Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus pneumoniae Pilus

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility

Inhibition of Sgk1 depresses the OxA-dependent induction of a small set of transcripts.

<p>GT1-7-OX₁ cells were treated with an Sgk1 inhibitor, GSK-650394, prior to addition of OxA. Total RNA was purified from lysates and used for qPCR analysis. The genes displayed had their level of orexin-induced transcription inhibited by GSK-650394. Bars represent averages (n = 1, reads done in triplicate) while error bars represent SEM.</p

Partial list of transcription factors regulated by OxA in GT1-7-OX₁ cells.

<p>Partial list of transcription factors regulated by OxA in GT1-7-OX₁ cells.</p

Genes regulated by both sleep deprivation and OX₁ signaling.

<p>Genes regulated by both sleep deprivation and OX₁ signaling.</p

Heat maps indicating the genes most highly regulated by OX₁ activation.

<p>(A) Vehicle-treated vs. 3-hour treatment with OxA. (B) Vehicle-treated vs. 8-hour treatment with OxA. The values (colors) shown are the regularized log transformations of the original count data.</p

Characterization of orexin receptor expression via qPCR.

<p>Characterization of orexin receptor expression via qPCR.</p

Characterization of cell lines previously reported to express orexin receptors.

<p>Each cell line was assayed for the presence of functional orexin receptors via the IP-One HTRF Assay. Cells were incubated with orexin A at various concentrations for 45 min. A CHO-based cell line stably expressing OX₁ (CHO-OX₁) was used as a positive control. The data are presented as a percentage of the baseline HTRF ratio (A₆₆₅/A₆₂₀ x 10000). Data points are mean (n = 3) and error bars represent standard error of the mean (SEM).</p

A partial set of transcription factors identified by PSCAN analysis of genes differentially regulated by OX₁ signaling.

<p>A partial set of transcription factors identified by PSCAN analysis of genes differentially regulated by OX₁ signaling.</p

Global analysis of gene expression mediated by OX₁ orexin receptor signaling in a hypothalamic cell line

<div><p>The orexins and their cognate G-protein coupled receptors have been widely studied due to their associations with various behaviors and cellular processes. However, the detailed downstream signaling cascades that mediate these effects are not completely understood. We report the generation of a neuronal model cell line that stably expresses the OX₁ orexin receptor (OX₁) and an RNA-Seq analysis of changes in gene expression seen upon receptor activation. Upon treatment with orexin, several families of related transcription factors are transcriptionally regulated, including the early growth response genes (<i>Egr</i>), the Kruppel-like factors (<i>Klf</i>), and the <i>Nr4a</i> subgroup of nuclear hormone receptors. Furthermore, some of the transcriptional effects observed have also been seen in data from <i>in vivo</i> sleep deprivation microarray studies, supporting the physiological relevance of the data set. Additionally, inhibition of one of the most highly regulated genes, serum and glucocorticoid-regulated kinase 1 (<i>Sgk1</i>), resulted in the diminished orexin-dependent induction of a subset of genes. These results provide new insight into the molecular signaling events that occur during OX₁ signaling and support a role for orexin signaling in the stimulation of wakefulness during sleep deprivation studies.</p></div

Generation of a GT1-7-based cell line stably expressing OX₁.

<p>A lentiviral transduction system was used to generate GT1-7 cells that stably express OX₁. (A) Presence of OX₁ mRNA in the transduced cells was verified via qPCR. Data were analyzed by the 2^-ΔΔC_T method, using mouse GAPDH as the reference, and are expressed as relative quantity (RQ), normalized to the parental cell line. (B) Parental, mock-transduced, and OX₁-transduced GT1-7 cells were tested for the presence of functional OX₁ via the IP-One HTRF Assay. (C) An orexin receptor antagonist, SB-334867, blocked orexin signaling in GT1-7-OX₁ cells in a concentration-dependent manner. Data points are mean (n = 3), error bars represent SEM.</p