Unraveling the mechanism of molecular doping in organic semiconductors

The mechanism by which molecular dopants donate free charge carriers to the host organic semiconductor is investigated and is found to be quite different from the one in inorganic semiconductors. In organics, a strong correlation between the doping concentration and its charge donation efficiency is demonstrated. Moreover, there is a threshold doping level below which doping simply has no electrical effect. Copyright cop. 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei

A Type VI secretion system encoding locus is required for Bordetella bronchiseptica immunomodulation and persistence in vivo.

Type VI Secretion Systems (T6SSs) have been identified in numerous gram-negative pathogens, but the lack of a natural host infection model has limited analysis of T6SS contributions to infection and pathogenesis. Here, we describe disruption of a gene within locus encoding a putative T6SS in Bordetella bronchiseptica strain RB50, a respiratory pathogen that circulates in a broad range of mammals, including humans, domestic animals, and mice. The 26 gene locus encoding the B. bronchiseptica T6SS contains apparent orthologs to all known core genes and possesses thirteen novel genes. By generating an in frame deletion of clpV, which encodes a putative ATPase required for some T6SS-dependent protein secretion, we observe that ClpV contributes to in vitro macrophage cytotoxicity while inducing several eukaryotic proteins associated with apoptosis. Additionally, ClpV is required for induction of IL-1β, IL-6, IL-17, and IL-10 production in J774 macrophages infected with RB50. During infections in wild type mice, we determined that ClpV contributes to altered cytokine production, increased pathology, delayed lower respiratory tract clearance, and long term nasal cavity persistence. Together, these results reveal a natural host infection system in which to interrogate T6SS contributions to immunomodulation and pathogenesis

Type Six Secretion System of <i>Bordetella bronchiseptica</i> and Adaptive Immune Components Limit Intracellular Survival During Infection

<div><p>The Type Six Secretion System (T6SS) is required for <i>Bordetella bronchiseptica</i> cytotoxicity, cytokine modulation, infection, and persistence. However, one-third of recently sequenced <i>Bordetella bronchiseptica</i> strains of the predominantly human-associated Complex IV have lost their T6SS through gene deletion or degradation. Since most human <i>B</i>. <i>bronchiseptica</i> infections occur in immunocompromised patients, we determine here whether loss of Type Six Secretion is beneficial to <i>B</i>. <i>bronchiseptica</i> during infection of immunocompromised mice. Infection of mice lacking adaptive immunity (Rag1^-/- mice) with a T6SS-deficient mutant results in a hypervirulent phenotype that is characterized by high numbers of intracellular bacteria in systemic organs. In contrast, wild-type <i>B</i>. <i>bronchiseptica</i> kill their eukaryotic cellular hosts via a T6SS-dependent mechanism that prevents survival in systemic organs. High numbers of intracellular bacteria recovered from immunodeficient mice but only low numbers from wild-type mice demonstrates that <i>B</i>. <i>bronchiseptica</i> survival in an intracellular niche is limited by B and T cell responses. Understanding the nature of intracellular survival during infection, and its effects on the generation and function of the host immune response, are important to contain and control the spread of <i>Bordetella</i>-caused disease.</p></div

The T6SS modulates virulence and bacterial dissemination.

<p>(A) Groups of Rag1^-/- mice (n = 8) were inoculated with 5x10⁵ CFU of RB50 (blue) or RB50Δ<i>clpV</i> (red) and were monitored for survival. (B) Groups of Rag1^-/- mice (n = 4 per group) were inoculated with 5x10⁵ CFU of RB50 (blue) versus RB50Δ<i>clpV</i> (red) and dissected on day 21 p.i. for bacterial enumeration in respiratory tract and systemic organs. The experiment was performed three times with similar results and a representative data set is shown. (C) Rag^-/- mice were inoculated with 5x10⁵ CFU RB50 (blue) versus RB50Δ<i>clpV</i> (red) and bacteria were enumerated from the spleen, liver, and kidney on days 3, 7, 21, and 35. With the exception of RB50Δ<i>clpV</i> on day 21 (n = 1), three mice were sacrificed per group per timepoint. * denotes p value <0.05. Grey dotted line indicates the limit of detection.</p

Deletion of <i>clpV</i> increases intracellular survival <i>in vitro</i>.

<p>(A) RAW264.7 macrophages were infected with RB50 (blue, n = 4) or RB50Δ<i>clpV</i> (red, n = 4) at an MOI of 100 and bacterial invasion and intracellular survival was determined at 1, 24, and 48 hour after addition of gentamicin. The experiment was conducted five times with similar results and a representative dataset is shown. (B) The cytotoxicity of RAW264.7 macrophages infected with RB50 (blue) or RB50Δ<i>clpV</i> (red) at an MOI of 100 was determined 1 hour and 24 hours after gentamicin application. The average percent cytotoxicity of four wells in three different experiments was measured by (LDH release from a well / LDH release from positive control well) x 100 ±SE is shown. * denotes p value <0.05. Grey line indicates limit of detection.</p

Clinical <i>B</i>. <i>bronchiseptica</i> strains that have lost their T6SS locus are aggregated in Complex IV.

<p>Genomes of 58 <i>B</i>. <i>bronchiseptica</i> clinical isolates were compared to prototypical RB50 genome. Presence of T6SS loci, as well as presence of pseudogenes in T6SS loci, was determined for all clinical isolates. Clinical strains containing an intact T6SS (blue), strains lacking a T6SS locus (red), and strains containing a pseudogene in the T6SS locus (pink) were divided based on whether they come from Complex I (A) or Complex IV (B).</p

Loss of <i>clpV</i> is required for systemic recovery.

<p>Groups of Rag1^-/- mice were either infected singly with RB50 (blue circles, n = 3) or RB50Δ<i>clpV</i> (red circles, n = 4) or co-infected with RB50 and RB50Δ<i>clpV</i> (red squares and blue squares, n = 4), and (A) respiratory tract bacterial numbers or (B) systemic organ bacterial numbers were enumerated on day 21 post-inoculation. This experiment was performed twice with similar results and a representative dataset is shown. ND signifies not detected. Grey line indicates the limit of detection.</p

<i>clpV</i> lowers inflammation and pathology <i>in vivo</i>.

<p>(A) Groups of four Rag1^-/- mice were inoculated with 5x10⁵ CFU of RB50 (blue) or RB50Δ<i>clpV</i> (red) and a histopathological analysis was conducted on the lung and liver of infected mice on day 21 p.i. for scoring of inflammation. (B) Representative H&E lung and liver sections from Rag1^-/- mice on day 21 p.i. after inoculation with 5x10⁵ CFU of RB50 (blue) or RB50Δ<i>clpV</i> (red) with average pathology scores in parentheses. (C) Groups of Rag1^-/- mice were inoculated with 5x10⁵ CFU RB50 (blue) and RB50Δ<i>clpV</i> (red) and elicited IL-1β levels were determined from the lung and spleen on day 21 p.i. This experiment was performed twice with similar results and a representative dataset is shown. ND signifies not detected. * denotes p value <0.05.</p

Confirmation of RB50Δ<i>clpV</i> construction by PCR and RT-PCR analysis.

<p>A. PCR analysis of <i>clpV</i> in RB50 (left) and RB50Δ<i>clpV</i> (right). Size markers are designated on the left. B. RT-PCR analysis of relative expression of <i>vgrG, hcp, clpV, icmF, bvgS,</i> and <i>fhaB</i> in RB50Δ<i>clpV</i> relative to RB50 expressed as mean ± standard deviation. Each gene was normalized to the expression of 16S RNA.</p