Inhibition of the human neutrophil NADPH oxidase by Coxiella burnetii - PubMed (original) (raw)

Comparative Study

Inhibition of the human neutrophil NADPH oxidase by Coxiella burnetii

Daniel W Siemsen et al. Microbes Infect. 2009 May-Jun.

Abstract

Coxiella burnetii is an obligate intracellular Gram-negative pathogen. A notable feature of C. burnetii is its ability to replicate within acidic phagolysosomes; however, the mechanisms utilized in evading host defenses are not well defined. Here, we investigated human neutrophil phagocytosis of C. burnetii (Nine Mile, phase II; NMII) and the effect of phagocytosed organisms on neutrophil reactive oxygen species (ROS) production. We found that opsonization with immune serum substantially enhanced phagocytosis of NMII. Human neutrophils phagocytosing opsonized NMII generated very little ROS compared to cells phagocytosing opsonized Staphylococcus aureus, Escherichia coli, or zymosan. However, phagocytosis of NMII did not affect the subsequent ROS response to a soluble agonist, indicating inhibition was localized to the phagolysosome and was not a global effect. Indeed, analysis of NADPH oxidase assembly in neutrophils after phagocytosis showed that translocation of cytosolic NADPH oxidase proteins, p47(phox) and p67(phox), to the membrane was absent in cells phagocytosing NMII, as compared to cells phagocytosing S. aureus or activated by phorbol myristate acetate. Thus, phagocytosed NMII is able to disrupt assembly of the human neutrophil NADPH oxidase, which represents a novel virulence mechanism for this organism and appears to be a common mechanism of virulence for many intracellular pathogens.

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Figures

Figure 1

Phagocytosis of NMII by human neutrophils is enhanced after opsonization with immune serum. Panel A: Alexa 488-labeled NMII were opsonized with non-immune and _Coxiella_-immune human serum, as described. As a positive control, Alexa 488-labeled S. aureus were opsonized with the same serum (S. aureus phagocytosis was similar for all sera; shown are bacteria opsonized with the _Coxiella_-immune serum). The treated S. aureus (20:1) and NMII (100:1) were incubated for 30 min with human neutrophils, and the samples were analyzed by flow cytometry. Cells alone received no bacteria. Panel B: Opsonized Alexa 488-labeled S. aureus (20:1) or opsonized Alexa 488-labeled NMII (500:1) were incubated for 30 min with human neutrophils for the indicated times, and the samples were analyzed by flow cytometry. Cells alone received no bacteria. In both panels, 10,000 events were collected for each sample, and the data are presented as the mean fluorescence intensity±SEM and are representative of at least 3 independent experiments. Statistically significant differences compared to NMII opsonized with nonimmune serum are indicated (*P<0.05; **P<0.001). Panels C and D: Human neutrophils were plated in quartz-bottom microtiter plate wells, stained with Alexa Fluor 594-conjugated cholera toxin B, and incubated for 30 min with opsonized Alexa 488-labeled C. burnetii NMII (100:1) or opsonized Alexa 488-labeled S. aureus (20:1). The cells were then washed, fixed, and analyzed by confocal microscopy. The cells shown are representative of three independent experiments.

Figure 2

Analysis of human neutrophil ROS production in cells phagocytosing NMII or E. coli using a microscopic NBT assay. Human neutrophils were plated into quartz-bottom microtiter plate wells and incubated for 30 min with control buffer (Panel A), opsonized Alexa 488-labeled E. coli (20:1, Panel B), or opsonized Alexa 488-labeled NMII (100:1, Panel C) in the presence of NBT, as described. The cells were analyzed by confocal microscopy. All panels show differential interference contrast (DIC) images, and panels B and C show merged images of DIC and Alexa 488 fluorescence. The cells shown are representative of three independent experiments.

Figure 3

Analysis of human neutrophil ROS production in cells phagocytosing NMII or zymosan using a quantitative NBT assay. Human neutrophils were incubated without bacteria (Control) or with the indicated concentrations of opsonized NMII or zymosan particles for 30 min at 37°C in the presence of NBT. The cells were then centrifuged, the supernatants (extracellular ROS) and pellets (intracellular ROS) were extracted, and absorbance at 620 nm was measured. The results are expressed as mean±SEM of triplicate samples and are representative of at least 3 independent experiments.

Figure 4

Analysis of human neutrophil ROS production in cells phagocytosing live and killed NMII. Human neutrophils were incubated without bacteria (Control), opsonized zymosan (20:1), opsonized live NMII (100:1), or opsonized ethanol-killed NMII (100:1) for 30 min at 37°C in the presence of NBT. The cells were then centrifuged, the supernatants (extracellular ROS) and pellets (intracellular ROS) were extracted, and absorbance at 620 nm was measured. Similar to what is shown in Figure 3, little extracellular ROS was generated by cells phagocytosing zymosan or any of the NMII samples (not shown). The results are expressed as mean±SEM of triplicate samples and are representative of at least 3 independent experiments. Statistically significant differences (P<0.001) compared to control cells (a) and cells treated with live NMII (b) are indicated.

Figure 5

Pre-exposure of human neutrophils to NMII does not diminish ROS production in response to subsequent stimuli. Human neutrophils were incubated without bacteria (Control) or with the indicated concentrations of S. aureus or NMII for 15 min at 37°C. The cells were washed, aliquotted into microtiter plate wells ±SOD, and activated by addition of 1 μM fMLF (upper and middle panels) or 100 ng/ml PMA (lower panel). ROS production was monitored using a chemiluminescence-based assay, and the results are expressed as SOD-inhibitable chemiluminescence, as described. Representative of 5 independent experiments.

Figure 6

Phagocytosis of NMII disrupts translocation of NADPH oxidase cytosolic proteins to the membrane. Human neutrophils were incubated with control buffer (lane 1), opsonized S. aureus (20:1, lane 2), or NMII (500:1, lane 3) for 30 min or with 100 ng/ml PMA (lane 4) for 10 min at 37°C, and membranes were prepared as described. Samples were normalized for protein content and analyzed by immunoblotting with antibodies against the indicated NADPH oxidase proteins, as described. The location of prestained molecular weight standards is shown for each blot. Representative of 3 independent experiments.

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Inhibition of the human neutrophil NADPH oxidase by Coxiella burnetii - PubMed (original) (raw)