The role of vascular smooth muscle Sirtuin-1 in aortic aneurysms

BACKGROUND: Sirtuin-1 (SirT1) is a NAD+-dependent deacetylase essential for maintaining the structure and function of the vasculature. Reduced SirT1 expression and activity has been correlated with the development of vascular diseases, mainly attributed to loss of SirT1’s anti-oxidant and anti-inflammatory beneficial effects. We previously found that deletion of vascular smooth muscle (VSM) SirT1 in mice is associated with increased matrix metalloproteinases (MMPs) and the subsequent development of aortic dissections or ruptures in response to the hypertensive peptide angiotensin II. Based on these previous findings, we hypothesize that loss of SirT1 activity is involved in the pathogenesis of AA. SirT1 is a stress response gene, its deacetylase activity can be impaired by excessive oxidative stress. We postulate that mutating three cysteine residues in SirT1’s catalytic domain can prevent its inactivation by oxidative insults and protect against AA and other vascular diseases. OBJECTIVES: assess the role of SirT1 in a genetic mouse model of Marfan Syndrome that develops AA; (2) Determine design and optimize an enzyme-based colorimetric ELISA to determine SirT1 activity in mouse VSM cells and aortas; (3) Produce an adeno-associated virus (AAV) expressing an oxidant-resistant triple mutant SirT1 in VSM cells that has the potential to mitigate the downstream outcomes derived from alterations in SirT1 activity, such as MMPs activation and development of AA in mgR-/- mice. METHODS: mgR-/- and littermate mgR+/+ (WT) mice aortas and VSM cells were cultured in conditioned medium and the activity of released MMPs was determined by in-gel zymography. For the development of the SirT1 activity assay, we designed a multi-step sandwich ELISA that captures a biotin- and FLAG-tagged acetylated p53 peptide, used as SirT1 deacetylase substrate. Amounts of acetylated and total p53 peptide were sequentially detected with antibodies and colorimetric substrates as index of SirT1 deacetylase activity. AAVs expressing a control or triple mutant SirT1 (3M) were produced in HEK293T cells; VSM cells were then infected with control or 3M AAV and SirT1 protein expression levels were measured by Western Blot. RESULTS: MMPs activity is increased in aortas and VSMC of mgR-/- mice; the first stage of optimization of the SirT1 activity assay successfully defined the assay conditions and experimental design, and it is ready to be optimized with mgR-/- cell and tissue samples; our novel control and SirT1 triple mutant AAVs were produced and successfully overexpressed in VSM cells.2024-03-14T00:00:00

A bistable prokaryotic differentiation system underlying development of conjugative transfer competence.

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7-4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements

Fitness loss of <i>P. putida</i> UWC1 (ICE<i>clc</i>) caused by the <i>mfsR</i> deletion.

<p>(A) Survival of <i>P. putida</i> UWC1 (ICE<i>clc</i>, 2737) and <i>P. putida</i> UWC1 (ICE<i>clc</i>-Δ<i>mfsR</i>, 4322) pregrown in suspended culture to stationary phase on 3-chlorobenzoate (3CBA) or succinate (succ), and plated from there on 3CBA or succinate agar. (E.g., 3CBA-3CBA, suspended culture on 3CBA, plated on 3CBA agar). Survival expressed as colony forming units (CFU) on the agar plate per ml of stationary phase culture. Data bars indicate the average from independent biological triplicates. Error bars indicate the calculated standard deviation from the average. (B) Phase-contrast micrographs at 1000-fold magnification of microcolonies of <i>P. putida</i> UWC1 (ICE<i>clc</i>) and <i>P. putida</i> (ICE<i>clc</i>-Δ<i>mfsR</i> P_int-<i>egfp</i>, 4612) growing on agarose surface supplemented with 0.1 mM 3CBA. Shown are a regular stationary phase microcolony of <i>P. putida</i> with wild-type ICE<i>clc</i> and a transfer competent microcolony (tcm), occurring at 1-3% frequency as reported previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004441#pgen.1004441-Reinhard1" target="_blank">[30]</a>. For comparison, massive lysis (white arrows) and cellular malformations formed in many microcolonies of <i>P. putida</i> UWC1 with the <i>mfsR</i> deletion.</p

Detailed view on the differential expression of the <i>mfsR</i> operon in <i>P. putida</i> ICE<i>clc</i> wild-type or mutants.

<p>(A) <i>MarR</i> deletion mutant versus wild-type. (B) <i>mfsR</i> deletion mutant versus wild type. (C) <i>tciR</i> deletion mutant versus wild-type. (D) <i>mfsR</i>-'<i>marR</i> deletion mutant versus wild-type. (E) <i>marR</i> versus <i>tciR</i> deletions. (F) <i>mfsR-</i>transposon insertion mutant versus wild-type. Panels show ²log-fold change of expression level per microarray probe in this region of ICE<i>clc</i> for exponential (dark dots) and stationary phase cells (white dots). Genetic map of the region drawn at the bottom of each section for clarity. Arrows represent genes, deleted regions are indicated by stippled bars and corresponding probes are within brackets.</p

Effect of mutations in the <i>mfsR</i> region on the expression of the P_mfsR-, P_int- and P_inR-promoters of ICE<i>clc</i> in <i>P. putida</i> UWC1.

<p>(A) mCherry expression from the <i>mfsR</i> promoter added in single copy to the chromosome of the indicated <i>P. putida</i> UWC1 strains (relevant genotypes and strain numbers specified below the graph). mCherry expression measured on individual cells (n = 1000) by epifluorescence microscopy in late exponential phase of cultures grown on 10 mM succinate and expressed as box plots (AU, arbitrary units at 20 ms exposure time). (B) Scatter plot of GFP and mcherry fluorescence in single cells of <i>P. putida</i> UWC1 (ICE<i>clc</i>) wild-type, Δ<i>mfsR</i>, Δ<i>tciR</i> or Δ<i>marR</i> deletions, equipped with a single copy mini-transposon containing the P_int-<i>egfp</i> and P_inR-<i>mcherry</i> fusions. Panels show expression of both markers at different growth phases, as indicated, with colors representing genotypes with independent mini-Tn<i>5</i> insertions. Note as example the subpopulation of wild-type cells (dotted ellips) expressing both reporters, compared to the majority of cells in the <i>mfsR</i> deletion mutant but a complete absence of such subpopulation in the <i>tciR</i> and <i>marR</i> deletion mutants.</p

Effect of <i>rpoS</i> interruption on reporter gene expression from two key bistable promoters (P<i>_int</i> and P<i>_inR</i>) controlling ICE<i>clc</i> activity in stationary phase <i>P. knackmussii</i> cells grown on 3CBA.

<p>(A) Relevant details of the P<i>_int</i>-<i>egfp</i>, P<i>_inR</i>-<i>echerry</i> mini-transposon reporter construct and of the mini-transposon introducing the native <i>rpoS</i> gene under control of its own promoter (only in the complemented strain B13-2993, orientation of this insert unknown). Transposon boundaries indicated by thick black lines. (B) Scatter plots showing eGFP (from P<i>_int</i>) and eCherry (from P<i>_inR</i>) fluorescence intensities in single cells (circles) of B13-2581 (wild-type), B13-2673 (<i>rpoS</i>), B13-2993 (<i>rpoS</i> complemented in <i>trans</i> by mini-Tn with <i>rpoS</i>), or B13-3091 (<i>rpoS</i>, <i>inrR^−/−</i>) at 24 h and 72 h in stationary phase. Note the camera saturation in the eCherry channel above 256 units (8-bits). For signal quantification and significance testing, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002818#pgen-1002818-t002" target="_blank">Table 2</a>.</p

Schematic representation of the ICE<i>clc</i> genetic layout and relevant regulatory features.

<p>(A) Excised and integrated ICE<i>clc</i> (not to scale) with its flanking <i>attL</i> and <i>attR</i> sites, and the 18-bp repeat sequences (black pentangle) that are the target of the IntB13 integrase. The ‘transfer region’ denotes the ∼50 kb ICE<i>clc</i> part largely conserved with other genomic islands <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002818#pgen.1002818-Gaillard1" target="_blank">[21]</a>. (B) The region of the open reading frames under control of P<i>_inR</i>, among which <i>inrR</i>, the product of which has been implicated in relaying bistable expression to the P<i>_int</i> promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002818#pgen.1002818-Dubnau1" target="_blank">[34]</a>. (C) The gene region around <i>rpoS</i> in strain B13.</p

Conservation of <i>tciR</i> analogues in putative ICE<i>clc</i>-like regions in a variety of other bacterial genomes.

<p>Illustration represents <i>tciR</i> analogues (identified on the basis of a BLASTN E-value lower than 1·10^-15), and surrounding relevant gene regions in the indicated bacterial genomes (species name, accession numbers) compared to ICE<i>clc</i>. Genes are indicated as in the respective genome accession. Rectangles show annotated genes and their orientation (top, orientation towards the left; bottom, gene orientation towards to right); common colors indicate similar predicted functions. Stippled rectangles indicate common gene regions inferred from Artemis comparison, but not present in the respective annotation. ICE were inferred from (i) more than 75% nucleotide identities across the complete core region of ICE<i>clc</i>, and within a 1-100 kb window from the <i>tciR</i> position, and (ii) the presence of an integrase gene (in brown) within a 5-20 kb window from the <i>tciR</i>-analogue. Note how some genomes carry multiple different ICE from the same family (e.g., <i>Achromobacter xylosoxidans</i>, <i>Acidovorax</i> sp. strain JS42), and further how pair-wise identical ICE regions (shaded in grey) occur between different genomes. Finally note how the <i>tciR</i>-analogues often co-occur with a <i>xer</i>-type regulatory gene on the other strand (light green), and a further <i>lysR</i> gene member (yellow), but in none of the cases shown here with an <i>mfsR</i> counterpart (in red).</p

Model for regulation of ICE<i>clc</i> transfer competence.

<p>MfsR autoregulates expression of itself and of the TciR activator, without which ICE<i>clc</i> transfer decreases by 2·10³-fold. TciR may activate specifically one or more promoters on ICE<i>clc</i>, such as the RpoS-dependent P_inR-promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004441#pgen.1004441-Miyazaki2" target="_blank">[29]</a>, or a promoter upstream of the <i>parA</i>-like gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004441#pgen.1004441-Gaillard2" target="_blank">[32]</a>. Expression of the P_inR-promoter occurs preferentially in cells having highest RpoS levels, and only 1-3% of cells in a population in stationary phase visibly express reporter gene from P_inR<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004441#pgen.1004441-Miyazaki2" target="_blank">[29]</a>. InrR transmits bistable activation through an unknown process to the <i>intB13</i> promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004441#pgen.1004441-Minoia1" target="_blank">[26]</a>, and possibly simultaneously to other promoters for the genes for the conjugative system in the ICE<i>clc</i> core region <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004441#pgen.1004441-Gaillard2" target="_blank">[32]</a>. The exact mechanism of arisal of bistability is unclear as yet. For gene locations on ICE<i>clc</i>, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004441#pgen-1004441-g001" target="_blank">Figure 1A</a>.</p

Effects of the <i>mfsR</i> deletion on the growth characteristics of <i>P. putida</i> UWC1 carrying ICE<i>clc</i>.

1)<p>Generation time was calculated as ln2/μ, whereby µ (min^-1) is the slope of the regression line on a plot of the log_{culture turbidity} versus time from at least 5 points during exponential phase. Coefficients of determination (R²) were >0.96 for each growth curve.</p>2)<p>Survival rate in stationary phase of cultures on the indicated media was calculated as the ratio of the number of CFU/ml counted on MM+3CBA and the number of CFU/ml on MM+succinate plates. Succ, succinate; 3CBA, 3-chlorobenzoate.</p>3)<p>Calculated standard deviation from triplicate measurements.</p>4)<p>Calculated p-value in a two-tailed Student's t-Test using equal variance.</p