35 research outputs found
Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum, the Syphilis Spirochete
Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure’s fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete’s complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator–P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG
Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission
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
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.
<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
Loss of RpoS and CoADR exacerbates round body formation <i>in vitro</i>.
<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
Round bodies within fed nymphal midguts recover into elongated spirochetes.
<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
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.
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
Contours of the RpoS<sub>Bb</sub> regulon in <i>I. scapularis</i>.
<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.
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