Aortic stenting in the growing sheep causes aortic endothelial dysfunction but not hypertension: Clinical implications for coarctation repair

Stent implantation is the treatment of choice for adolescents and adults with aortic coarctation (CoAo). Despite excellent short-term results, 20%-40% of the patients develop arterial hypertension later in life, which was attributed to inappropriate response of the aortic baroreceptors to increased stiffness of the ascending aorta (ASAO), either congenital or induced by CoAo repair. In particular, it has been hypothesized that stent itself may cause or sustain hypertension. Therefore, we aimed to study the hemodynamic and structural impact following stent implantation in the normal aorta of a growing animal

Genetic plasticity of the Shigella virulence plasmid is mediated by intra- and inter-molecular events between insertion sequences

Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segrega tional killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecula r recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS 1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV

Virulence plasmid pINV as a genetic signature for Shigella flexneri phylogeny

Shigella flexneri is a major health burden in low- and middle-income countries, where it is a leading cause of mortality associated with diarrhoea in children, and shows an increasing incidence among travellers and men having sex with men. Like all Shigella spp., S. flexneri has evolved from commensal Escherichia coli following the acquisition of a large plasmid pINV, which contains genes essential for virulence. Current sequence typing schemes of Shigella are based on combinations of chromosomal genetic loci, since pINV-encoded virulence genes are often lost during growth in the laboratory, making these elements inappropriate for sequence typing. By performing comparative analysis of pINVs from S. flexneri strains isolated from different geographical regions and belonging to different serotypes, we found that in contrast to plasmid-encoded virulence genes, plasmid maintenance genes are highly stable pINV-encoded elements. For the first time, to our knowledge, we have developed a S. flexneri plasmid multilocus sequence typing (pMLST) method based on different combinations of alleles of the vapBC and yacAB toxin-antitoxin (TA) systems, and the parAB partitioning system. This enables typing of S. flexneri pINV plasmids into distinct 'virulence sequence types' (vSTs). Furthermore, the phylogenies of vST alleles and bacterial host core genomes suggests an intimate co-evolution of pINV with the chromosome of its bacterial host, consistent with previous findings. This work demonstrates the potential of plasmid maintenance loci as genetic characteristics to study as well as to trace the molecular phylogenesis of S. flexneri pINV and the phylogenetic relationship of this plasmid with its bacterial host

Chromosomal integration of pINV occurs <i>via</i> distinct copies of IS<i>1294</i>.

<p>(A) Agarose gel electrophoresis of pINV DNA purified from nine independent CR^- colonies (lanes 1–9) emerging from native <i>mvp</i>^WT at 21°C that retained all the virulence-related genes tested by multiplex PCR. Lanes: (lane labelled M) M90T, (B) BS176, (D) T3SS PAI-deleted M90T. (B) Schematic representation of chromosomal pINV integration in isolate 4 (left) and isolate 6 (right). The chromosomal and plasmid origins of replication are shown in yellow. ORFs are shown with the same colour coding as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007014#pgen.1007014.g002" target="_blank">Fig 2</a>.</p

pINV integration results in downregulation of T3SS PAI gene expression.

<p>qRT-PCR analysis of <i>ipaB</i> (A), <i>virB</i> (B) and <i>virF</i> (C) in M90T, and isolates 4 and 6. Relative gene expression was determined by the 2^-ΔΔ method. Error bars show mean + S.E.M. of four biological replicates. **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001 ****, <i>p</i> ≤ 0.0001. Values analysed with one-way ANOVA, Tukey multiple comparisons test.</p

The position of <i>mvpAT</i> influences loss of CR binding.

<p>(A) Schematic representation of the repositioning of <i>mvpAT</i> near the T3SS PAI in ectopic <i>mvp</i> strains. (B) Proportion of CR^- colonies in <i>S</i>. <i>flexneri</i> native <i>mvp</i>^WT (reproduced from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007014#pgen.1007014.g001" target="_blank">Fig 1A</a> for statistical comparison), native, ectopic <i>mvp</i>^WT and ectopic <i>mvp</i>^D7A after approximately 50 generations growth at 37°C. Solid line: mean of six biological replicates. (C) Multiplex PCR analysis was performed as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007014#pgen.1007014.g001" target="_blank">Fig 1B</a>. Results are shown as mean (n = six biological replicates). ****, <i>p</i> ≤ 0.0001; values analysed with one-way ANOVA, Tukey multiple comparisons test.</p

Excision of pINV restores type III secretion.

<p>(A) CR-TSA plates of strains grown at 37°C overnight: M90T (M), BS176 (B), T3SS PAI-deleted M90T (D), isolate 4 and its revertants (middle row), and isolate 6 and its revertants (lower row). (B) Agarose gel electrophoresis of purified plasmid DNA from control strains as for Panel A and plasmid-integrated isolates 4 and 6 (CR^-), and two revertants (r1 and r2) derived from isolates 4 and 6. (C) Secretion through the T3SS induced by CR. Legend as for panel B. The bands corresponding to secreted effectors are indicated.</p

Emergence of CR^- colonies in <i>S</i>. <i>flexneri</i> results from loss of the PAI at 37°C.

<p>(A) Proportion of CR^- colonies in <i>S</i>. <i>flexneri</i> M90T, native <i>mvp</i>^WT <i>and</i> native <i>mvp</i>^D7A after approximately 50 generations at 37°C. Solid line: mean (n = six biological replicates). (B) Percentage of CR^- colonies lacking specified virulence-related genes. Loss of the entire plasmid is inferred by loss of the origin of replication; “others” refers to CR^- colonies that contain <i>virB</i>, <i>virF</i> and the origin of replication. Eight independent CR^- colonies obtained from native <i>mvp</i>^WT grown in six biological repeats (total 48 colonies) were analysed by multiplex PCR. ***, <i>p</i> ≤0.001; ****, <i>p</i> ≤0.0001; n.s., not significant; values analysed with one-way ANOVA, Tukey multiple comparisons test. (C) BLAST Ring Image Generator (BRIG 0.95 and BLASTN v2.2.29) alignment of plasmid sequences from twenty CR^- colonies that had emerged from native <i>mvp</i>^WT at 37°C; each ring represents the plasmid from an independent CR^- colony. <i>S</i>. <i>flexneri</i> M90T pWR100 (inner black ring) shown as the reference.</p

Characterization of CR^- colonies emerging at 21°C.

<p>(A) Proportion of CR^- colonies in <i>S</i>. <i>flexneri</i> native <i>mvp</i>^WT, native <i>mvp</i>^D7A, ectopic <i>mvp</i>^WT and ectopic <i>mvp</i>^D7A after approximately 50 generations at 21°C. Solid line: mean of six biological replicates. (B) Multiplex PCR analysis was performed as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007014#pgen.1007014.g001" target="_blank">Fig 1B</a>. Results are shown as mean (n = six biological replicates). *, <i>p</i> ≤ 0.05; ****, <i>p</i> ≤ 0.0001; n.s., not significant; values analysed with one-way ANOVA, Tukey multiple comparisons test.</p