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Daniel D Rockey - One of the best experts on this subject based on the ideXlab platform.

  • horizontal transfer of tetracycline resistance among chlamydia spp in vitro
    Antimicrobial Agents and Chemotherapy, 2009
    Co-Authors: Robert J Suchland, Walter E. Stamm, Kelsi M Sandoz, Brendan M Jeffrey, Daniel D Rockey
    Abstract:

    Members of the chlamydiae are obligately intracellular bacteria that cause serious diseases in a wide variety of hosts (19). In humans, Chlamydia trachomatis causes trachoma and a variety of sexually transmitted conditions, diseases that affect millions of people around the world (25). Two related species, Chlamydia suis and Chlamydia muridarum, cause diseases of mucosal membranes in pigs and mice, respectively. A second genus within the chlamydiae includes the more distantly related guinea pig pathogen Chlamydophila caviae and the human pathogen Chlamydophila pneumoniae. While the members of the chlamydiae are generally similar in many aspects of basic biology, including genome order and gene content, there are differences in their intracellular survival strategies. One of these differences is the ability to form fusogenic inclusions. Wild-type strains of the Chlamydia spp. form intracellular vacuoles (termed inclusions) that undergo homotypic fusion (30), while no tested strains of these species form inclusions that fuse with inclusions of Chlamydophila caviae or Chlamydophila pneumoniae (23) (see Fig. ​Fig.1)1) (unpublished data). FIG. 1. Immunofluorescence images of McCoy cells coinfected with C. suis R19/tetR and C. muridarum MoPn/oflR (A) or with C. suis R19/tetR and Chlamydophila caviae GPIC/rifR (B). In both panels, C. suis R19/tetR is labeled red, while the alternate species is labeled ... The genome sequences of most chlamydial species do not contain examples of recent horizontal acquisition of DNA. To date, the only reported example of a genomic island within any species of Chlamydia is the tet(C) island of C. suis. This island contains a C. suis-specific insertion element (IScs605), plasmid sequence with roots in gram-negative bacteria, and a tet(C) resistance gene (10). The tetracycline-resistant strains are common in swine herds in the United States (1) and recently have been identified in pigs from Italy (8). The strains carrying this island are the only examples of naturally acquired antibiotic resistance in any chlamydial species. The C. suis tetracycline-resistant strains are also resistant to doxycycline, one of two contemporary front-line drugs of choice against chlamydial infection in humans. Because of its low cost, tetracycline is also used to treat millions of cases of trachoma in developing countries (20). Although there are reports of drug resistance in strains collected from patients suffering treatment failure, documented stable homotypic drug resistance to antibiotics used for treating acute human chlamydial infections is controversial (18, 24, 28). Transfer of a stable tetracycline-resistant phenotype to human clinical chlamydiae would represent a significant public health challenge. The very limited examples of horizontally acquired DNA in the chlamydiae support a hypothesis that recombination might be rare in this system. However, accumulating sequence data indicate that Chlamydia spp. are actively recombinogenic within a species. Early sequencing studies identified ompA variants that encode protein sequences from different classical serovars and showed that recombination may also occur at other locations in the chromosome (2, 11, 17, 21). Additionally, recent studies demonstrated conclusively that lateral gene transfer can be selected for in C. trachomatis in cell culture following coinfection with strains that have dissimilar drug resistance markers (6, 7). The mechanism of transfer in any of these recombination events remains to be elucidated. In order to explore possible tools for the development of a workable genetic system for directed mutagenesis of Chlamydia DNA, and to investigate the possibility of tetracycline resistance acquisition in human strains of C. trachomatis, we conducted in vitro recombination experiments using rifampin (rifampicin)-, ofloxacin-, and tetracycline-resistant strains as donors and recipients. Our results demonstrate that recombination can be readily demonstrated within and among species of the genus Chlamydia and that tetracycline resistance can be manifested by C. trachomatis following coculture with tetracycline-resistant C. suis strains. Tetracycline resistance was not successfully transferred to Chlamydophila caviae, suggesting a biological barrier that does not allow recombination across these genera.

  • Horizontal Transfer of Tetracycline Resistance among Chlamydia spp. In Vitro
    Antimicrobial agents and chemotherapy, 2009
    Co-Authors: Robert J Suchland, Walter E. Stamm, Kelsi M Sandoz, Brendan M Jeffrey, Daniel D Rockey
    Abstract:

    There are no examples of stable tetracycline resistance in clinical strains of Chlamydia trachomatis. However, the swine pathogen Chlamydia suis is commonly tetracycline resistant, both in America and in Europe. In tested U.S. strains, this resistance is mediated by a genomic island carrying a tet(C) allele. In the present study, the ability of C. suis to mobilize tet(C) into other chlamydial species was examined. Differently antibiotic resistant strains of C. suis, C. trachomatis, and Chlamydia muridarum were used in coculture experiments to select for multiply antibiotic resistant progeny. Coinfection of mammalian cells with a naturally occurring tetracycline-resistant strain of C. suis and a C. muridarum or C. trachomatis strain containing selected mutations encoding rifampin (rifampicin) or ofloxacin resistance readily produced doubly resistant recombinant clones that demonstrated the acquisition of tetracycline resistance. The resistance phenotype in the progeny from a C. trachomatis L2/ofl(R)-C. suis R19/tet(R) cross resulted from integration of a 40-kb fragment into a single ribosomal operon of a recipient, leading to a merodiploid structure containing three rRNA operons. In contrast, a cross between C. suis R19/tet(R) and C. muridarum MoPn/ofl(R) led to a classical double-crossover event transferring 99 kb of DNA from C. suis R19/tet(R) into C. muridarum MoPn/ofl(R). Tetracycline resistance was also transferred to recent clinical strains of C. trachomatis. Successful crosses were not obtained when a rifampin-resistant Chlamydophila caviae strain was used as a recipient for crosses with C. suis or C. trachomatis. These findings provide a platform for further exploration of the biology of horizontal gene transfer in Chlamydia while bringing to light potential public health concerns generated by the possibility of acquisition of tetracycline resistance by human chlamydial pathogens.

  • Host α-adducin is redistributed and localized to the inclusion membrane in Chlamydia- and Chlamydophila-infected cells
    Microbiology, 2008
    Co-Authors: Hencelyn G Chu, Sara K Weeks, Diana M Gilligan, Daniel D Rockey
    Abstract:

    A large-scale analysis of proteins involved in host-cell signalling pathways was performed using chlamydia-infected murine cells in order to identify host proteins that are differentially activated or localized following infection. Two proteins whose distribution was altered in Chlamydia trachomatis-infected cells relative to mock-infected cells were the actin-binding protein adducin and the regulatory kinase Raf-1. Immunoblot analysis with antibodies to both phosphorylated and non-phosphorylated forms of these proteins demonstrated that the abundance of each protein was markedly reduced in the cytosolic fraction of C. trachomatis- and Chlamydophila caviae-infected cells, but the total cellular protein abundance remained unaffected by infection. Fluorescence microscopy of chlamydia-infected cells using anti-α-adducin antibodies demonstrated labelling at or near the chlamydial inclusion membrane. Treatment of infected cells with nocodazole or cytochalasin D did not affect α-adducin that was localized to the margins of the inclusion. The demonstration of α-adducin and Raf-1 redistribution within cells infected by different chlamydiae provides novel opportunities for analysis of host–pathogen interactions in this system.

  • Host alpha-adducin is redistributed and localized to the inclusion membrane in chlamydia- and Chlamydophila-infected cells.
    Microbiology (Reading England), 2008
    Co-Authors: Hencelyn G Chu, Sara K Weeks, Diana M Gilligan, Daniel D Rockey
    Abstract:

    A large-scale analysis of proteins involved in host-cell signalling pathways was performed using chlamydia-infected murine cells in order to identify host proteins that are differentially activated or localized following infection. Two proteins whose distribution was altered in Chlamydia trachomatis-infected cells relative to mock-infected cells were the actin-binding protein adducin and the regulatory kinase Raf-1. Immunoblot analysis with antibodies to both phosphorylated and non-phosphorylated forms of these proteins demonstrated that the abundance of each protein was markedly reduced in the cytosolic fraction of C. trachomatis- and Chlamydophila caviae-infected cells, but the total cellular protein abundance remained unaffected by infection. Fluorescence microscopy of chlamydia-infected cells using anti-alpha-adducin antibodies demonstrated labelling at or near the chlamydial inclusion membrane. Treatment of infected cells with nocodazole or cytochalasin D did not affect alpha-adducin that was localized to the margins of the inclusion. The demonstration of alpha-adducin and Raf-1 redistribution within cells infected by different chlamydiae provides novel opportunities for analysis of host-pathogen interactions in this system.

  • Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA
    BMC Microbiology, 2004
    Co-Authors: Damir Alzhanov, Jennifer Barnes, Dennis E Hruby, Daniel D Rockey
    Abstract:

    Background Chlamydiae produce a set of proteins, termed Inc proteins, that are localized to the inclusion membrane and exposed to the host cell cytosol. Little information exists regarding the interaction of Inc proteins with the eukaryotic cell. To examine these interactions, Vaccinia virus vectors and mammalian plasmid-based systems were used to express inc genes in mammalian cells. Results Cells transfected with plasmids expressing Chlamydophila caviae incA were not productively infected by C. caviae . Expression of C. caviae incA also reduced inclusion formation by Chlamydia trachomatis , but not to the degree seen for C. caviae . Chlamydia trachomatis incA did not block development of either C. trachomatis or C. caviae . Deletion mutagenesis was used to demonstrate that plasmids encoding either the amino or carboxy-terminal regions of the protein, as well as the changing of a single amino acid within IncA (serine 17) could not block C. caviae infection. Immunoblot analysis of truncated IncA in a Vaccinia virus system provided evidence that serine 17 of C. caviae IncA is a target for phosphorylation. Conclusions These experiments provide insight into the interaction of Inc proteins with the host cell and introduce a model system where these interactions can be explored further.

Andrew W. B. Johnston - One of the best experts on this subject based on the ideXlab platform.

  • Screening a wide host‐range, waste‐water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes
    Environmental microbiology, 2005
    Co-Authors: Margaret Wexler, David J. Richardson, Philip L. Bond, Andrew W. B. Johnston
    Abstract:

    A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

Margaret Wexler - One of the best experts on this subject based on the ideXlab platform.

  • Screening a wide host‐range, waste‐water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes
    Environmental microbiology, 2005
    Co-Authors: Margaret Wexler, David J. Richardson, Philip L. Bond, Andrew W. B. Johnston
    Abstract:

    A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

David J. Richardson - One of the best experts on this subject based on the ideXlab platform.

  • Screening a wide host‐range, waste‐water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes
    Environmental microbiology, 2005
    Co-Authors: Margaret Wexler, David J. Richardson, Philip L. Bond, Andrew W. B. Johnston
    Abstract:

    A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

Philip L. Bond - One of the best experts on this subject based on the ideXlab platform.

  • Screening a wide host‐range, waste‐water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes
    Environmental microbiology, 2005
    Co-Authors: Margaret Wexler, David J. Richardson, Philip L. Bond, Andrew W. B. Johnston
    Abstract:

    A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.