Rapid identification of bacterial pathogens using a PCR- and microarray-based assay.

BACKGROUND: During the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples. RESULTS: Comparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence. CONCLUSION: The assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.

Concordant gene regulation related to perturbations of three GDP-mannose-related genes.

Glycosylation of proteins is one of the most crucial post-translational modifications. In order to access system-level and state-dependent data related to the regulation of glycosylation events, we cultivated yeast cell strains each harboring a selected conditional knockdown construct for a gene (either SEC53, VRG4 or DPM1) related to GDP-mannose synthesis or its utilization in glycan biosynthesis. In order to carry this out efficiently, we developed automated sampling from bioreactor cultivations, a collection of in silico workflows for data analysis as well as their integration into a large data warehouse. Using the above-mentioned approaches, we could show that conditional knocking down of transcripts related to GDP-mannose synthesis or transportation led to altered levels of over 300 transcripts. These transcripts and their corresponding proteins were characterized by their gene ontology (GO) annotations, and their putative transcriptional regulation was analyzed. Furthermore, novel pathways were generated indicating interactions between GO categories with common proteins, putative transcriptional regulators of such induced GO categories, and the large protein-protein interaction network among the proteins whose transcripts indicated altered expression levels. When these results are always added to an ever-expanding data warehouse as annotations, they will incrementally increase the knowledge of biological systems.

Cystatin B: mutation detection, alternative splicing and expression in progressive myclonus epilepsy of Unverricht-Lundborg type (EPM1) patients.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion of a dodecamer repeat in the CSTB promoter accounts for the majority of EPM1 disease alleles worldwide. We here describe a novel PCR protocol for detection of the dodecamer repeat expansion. We describe two novel EPM1-associated mutations, c.149G > A leading to the p.G50E missense change and an intronic 18-bp deletion (c.168+1_18del), which affects splicing of CSTB. The p.G50E mutation that affects the conserved QVVAG amino acid sequence critical for cathepsin binding fails to associate with lysosomes. This further supports the previously implicated physiological importance of the CSTB-lysosome association. Expression of CSTB mRNA and protein was markedly reduced in lymphoblastoid cells of the patients irrespective of the mutation type. Patients homozygous for the dodecamer expansion mutation showed 5-10% expression compared to controls. By combining database searches with RT-PCR we identified several alternatively spliced CSTB isoforms. One of these, CSTB2, was also present in mouse and was analyzed in more detail. In real-time PCR quantification, CSTB2 expression was less than 5% of total CSTB expression in all human adult and fetal tissues analyzed. In patients homozygous for the minisatellite mutation, the level of CSTB2 was reduced similarly to that of CSTB implicating regulation from the same promoter. The physiological significance of CSTB2 remains to be determined.

Excess mannose limits the growth of phosphomannose isomerase PMI40 deletion strain of Saccharomyces cerevisiae.

Phosphomannose isomerase (PMI40) catalyzes the conversion between fructose 6-phosphate and mannose 6-phosphate and thus connects glycolysis, i.e. energy production and GDP-mannose biosynthesis or cell wall synthesis in Saccharomyces cerevisiae. After PMI40 deletion (pmi(-)) the cells were viable only if fed with extracellular mannose and glucose. In an attempt to force the GDP-mannose synthesis in the pmi(-) strain by increasing the extracellular mannose concentrations, the cells showed significantly reduced growth rates without any alterations in the intracellular GDP-mannose levels. To reveal the mechanisms resulting in reduced growth rates, we measured genome-wide gene expression levels, several metabolite concentrations, and selected in vitro enzyme activities in central metabolic pathways. The increasing of the initial mannose concentration led to an increase in the mannose 6-phosphate concentration, which inhibited the activity of the second enzyme in glycolysis, i.e. phosphoglucose isomerase converting glucose 6-phosphate to fructose 6-phosphate. As a result of this limitation, the flux through glycolysis was decreased as was the median expression of the genes involved in glycolysis. The expression levels of RAP1, a transcription factor involved in the regulation of the mRNA levels of several enzymes in glycolysis, as well as those of cell cycle regulators CDC28 and CLN3, decreased concomitantly with the growth rates and expression of many genes encoding for enzymes in glycolysis.

Inflammation-induced transcriptional regulation of Golgi transporters required for the synthesis of sulfo sLex glycan epitopes.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3'-phosphate 5'-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.

Diazepam-induced adaptive plasticity revealed by alpha1 GABAA receptor-specific expression profiling.

Benzodiazepines are in wide clinical use for their sedative and tranquilizing actions, the former being mediated via alpha1-containing GABAA receptors. The signal transduction pathways elicited beyond the receptor are only poorly understood. Changes of transcript levels in cerebral cortex induced by acute diazepam administration were therefore compared by microarray analysis between wild-type and point mutated alpha1(H101R) mice, in which the alpha1 GABAA receptor subunit had been rendered insensitive to diazepam. In wild-type animals, diazepam reduced the expression levels of the alpha subunit of the calcium/calmodulin-dependent protein kinase II, as well as brain-derived neurotrophic factor, MAP kinase phosphatase, transcription factor GIF, c-fos and nerve growth factor induced gene-A. None of these transcripts was changed in the alpha1(H101R) mice after treatment with diazepam. Thus, the sedative action of diazepam is correlated with a selective down-regulation of transcripts involved in the regulation of neuronal plasticity and neurotrophic responses. Most transcript changes were transient except for the decrease of the CaMKIIalpha transcript which persisted even 40 h after the single dose of diazepam. This long-term alteration is likely to contribute to the resetting of the neuronal responsiveness, which may be involved in rebound phenomena and, under chronic treatment, in the development of tolerance and dependence.

Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose.

In the salvage pathway of GDP-L-fucose, free cytosolic fucose is phosphorylated by L-fucokinase to form L-fucose-L-phosphate, which is then further converted to GDP-L-fucose in the reaction catalyzed by GDP-L-fucose pyrophosphorylase. We report here the cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. Murine L-fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of L-fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP-L-fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP-L-fucose, the reaction product of GDP-L-pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine L-fucokinase and GDP-L-fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of L-fucokinase and GDP-L-fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP-L-fucose in the cytosol.