Fig 1.
LPS types present in B. pseudomallei strains used in this study.
The O-antigen structures shown here are based on previously published structures in the case of Type A and Type AV and unpublished data in the cases of Type B and B2. Inner core is based on structures found in Burkholderia cenocepacia and lipid A is a composite of structures found in this work and others referenced in the text. B. mallei is used as an example of the Type AV LPS and was not investigated in this study. Red circles indicate variable methylation and acetylation of the L-6dTalp residues of Type AV LPS.
Table 1.
Glycosyl composition of type B, B2, and rough Burkholderia LPS.
Fig 2.
Bp LPS differentially induce inflammatory mediators NOS and TNF-α.
RAW264.7 macrophages were treated with LPS isolated from the indicated strains. At the indicated time points iNOS (A) and TNF-α (B) secretion was measured (iNOS was assayed as a measure of total NO). (C), NO induction data in a dose-dependent format. (D), TNF-α secretion presented in a dose-dependent format. Red dashed lines indicated basal levels of NO. Differences between samples when compared to 1026b samples were significant as determined by two way ANOVA analysis, * = p<0.05, ** = p<0.005, *** = p<0.0005, **** = p<0.00005.
Fig 3.
Dissimilar induction of innate and adaptive immunity by Type A and B LPS in RAW264.7 macrophages.
Macrophages were treated for 2 h with either 10 ng/mL Type A or Type B LPS before isolation of total RNA. Gene expression profiles were assessed using innate and adaptive mouse RT2 Profiler qPCR arrays. Panels A and B show genes that are either upregulated more than 2-fold (red dots) unchanged (black dots) or downregulated more than 2-fold (blue dots) in response to macrophage treatment with Type A LPS (derived from 1026b) or Type B LPS (derived from 576a), when compared to untreated macrophages. C), Heat-map of all genes analyzed and grouped by expression trends. Genes with significantly different expression are listed in S1 Table.
Fig 4.
Differential induction of autophagy by purified Bp LPS and live bacteria.
RAW264.7 cells were treated with LPS for 16 h and LC3-II+ puncta were observed (A) and LC3-II+ nuclei counted (B). The experiment was conducted in triplicate and 3 random fields of view were imaged for puncta analysis. Signal thresholding and particle analysis was used to correlate each nucleus with high levels of LC3 puncta. The Type B LPS from strain 576a and the rough strain MSHR435 HMW LPS were potent inducers of autophagy while type A from 1026b and type B2 from MSHR840 were weak inducers. RAW264.7 cells were infected with live Bp at an MOI of 10:1 for 6 h after which LC3-II+ phagosomes were observed (C) and percent of LC3-II+ cells counted (D). ns = not significant, * = p<0.05, *** = p<0.001, **** = p<0.0001.
Fig 5.
Dissimilar induction of innate and adaptive immunity by Type A and B LPS in human PBMCs.
RT2 qPCR arrays were used to look at differences between Type A and B LPS in their ability to induce innate and adaptive immunity in healthy human derived PBMCs. Heat maps of expression levels of innate and adaptive immune targets from human PBMCs of two donors after 24 h treatment in mock treated (-), Type A LPS (from 1026b) treated (A), and Type B LPS (from 576a) treated (B) PBMCs. Red is the highest fold change in expression and green is the least. Log2 gene expression scatterplots are shown below the heat-maps.
Table 2.
Gene expression ratios in LPS treated human PBMCs.
Fig 6.
Cytokine secretion by human PBMCs in response to treatment with B. pseudomallei LPS.
Levels of cytokines secreted by human PBMCs were measured at 2 and 24 h post LPS treatment for Donor 1 and 24 h post treatment from Donor 2. The cytokines analyzed are indicated above each plot with the sample origins indicated on the X-axis. Data for each sample set are divided by a dashed line. Mock treated negative control replicates are indicated by black circles, data obtained for Type A (1026b) LPS treatment are indicated by blue squares, and data obtained for Type B (576a) LPS treatment are indicated by green triangles. Salmon-colored triangles indicate data from positive control S. minnesota S-LPS treated samples. A), GM-CSF; B), IFN-γ; C), IL-1β; D), IL-2; E), IL-4; F), IL-5; G), IL-6; H), IL-8; I), IL-10; J), TNF-α. Scales are either linear or log depending on the cytokine for clarity. The means of all replicates and standard error of the means are indicated by error bars and lines. Differences between 576a compared to 1026b and Salmonella compared to 1026b treated were significant as determined by multiple one-way ANOVAs as shown above the datasets. ns = not significant, * = p<0.05, ** = p<0.005, *** = p<0.0005, **** = p<0.0001.
Fig 7.
MALDI-TOF/TOF comparisons and predicted structures.
The lipid A portion of the LPS from the indicated strains was purified and analyzed by MALDI-TOF/TOF. All lipid A scans are in the negative ion mode with mass from 1,000 to 2,000 Da on the x-axes and relative intensity on the y-axes. Scan traces are color-coded: 1026b scans are blue; 576a scans are orange, MSHR840 scans are green; MSHR435LMW scans are magenta; and MSHR435HMW scans are salmon. Panels A-F show comparisons of trace scans: A, 1026b (top) compared to 576a (bottom), B, 1026b (top) compared to MSHR840 (bottom), C, 576a (top) compared to MSHR840 (bottom), D, 1026b (top) compared to MSHR435LMW (bottom), E, MSHR435HMW (top) compared to MSHR435 LMW (bottom), and F, 576a (top) compared to MSHR435 LMW (bottom). Panel G shows the predicted structure of the predominant penta-acylated lipid A in the 1026b sample with the non-stoichiometric hydroxyl group not present in the 576a penta-acylated lipid A shown in red. The table in panel H, summarizes expected and observed masses of the indicated lipid A substituents and acyl chains.