Stkp and PhpP protein expression in T4Δ<i>stkP</i> and StkP complemented strains.

<p>Western blotting analysis was performed on TIGR4, T4Δ<i>stkP</i>, T4Δ<i>stkP</i>∇ST, T4Δ<i>stkP</i>∇XST and T4Δ<i>rrgB</i> at OD 0.6 (OD_600nm) looking at expression of StkP and PhpP in all strains (α-StkP/ -PhpP antibody). Equal protein loading was confirmed by equal expression of GroEL (α-GroEL antibody).</p

A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells <i>In Vitro</i>

<div><p>The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin.</p></div

RrgB surface expression in T4Δ<i>stkP</i> and StkP complemented strains.

<p>Flow cytometry was performed on T4Δ<i>stkP</i>, TIGR4, T4Δ<i>stkP</i>∇ST, T4Δ<i>stkP</i>∇XST, T4Δ<i>stkP</i>Δ<i>rrgB</i> and T4Δ<i>rrgB</i>. All samples were initially gated on for being capsule positive (data not shown). This population was then gated on for RrgB positive. Histograms show negative (left) and positive (right) RrgB populations in each strain. Table shows the percentage RrgB positive and negative cells in a growing bacterial population.</p

RrgB protein expression in T4Δ<i>stkP</i> and StkP complemented strains.

<p>Western blotting analysis was performed on TIGR4, T4Δ<i>stkP</i>, T4Δ<i>stkP</i>∇ST, T4Δ<i>stkP</i>∇XST and T4Δ<i>rrgB</i> at OD 0.6 (OD_600nm) looking at expression of RrgB in all strains (α-RrgB antibody). Equal protein loading was confirmed by equal expression of GroEL (α-GroEL antibody).</p

RrgA protein expression in T4Δ<i>stkP</i> and StkP complemented strains.

<p>Western blotting analysis was performed on TIGR4, T4Δ<i>stkP</i>, T4Δ<i>stkP</i>∇ST, T4Δ<i>stkP</i>∇XST and T4Δ<i>rrgB</i> at OD 0.6 (OD_600nm) looking at expression of RrgA in all strains (α-RrgA antibody). Equal protein loading was confirmed by equal expression of GroEL (α-GroEL antibody).</p

Pilus gene expression in T4Δ<i>stkP</i> and StkP complemented strains.

<p>Graph shows RT-PCR expression of the genes present on the whole pilus islet (<i>rlrA</i>, <i>rrgA</i>, <i>rrgB</i>, <i>rrgC</i>, <i>srtB</i>, <i>srtC</i>, <i>srtD</i>) in T4Δ<i>stkP</i>, T4Δ<i>stkP</i>∇ST and T4Δ<i>stkP</i>∇XST compared to TIGR4. Fold change represents that of the mutant strain compared to TIGR4. Each bar represents the average of three replicas and errors bars the standard deviation.</p

Adherence of T4Δ<i>stkP</i> and StkP complemented strains to different cell lines.

<p>Adherence of strains TIGR4, T4Δ<i>stkP</i>, T4Δ<i>stkP</i>Δ<i>rrgB</i>, T4Δ<i>stkP</i>∇ST, T4Δ<i>stkP</i>∇XST and <i>T4</i>Δ<i>rrgB</i> was assessed to HBMEC (A) and A549 (B) cell lines. Data is represented as percentage adherence relative to that of TIGR4 (100%, dashed line), each bar is an average of three replicas and the error bars represent the standard error of the mean. Statistical analysis was performed using a 1-way ANOVA with a Tukeys testing correction, * P<0.05. * above the bar represent statistical significance compared to TIGR4 (not represented as a bar on the graphs).</p

Expression of the <i>lux</i> genes in <i>Streptococcus pneumoniae</i> modulates pilus expression and virulence

<div><p>Bioluminescence has been harnessed for use in bacterial reporter systems and for <i>in vivo</i> imaging of infection in animal models. Strain Xen35, a bioluminescent derivative of <i>Streptococcus pneumoniae</i> serotype 4 strain TIGR4 was previously constructed for use for <i>in vivo</i> imaging of infections in animal models. We have shown that strain Xen35 is less virulent than its parent TIGR4 and that this is associated with the expression of the genes for bioluminescence. The expression of the <i>luxA-E</i> genes in the pneumococcus reduces virulence and down regulates the expression of the pneumococcal pilus.</p></div

Survival and weight loss of mice infected with Xen35.

<p>(A) Percentage survival of mice intra-nasally inoculated with TIGR4 or Xen35 over time, statistical analysis was performed using a logrank Test, * P<0.01. * above the strain indicated a statistical difference compared to Xen35. Only the survival group was used for analysis (n = 5 per bacterial strain) (B) Percentage weight loss of mice infected with either Xen35 or TIGR4. All groups were used for analysis, later groups have smaller numbers due to some mice being sacrificed. Statistical analysis was performed using a non-parametric Mann-Whitney two sample rank test, *P<0.001.</p

Energy production and consumption.

<p>(A) Reaction catalysed by luciferase enzyme (Flavin reductase) leading to light emission (bioluminescence). Reaction requires reduced riboflavin (FMNH₂), long chain aldehyde (RCHO) and oxygen (O₂) resulting in flavin mononucleotide (FMN), Water (H₂O), fatty acids (RCOOH) and light [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189426#pone.0189426.ref003" target="_blank">3</a>] (B) Diagram showing adenosine triphosphate (ATP), reduced nicotinamide adenine dinucleotide (NADH), adenosine diphosphate (ADP) and oxidised nicotinamide adenine dinucleotide (NAD⁺) production and utilisation during glycolysis and pyruvate metabolism in <i>S</i>. <i>pneumoniae</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189426#pone.0189426.ref046" target="_blank">46</a>]. (C) Reaction catalysed by the fatty acid reductase complex required to produce the long chain aldehyde substrate for the luciferase enzyme. Reaction requires RCOOH, ATP, reduced nicotinamide adenine dinucleotide phosphate (NADPH) resulting in nicotinamide adenine dinucleotide phosphate (NADP), adenosine monophosphate (AMP), pyrophosphate (PPi) and RCHO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189426#pone.0189426.ref003" target="_blank">3</a>].</p