Dec 16, 2013 ... 29 membranes. While Gc expressing opacity-associated (Opa) proteins can
induce. 30 ... (8, 9). p67phox then interacts with GTP-bound Rac2 (10, 11). 70 ....
DPBS without calcium and magnesium containing 0.1% dextrose at a. 151 ......
McCaw SE, Schneider J, Liao EH, Zimmermann W, Gray-Owen SD.
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.
1
Assembly of NADPH oxidase in human neutrophils is modulated by the
2
opacity-associated protein expression state of Neisseria gonorrhoeae
3
Asya Smirnov, Kylene P. Daily, and Alison K. Criss*
5
Department of Microbiology, Immunology, and Cancer Biology, University of
6
Virginia, Charlottesville, Virginia, USA
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
4
7 8
Running title: Gonococcal Opa-mediated NADPH oxidase assembly in PMNs
9
*Correspondence:
10
Alison K. Criss
11
Department of Microbiology, Immunology, and Cancer Biology
12
Box 800734
13
University of Virginia Health Sciences Center
14
Charlottesville, VA 22908-0734, USA
15
[email protected]
16
Phone: (434) 243-3561
17
Fax: (434) 982-1071
18 19
Keywords: Neisseria gonorrhoeae, neutrophils, polymorphonuclear leukocytes,
20
NADPH oxidase, reactive oxygen species, opacity associated proteins 1
21 22
ABSTRACT Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc
24
survives exposure to neutrophils despite these cells’ antimicrobial products, such
25
as reactive oxygen species (ROS). ROS production in neutrophils is initiated by
26
NADPH oxidase, which converts oxygen into superoxide. The subunits of
27
NADPH oxidase are spatially separated between granules (gp91phox/p22phox) and
28
the cytoplasm (p47phox, p67phox and p40phox). Activation of neutrophils promotes
29
the coassembly of NADPH oxidase subunits at phagosome and/or plasma
30
membranes. While Gc expressing opacity-associated (Opa) proteins can induce
31
neutrophils to produce ROS, Opa-negative Gc does not stimulate neutrophil ROS
32
production. Using constitutively Opa- and OpaD+ Gc in strain FA1090, we now
33
show that the difference in ROS production in primary human neutrophils
34
between these backgrounds can be attributed to differential assembly of NADPH
35
oxidase. Neutrophils infected with Opa- Gc showed limited translocation of
36
NADPH oxidase cytoplasmic subunits to cellular membranes, including the
37
bacterial phagosome. In contrast, these subunits rapidly translocated to
38
neutrophil membranes following infection with OpaD+ Gc. gp91phox and p22phox
39
were recruited to Gc phagosomes regardless of bacterial Opa expression. These
40
results suggest that Opa- Gc interferes with the recruitment of neutrophil NADPH
41
oxidase cytoplasmic subunits to membranes - in particular the p47phox 2
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
23
42
“organizing” subunit - to prevent assembly of the holoenzyme, resulting in an
43
absence of the oxidative burst.
44 45
The obligate human pathogen Neisseria gonorrhoeae (the gonococcus or
47
Gc) causes the sexually transmitted disease gonorrhea, with over 106 million
48
cases estimated each year worldwide (1). Gonorrhea remains a major public
49
health concern due to increasing Gc resistance to antibiotics, lack of protective
50
immunity from prior infection, and no protective vaccine (2, 3). The inflammatory
51
response associated with gonorrhea leads to serious complications such as
52
pelvic inflammatory disease and infertility (4).
53
Inflammation during gonorrheal infection is characterized by the influx of
54
neutrophils to the site of infection (4). Neutrophils are professional phagocytes
55
whose antimicrobial components are found within different subsets of
56
cytoplasmic granules (5). One feature of neutrophils is their production of reactive
57
oxygen species (ROS), or the oxidative burst. Neutrophil NADPH oxidase
58
catalyzes the initial requisite step of ROS production, by formation of superoxide
59
through the transfer of an electron from NADPH to oxygen (6). The activity of
60
NADPH oxidase in human neutrophils is regulated by separation of the catalytic
61
complex. In resting neutrophils, the gp91phox/p22phox subunits are found in the
62
membranes of secondary and tertiary granules, while p47phox, p67phox, and 3
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
46
INTRODUCTION
p40phox, as well as the GTPase Rac2, are in the cytoplasm. During phagocytosis
64
or stimulation with soluble bacterial products, granules containing
65
gp91phox/p22phox accumulate at the phagosome or plasma membrane.
66
Concomitant activation of serine/threonine and tyrosine kinase phosphorylation
67
pathways leads to p47phox phosphorylation and its translocation to the membrane
68
(7). Phosphorylated p47phox serves as the “organizing” subunit facilitating
69
translocation of p67phox and p40phox to the membrane and association with p22phox
70
(8, 9). p67phox then interacts with GTP-bound Rac2 (10, 11).
71
Despite the presence of neutrophils at sites of Gc infection, viable Gc are
72
found inside neutrophil phagosomes and can be cultured from gonorrheal
73
purulent exudates (4, 12). The mechanisms underlying the inability of neutrophils
74
to clear Gc infection are incompletely understood. Although Gc expresses many
75
antioxidant gene products, implying a role for neutrophil-derived ROS during
76
human infection, resistance of Gc to neutrophil killing is independent of the
77
neutrophil oxidative burst (13-17). Instead, ROS production may exacerbate Gc
78
infectivity and symptomatic infection. Gc responds to sublethal concentrations of
79
ROS by upregulating the expression of gene products such as NGO1686/Mpg
80
and RecN, which protect the bacteria from killing by neutrophils (18, 19).
81
Moreover, neutrophil-derived ROS may incur local tissue damage, contributing to
82
the inflammation and scarring associated with gonorrheal disease (20).
4
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
63
83
Gc modulation of the neutrophil oxidative burst is associated with bacterial expression of certain outer membrane opacity-associated proteins
85
(Opa). Opa proteins interact with heparan sulfate proteoglycans, extracellular
86
matrix components, and/or human carcinoembryonic antigen-related cell
87
adhesion molecule (CEACAM) receptors on the surface of neutrophils and other
88
cells (21, 22). Bacteria expressing Opa proteins that engage neutrophil
89
CEACAMs, particularly the granulocyte-specific CEACAM3, induce a potent
90
oxidative burst in neutrophils ex vivo (23-27). In contrast, Opa- Gc not only fails
91
to induce ROS production in neutrophils, it also suppresses the neutrophil
92
oxidative burst in response to other stimuli (28-32). Gc strains typically possess
93
up to 11 opa-encoding genes per genome, each of which undergoes phase
94
variation between expressed and unexpressed states (22). Thus a Gc population
95
consists of bacteria of different Opa expression profiles, making it difficult to
96
assess how the presence or absence of Opa proteins affects Gc pathogenesis.
97
To circumvent this issue, we generated isogenic Gc of strain FA1090 in which all
98
opa genes were deleted (Opaless) and into which we reintroduced a CEACAM-
99
binding Opa that cannot phase vary (OpaD+nv). OpaD was selected for these
100
studies because it induced the most potent neutrophil ROS response among all
101
the FA1090 Opa proteins tested(25, 28).
102 103
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
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
84
the oxidative burst in primary human neutrophils. Infection with OpaD+nv Gc
105
stimulated the assembly of the NADPH oxidase complex on neutrophil
106
membranes, including the Gc-containing phagosome. In contrast, there was
107
limited assembly of NADPH oxidase in neutrophils exposed to Opaless Gc, due
108
to reduced recruitment of the cytoplasmic subunits to the NADPH oxidase
109
complex. These results indicate that the variations in ROS production in
110
neutrophils exposed to Opa+ vs. Opa- Gc is primarily attributable to differences in
111
formation of the NADPH oxidase holoenzyme.
112 113
MATERIALS AND METHODS
114 115
Bacterial strains and growth conditions. All Gc in this study were derived from
116
the FA1090 strain background (28) and are otherwise isogenic for expression of
117
LOS and the 1-81-S2 pilin (33). Piliated, Opa-deficient (ΔopaA-K, Opaless) and
118
isogenic, constitutively Opa-expressing (OpaD+nv) Gc were previously described
119
(28) . A FA1090 Δkat::aph mutant was a kind gift of A. Jerse (Uniformed Services
120
University of the Health Sciences). The insertion region was amplified by PCR
121
using forward (5’-GGGCAGGCGTTTTTTATTCGC) and reverse (5’-
122
TGCCGAACAATACGCCAAAAGC) primers. The PCR product was transformed
123
into phenotypically Opa- ΔopaBEGK or predominantly OpaD+ ΔopaBEGK strain
6
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
104
FA1090 Gc (28) by natural transformation, clones resistant to 40 µg ml-1
125
kanamycin were selected, and replacement of the parental kat gene with the
126
mutation was confirmed by the absence of oxygen bubbles after the mutants
127
were exposed to H2O2. Gc was grown on gonococcal medium base agar (GCB)
128
containing Kellogg’s supplements I and II (34) at 37 °C, 5% CO2 for 8 to 10 h.
129
Viable, exponential-phase Gc were obtained by sequential dilutions in rich liquid
130
medium as described (29). For immunofluorescence experiments, where
131
indicated, bacteria were labeled with 5 µg ml-1 carboxylfluorescein diacetate
132
succinimidyl ester (CFSE) (Life Technologies) in PBS containing 5 mM MgSO4, at
133
37°C for 20 min.
134
For serum opsonization experiments, Gc was exposed to 50% normal
135
human serum in Morse’s defined medium (MDM; (35)) for 15 min at 37°C.
136
Complement deposition on the surface of bacteria was analyzed by
137
immunofluorescence of PFA-fixed bacteria using monoclonal antibody against
138
iC3b (3E7) (gift of R. P. Taylor, University of Virginia).
139
Δspa S. aureus strain Newman (provided by E. Skaar, Vanderbilt
140
University) was grown in liquid LB media for 16-18h at 37 °C. S. aureus was
141
opsonized in 20% normal human serum in MDM for 20 min at 37 °C. For
142
immunofluorescence experiments, opsonized S. aureus was simultaneously
143
labeled with 10µg ml-1 4′- 6-diamidino-2-phenylindole (DAPI, Sigma).
144 7
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
124
Isolation of human neutrophils. Peripheral venous blood was collected from
146
healthy human donors into heparinized tubes. Each donor gave written informed
147
consent and the procedure was approved by the University of Virginia
148
Institutional Review Board for Health Science Research. Neutrophils were
149
purified using Ficoll-Hypaque gradient followed by hypotonic erythrocyte lysis as
150
described previously (18). Neutrophils were pelleted and resuspended in ice-cold
151
DPBS without calcium and magnesium containing 0.1% dextrose at a
152
concentration of 1x107 to 2x107 cell ml-1 , kept on ice and used for experiments
153
within 30 min. Neutrophil content in suspension was >95% as monitored by
154
phase-contrast microcopy. Replicate experiments were conducted using
155
neutrophils from different donors to avoid any donor-specific effects.
156 157
Detection of reactive oxygen species (ROS). Luminol-dependent
158
chemiluminescence was used to detect ROS production by primary human
159
neutrophils as previously described (29), except that the assay was performed in
160
a 96-well plate, in a total volume of 200 µl, containing 2x105 neutrophils per well
161
in suspension. Bacteria were washed into MDM and incubated at 37 °C in the
162
presence of 20 µM luminol (Sigma) at MOI ~150. Fusion of primary granules with
163
neutrophil membranes was induced by pretreating neutrophils with 30 μM
164
lysophosphatidylcholine (18:0) (LPC, Sigma-Aldrich) prior to addition of bacteria.
165
Luminescence measurements from triplicate wells for each condition were taken 8
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
145
166
on a VICTOR3TM Wallac luminometer (Perkin-Elmer) every 3 min over 1 h.
167
Experiments were performed 3 times, with a single representative experiment
168
shown.
169
Neutrophil cytosol-membrane fractionation. 1x107 neutrophils were
171
suspended in DPBS containing 0.1% dextrose, 1.25 mM CaCl2, 0.5 mM MgCl2,
172
0.4 mM MgSO4 in the absence or presence of bacteria. Neutrophils were
173
incubated for indicated times in a 37°C water bath, with mixing by inversion every
174
5 min. The reactions were stopped by adding 5 volumes of ice-cold DPBS
175
containing 0.1% dextrose. Membrane/cytosol fractionation was performed
176
according to method described (36) with the following modifications. Neutrophils
177
were pelleted and resuspended in relaxation buffer (10 mM PIPES pH 7.3, 100
178
mM KCl, 3 mM NaCl, 1.25 mM EGTA, 5 mM EDTA, 1 mM PMSF, 20 µg ml-1
179
leupeptin, 20 µg ml-1 pepstatin). Samples were sonicated at 50% amplitude for
180
15 sec on ice using Sonic Dismembranator Model 120 (Fisher Scientific), and
181
unbroken cells and nuclei were pelleted by centrifugation at 800 g for 10 min at
182
4°C. The supernatant was centrifuged at 50,000 g for 12 min at 4°C in an
183
OptimaTM TLX bench top ultracentrifuge (Beckman Coulter) to pellet membranes.
184
The pellet was resuspended in solubilization buffer (20 mM Tris pH 7.5, 1% SDS,
185
1 mM PMSF, 20 µg ml-1 leupeptin and 20 µg ml-1 pepstatin) in volume equal to
186
the initial total volume. Equal volumes of each fraction, in 1x SDS sample buffer, 9
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
170
were separated by 12% SDS-PAGE and transferred to PVDF (Millipore). After
188
blocking with 5% BSA (Sigma), membranes were probed with mouse anti-p47phox
189
(BD Biosciences), rabbit anti-p67phox (Epitomics), rabbit anti-p40phox (Upstate),
190
mouse anti-p22phox (Santa Cruz), or mouse anti-GAPDH (Santa Cruz), and
191
followed by horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-
192
rabbit IgG (Thermo Scientific). Blots were developed with the Super Signal West
193
Pro detection kit (Thermo Scientific). Images were acquired on a Gel DocTM XR+
194
System using ImageLabTM software (Bio Rad).
195 196
Immunofluorescence and image acquisition. Neutrophils were suspended in
197
RPMI (Mediatech) containing 10 nM interleukin-8 (R&D Systems) and allowed to
198
attach to glass coverslips for 30 min at 37 °C, 5% CO2. The coverslips were
199
coated with 50% pooled human serum (Sigma) as described (12). Neutrophils
200
were then infected with non-opsonized Opaless Gc, non-opsonized OpaD+nv Gc,
201
or serum-opsonized S. aureus as described (12). Under these conditions
202
neutrophils can phagocytose unopsonized, Opa-negative Gc (12, 13, 28).At 15,
203
30, and 60 min post infection, cells were fixed with 4% PFA in PBS for 10 min,
204
followed by blocking in 10% normal goat serum for 15 min. Immunofluorescence
205
staining to discriminate internalized and external bacteria was performed as
206
described (12). Permeabilized neutrophils were also stained with antibodies
207
against p47phox (provided by W. M. Nauseef, University of Iowa), p67phox , or a 10
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
187
208
mix of monoclonal antibodies against gp91phox (54.1) and p22phox (44.1) (Santa
209
Cruz), followed by Alexa Fluor-coupled goat anti-rabbit or goat anti-mouse
210
antibodies (Life Technologies). For some experiments total Gc was prelabeled
211
with CFSE; this did not affect bacterial uptake or trafficking in neutrophils (data
212
not shown). Images of 10-20 63x fields for each condition were acquired on a Nikon
214
Eclipse E800 UV/visible fluorescence microscope with Hamamatsu Orca-ER
215
digital camera using Openlab 5.5.0 software (Improvision). For consistency in
216
presentation, images were false colored using Adobe Photoshop CS5 as follows.
217
In phagocytosis experiments, external bacteria appear red and green, while
218
internalized bacteria appear green only. In experiments assessing NADPH
219
oxidase gp91phox/ p22phox subunit recruitment to phagosomes, external bacteria
220
appear blue and red, internalized bacteria appear red only, and gp91phox/ p22phox
221
subunits appear green. In experiments assessing NADPH oxidase p47phox and
222
p67phox subunit recruitment to phagosomes, external bacteria appear blue, red
223
and green, internalized bacteria appear red only, and p47phox and p67phox
224
subunits appear green. For each condition, 50-200 bacteria or phagosomes
225
were analyzed. Phagosomes were considered positive for presence of the
226
subunits of NADPH oxidase when fluorescence staining was found at 50% or
227
more of the phagosome perimeter. Values are expressed as a mean ± SEM from
228
three replicate experiments. Statistical analysis was performed using Student’s 11
Downloaded from http://iai.asm.org/ on November 13, 2017 by guest
213
229
two tailed t-test, and differences were considered statistically significant at P
230