modulating its extracellular proteome. For B31 cultivated at the more restrictive UT condition, microarray data provided evidence of a bacterial stringent response and factors that restrict cell division. A large proportion of genes were responsive to the FT growth condition, wherein increased temperature and reduced pH were prominent environmental parameters. A surprising theme, supported by cluster analysis, was that many of the gene expression changes induced during the FT growth condition were transient and largely tempered as B31 adapted to the mammalian host, suggesting that once Bb gains entry and adapts to mammalian tissues, fewer differentially regulated genes are exploited. It therefore would seem that although widely dissimilar, the UT and dialysis membrane chamber growth conditions promote more static patterns of gene expression in Bb. The microarray data thus provide a basis for formulating new testable hypotheses regarding the life cycle of Bb and attaining a more complete understanding of many aspects of Bb's complex parasitic strategies.

Animals↗
PubMed2002

Priming reverse transcription with oligo(dT) does not yield representative samples of Mycobacterium tuberculosis cDNA.

Several recent publications have suggested that oligo(dT) can prime reverse transcription of several mycobacterial mRNAs. To determine if this is the case for most Mycobacterium tuberculosis mRNA species, reverse transcription reactions of M. tuberculosis RNA were primed with oligo(dT) or with other primers that did not target polyadenylylated sequences, and the resultant cDNA product was evaluated. Priming with oligo(dT) yielded more cDNA than priming with an arbitrary primer for only 1 of 12 unrelated M. tuberculosis genes, as measured by competitive PCR. Priming with oligo(dT) yielded cDNA for only 30% of the genes primed for by 37 M. tuberculosis genome-directed oligonucleotides, as assessed by hybridization of cDNA with an M. tuberculosis microarray. These data demonstrate that priming of reverse transcription of mycobacterial mRNA with oligo(dT) does not yield representative samples of cDNA.

DNA Primers↗
PubMedDate not supplied

Genetic immunization: what's in a name?

The concept and demonstration of genetic immunization (GI) was first introduced in 1992. At the time it appeared to be a revolutionary new approach in vaccinology. Since then, genetic immunization has been applied with much success in a wide variety of model and natural systems. It has also been used in several human clinical trials. Currently there is a general impression that genetic immunization has limitations inhibiting its broad use. The technique is thought to be poor at antibody production and more importantly not to work well in primates and humans (simian barrier). However, recent reports addressing these issues (poor antibody production and the simian barrier) showed improvements of GI to produce protective immune responses in humans. We propose that the apparent limitations of gene vaccines may arise from not using the technologies' potential to manipulate the immune system. This dearth of imaginative use is manifested in the tendency by some to term the technique DNA immunization. The apparent limitations of DNA vaccines may not be limitations for gene vaccines.

Adjuvants, Immunologic↗