Strains, plasmids and cell lines used in this study.

<p>Strains, plasmids and cell lines used in this study.</p

Mice infected with the <i>bbfA</i> mutant demonstrated a delay to death in comparison to those challenged with the parental strain.

<p>Groups of six BALB/c mice were challenged via the intra-peritoneal route and the median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection. Mice inoculated with the <i>bbfA</i> mutant had a mean survival time of 3.5 days versus 2 days for those dosed with the wild-type (<i>p</i> = 0.03).</p

The <i>bpss1439</i> mutant displayed reduced biofilm formation which was complemented by <i>in trans bpss1439</i> expression.

<p>a. The wild-type and <i>bpss1439</i> mutant were grown in LB under static conditions at 37°C for 48 hours. Biofilms were stained with 1% w/v crystal violet, solubilised and quantified using an optimal density reading at 595 nm. Results are plotted with standard deviation error bars from triplicate experiments each consisting of eight experimental replicates; the <i>p</i> value was calculated using a paired Student's T test. b. The trans-complemented <i>bpss1439</i> mutant (pME-<i>1439</i>), as well as the wild-type and <i>bpss1439</i> pME strain, were also assessed for biofilm production as described previously.</p

Comparison of the <i>bbfA</i> mutant with other reported biofilm reduced strains.

†<p>EPS =  exopolysaccharide.</p

The arrangement of the <i>bpss1439</i> operon.

<p>The <i>bpss1439</i> gene is located in an operon with two downstream genes, <i>bpss1442</i> and <i>bpss1443</i>; both which encode hypothetical proteins of no significant database hits. The closest orthologue to <i>bpss1439</i> is the upstream gene (<i>bpss1434</i>) encoding another unstudied predicted TAA.</p

The <i>bbfA</i> mutant demonstrated reduced adhesion and microcolony formation.

<p>A. The wild-type, <i>bbfA</i> mutant and trans-complemented bbfA mutant (pME-<i>1439</i>), along with the control wild-type and <i>bbfA</i> mutant strains harbouring pME, were grown in LB under static conditions at 37°C for 48 hours. Biofilms were stained for exopolysaccharide using the periodic acid-Schiff protocol and examined by light microscopy. B. The wild-type and <i>bbfA</i> mutant biofilms were also fixed with 2.5% v/v glutaldehyde and examined by scanning electron microscopy.</p

<i>In Vivo</i> Manipulation of γ9⁺ T Cells in the Common Marmoset (<i>Callithrix Jacchus</i>) with Phosphoantigen and Effect on the Progression of Respiratory Melioidosis

<div><p><i>Burkholderia pseudomallei</i> is a dangerous human pathogen. Phosphoantigens specifically the target primate specific γ9⁺δ2⁺ T cells subset and some have been developed as potential immunotherapeutics. Previously, we demonstrated that, when stimulated with the phosphoantigen CHDMAPP, γ9⁺δ2⁺ T cells aid in the killing of intracellular <i>B. pseudomallei</i> bacteria. Moreover, we found that common marmoset (<i>Callithrix Jacchus</i>) γ9⁺ T cells increase in frequency and respond to the phosphoantigen CHDMAPP and/or <i>B. pseudomallei</i>, in combination with IL-2, in a similar manner to human γ9⁺δ2⁺ T cells. Here we evaluate the efficacy of the phosphoantigen CHDMAPP, in combination with IL-2, as a therapy against <i>B. pseudomallei</i> infection, <i>in vivo</i>. We found that the previous studies predicted the <i>in vivo</i> responsiveness of γ9⁺ T cells to the CHDMAPP+IL-2 treatment and significant expansion of the numbers of peripheral and splenic γ9⁺ T cells were observed. This effect was similar to those reported in other primate species treated with phosphoantigen. Furthermore, splenocytes were retrieved 7 days post onset of treatment, restimulated with CHDMAPP or heat-killed <i>B. pseudomallei</i> and the cultured γ9⁺ T cells demonstrated no reduction in IFN-γ response when CHDMAPP+IL-2 animals were compared to IL-2 only treated animals. Using an established model of <i>B. pseudomallei</i> infection in the marmoset, we assessed the potential for using phosphoantigen as a novel immunotherapy. The CHDMAPP treatment regime had no effect on the progression of respiratory melioidosis and this was despite the presence of elevated numbers of γ9⁺ T cells in the spleen, liver and lung and an increased proportion of IFN-γ⁺ cells in response to infection. We therefore report that the common marmoset has proven a good model for studying the effect <i>in vivo</i> of γ9⁺ T cell stimulation; however, γ9⁺ T cells have little or no effect on the progression of lethal, respiratory <i>B. pseudomallei</i> infection.</p></div

A variety of interactions between several parameters were observed in marmosets pre-treated with CHMDAPP+IL-2 and infected with <i>B. pseudomallei</i>.

<p>Animals were treated with CHMDAPP and IL-2 (n = 7), and group treated with IL-2 only (n = 7). Animals received a single dose of CHMDAPP (day 0, at 2.5 mg/kg) and five doses of IL-2 (days 0, 1, 2, 3, 4 and 5, at 0.18 U×10⁶ per kg) or only received the five doses of IL-2 or received PBS only. Animals were then challenged with 77–1,081 CFU of <i>B. pseudomallei</i> strain K96423 at day 5 post onset of treatment. At the lethal end point multiple parameters were read and correlation matrices of liver pathology scores, time-to-death, IL-6 concentration within the liver post mortem, IL-1β concentration within the liver post mortem, and proportion of γ9⁺ T cells in the blood pre and post mortem and in the liver, lung and spleen post mortem were performed. Panel A shows a matrix scatter plot showing the relationship between these parameters. Panel B shows the statistical analysis of these relationships. These two panels directly relate to each other. Pearson's correlations were performed with the exception of correlation to liver pathology scores, where the Spearman's method was used. White boxes indicate significant negative correlations, and dark grey boxes indicate significant positive correlations.</p

The liver of marmosets, some pre

<p>-<b>treated with the immuno</b>-<b>stimulant CHMDAPP and IL-2 after infection with an aerosol challenge of </b><b><i>B. pseudomallei</i></b><b> show a variety of levels of pathology.</b> Liver pathology was measured in animals treated with CHMDAPP and IL-2 (n = 7), and group treated with IL-2 only (n = 7) using samples take post lethal endpoint. Animals received a single dose of CHMDAPP (day 0, at 2.5 mg/kg) and five doses of IL-2 (days 0, 1, 2, 3, 4 and 5, at 0.18 U×10⁶ per kg) or only received the five doses of IL-2 or received PBS only. Animals were then challenged with 77–1,081 CFU of <i>B. pseudomallei</i> strain K96423 at day 5 post onset of treatment. Liver samples were investaged by tissue sectioning and H&E stain. Liver pathology in these animals was blindly set against a scale from 0 (no pathology) to 3 (severe pathology). Panel A shows a chart depicting the frequency of the different levels of liver pathology observed in this experiment. Panel B shows an example of liver pathology in an animal set a score of 1 (x200 magnification), panel C a score of 2 (x200 magnification) and panel D a score of 3 (x400 magnification).</p

Treatment with CHMDAPP+IL-2 does not affect bacterial colonisation in marmoset tissues following aerosol challenge with <i>B. pseudomallei</i>.

<p>Bacterial colonisation in marmoset tissues treated with CHMDAPP and IL-2 (n = 7), and group treated with IL-2 only (n = 7). Animals received a single dose of CHMDAPP (day 0, at 2.5 mg/kg) and five doses of IL-2 (days 0, 1, 2, 3, 4 and 5, at 0.18 U×10⁶ per kg) or only received the five doses of IL-2 or received PBS only. Animals were then challenged with 77–1,081 CFU of <i>B. pseudomallei</i> strain K96423 at day 5 post onset of treatment. The graph shows bacterial colonisation measured using standard viable counting. The sham group (PBS) includes some legacy data, from previously published experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074789#pone.0074789-Nelson3" target="_blank">[22]</a>. Each data point represents a reading of a single organ from a single marmoset and the line represents the population median.</p