Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase
Signaling during Growth in Macrophages
Laura J. MacDonald,aRichard C. Kurten,b,cand Daniel E. Votha
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USAa; Department of Physiology and Biophysics,
University of Arkansas for Medical Sciences, Little Rock, Arkansas, USAb; and Arkansas Children’s Hospital Research Institute, Little Rock, Arkansas, USAc
Coxiella burnetii is the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in im-
munocompromised individuals. Following aerosol-mediated transmission, C. burnetii replicates in alveolar macrophages in a
unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms of C. burnetii intracellular
probed the role of PKA signaling during C. burnetii infection of macrophages. Using PKA-specific inhibitors, we found the ki-
nase was needed for biogenesis of prototypical PV and C. burnetii replication. PKA and downstream targets were differentially
gered PKA activation, which was also required for PV formation by virulent C. burnetii isolates during infection of primary hu-
man alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated during C. burnetii infection,
satile role for PKA in C. burnetii infection and indicate virulent organisms usurp host kinase cascades for efficient intracellular
gen can also establish a chronic infection resulting in potentially
ing cause of noncultivatable infectious endocarditis, a condition
that is notoriously difficult to treat with current antibiotics (36).
C. burnetii is naturally spread by contaminated aerosols, and live-
stock workers are often exposed to the organism while working
with infected animals, particularly during parturition. This expo-
sure risk was recently highlighted by a major Q fever outbreak in
the Netherlands that resulted in over 4,000 cases and 11 deaths
C. burnetii has historically been difficult to study and pathogenic
determinants are not well understood.
In vivo, C. burnetii targets alveolar phagocytic cells, with mac-
ing uptake into a host cell, the pathogen is housed for 4 to 6 h in a
tight-fitting phagosome that decorates with early endosomal
early vacuole also interacts with autophagosomes (20, 37) and
fluid-phase endosomes before ultimately fusing with lysosomes
(43). Although antibacterial acid hydrolases are present in the
vacuole, lysosomal fusion triggers acid pH-dependent activation
of C. burnetii metabolism (21, 32), and the organism replicates in
tious cycle necessitates regulation of host cell survival to ensure a
intrinsic apoptosis by triggering a prosurvival transcriptional re-
vival kinases Akt and Erk1/2 to promote cell survival (44). These
events are likely controlled by the organism’s Dot/Icm type IV
oxiella burnetii is the highly infectious bacterial agent of hu-
man Q fever, a zoonotic disease that typically presents as an
secretion system that translocates effector proteins into the host
cytosol, where they regulate intracellular replication and inhibi-
tion of apoptosis (4, 10, 29). However, host signaling pathways
that regulate parasitophorous vacuole (PV) formation and main-
tenance have not been defined.
Previous studies uncovered the presence of host vacuolar fu-
sogenic proteins, such as Rab GTPases, on the PV membrane but
did not assess the role of kinase-dependent signaling cascades in
vacuole biogenesis (7, 9, 37). We recently performed a directed
for PV generation (25). This study uncovered a role for many
kinases in PV formation and suggests signaling cascades may be
intimately linked to biogenesis of pathogen replication vacuoles.
Eleven kinases were involved in PV formation, including protein
kinase C (PKC), myosin light chain kinase, calmodulin-depen-
(PKA). PKA is a versatile host protein that directs many host re-
sponses, including cell survival, cytokine production, and cyto-
skeletal organization (34). PKA target proteins are differentially
phosphorylated during C. burnetii infection, and the effects of
PKA inhibition are reversible (25), indicating the pathogen con-
tinually modulates this pathway during intracellular growth.
Received 30 January 2012 Returned for modification 1 March 2012
Accepted 13 March 2012
Published ahead of print 2 April 2012
Editor: F. C. Fang
Address correspondence to Daniel E. Voth, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
iai.asm.orgInfection and Immunityp. 1980–1986June 2012 Volume 80 Number 6
macologic inhibitors, we show that PKA activity is critical for
proper PV formation and robust bacterial replication. PKA phos-
Importantly, PKA activity is required for virulent C. burnetii in-
the pathogen’s in vivo target cell. Collectively, these results impli-
cate the PKA signaling cascade as a major determinant of C. bur-
netii-host cell interactions.
MATERIALS AND METHODS
C. burnetii and mammalian cell culture. C. burnetii phase II (RSA439)
Culture Collection [ATCC], Manassas, VA) as previously described (14).
C. burnetii Nine Mile phase I (RSA493) and G (Q212) isolates were cul-
tured in acidified citrate cysteine medium (33) for 7 days at 37°C in 5%
CO2and 2.5% O2and then collected by centrifugation and washed with
sucrose phosphate buffer prior to use. Human monocytic THP-1 cells
(ATCC) were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA)
supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C in
5% CO2. THP-1 cells were cultured in 24- or 6-well tissue culture plates
for infections. Cells were incubated with 200 nM phorbol 12-myristate
13-acetate (PMA; EMD Biosciences, San Diego, CA) overnight to stimu-
late differentiation into adherent, macrophage-like cells (45). Before in-
fection, medium containing PMA was replaced with PMA-free medium.
THP-1 cells were infected with C. burnetii in phase I or phase II at a
multiplicity of infection (MOI) of 100 or 10, respectively, by addition of
organisms to the culture medium. C. burnetii in phase II was used at a
lower MOI due to increased infectivity of these organisms compared to
phase I bacteria (43). All work with virulent C. burnetii was performed in
the Centers for Disease Control and Prevention-approved biosafety lev-
el-3 facility at the University of Arkansas for Medical Sciences. Where
indicated, chloramphenicol (10 ?g/ml) was added to cell cultures to in-
hibit bacterial protein synthesis.
Primary human alveolar macrophages were isolated by bronchioal-
veolar lavage from lung tissue obtained postmortem from the National
Disease Research Interchange. Lavage fluid was filtered, subjected to a
Ficoll gradient, and cells collected by centrifugation. After processing,
cells were placed in 24-well plates in Dulbecco modified Eagle medium/
sulfate (50 ?g/ml), and gentamicin sulfate (50 ?g/ml) (Invitrogen) and
incubated for 2 h at 37°C in 5% CO2. Nonadherent cells were then re-
rophages were infected as described above for THP-1 cells in the absence
of penicillin, streptomycin, and gentamicin. Alveolar macrophages were
routinely assessed for homogeneity by immunofluorescence and immu-
noblot analysis (data not shown) with antibodies directed against the
macrophage-specific markers CD11/18b, CD14, and CD68 (Abcam,
in 24-well plates on 12-mm glass coverslips for microscopy analysis. Two
hours before infection, PKA was inactivated by pharmacologic inhibition
with H-89 (10 ?M; Sigma-Aldrich, St. Louis, MO) or Rp-adenosine-
3=,5=-cyclic mono-phosphorothioate triethylamine salt (Rp-cAMPS; 100
to 250 ?M; Enzo Life Sciences, Plymouth Meeting, PA). Cells were in-
fected with C. burnetii isolates as described above and processed for mi-
were considered to contain typical PVs when a large vacuole (?10 ?m in
diameter) was present and contained replicating organisms.
FFU assays. THP-1 cells were seeded in 24-well plates and differenti-
to 250 ?M) for 2 h. The cells were then infected with C. burnetii as de-
scribed above. At 48 hpi, the cells were washed and harvested by scraping
icated to release intracellular bacteria, and the samples were applied to
Vero cells in 24-well plates. Infected Vero cells were incubated for 120 h
and then processed for fluorescence microscopy using an anti-C. burnetii
ary antibody (Invitrogen) to detect infectious foci. Focus-forming units
(FFU) were visualized using a ?40 objective lens and quantified as the
FFU/ml as previously described (13, 14).
tion of THP-1 cells or human alveolar macrophages as described above,
the cells were fixed and permeabilized with 100% ice-cold methanol for 3
min, then blocked for 1 h in phosphate-buffered saline (PBS) containing
0.5% bovine serum albumin (BSA; Cell Signaling, Danvers, MA) at room
temperature. The cells were incubated with mouse anti-CD63 (BD Bio-
sciences, San Jose, CA) and rabbit anti-C. burnetii primary antibodies for
and incubated in PBS containing 0.5% BSA with Alexa Fluor 488-conju-
gated anti-mouse and Alexa Fluor 594-conjugated anti-rabbit secondary
antibodies (Invitrogen) for 1 h at room temperature. The cells were incu-
bated with DAPI (4=,6=-diamidino-2-phenylindole dilactate; Invitrogen)
for 5 min at room temperature to stain eukaryotic and bacterial DNA.
Fluorescence microscopy was performed using a Ti-U microscope with
a ?60 oil immersion objective (Nikon, Melville, NY). Images were ob-
tained with a D5-QilMc digital camera and analyzed using NIS-Elements
Immunoblot analysis. C. burnetii-infected mammalian cells in six-
well plates were directly lysed in buffer containing 1% sodium dodecyl
sulfate (SDS) by 10 passages through a 26-gauge needle and boiled for 5
ferred to a 0.2-?m-pore-size polyvinylidene fluoride membrane (Bio-
Rad, Hercules, CA), and blocked in Tris-buffered saline (150 mM NaCl,
100 mM Tris-HCl [pH 7.6]) containing 0.1% Tween 20 and 5% nonfat
milk for 1 h at room temperature. After blocking, the membranes were
probed for equal protein loading using a mouse anti-?-tubulin primary
antibody (Sigma-Aldrich) overnight at 4°C. The lysates were probed for
or phosphorylated PKA (Thr197) (Cell Signaling). The lysates were then
probed for total phosphorylated PKA substrates using rabbit primary an-
tibody to phospho-(Ser/Thr) PKA substrates, which detects phosphory-
lation of S/T residues with arginine at the ?3 position (Cell Signaling).
Lysates were probed for specific downstream PKA substrates using rabbit
primary antibodies directed against total CREB, phospho-CREB (Ser133),
horseradish peroxidase (Cell Signaling), which were was used to detect the
PKA activity is required for C. burnetii PV formation and bac-
terial replication. Our previous studies showed that the PKA in-
hibitor H-89 prevents prototypical PV formation (25). H-89 is
commonly used as a specific inhibitor of PKA (3) but can less
and stress-activated kinase 1 (MSK1) and p70 ribosomal protein
itor. Rp-cAMPS is a PKA-specific inhibitor that competes with
endogenous cAMP for binding sites on PKA, efficiently prevent-
ing activation (6). It is important to note that the effective dose of
Rp-cAMPS is higher than that of H-89 due to lower cell permea-
bility (6, 8), and the inhibitor was therefore tested at 100 to 500
Coxiella burnetii Alters Host PKA Activity
June 2012 Volume 80 Number 6iai.asm.org 1981
?M. For these studies, we used PMA-differentiated human mac-
rophage-like THP-1 cells, which are a reliable in vitro model of C.
burnetii-macrophage interactions (45). The cells were pretreated
with H-89 or Rp-cAMPS for 2 h and then infected with avirulent
C. burnetii NMII for 48 h in the presence of inhibitors. The cells
were processed for fluorescence microscopy using an antibody
directed against the late phagosome/lysosome marker CD63 to
label PV (Fig. 1A). Single, large PVs were present in fewer than
(Fig. 1B), indicating PKA is required for optimal vacuole forma-
tion. Rp-cAMPS-treated cultures contained significantly more
cells harboring multiple, small PVs with fewer organisms in each
vacuole. In addition, typical PV were present in ?5% of H-89-
treated cells (Fig. 1B), confirming our previous results.
We next assessed the requirement of PKA activity for C. bur-
netii replication using a standard FFU assay (13, 14). THP-1 cells
were infected for 48 h in the presence of H-89 or Rp-cAMPS and
then sonicated to release intracellular organisms. Sonicated sam-
ples were applied to a monolayer of Vero cells for 120 h, and PVs
indicative of infectious foci were enumerated. As shown in Fig. 2,
FFU significantly decreased in inhibitor-treated, infected cells
sulted in ca. 50% fewer FFU and H-89-treated cells resulted in an
ca. 70% decrease in FFU. Together, these results indicate PKA
activity is critical for C. burnetii replication that relies on forma-
tion and expansion of the phagolysosome-like PV.
PKA is activated during C. burnetii infection. Because PKA
dicted that C. burnetii activates PKA and downstream signaling
during intracellular growth. To assess PKA activity, we examined
phosphorylation of the kinase during infection by immunoblot.
phorylated on Thr197 by either the upstream kinase phosphoi-
nositide-dependent kinase 1 or autophosphorylation (34). As
shown in Fig. 3, PKA phosphorylation levels in C. burnetii-in-
infected in the presence of chloramphenicol to inhibit bacterial
protein synthesis (Fig. 3B), indicating C. burnetii actively triggers
PKA activation. In addition, PKA phosphorylation levels did not
increase during infection of cells treated with H-89 (data not
mary human alveolar macrophages. Most studies of C. burnetii-
Rp-cAMPS (100 or 250 ?M) 2 h prior to infection. Inhibitors were present
through 48 hpi, and then the cells were prepared for microscopy. (A) The
stained with DAPI (blue), and bacteria were detected using a C. burnetii-
hpi, and arrows denote small, atypical PV. Bar, 10 ?m. (B) PV in at least five
fields of 20 cells/field were counted for each treatment condition, and the
percentages of cells forming large vacuoles (?10 ?m) containing replicating
asterisk indicates a P value of ?0.005 compared to untreated cells as deter-
mined using the Student t test. These results show that H-89 and Rp-cAMPS
antagonize PV formation.
FIG 2 PKA inhibitors antagonize C. burnetii replication. THP-1 cells were
infected with NMII C. burnetii after treatment with H-89 (10 ?M) or Rp-
cAMPS (100 ?M) 2 h prior to infection. Inhibitors were present throughout
the infection time course and, at 48 hpi, the cells were sonicated to release
intracellular organisms. Harvested organisms were incubated with Vero cells
for 120 h and then processed for immunofluorescence microscopy. The vacu-
oles representative of infectious foci were enumerated as FFU/ml. Error bars
of ?0.05 compared to untreated cells as determined using the Student t test.
Samples were analyzed in triplicate and are representative of at least two inde-
pendent experiments. Treatment with either PKA inhibitor prevents C. bur-
MacDonald et al.
iai.asm.org Infection and Immunity
vitro host cell interactions, it is important to examine infection-
these events occur during natural infection. We recently devel-
oped a new in vitro model for C. burnetii-host cell interactions
chioalveolar lavage samples. During natural infection, C. burnetii
targets alveolar macrophages for replication. The pathogen repli-
cates efficiently in a large PV in these cells in vitro (J. G. Graham
and D. E. Voth, unpublished results), representing the most ap-
propriate model of in vivo cellular interactions. Here, alveolar
macrophages were treated with H-89 and then infected with vir-
positive PV in primary alveolar macrophages. However, macro-
phages treated with H-89 do not support PV expansion, suggest-
ing that PKA activity is required for natural C. burnetii infection.
Inhibitor treatment results correlated well with increased PKA
phosphorylation levels in infected macrophages at 72 to 96 hpi
(Fig. 4B). Together, these results implicate a major role for PKA
during in vivo infection.
PKA substrates are differentially phosphorylated during C.
burnetii infection. PKA regulates numerous downstream targets
to efficiently control the host response to infection (34). We pre-
viously showed that PKA substrates are differentially phosphory-
lated during infection using a pan-phospho-PKA substrate anti-
Lysates were harvested from THP-1 cells infected in the presence
or absence of chloramphenicol. As shown in Fig. 5, C. burnetii
treatment, indicating the pathogen actively modulates PKA sig-
(25), infection in the absence of chloramphenicol resulted in dif-
ferential phosphorylation of 8 PKA substrates. This response was
also negated when infected cells were treated with H-89 (data not
shown), indicating C. burnetii activates a specific PKA signaling
quences of this differential regulation are not known. To address
this gap in our understanding of C. burnetii-regulated PKA activ-
teins during infection. Increased phosphorylation of cAMP re-
sponse element binding protein (CREB) indicates activation of
the protein ((5, 40), while phosphorylation of Bad, p105 (an
NF-?B family member), and glycogen synthase kinase-3? (GSK-
3?) correlates with inactivation (5, 12, 41). We first monitored
phosphorylation of CREB, which is a common readout of PKA
activity and is generally phosphorylated when PKA activity in-
creases (5, 19, 40). Interestingly, phosphorylated levels of CREB
postinfection (Fig. 6A). Similar to CREB, GSK-3? phosphoryla-
with NMII C. burnetii in the absence (? Cm) or presence (? Cm) of chlor-
indicated times postinfection and subjected to immunoblot analysis with an-
tibodies directed against total or phosphorylated PKA. C, control, uninfected
cells; p-, phosphorylated. PKA phosphorylation levels continually increase
from 24 to 96 hpi, suggesting the kinase is activated throughout intracellular
growth. Treatment with chloramphenicol abrogates increased phosphoryla-
tion of PKA, indicating C. burnetii actively triggers activation of the kinase.
I (NMI) or G. Infections proceeded for 48 h, and then the cells were prepared
for microscopy. The lysosomal marker CD63 (green) was used to confirm PV
formation, DNA was stained with DAPI (blue), and bacteria were detected
using a C. burnetii-specific antibody (red). Arrowheads indicate typical PV
(?10 ?m) at 48 hpi, and arrows denote small, atypical PV. Bar, 10 ?m. PKA
macrophages were infected with C. burnetii NMI or G for the indicated times,
and then cell lysates were harvested and subjected to immunoblot analysis
using primary antibodies to detect total or phosphorylated PKA. C, control,
uninfected cells; p-, phosphorylated. Similar to the in vitro THP-1 model,
virulent C. burnetii isolates trigger increasing levels of phosphorylated PKA
indicative of activation at 72 to 96 hpi.
Coxiella burnetii Alters Host PKA Activity
June 2012 Volume 80 Number 6iai.asm.org 1983
tion levels did not change compared to uninfected cells (Fig. 6B).
In contrast, increased phosphorylation was observed for Bad be-
tween 24 and 96 hpi and for p105 between 2 and 96 hpi (Fig. 6B).
Taken together, these results suggest C. burnetii regulates PKA
all PKA-dependent responses. Furthermore, levels of phosphory-
lated Bad and p105 also increased during virulent C. burnetii in-
fection of primary human alveolar macrophages (data not
shown), further supporting a role for PKA-dependent responses
in natural infection.
Eukaryotic PKA is required for PV formation and bacterial repli-
cation during virulent and avirulent C. burnetii infection of mac-
rophages. An increase in PKA phosphorylation indicative of acti-
vation occurs throughout infection, suggesting prolonged
stimulation of the signaling cascade. C. burnetii-directed altera-
tion of PKA activity triggers downstream phosphorylation of dis-
tinct targets. Interestingly, C. burnetii does not activate all PKA-
dependent events, indicating the pathogen cleverly usurps a
kinase cascade to elicit specific downstream cellular responses.
alters host cell physiology via signaling cascades to survive in a
degradative phagolysosomal compartment.
H-89, we used a more specific antagonist, Rp-cAMPS. Although
Rp-cAMPS negatively impacts the formation of large, prototypi-
cal PVs and C. burnetii replication, it is less efficient than H-89
treatment. One explanation for this discrepancy may be differ-
ences in cell permeability of the two inhibitors, with H-89 more
efficiently crossing cellular membranes to access PKA. A second,
more intriguing possibility is the involvement of other kinases
reportedly inhibited by H-89 in certain systems. For example,
H-89 can influence activity of the kinases MSK and S6K (15).
ence PV formation, we are currently probing the potential role of
these proteins in C. burnetii infection. We do not predict S6K
involvement in PV formation, since the kinase is not activated
ity is often analyzed in cell-free biochemical assays or in specific
cell lines. Thus, non-PKA effects of H-89 must be determined in
macrophages to understand their role in C. burnetii infection.
nipulated by other intracellular pathogens. Brucella suis causes
increased cAMP accumulation and CREB phosphorylation in
macrophages and H-89 treatment prevents bacterial replication
(19). Similarly, treating J774 macrophages with H-89 prevents
intracellular replication of Mycobacterium smegmatis and M. tu-
berculosis (26). These results are similar to our data showing that
H-89 or Rp-cAMPS treatment prevent typical PV formation and
bacterial replication. cAMP and PKA control many events that
alter eukaryotic cellular physiology, including phagocytosis and
phagosome biogenesis (35). Indeed, cAMP signaling controls en-
dosome acidification in some systems (42) and PKA regulates au-
tophagosome formation through interactions with LC3 (11, 31).
netii infection (L. J. MacDonald and D. E. Voth, unpublished re-
sults). However, cAMP compartmentalization is critical for con-
trolling distinct downstream signaling events (35). PKA activity
(23). Therefore, proper cAMP and PKA localization may be im-
portant for C. burnetii infection and this possibility is under in-
FIG 6 Distinct downstream PKA targets are phosphorylated during C. bur-
netii infection. THP-1 cells were infected with NMII C. burnetii and lysates
harvested at the indicated points of time. Lysates were probed for total or
phosphorylated forms of CREB (A) or Bad, GSK-3?, or p105 (B). C, control,
uninfected cells; p-, phosphorylated. C. burnetii infection does not induce
substantial changes in CREB or GSK-3? phosphorylation. In contrast, the
phosphorylated levels of Bad and p105 increase at 24 and 2 hpi, respectively,
and the levels remained elevated above those of control cells throughout in-
fection, indicating continual altered regulation of these PKA targets.
FIG 5 C. burnetii actively regulates PKA substrate phosphorylation. THP-1
cells were infected with NMII C. burnetii in the presence or absence of chlor-
strates and tubulin as a loading control. C, control, uninfected cells. The mo-
lecular mass (MM) is shown in kilodaltons. Asterisks indicate proteins with
increased phosphorylation levels at 24 to 96 hpi and circles denote proteins
with decreased phosphorylation levels after 48 hpi that are distinct from pro-
files observed in chloramphenicol-treated cells. PKA substrates are differen-
tially phosphorylated during infection and chloramphenicol treatment abro-
gates these changes, further indicating C. burnetii actively modulates PKA
MacDonald et al.
iai.asm.orgInfection and Immunity
eton-related proteins. F-actin is recruited to the PV early during
infection and actin depolymerizing agents disrupt vacuole matu-
ration (1). The small GTPases RhoA and Cdc42 are also recruited
to the PV and are predicted to control actin assembly on the ma-
turing vacuole. PKA is known to regulate activity of RhoA and
Cdc42. PKA inactivates RhoA by phosphorylation, promoting
RhoA interaction with Rho guanine dissociation inhibitor pro-
teins that maintain the protein in an inactive cytosolic form (16,
17). Conversely, PKA promotes the activation of Cdc42, allowing
ization (18). AKAPs can tether PKA to cytoskeletal components
(23), further suggesting the importance of PKA localization dur-
regulating actin polymerization around the maturing vacuole.
We predict that PKA has a dual role in C. burnetii infection.
First, PKA is involved in PV expansion and thus must act during
target proteins increases throughout infection at times when the
PV has fully matured and expanded. Specific downstream targets
altered during infection, including Bad and p105, do not have a
predicted role in phagosome maturation but may be critical for
other aspects of infection following PV establishment. Bad is a
mitochondrial proapoptotic protein that regulates cytochrome c
release (27). C. burnetii potently inhibits host cell apoptosis in a
cytochrome c- and effector-dependent manner (29, 30, 45); how-
ever, the host components involved have not been fully defined.
Bad phosphorylation by PKA on Ser155, which inactivates Bad,
likely contributes to preventing apoptosis. Bad can also be phos-
phorylated on Ser136 by Akt, a kinase activated during infection
(44), to regulate mitochondrial-dependent apoptosis, suggesting
that C. burnetii uses both PKA and Akt to target Bad and prevent
production (5). After phosphorylation and proteolysis of p105, the
production by macrophages in response to M. smegmatis (47), sup-
porting a role for PKA in regulating the cytokine response to infec-
(38), suggesting the p105 pathway may regulate this cytokine re-
PKA activity is also required for optimal PV formation in pri-
mary macrophages. Alveolar macrophages are central to C. bur-
netii infection, representing the pathogen’s initial target cell upon
aerosol-mediated uptake by a host. Virulent C. burnetii efficiently
infects and replicates in primary human alveolar macrophages in
vitro (Graham and Voth, unpublished) and increased PKA phos-
phorylation is apparent at 3 to 4 days postinfection. PKA alters
alveolar macrophage responses to microbial pathogens by regu-
lating antimicrobial molecule production. Specifically, PKA reg-
ulates production of TNF-? and H2O2(46), defenses used by eu-
karyotic cells to degrade intracellular organisms. Avirulent C.
burnetii disrupts assembly of the NADPH oxidase complex in
neutrophils and mouse macrophages, lowering reactive oxygen
species (ROS) levels (22, 39). Thus, it is tempting to predict the
pathogen stimulates PKA activity to alter ROS-directed signaling.
In conclusion, we have uncovered a role for host PKA in C.
burnetii PV formation. Similar to previous findings on Akt and
Erk1/2 (44), PKA activity is stimulated throughout infection, fur-
ther indicating that C. burnetii continually manipulates host sig-
naling long after uptake by macrophages. Modulation of PKA
the kinase is activated and involved in PV generation in primary
human alveolar macrophages. Our results present yet another ex-
ample of how intracellular pathogens adeptly subvert host cell
functions using complex kinase signaling pathways. Future stud-
ies on PKA control of cytoskeletal organization, inhibition of
apoptosis, and cytokine production will provide a better under-
standing of the scope of host signaling required for C. burnetii
We thank Joseph Graham for critical reading of the manuscript.
This research was supported by funding to D.E.V. from the American
Heart Association (BGIA3080001) and NIH/NIAID (R01AI087669) and to
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