Assembly of NADPH oxidase in human neutrophils is modulated by ...

IAI Accepts, published online ahead of print on 16 December 2013 Infect. Immun. doi:10.1128/IAI.00881-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Assembly of NADPH oxidase in human neutrophils is modulated by the

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opacity-associated protein expression state of Neisseria gonorrhoeae

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Asya Smirnov, Kylene P. Daily, and Alison K. Criss*

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Department of Microbiology, Immunology, and Cancer Biology, University of

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Virginia, Charlottesville, Virginia, USA

Downloaded from http://iai.asm.org/ on November 13, 2017 by guest

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Running title: Gonococcal Opa-mediated NADPH oxidase assembly in PMNs

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*Correspondence:

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Alison K. Criss

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Department of Microbiology, Immunology, and Cancer Biology

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Box 800734

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University of Virginia Health Sciences Center

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Charlottesville, VA 22908-0734, USA

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[email protected]

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Phone: (434) 243-3561

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Fax: (434) 982-1071

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Keywords: Neisseria gonorrhoeae, neutrophils, polymorphonuclear leukocytes,

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NADPH oxidase, reactive oxygen species, opacity associated proteins 1

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ABSTRACT Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc

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survives exposure to neutrophils despite these cells’ antimicrobial products, such

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as reactive oxygen species (ROS). ROS production in neutrophils is initiated by

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NADPH oxidase, which converts oxygen into superoxide. The subunits of

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NADPH oxidase are spatially separated between granules (gp91phox/p22phox) and

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the cytoplasm (p47phox, p67phox and p40phox). Activation of neutrophils promotes

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the coassembly of NADPH oxidase subunits at phagosome and/or plasma

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membranes. While Gc expressing opacity-associated (Opa) proteins can induce

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neutrophils to produce ROS, Opa-negative Gc does not stimulate neutrophil ROS

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production. Using constitutively Opa- and OpaD+ Gc in strain FA1090, we now

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show that the difference in ROS production in primary human neutrophils

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between these backgrounds can be attributed to differential assembly of NADPH

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oxidase. Neutrophils infected with Opa- Gc showed limited translocation of

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NADPH oxidase cytoplasmic subunits to cellular membranes, including the

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bacterial phagosome. In contrast, these subunits rapidly translocated to

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neutrophil membranes following infection with OpaD+ Gc. gp91phox and p22phox

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were recruited to Gc phagosomes regardless of bacterial Opa expression. These

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results suggest that Opa- Gc interferes with the recruitment of neutrophil NADPH

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oxidase cytoplasmic subunits to membranes - in particular the p47phox 2


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“organizing” subunit - to prevent assembly of the holoenzyme, resulting in an

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absence of the oxidative burst.

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The obligate human pathogen Neisseria gonorrhoeae (the gonococcus or

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Gc) causes the sexually transmitted disease gonorrhea, with over 106 million

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cases estimated each year worldwide (1). Gonorrhea remains a major public

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health concern due to increasing Gc resistance to antibiotics, lack of protective

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immunity from prior infection, and no protective vaccine (2, 3). The inflammatory

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response associated with gonorrhea leads to serious complications such as

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pelvic inflammatory disease and infertility (4).

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Inflammation during gonorrheal infection is characterized by the influx of

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neutrophils to the site of infection (4). Neutrophils are professional phagocytes

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whose antimicrobial components are found within different subsets of

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cytoplasmic granules (5). One feature of neutrophils is their production of reactive

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oxygen species (ROS), or the oxidative burst. Neutrophil NADPH oxidase

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catalyzes the initial requisite step of ROS production, by formation of superoxide

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through the transfer of an electron from NADPH to oxygen (6). The activity of

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NADPH oxidase in human neutrophils is regulated by separation of the catalytic

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complex. In resting neutrophils, the gp91phox/p22phox subunits are found in the

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membranes of secondary and tertiary granules, while p47phox, p67phox, and 3


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INTRODUCTION

p40phox, as well as the GTPase Rac2, are in the cytoplasm. During phagocytosis

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or stimulation with soluble bacterial products, granules containing

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gp91phox/p22phox accumulate at the phagosome or plasma membrane.

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Concomitant activation of serine/threonine and tyrosine kinase phosphorylation

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pathways leads to p47phox phosphorylation and its translocation to the membrane

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(7). Phosphorylated p47phox serves as the “organizing” subunit facilitating

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translocation of p67phox and p40phox to the membrane and association with p22phox

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(8, 9). p67phox then interacts with GTP-bound Rac2 (10, 11).

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Despite the presence of neutrophils at sites of Gc infection, viable Gc are

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found inside neutrophil phagosomes and can be cultured from gonorrheal

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purulent exudates (4, 12). The mechanisms underlying the inability of neutrophils

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to clear Gc infection are incompletely understood. Although Gc expresses many

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antioxidant gene products, implying a role for neutrophil-derived ROS during

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human infection, resistance of Gc to neutrophil killing is independent of the

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neutrophil oxidative burst (13-17). Instead, ROS production may exacerbate Gc

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infectivity and symptomatic infection. Gc responds to sublethal concentrations of

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ROS by upregulating the expression of gene products such as NGO1686/Mpg

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and RecN, which protect the bacteria from killing by neutrophils (18, 19).

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Moreover, neutrophil-derived ROS may incur local tissue damage, contributing to

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the inflammation and scarring associated with gonorrheal disease (20).

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Gc modulation of the neutrophil oxidative burst is associated with bacterial expression of certain outer membrane opacity-associated proteins

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(Opa). Opa proteins interact with heparan sulfate proteoglycans, extracellular

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matrix components, and/or human carcinoembryonic antigen-related cell

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adhesion molecule (CEACAM) receptors on the surface of neutrophils and other

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cells (21, 22). Bacteria expressing Opa proteins that engage neutrophil

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CEACAMs, particularly the granulocyte-specific CEACAM3, induce a potent

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oxidative burst in neutrophils ex vivo (23-27). In contrast, Opa- Gc not only fails

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to induce ROS production in neutrophils, it also suppresses the neutrophil

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oxidative burst in response to other stimuli (28-32). Gc strains typically possess

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up to 11 opa-encoding genes per genome, each of which undergoes phase

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variation between expressed and unexpressed states (22). Thus a Gc population

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consists of bacteria of different Opa expression profiles, making it difficult to

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assess how the presence or absence of Opa proteins affects Gc pathogenesis.

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To circumvent this issue, we generated isogenic Gc of strain FA1090 in which all

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opa genes were deleted (Opaless) and into which we reintroduced a CEACAM-

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binding Opa that cannot phase vary (OpaD+nv). OpaD was selected for these

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studies because it induced the most potent neutrophil ROS response among all

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the FA1090 Opa proteins tested(25, 28).

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Since Opa expression varies during the course of human infection, in this study we sought to understand how Opa expression modulates the induction of 5


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the oxidative burst in primary human neutrophils. Infection with OpaD+nv Gc

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stimulated the assembly of the NADPH oxidase complex on neutrophil

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membranes, including the Gc-containing phagosome. In contrast, there was

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limited assembly of NADPH oxidase in neutrophils exposed to Opaless Gc, due

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to reduced recruitment of the cytoplasmic subunits to the NADPH oxidase

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complex. These results indicate that the variations in ROS production in

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neutrophils exposed to Opa+ vs. Opa- Gc is primarily attributable to differences in

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formation of the NADPH oxidase holoenzyme.

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MATERIALS AND METHODS

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Bacterial strains and growth conditions. All Gc in this study were derived from

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the FA1090 strain background (28) and are otherwise isogenic for expression of

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LOS and the 1-81-S2 pilin (33). Piliated, Opa-deficient (ΔopaA-K, Opaless) and

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isogenic, constitutively Opa-expressing (OpaD+nv) Gc were previously described

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(28) . A FA1090 Δkat::aph mutant was a kind gift of A. Jerse (Uniformed Services

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University of the Health Sciences). The insertion region was amplified by PCR

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using forward (5’-GGGCAGGCGTTTTTTATTCGC) and reverse (5’-

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TGCCGAACAATACGCCAAAAGC) primers. The PCR product was transformed

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into phenotypically Opa- ΔopaBEGK or predominantly OpaD+ ΔopaBEGK strain

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FA1090 Gc (28) by natural transformation, clones resistant to 40 µg ml-1

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kanamycin were selected, and replacement of the parental kat gene with the

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mutation was confirmed by the absence of oxygen bubbles after the mutants

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were exposed to H2O2. Gc was grown on gonococcal medium base agar (GCB)

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containing Kellogg’s supplements I and II (34) at 37 °C, 5% CO2 for 8 to 10 h.

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Viable, exponential-phase Gc were obtained by sequential dilutions in rich liquid

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medium as described (29). For immunofluorescence experiments, where

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indicated, bacteria were labeled with 5 µg ml-1 carboxylfluorescein diacetate

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succinimidyl ester (CFSE) (Life Technologies) in PBS containing 5 mM MgSO4, at

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37°C for 20 min.

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For serum opsonization experiments, Gc was exposed to 50% normal

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human serum in Morse’s defined medium (MDM; (35)) for 15 min at 37°C.

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Complement deposition on the surface of bacteria was analyzed by

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immunofluorescence of PFA-fixed bacteria using monoclonal antibody against

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iC3b (3E7) (gift of R. P. Taylor, University of Virginia).

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Δspa S. aureus strain Newman (provided by E. Skaar, Vanderbilt

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University) was grown in liquid LB media for 16-18h at 37 °C. S. aureus was

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opsonized in 20% normal human serum in MDM for 20 min at 37 °C. For

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immunofluorescence experiments, opsonized S. aureus was simultaneously

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labeled with 10µg ml-1 4′- 6-diamidino-2-phenylindole (DAPI, Sigma).

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Isolation of human neutrophils. Peripheral venous blood was collected from

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healthy human donors into heparinized tubes. Each donor gave written informed

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consent and the procedure was approved by the University of Virginia

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Institutional Review Board for Health Science Research. Neutrophils were

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purified using Ficoll-Hypaque gradient followed by hypotonic erythrocyte lysis as

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described previously (18). Neutrophils were pelleted and resuspended in ice-cold

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DPBS without calcium and magnesium containing 0.1% dextrose at a

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concentration of 1x107 to 2x107 cell ml-1 , kept on ice and used for experiments

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within 30 min. Neutrophil content in suspension was >95% as monitored by

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phase-contrast microcopy. Replicate experiments were conducted using

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neutrophils from different donors to avoid any donor-specific effects.

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Detection of reactive oxygen species (ROS). Luminol-dependent

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chemiluminescence was used to detect ROS production by primary human

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neutrophils as previously described (29), except that the assay was performed in

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a 96-well plate, in a total volume of 200 µl, containing 2x105 neutrophils per well

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in suspension. Bacteria were washed into MDM and incubated at 37 °C in the

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presence of 20 µM luminol (Sigma) at MOI ~150. Fusion of primary granules with

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neutrophil membranes was induced by pretreating neutrophils with 30 μM

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lysophosphatidylcholine (18:0) (LPC, Sigma-Aldrich) prior to addition of bacteria.

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Luminescence measurements from triplicate wells for each condition were taken 8


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on a VICTOR3TM Wallac luminometer (Perkin-Elmer) every 3 min over 1 h.

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Experiments were performed 3 times, with a single representative experiment

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shown.

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Neutrophil cytosol-membrane fractionation. 1x107 neutrophils were

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suspended in DPBS containing 0.1% dextrose, 1.25 mM CaCl2, 0.5 mM MgCl2,

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0.4 mM MgSO4 in the absence or presence of bacteria. Neutrophils were

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incubated for indicated times in a 37°C water bath, with mixing by inversion every

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5 min. The reactions were stopped by adding 5 volumes of ice-cold DPBS

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containing 0.1% dextrose. Membrane/cytosol fractionation was performed

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according to method described (36) with the following modifications. Neutrophils

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were pelleted and resuspended in relaxation buffer (10 mM PIPES pH 7.3, 100

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mM KCl, 3 mM NaCl, 1.25 mM EGTA, 5 mM EDTA, 1 mM PMSF, 20 µg ml-1

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leupeptin, 20 µg ml-1 pepstatin). Samples were sonicated at 50% amplitude for

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15 sec on ice using Sonic Dismembranator Model 120 (Fisher Scientific), and

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unbroken cells and nuclei were pelleted by centrifugation at 800 g for 10 min at

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4°C. The supernatant was centrifuged at 50,000 g for 12 min at 4°C in an

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OptimaTM TLX bench top ultracentrifuge (Beckman Coulter) to pellet membranes.

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The pellet was resuspended in solubilization buffer (20 mM Tris pH 7.5, 1% SDS,

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1 mM PMSF, 20 µg ml-1 leupeptin and 20 µg ml-1 pepstatin) in volume equal to

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the initial total volume. Equal volumes of each fraction, in 1x SDS sample buffer, 9


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were separated by 12% SDS-PAGE and transferred to PVDF (Millipore). After

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blocking with 5% BSA (Sigma), membranes were probed with mouse anti-p47phox

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(BD Biosciences), rabbit anti-p67phox (Epitomics), rabbit anti-p40phox (Upstate),

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mouse anti-p22phox (Santa Cruz), or mouse anti-GAPDH (Santa Cruz), and

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followed by horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-

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rabbit IgG (Thermo Scientific). Blots were developed with the Super Signal West

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Pro detection kit (Thermo Scientific). Images were acquired on a Gel DocTM XR+

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System using ImageLabTM software (Bio Rad).

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Immunofluorescence and image acquisition. Neutrophils were suspended in

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RPMI (Mediatech) containing 10 nM interleukin-8 (R&D Systems) and allowed to

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attach to glass coverslips for 30 min at 37 °C, 5% CO2. The coverslips were

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coated with 50% pooled human serum (Sigma) as described (12). Neutrophils

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were then infected with non-opsonized Opaless Gc, non-opsonized OpaD+nv Gc,

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or serum-opsonized S. aureus as described (12). Under these conditions

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neutrophils can phagocytose unopsonized, Opa-negative Gc (12, 13, 28).At 15,

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30, and 60 min post infection, cells were fixed with 4% PFA in PBS for 10 min,

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followed by blocking in 10% normal goat serum for 15 min. Immunofluorescence

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staining to discriminate internalized and external bacteria was performed as

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described (12). Permeabilized neutrophils were also stained with antibodies

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against p47phox (provided by W. M. Nauseef, University of Iowa), p67phox , or a 10


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mix of monoclonal antibodies against gp91phox (54.1) and p22phox (44.1) (Santa

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Cruz), followed by Alexa Fluor-coupled goat anti-rabbit or goat anti-mouse

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antibodies (Life Technologies). For some experiments total Gc was prelabeled

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with CFSE; this did not affect bacterial uptake or trafficking in neutrophils (data

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not shown). Images of 10-20 63x fields for each condition were acquired on a Nikon

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Eclipse E800 UV/visible fluorescence microscope with Hamamatsu Orca-ER

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digital camera using Openlab 5.5.0 software (Improvision). For consistency in

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presentation, images were false colored using Adobe Photoshop CS5 as follows.

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In phagocytosis experiments, external bacteria appear red and green, while

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internalized bacteria appear green only. In experiments assessing NADPH

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oxidase gp91phox/ p22phox subunit recruitment to phagosomes, external bacteria

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appear blue and red, internalized bacteria appear red only, and gp91phox/ p22phox

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subunits appear green. In experiments assessing NADPH oxidase p47phox and

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p67phox subunit recruitment to phagosomes, external bacteria appear blue, red

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and green, internalized bacteria appear red only, and p47phox and p67phox

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subunits appear green. For each condition, 50-200 bacteria or phagosomes

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were analyzed. Phagosomes were considered positive for presence of the

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subunits of NADPH oxidase when fluorescence staining was found at 50% or

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more of the phagosome perimeter. Values are expressed as a mean ± SEM from

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three replicate experiments. Statistical analysis was performed using Student’s 11


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two tailed t-test, and differences were considered statistically significant at P

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