Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia pseudomallei Clinical Isolates from Thailand

Burkholderia pseudomallei is the etiologic agent of melioidosis, an emerging tropical disease. Because of low infectious dose, broad-host-range infectivity, intrinsic antibiotic resistance and historic precedent as a bioweapon, B. pseudomallei was listed in the United States as a Select Agent and Priority Pathogen of biodefense concern by the US Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. The mechanisms governing antibiotic resistance and/or susceptibility and virulence in this bacterium are not well understood. Most clinical and environmental B. pseudomallei isolates are highly resistant to aminoglycosides, but susceptible variants do exist. The results of our studies with three such variants from Thailand reveal that lack of expression or deletion of an efflux pump is responsible for this susceptibility. The large deletion present in one strain not only removes an efflux pump but also several putative virulence genes, including an entire siderophore gene cluster. Despite this deletion, the strain is fully virulent in an acute mouse melioidosis model. In summary, our findings shed light on mechanisms of antibiotic resistance and pathogenesis. They also validate the previously advocated use of laboratory-constructed, aminoglycoside susceptible efflux pump mutants in genetic manipulation experiments

Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses

Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4(+) T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4(+) T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster

Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei▿ †

Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZα gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-PBAD-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Δasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme

Global Gene Expression Profiles Suggest an Important Role for Nutrient Acquisition in Early Pathogenesis in a Plant Model of Pseudomonas aeruginosa Infection▿ †

Although Pseudomonas aeruginosa is an opportunistic pathogen that does not often naturally infect alternate hosts, such as plants, the plant-P. aeruginosa model has become a widely recognized system for identifying new virulence determinants and studying the pathogenesis of the organism. Here, we examine how both host factors and P. aeruginosa PAO1 gene expression are affected in planta after infiltration into incompatible and compatible cultivars of tobacco (Nicotiana tabacum L.). N. tabacum has a resistance gene (N) against tobacco mosaic virus, and although resistance to PAO1 infection is correlated with the presence of a dominant N gene, our data suggest that it is not a factor in resistance against PAO1. We did observe that the resistant tobacco cultivar had higher basal levels of salicylic acid and a stronger salicylic acid response upon infiltration of PAO1. Salicylic acid acts as a signal to activate defense responses in plants, limiting the spread of the pathogen and preventing access to nutrients. It has also been shown to have direct virulence-modulating effects on P. aeruginosa. We also examined host effects on the pathogen by analyzing global gene expression profiles of bacteria removed from the intracellular fluid of the two plant hosts. We discovered that the availability of micronutrients, particularly sulfate and phosphates, is important for in planta pathogenesis and that the amounts of these nutrients made available to the bacteria may in turn have an effect on virulence gene expression. Indeed, there are several reports suggesting that P. aeruginosa virulence is influenced in mammalian hosts by the availability of micronutrients, such as iron and nitrogen, and by levels of O2

IFN-γ expression in CD4⁺ T cells.

<p>Panel A shows representative flow cytometry plots demonstrating positive staining for CD4 and IFN-γ staining in cells recovered from co cultures of autologous T cells and cDCs with fraction F4 extracted from <i>B. pseudomallei</i>. Panel B represents the Mean Fluorescence Channel (MFC) for CD4/IFN-γ positive cells in each cell culture. Panel C represents the % CD4/IFN-γ positive cells over the percentage of positive cells expressing CD4/IFN-γ cultured in media alone.</p

Mass spectrometry of the four major fractions of polar lipids.

<p>Lipids derived from the second lipid extract of <i>B. pseudomallei</i>. Each mass spectrometric trace has the lipid composition of its fraction indicated by a 2D-TLC plate in the upper right corner. Lipid spot numbers correspond to those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080368#pone-0080368-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080368#pone-0080368-g002" target="_blank">2</a>. Major ions of each spot are highlighted in colored boxed. Molecular ions are in bold and enlarged in panels they first appeared. Panel A represents lipid fraction V (F4); panel B represents lipid fraction VI (F5); panel C represents lipid fraction VII (F6); and panel D represents lipid fraction VIII (F7).</p

IL-10 expression in CD4⁺ T cells.

<p>Panel A shows the representative flow cytometry plots demonstrating positive staining for CD4 and IL-10 staining in cells recovered from co cultures of autologous T cells and cDCs with media alone, fraction F4 and F7 extracted from <i>B. pseudomallei</i>. Panel B represents the mean fluorescence channel (MFC) for CD4/IL-10 positive cells in each cell culture. Panel C represents the percentage CD4/IL-10 positive cells over the percentage of positive cells expressing CD4/IL-10 cultured in media alone.</p

Composite sketch of total lipids of <i>B. pseudomallei</i>.

<p>Lipids shown were found within the first extract in five different solvent systems from very polar to highly non-polar (B, A, E, C, D). Colors of various lipid spots represent different staining and detection methods: Maroon Red: Spots detected as dark spot under UV light; Strawberry Red: Spots detected as a bright spot under UV light; Steel gray: Spots detected by spraying with CuSO₄ in H₃PO₄; Blue: Spots detected by spraying with Dittmer-Lester reagent; Plum purple: Spots detected by spraying with ninhydrin as molecules containing primary amino groups; Red: Spots detected by spraying with ninhydrin as molecules containing secondary amino groups; Orange: Spots detected by spraying with ninhydrin as molecules containing tertiary amino groups/proline; Magenta: Spots detected by spraying with α-naphthol.</p

Characterization of caprine peripheral blood monocyte-derived dendritic cells.

<p>Panel A shows a photograph of a classic morphology of a caprine dendritic cell. The same cultures were examined for cell surface expression of myeloid dendritic cell markers: CD14, MHC class II DR, and CD1w2 antigens. In panel B are representative histograms for cell surface expression of CD1w2 antigen (dark line) or isotype matched control (dotted line) in caprine dendritic cells from goat #19 (top and framed in red) and goat #29 (bottom and framed in blue) when cultured in media alone. Panels C and D correspond to cell surface expression of CD1w2 antigen in caprine dendritic cells when cultured for 16 hours in culture media alone or culture media with ConA, first extraction of total cell lipids of <i>B. pseudomallei</i> (F1), second lipid extract (F2), or lipid fraction of the second lipid extract (F4 to F7). Panel C represents the mean Fluorescence Channel (MFC) for CD1w2 expression in each culture. Panel D represents the percentage of positive cells for CD1w2 when compared to media alone.</p