Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils.

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD(50) via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo

Serum opsonization enhances internalization and clearance of <i>B. pseudomallei</i> and <i>B. thailandensis</i> by neutrophils.

<p>Isolated human neutrophils were treated with DPI or DMSO (vehicle control) for 20 min prior to bacterial infection. Neutrophils were incubated with <i>B. pseudomallei</i> and <i>B. thailandensis</i> for (A) 10 min to measure bacterial uptake or for (B) 2 h to measure bacterial survival. The bars represent the mean±SEM of three separate experiments using neutrophils from different donors and performed in duplicate. The single asterisk (*) indicates a significant difference (<i>P</i>≤0.05) compared to unopsonized bacteria. The double asterisks (**) indicate a significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i>. The triple asterisks (***) indicate a significant difference (<i>P</i>≤0.05) between 20% NHS opsonized <i>Burkholderia</i> species and all other conditions.</p

<i>B. pseudomallei</i> are resistant to C3 deposition by the alternative pathway of complement.

<p><i>B. pseudomallei</i> and <i>B. thailandensis</i> were incubated with the indicated serum concentrations in the presence or absence of MgEGTA or EDTA, washed, and stained with an anti-human C3 FITC-conjugated antibody. Samples were analyzed by flow cytometry and the data reported as mean fluorescence intensity (MFI). The bars represent the mean±SEM of three separate experiments performed with duplicate samples. The single asterisk (*) indicates a statistically significant increase (<i>P</i>≤0.05) over unopsonized bacteria. The double asterisks (**) indicate a statistically significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i> values.</p

Levels of endogenous <i>Burkholderia</i>-reactive antibodies present in normal human serum.

<p><i>B. pseudomallei,</i> B. <i>thailandensis</i>, and <i>E. coli</i> were incubated with the indicated serum concentrations, washed, and stained with both an APC-labeled anti-human IgM antibody and R-PE-labeled anti-human IgG antibody. Samples were analyzed by flow cytometry and the data reported as mean fluorescence intensity (MFI). The bars represent the mean±SEM of two separate experiments performed with duplicate samples. The single asterisk (*) indicates a statistically significant increase (<i>P</i>≤0.05) over unopsonized bacteria. The double asterisks (**) indicate a statistically significant difference (<i>P</i>≤0.05) over 20% NHS-opsonized <i>B. pseudomallei</i> and <i>B. thailandensis</i> values.</p

<i>B. pseudomallei</i> and <i>B. thailandensis</i> are inherently resistant to killing by human neutrophils.

<p>(A) <i>B. pseudomallei</i> and <i>B. thailandensis</i> were either incubated with neutrophils for 10 min to measure bacterial uptake (% bacterial input) or (B) for 2 h to measure bacterial survival (fold change from respective uptake values) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052276#s2" target="_blank">Materials and Methods</a>. The bars represent the mean±SEM of three separate experiments using neutrophils from different donors, each performed in duplicate. * indicates a statistically significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i> values.</p

<i>B. pseudomallei</i> acquire less complement C3 on its surface than <i>B. thailandensis.</i>

<p>(A) <i>B. pseudomallei</i> 1026b and <i>B. thailandensis</i> E264, or (B) <i>B. pseudomallei</i> DD503, <i>B. pseudomallei</i> ΔCPS, and <i>B. pseudomallei</i> ΔLPS were opsonized for 30 min at the indicated serum concentrations, washed, and stained with an anti-human C3 FITC-conjugated antibody. Samples were analyzed by flow cytometry and the data reported as mean fluorescence intensity (MFI). The bars represent the mean±SEM of three separate experiments performed with duplicate samples. (A) * indicates a statistically significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i> values; ns  =  not significant. (B) * indicates a statistically significant difference (<i>P</i>≤0.05) compared to <i>B. pseudomallei</i> DD503 (wild-type).</p

<i>B. pseudomallei</i> and <i>B. thailandensis</i> are resistant to direct complement-mediated killing.

<p><i>B. pseudomallei</i> 1026b, <i>B. thailandensis, B. pseudomallei</i> DD503, <i>B. pseudomallei</i> ΔCPS, <i>B. pseudomallei</i> ΔLPS and <i>E. coli</i> were incubated at the indicated serum concentrations at 37°C. At the indicated times, an aliquot of each sample was serially diluted and plated on TSA to determine CFU/ml. Each condition was assessed as duplicate samples in all experiments. * indicates a significant difference (<i>P</i>≤0.05) between <i>E. coli</i> and both <i>Burkholderia</i> species.</p