<i>In planta</i> expression of the <i>cps3S</i> gene and formation of serotype 3 polysaccharide.

<p><b>A.</b> Reverse transcriptase PCR to detect <i>cps3S</i> mRNA in transgenic tobacco plants. RNA was extracted from a wildtype (Lanes 1 and 3) and a transgenic <i>N. tabacum</i> containing <i>cps3S</i> (Lanes 2 and 4). PCR products, using <i>cps3S</i> specific primers were analysed by agarose gel electrophoresis. Lanes 1 and 2 showed the absence of <i>cps3S</i> DNA in the RNA. RT-PCR on the same samples showed the presence of <i>cps3S</i> mRNA in the transgenic plant (Lane 4) but not in the wildtype (Lane 3). Lane 5 PCR of pCMS4 containing <i>cps3S</i>, done as before. The 1.3 kb amplicon in Lanes 4 and 5 shows a full-length transcript of <i>cps3S</i> is expressed in the transgenic plant. <b>B.</b> Double immunodouble diffusion. Well 1∶10 µg purified serotype 3 polysaccharide; Wells 2-4: extract from tobacco plants shown to express <i>cps3S</i>: Wells 5 and 6: extract from a wildtype tobacco plant. Well A: type 3 polysaccharide specific antiserum. The preciptin lines identify the presence of type 3 polysaccharide. <b>C.</b> Western blotting using type 3 polysaccharide specific antiserum. Lane 1: purified type 3 polysaccharide; Lane 2: wildtype plant extract; Lane 3: transgenic plant extract. <b>D</b>. High-voltage paper electrophoresis of tobacco leaf acid hydrolysates. Lanes 1-3: 25 µg of each marker, (Lane 1) galacturonic acid (GalA) and glucose, (Lane 2) glucose and β-d-glucuronosyl-(1→4)-d-glucose (GlcA–Glc) (partial hydrolysate of 10 µg type 3 pneumococcal polysaccharide) and (Lane 3) 10 µg of a mixture of mannose, α-d-glucuronosyl-(1→2)-<i>myo</i>-inositol (GlcA–Ins) and a trace of α-d-mannosyl-(1→4)-α-d-glucuronosyl-(1→2)-<i>myo</i>-inositol (Man–GlcA–Ins). Lanes 4–10: hydrolysate of polysaccharides cold-acid-extracted from 32 mg fresh weight of wildtype (Lanes 4 and 5) or transgenic (Lanes 6–10) tobacco leaves. Each lane also contains a trace of Orange G (coloured internal marker). All lanes show similar levels of staining for neutral sugars (co-migrating with glucose, near the origin). The samples were electrophoresed at pH 6.5, at 3.0 kV for 60 min (anode at top) and stained with AgNO₃. Spots of the disaccharide, GlcA–Glc, diagnostic of type 3 pneumococcal polysaccharide, are highlighted by the dashed box; these spots were quantified for grey density in PhotoShop (see histogram).</p

Immunogenicity and protective efficacy of serotype 3 pneumococcal polysaccharide produced <i>in planta</i>.

<p><b>A.</b> Concentration of serotype 3 polysaccharide-specific IgG in serum of mice immunised with extracts from tobacco plants expressing <i>cps3S</i> (black bars) or wildtype plant (white bars); n  =  5. <b>B.</b> Survival of mice challenged with virulent type 3 pneumococci 230 days after the final immunisation with transgenic plant extract (closed triangles), wildtype extracts (open triangles) or sham-immunised mice (closed circles). Mice alive at 240h post-infection were considered to have survived the infection.</p

Detection of the <i>cps3S</i> gene in transformed tobacco plants.

<p><b>A.</b> DNA was used as a template for PCR (Lanes 2, 3: wild type plants; Lanes 4 – 7: transformed plants.) using <i>cps3S</i>-specific primers. PCR products were analysed by agarose gel electrophoresis. The results show the presence of the <i>cps3S</i> gene in the transformed plants (Lanes 4 - 7) but not the wild type plants. The PCR reaction in Lane 9 contained purified plasmid DNA containing <i>cps3S</i> (pCMS4) as a positive control and Lane 8 contained no template DNA. Molecular sizes are indicated. <b>B</b>. PCR showing the absence of <i>Agrobacterium</i> DNA contaminating DNA preparations from wild type (Lanes 2, 3) and transformed (Lanes 4 - 7) tobacco plants. PCR was done with <i>Agrobacterium</i>-specific primers. The results show that there was no <i>Agrobacterium</i> DNA present in the transgenic plant samples. The PCR reaction in Lane 9 contained <i>Agrobacterium</i> DNA as a positive control and shows the expected 730bp band and Lane 8 contained no template. Molecular sizes are indicated. DNA was extracted from the same six <i>N. tabacum</i> plants for the PCRs shown in A and B.</p

Construction of the pCMS4 vector.

<p><b>A.</b> The digested products of three separate restriction digests of pCMS3 and pCambia 2301. Lane 1 shows the expected 3 fragments of pCMS3 when digested with EcoR I and Kpn I. Lane 2 shows the expected 2 fragments produced from the digestion of pCMS3 with Kpn I and HinD III. Lane 3 shows the expected band of 11.5 Kbp when pCambia 2301 was digested with EcoR I and HinD III. The DNA fragments of 864bp (Lane 1) and 1978bp (Lane 2), indicated by the arrows, were used to reconstruct the pCMS3 T-DNA fragment containing cps3S and these were ligated to the 11.5 Kb pCambia fragment (Lane 3). This plasmid was termed pCMS4. Undigested DNA is not shown. A 1 Kb ladder (New England Biolabs) was used as a molecular size marker. <b>B.</b> The expression vector pCHF2. A CaMV35S promoter, rbcS terminator region, and a PR1B signal sequence were cloned into the T-DNA region of the pPZP222 vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088144#pone.0088144-Hajdukiewicz1" target="_blank">[17]</a>. The unique restriction endonuclease sites are also shown.</p

Oleoyl coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection