Image_1_The Structural Integrity of Plasmid-Encoded Pgp3 Is Essential for Induction of Hydrosalpinx by Chlamydia muridarum.pdf

Pgp3 consists of globular N- and C-terminal domains connected by a triple-helical coiled-coil middle domain. We demonstrated previously that Pgp3 is required for induction of hydrosalpinx by Chlamydia muridarum. We constructed C. muridarum transformants harboring deletion of the Pgp3 N-terminus (pgp3Δn), C-terminus (pgp3Δc), or middle domain (pgp3Δm). C3H/HeJ and CBA/J mice infected with pgp3Δn or pgp3Δm failed to induce hydrosalpinx in oviduct tissue. However, the pgp3Δc transformant induced mild hydrosalpinx in 20% of C3H/HeJ mice (severity score 0.2 ± 0.6) and in 40% of CBA/J mice (severity score 0.8 ± 1.3). The attenuated pathogenicity of the transformants harboring Pgp3 domain deletions was correlated with impaired in vitro growth and significantly reduced infectivity in the mouse lower genital tract. Moreover, the oviduct tissue of C3H/HeJ and CBA/J mice infected with the Pgp3-domain-deficient transformants displayed less inflammatory cell infiltration. Thus, the structural integrity of plasmid-encoded Pgp3 is essential for induction of hydrosalpinx by C. muridarum.</p

The Role of TRAPγ/SSR3 in Preproinsulin Translocation into the Endoplasmic Reticulum

In the endoplasmic reticulum (ER), the Translocation-Associated Protein complex (TRAP, also called Signal sequence receptor, SSR) includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2 and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2 or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting co-translational and post-translational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels — observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkable, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis

Urolithin A Alleviates Colitis in Mice by Improving Gut Microbiota Dysbiosis, Modulating Microbial Tryptophan Metabolism, and Triggering AhR Activation

Urolithin A (UroA) is a microbial metabolite derived from ellagitannins and ellagic acid with good bioavailability. In this study, we explored the anticolitis activity of UroA and clarified the mechanism by 16S rDNA sequencing and metabonomics. UroA alleviated dextran sulfate sodium (DSS)-induced colitis in mice, characterized by a decreased disease activity index, increased colon length, and improved colonic histopathological lesions, along with inhibited phosphorylation of the mitogen-activated protein kinase signaling pathway. In addition, UroA improved gut microbiota dysbiosis and modulated the microbiota metabolome. Furthermore, targeted metabolomics focused on tryptophan catabolites showed that UroA significantly increased the production of indole-3-aldehyde (IAld) and subsequently led to increased colonic expression of aryl hydrocarbon receptor (AhR) and promoted the serum content of IL-22 in mice with colitis. Collectively, our data identified a novel anticolitis mechanism of UroA by improving gut microbiota dysbiosis, modulating microbial tryptophan metabolism, promoting IAld production, and triggering AhR/IL-22 axis activation. However, a limitation noted in this study is that these beneficial effects of UroA were found at 50 μM in vitro and 20 mg/kg in vivo, which were nonphysiological concentrations

Construction and characterization of luciferase-expressing C. muridarum.

<p>(A) The DNA fragment consisting of pgp4 promoter (p4, orange), luciferase gene (luc, red) and transcriptional termination signal (not shown) was inserted in front of the GFP-CAT gene (green) of the pGFP::CM shuttle vector to create a new recombinant plasmid pGFP-luc-CM. (B) The newly created recombinant plasmid pGFP-luc-CM was used for transforming a plasmid-free <i>C. muridarum</i> organism to produce a plasmid-competent and luciferase-expressing <i>C. muridarum</i> (grey inclusions in panel a and green in b). (C) HeLa cells infected with the luciferase-expressing <i>C. muridarum</i> organisms were used for both measuring luciferase transcripts using qRT-PCR (panel a) and detecting luciferase activity by quantitating intensity of bioluminescence generated from luciferase-catalyzed luciferin (panel b). Note that both luciferase gene expression and activity became detectable at 12, peaked at 15 and significantly decreased by 24 hours after infection, with a coefficient (r_s) of 0.8333 and p value of 0.0053 (student <i>t</i>-Test).</p

Correlation of live organism recovery with bioluminescence intensity.

<p>The live organisms recovered from the lower genital tract (swab IFUs, diamond) was analyzed against bioluminescence intensity or luciferase activity measured in the lower (circle) or upper (triangle) genital tracts (both in log₁₀ shown along Y-axis) along the entire infection course after intravaginal inoculation (X-axis) using Spearman Correlation followed by student <i>t</i>-Test. Note that a correlation coefficient (r_s) of 0.81 with a p value of 0.049 (<i>t</i>-Test) was found between the IFUs and the upper genital tract bioluminescence while 0.89 with p = 0.017 found between the IFUs and the lower genital tract bioluminescence.</p

Comparison of plaque sizes among <i>Chlamydia muridarum</i> transformants.

<p>(A) The following <i>C</i>. <i>muridarum</i> organisms were inoculated onto McCoy monolayers grown in 6-well plate: Wild type <i>C</i>. <i>muridarum</i> [CM(wt), image a], plasmid-free <i>C</i>. <i>muridarum</i> [CMUT3 (pf), b], CMUT3 transformed with the intact plasmid pGFP::CM (CM-pGFP::CM, c) or the pGFP::CM with a premature stop codon in pgp3 (CM-pgp3S, d), 4 (CM-pgp4S, e) or 5 (CM-pgp5S, f) or deletion of pgp5 (CM-Δpgp5, g) or 7 (CM-Δpgp7, h). The cultures were allowed to grow in 0.55% of argarose-containing medium for 5 days before neutral red staining and picture taking. (B) For quantitating the plaque sizes, the stained plates were scanned and the plaque sizes were measured in pixels using the customer-designed software PlaqueDetector. All plaque sizes were normalized using the areas of the corresponding wells so that plaque areas from different wells became comparable. The mean areas and standard deviations were used for comparing between different groups. Note that the plasmid-free CMUT3 and CM-pgp4S produced significantly smaller plaques (p<0.01, Student <i>t</i>-test).</p

Oviduct inflammatory infiltration induced by Pgp5-deficient <i>C</i>. <i>muridarum</i>.

<p>The oviduct tissues of mice infected with <i>C</i>. <i>muridarum</i> organisms as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124840#pone.0124840.g002" target="_blank">Fig 2</a> legend were harvested and subjected to H&E staining for microscopic evaluation of inflammatory infiltration. (A) Representative images from each group taken under 4X (left, panels a-f) and 100X (right, panels a1-f1) objective lens. White rectangles in the 4X lens images indicated the same areas from which the right images were taken under 100X lens. (B) The severity of inflammatory infiltration in oviduct tissue was scored as described in Materials and Methods and listed along the Y- axis [solid circle for CM(wt), star for CMUT3(pf), solid upright triangle for CM-pGFP::CM, solid upside down triangle for CM-pgp5S, solid diamond for CM-Δpgp5 and open circle for CM-Δpgp7). Note that mice infected with CM-pgp5S or CM-Δpgp5 but not CM-Δpgp7 developed significantly lower levels of inflammatory infiltration. *p<0.05 while **p<0.01 (Wilcoxon rank-sum test, all groups were compared against the full plasmid-complemented CM-pGFP::CM).</p

Comparison of the primary growth curves among <i>Chlamydia muridarum</i> transformants.

<p>All <i>C</i>. <i>muridarum</i> organisms were inoculated onto McCoy monolayers grown in 24-well plates at an MOI of 0.5 (to avoid over infection). The infected cultures were harvested at 6, 12, 18, 24, 30 & 36 hours after infection as shown along the X-axis for titrating the number of live organisms on fresh monolayers of HeLa cells. The number of live organisms recovered from each culture at each time point was calculated into progeny IFU (inclusion forming unit) per culture as listed along the Y-axis in Log scale. The experiment was repeated 3 three times with duplicate in each. There were no significant differences in the live organism yields between any two organisms at any time points measured (Krustal-Wallis).</p

Live organism shedding from mouse lower genital tract following infection with Pgp5-deficient <i>C</i>. <i>muridarum</i>.

<p>The C3H/Hej mice were intravaginally infected with <i>C</i>. <i>muridarum</i> organisms as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124840#pone.0124840.g002" target="_blank">Fig 2</a> legend. On different days after infection as shown along the X-axis, vaginal swabs were taken for titrating live organisms on HeLa cell monolayers. The live organisms recovered from each swab are expressed as Log₁₀ IFUs along the Y-axis. Note that mice infected with the plasmid free CMUT3 but not other groups displayed a significantly reduced shedding course than the group infected with CM-pGFP::CM. *p<0.05 (Wilcoxon rank-sum test).</p

Cytokines from oviduct tissues of mice infected with <i>C</i>. <i>muridarum</i> with or without Pgp5 deficiency.

<p>Oviduct tissue homogenates were produced from mice infected with CM-pGFP::CM (2^nd column, n = 5) or CM-pgp5S (3^rd column, n = 5) on day 10 after intravaginal inoculation for simultaneously measuring 27 cytokines using a multiplex bead array assay. All cytokines are expressed in pg/mL as mean plus/minus standard deviation. The means were used for calculating ratio (2^nd last column) and Student’s t-test (last column). P values equal to or under 0.05 were listed in bold face. Note that 20 of the 27 cytokines were significantly lower in the oviducts of mice infected with CM-pgp5S than with CM-pGFP::CM.</p><p>Cytokines from oviduct tissues of mice infected with <i>C</i>. <i>muridarum</i> with or without Pgp5 deficiency.</p