20 research outputs found

    Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission

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    While the roles of rpoSBb and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable

    Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission

    Get PDF
    While the roles of rpoSBb and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable

    <i>ΔrpoS Bb</i> form round bodies within nymphal midguts during the later stages of feeding.

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    <p>Representative TEM images of nymphal midguts containing WT-<i>gfp</i> and <i>ΔrpoS-gfp</i> spirochetes isolated at (A–B) 48 or (C-D) 72 h post-placement. Color overlays are used to highlight normal spirochetes (red), round bodies (blue), and peritrophic membranes (PM, green); scale bars = 2 µm.</p

    Factors Controlling Fibroblast Growth Factor Receptor-1's Cytoplasmic Trafficking and Its Regulation as Revealed by FRAP Analysis

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    Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) “nuclear pathway,” where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) “membrane pathway,” in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1

    Loss of RpoS and CoADR exacerbates round body formation <i>in vitro</i>.

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    <p><i>Bb</i> were incubated in RPMI for 1–4 days. A minimum of 300 organisms were counted per strain for each time point. Experiments were performed in triplicate; error bars represent means ± SEM. Representative images of fields used to quantify round body formation are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002532#ppat.1002532.s007" target="_blank">Figure S7</a>. The percentages of round bodies formed by Δ<i>rpoS-gfp</i> and Δ<i>cdr</i> isolates were significantly greater than WT on days 1 through 4 (<i>p</i>≤0.002). Round body formation by Δ<i>rpoS-gfp</i> and Δ<i>cdr</i> isolates was significantly different on days 3 and 4 (<i>p</i>≤0.002).</p

    One or more RpoS-dependent gene products, independent of <i>ospC</i>, are required for spirochete's to penetrate into the hemolymph; see Figure S1 for spirochete burden analyses.

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    a<p>Hemolymph was collected from feeding nymphs 72 h post-placement and cultured in BSK-II. The denominator represents the total number of ticks analyzed.</p>b<p>Cultures were monitored for the presence of spirochetes by dark field microscopy for 8 weeks.</p>c<p>The time frames in which organisms (WT, WT-<i>gfp</i>, <i>ΔospC</i>, <i>ΔrpoS</i>+<i>rpoS</i>, and <i>ΔrpoS-gfp</i>+<i>rpoS</i>) were recovered from the hemolymph was highly similar in each experiment (n = 3).</p>d<p>Skin of a C3H/HeJ mouse (4–6 sites per mouse) was excised from the site where a nymph was attached at the indicated time post-repletion. A minimum of 3 mice were tested per isolate. The denominator represents the total number of bite sites analyzed; ND = not determined.</p>e<p>Mice were sacrificed 4 weeks post-inoculation and the indicated tissues cultured in BSK-II. The denominator represents the total number of mice analyzed per isolate.</p>f<p>Ear punches were performed at 2 and 4 weeks post-feeding and cultured in BSK-II.</p

    Round bodies within fed nymphal midguts recover into elongated spirochetes.

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    <p><i>ΔrpoS</i>-infected nymphs were removed at 72 h post-placement and midguts dissected into RPMI. (A) The addition of BSK-II induced the recovery of round bodies into elongated spirochetes. (B) Round bodies do not recover when midguts were submerged in RPMI. Scale bars = 10 µm. See also the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002532#ppat.1002532.s011" target="_blank">Video S1</a> of the rapid recovery of <i>in vitro</i>-derived organisms.</p

    Contours of the RpoS<sub>Bb</sub> regulon in <i>I. scapularis</i>.

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    <p>qRT-PCR analysis of (A) absolutely and (B) partially RpoS-dependent upregulated genes and (C) RpoS-repressed genes selected from microarray data derived from <i>Bb</i> cultivated within DMCs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002532#ppat.1002532-Caimano1" target="_blank">[13]</a>. A representative sample of genes is shown; data for the remaining genes are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002532#ppat.1002532.s002" target="_blank">Figure S2</a>. Expression profiling was performed using fed larvae, unfed and fed nymphs that had been naturally-infected with WT <i>Bb</i> as well as fed nymphs that had been infected as larvae by immersion with either WT-<i>gfp</i> or Δ<i>rpoS-gfp</i> isolates. Values represent the average <i>flaB</i>-normalized transcript copy number ± standard error of the mean (SEM) for each gene; values are considered significantly different when <i>p</i> is ≤0.05 (indicated by asterisks). Composite confocal images through the full thickness of nymphal midguts at 72 h post-placement showing the distribution of spirochetes expressing <i>gfp</i> under the control of the (D) <i>flaB</i> or (E) <i>ospA</i> promoter. A detailed schematic indicating how confocal images of fed midguts were acquired is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002532#ppat.1002532.s003" target="_blank">Figure S3</a>. Here and elsewhere, green represents GFP<sup>+</sup> spirochetes while red indicates midgut epithelial cells labeled with FM4-64; scale bars = 25 µm.</p

    <i>Borrelia burgdorferi</i> strains used in this study.

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    1<p>Antibiotic resistance determined by growing spirochetes in the presence of the following antibiotics: erythromycin (Erm, 0.06 µg/ml); kanamycin (Kan, 400 µg/ml); and/or gentamycin (Gent, 50 µg/ml).</p
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