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At least 1,279 records · Page 71Linked to original sources
PubMed2004

Phosphonoformate: a minimal transition state analogue inhibitor of the fosfomycin resistance protein, FosA.

Fosfomycin [(1R,2S)-epoxypropylphosphonic acid] is a simple phosphonate found to have antibacterial activity against both Gram-positive and Gram-negative microorganisms. Early resistance to the clinical use of the antibiotic was linked to a plasmid-encoded resistance protein, FosA, that catalyzes the addition of glutathione to the oxirane ring, rendering the antibiotic inactive. Subsequent studies led to the discovery of a genomically encoded homologue in the pathogen Pseudomonas aeruginosa. The proteins are Mn(II)-dependent enzymes where the metal is proposed to act as a Lewis acid stabilizing the negative charge that develops on the oxirane oxygen in the transition state. Several simple phosphonates, including the antiviral compound phosphonoformate (K(i) = 0.4 +/- 0.1 microM, K(d) approximately 0.2 microM), are shown to be inhibitors of FosA. The crystal structure of FosA from P. aeruginosa with phosphonoformate bound in the active site has been determined at 0.95 A resolution and reveals that the inhibitor forms a five-coordinate complex with the Mn(II) center with a geometry similar to that proposed for the transition state of the reaction. Binding studies show that phosphonoformate has a near-diffusion-controlled on rate (k(on) approximately 10(7)-10(8) M(-1) s(-1)) and an off rate (k(off) = 5 s(-1)) that is slower than that for fosfomycin (k(off) = 30 s(-1)). Taken together, these data suggest that the FosA-catalyzed reaction has a very early transition state and phosphonoformate acts as a minimal transition state analogue inhibitor.

Bacterial Proteins↗
PubMed2003

Disease proteomics.

The sequencing of the human genome and that of numerous pathogens has opened the door for proteomics by providing a sequence-based framework for mining proteomes. As a result, there is intense interest in applying proteomics to foster a better understanding of disease processes, develop new biomarkers for diagnosis and early detection of disease, and accelerate drug development. This interest creates numerous opportunities as well as challenges to meet the needs for high sensitivity and high throughput required for disease-related investigations.

Disease↗
PubMed2005

The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J.

The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.

Chromosomes, Human, Pair 17↗
PubMed2005

Mapping of interactions and mouse congenic strains identified novel epistatic QTLs controlling the persistence of Salmonella Enteritidis in mice.

The host response to infection in humans is multifactorial and involves the complex interaction between two genomes (the host and the pathogen) and the environment. Using an experimental mouse model of chronic infection, we have previously identified the individual effect of three significant and one suggestive quantitative trait loci (QTLs) (Ses1, Ses2, Ses3 and Ses1.1) on Salmonella Enteritidis persistence in target organs of 129S6/SvEvTac mice. Congenic strain construction was performed by transferring each of these QTLs from C57BL/6J onto the 129S6/SvEvTac background, and phenotypic analysis confirmed that Ses1 and Ses1.1 contribute to bacterial clearance. Additional QTLs regulating Salmonella carriage in 129S6/SvEvTac mice were identified using a two-locus epistasis QTL linkage mapping approach conducted separately in females and males. The epistatic model for females included the individual effect of Ses3 and two significant interactions (Ses1-D7Mit267 and Ses1-DXMit48) accounting for 47% of the total phenotypic variance. The model for males included the individual effect of Ses1.1, three interactions (Ses1-D9Mit218, D2Mit197-D4Mit2 and D3Mit256-D13Mit36) and explained 47% of the phenotypic variance. Our results suggest that the oligogenic nature of Salmonella persistence and epistasis are important constituents of the genetic architecture of the host response to chronic Salmonella infection.

Animals↗
PubMed2006

Gene therapy progress and prospects--vectorology: design and production of expression cassettes in AAV vectors.

Adeno-associated virus (AAV) derived vectors are considered highly eligible vehicles for human gene therapy. Not only do they possess many great potential for clinical applications due to their wide range of tissue targets but also their excellent preclinical safety profile makes them particularly suitable candidates for treating serious diseases. Initial clinical trials have yielded encouraging results and prompted further improvements in their design and methods of production. Many studies have been performed to modify the tropism of recombinant (r)AAV by capsid modification. However, the precise control of spatial and temporal gene expression, which may be important in determining the safety and efficacy of gene transfer, lies in a rational choice and a subtle combination of various regulatory genetic elements to be inserted into the expression cassette. Moreover, new strategies based on such genetic sequences open new perspectives for enhancing vector genome persistence, disrupting or reducing pathogenic gene expression and even targeting genes.

Animals↗
PubMed2006

The G(-248)A polymorphism in the promoter region of the Bax gene does not correlate with prognostic markers or overall survival in chronic lymphocytic leukemia.

The G(-248)A polymorphism in the promoter region of the Bax gene was recently associated with low Bax expression, more advanced stage, treatment resistance and short overall survival in B-cell chronic lymphocytic leukemia (CLL), the latter particularly in treated patients. To investigate this further, we analyzed 463 CLL patients regarding the presence or absence of the G(-248)A polymorphism and correlated with overall survival, treatment status and known prognostic factors, for example, Binet stage, VH mutation status and genomic aberrations. In this material, similar allele and genotype frequencies of the Bax polymorphism were demonstrated in CLL patients and controls (n=207), where 19 and 21% carried this polymorphism, respectively, and no skewed distribution of the polymorphism was evident between different Binet stages and VH mutated and unmutated CLLs. Furthermore, no difference in overall survival was shown between patients displaying the G(-248)A polymorphism or not (median survival 85 and 102 months, respectively, P=0.21), and the polymorphism did not influence outcome specifically in treated CLL. Neither did the polymorphism affect outcome in prognostic subsets defined by VH mutation status or genomic aberrations. In conclusion, the pathogenic role and clinical impact of the Bax polymorphism is limited in CLL.

Adult↗
PubMed2002

Molecular microbiology and pathogenesis of Helicobacter and Campylobacter updated: a meeting report of the 11th conference on Campylobacter, Helicobacter and related organisms.

The genome analysis of the gastrointestinal pathogens Helicobacter and Campylobacter has stimulated a wealth of new research activities, which are presented every 2 years at the international conferences on Campylobacter, Helicobacter and Related Organisms (CHRO). Both organisms represent excellent models for the identification of new molecular mechanisms involved in pathogenesis, host response and physiological adaptation in course of acute and chronic infectious diseases. The investigation of their global distribution, pronounced genetic and antigenic diversity as well as the molecular mechanisms allowing long-term persistence in hostile and unusual microbial habitats, is a challenge for scientists of many different disciplines world-wide. With a focus on the molecular microbiology aspects, this review summarizes recent trends in Helicobacter and Campylobacter research by highlighting selected presentations at the 11th CHRO conference. The topics include the discovery of new virulence factors, functional analysis of protein secretion systems, host signalling pathways, adaptation to stress conditions, global gene regulation, and genetic variability.

Animals↗
PubMed2005

Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.

Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naive mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.

Animals↗
PubMed2005

Imaging bacterial infections with radiolabeled 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouracil.

Bacterial infections provide diagnostic dilemmas that could be enlightened by modern imaging technologies. We have developed a simple method for imaging bacterial infections in mice that relies on the phosphorylation and trapping of the thymidine kinase (TK) substrate 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-[125I] iodouracil ([125I]FIAU) within bacteria. FIAU was found to inhibit the growth of WT Escherichia coli but not a TK- strain, indicating that WT E. coli could metabolize this compound. In silico analyses demonstrated that all pathogenic strains of bacteria whose genomes have been sequenced contain a TK gene highly homologous to the E. coli TK. Accordingly, we demonstrated that localized infections caused by representatives of five genera of bacteria could be readily imaged with [125I]FIAU. Such imaging provides a general method for the diagnosis of localized bacterial infections that could be translatable to the clinic.

Amino Acid Sequence↗
PubMed2005

Origin and evolution of the chicken leukocyte receptor complex.

In mammals, the cell surface receptors encoded by the leukocyte receptor complex (LRC) regulate the activity of T lymphocytes and B lymphocytes, as well as that of natural killer cells, and thus provide protection against pathogens and parasites. The chicken genome encodes many Ig-like receptors that are homologous to the LRC receptors. The chicken Ig-like receptor (CHIR) genes are members of a large monophyletic gene family and are organized into genomic clusters, which are in conserved synteny with the mammalian LRC. One-third of CHIR genes encode polypeptide molecules that contain both activating and inhibitory motifs. These genes are present in different phylogenetic groups, suggesting that the primordial CHIR gene could have encoded both types of motifs in a single molecule. In contrast to the mammalian LRC genes, the CHIR genes with similar function (inhibition or activation) are evolutionarily closely related. We propose that, in addition to recombination, single nucleotide substitutions played an important role in the generation of receptors with different functions. Structural models and amino acid analyses of the CHIR proteins reveal the presence of different types of Ig-like domains in the same phylogenetic groups, as well as sharing of conserved residues and conserved changes of residues between different CHIR groups and between CHIRs and LRCs. Our data support the notion that the CHIR gene clusters are regions homologous to the mammalian LRC gene cluster and favor a model of evolution by repeated processes of birth and death (expansion-contraction) of the Ig-like receptor genes.

Animals↗
PubMed2001

Structural features of an influenza virus promoter and their implications for viral RNA synthesis.

The influenza A virus, a severe pandemic pathogen, has a segmented RNA genome consisting of eight single-stranded RNA molecules. The 5' and 3' ends of each RNA segment recognized by the influenza A virus RNA-dependent RNA polymerase direct both transcription and replication of the virus's RNA genome. Promoter binding by the viral RNA polymerase and formation of an active open complex are prerequisites for viral replication and proliferation. Here we describe the solution structure of this promoter as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. Our studies show that the viral promoter has a significant dynamic nature and reveal an unusual displacement of an adenosine that forms a novel (A-A) x U motif and a C-A mismatch stacked in a helix. The characterized structural features of the promoter imply that the specificity of polymerase binding results from an internal RNA loop. In addition, an unexpected bending (46 +/- 10 degrees ) near the initiation site suggests the existence of a promoter recognition mechanism similar to that of DNA-dependent RNA polymerase and a possible regulatory function for the terminal structure during open complex formation.

Influenza A virus↗
PubMed2003

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

Escherichia coli↗
PubMed1997

Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi.

Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics.

Amino Acid Sequence↗
PubMed1998

Prions.

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.

Animals↗
PubMed1998

The largest subunits of RNA polymerase from gastric helicobacters are tethered.

The rpoB and rpoC genes of eubacteria and archaea, coding respectively for the beta- and beta'-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. The genome sequence of the gastric pathogen Helicobacter pylori (strain 26695) revealed homologs of two genes in one continuous open reading frame that potentially could encode one 2890-amino acid-long beta-beta' fusion protein. Here, we show that this open reading frame does in fact encode a fused beta-beta' polypeptide. In addition, we establish by DNA sequencing that rpoB and rpoC are also fused in each of four other unrelated strains of H. pylori, as well as in Helicobacter felis, another member of the same genus. In contrast, the rpoB and rpoC genes are separate in two members of the related genus Campylobacter (Campylobacter jejuni and Campylobacter fetus) and encode separate RNA polymerase subunits. The Campylobacter genes are also unusual in overlapping one another rather than being separated by a spacer as in other Gram-negative bacteria. We propose that the unique organization of rpoB and rpoC in H. pylori may contribute to its ability to colonize the human gastric mucosa.

Amino Acid Sequence↗
PubMed2005

Mitogen activated protein kinases of Aspergillus fumigatus.

In microbial eukaryotes, mitogen activated protein kinase (MAPK) pathways play a pivotal role in regulating cellular physiology. In fungi MAPK pathways have established functions in mating-pheromone responses, maintaining cell wall integrity, responding to changes in osmolarity and nutrient sensing. We have been studying MAPK functions in the human pathogenic fungus Aspergillus fumigatus. The genome of A. fumigatus has four MAPK genes, sakA/hogA, mpkA, mpkB and mpkC. Deletion of the sakA gene produces a strain that does not correctly regulate conidial germination, sense environmental nitrogen or responds to hypertonic stress. The function of the remaining MAPK genes is still under investigation, but by analogy to work in other filamentous fungi, we speculate as to their possible functions in A. fumigatus.

Aspergillus fumigatus↗
PubMed2026

Artificial intelligence in molecular diagnostics for pandemic preparedness.

INTRODUCTION: Molecular diagnostics focusing on the detection and analysis of nucleic acids are indispensable tools for early pathogen identification, transmission monitoring, and genomic surveillance during pandemics. Recent technological advances have broadened the diagnostic landscape, incorporating PCR-based methods, isothermal amplification, high-CRISPR-based amplification detection, and sequencing. Despite their diagnostic potential, widespread implementation remains limited by high validation costs, time and logistical constraints, the need for specialized professional knowledge, and a lack of adaptability in resource-limited settings. Artificial intelligence (AI) is increasingly recognized as a promising but challenging approach, offering tools that streamline assay development, automate data interpretation, and optimize real-time diagnostic performance. AREAS COVERED: This review introduces recently published AI tools with potential to enhance the in-silico design validation process of oligonucleotides for molecular assays. These cover tools for initial assay design and optimization to validation and continuous assay updates. The limitations, including concerns regarding data accuracy, the lack of transparency in data processing ('black box' models), and unresolved licensing and regulatory issues, are highlighted for each tool and as expert opinion. EXPERT OPINION: Collectively, these challenges currently confine most AI-based approaches to research settings and prevent their routine implementation in clinical molecular diagnostics. Their widespread adoption depends on addressing remaining technical, regulatory, and practical challenges.

Humans↗
PubMed2005

The "domino theory" of gene death: gradual and mass gene extinction events in three lineages of obligate symbiotic bacterial pathogens.

During the adaptation of an organism to a parasitic lifestyle, various gene functions may be rendered superfluous due to the fact that the host may supply these needs. As a consequence, obligate symbiotic bacterial pathogens tend to undergo reductive genomic evolution through gene death (nonfunctionalization or pseudogenization) and deletion. Here, we examine the evolutionary sequence of gene-death events during the process of genome miniaturization in three bacterial species that have experienced extensive genome reduction: Mycobacterium leprae, Shigella flexneri, and Salmonella typhi. We infer that in all three lineages, the distribution of functional categories is similar in pseudogenes and genes but different from that of absent genes. Based on an analysis of evolutionary distances, we propose a two-step "domino effect" model for reductive genome evolution. The process starts with a gradual gene-by-gene-death sequence of events. Eventually, a crucial gene within a complex pathway or network is rendered nonfunctional triggering a "mass gene extinction" of the dependent genes. In contrast to published reports according to which genes belonging to certain functional categories are prone to nonfunctionalization more frequently and earlier than genes belonging to other functional categories, we could discern no characteristic regularity in the temporal order of function loss.

Bacteria↗