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Mobius N.,Heinrich Heine University Düsseldorf | Brenneisen W.,Institute of Medical Microbiology and Hospital Hygiene | Schaeffer A.,Institute of Medical Microbiology and Hospital Hygiene | Henrich B.,Heinrich Heine University Düsseldorf
Methods in Molecular Biology | Year: 2012

Urogenital tract infections can be caused by a number of pathogens, some of which, like the obligate intracellular Chlamydia trachomatis, are difficult to culture, or the cell wall-less mollicutes, like M. hominis or Ureaplasma spp. Real-time PCR (qPCR) has become an important diagnostic tool as it enables not only the species-specific detection of the organism but also the quantification essential to define the etiological relevance of a facultative pathogenic bacterium. We developed a set of TaqMan qPCRs for the detection of the species M. genitalium and M. hominis (Mh/Mg-duplex qPCR), U. parvum and U. urealyticum (Uu/Up duplex-PCR), and C. trachomatis (CT-qPCR), and for typing of lymphogranuloma venereum-associated L-serovars of C. trachomatis (LGV-qPCR) as well as a sub-typing of L1, L2, and L3. In addition, the human gap-gene was amplified as quality control of the specimen, and a cryptic plasmid co-amplified in CT-qPCR as an inhibition control. The present protocol focuses on the step-by-step description for the establishment of these TaqMan multiplex qPCRs. © 2012 Springer Science+Business Media New York.

Kravets E.,Institute of Medical Microbiology and Hospital Hygiene | Degrandi D.,Institute of Medical Microbiology and Hospital Hygiene | Weidtkamp-Peters S.,Center for Advanced Imaging | Ries B.,Institute of Biochemistry | And 9 more authors.
Journal of Biological Chemistry | Year: 2012

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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