Host cell-free growth of the Q fever bacterium
Anders Omslanda, Diane C. Cockrella, Dale Howea, Elizabeth R. Fischerb, Kimmo Virtanevac, Daniel E. Sturdevantc,
Stephen F. Porcellac, and Robert A. Heinzena,1
aCoxiella Pathogenesis Section, Laboratory of Intracellular Parasites,bElectron Microscopy Unit, andcGenomics Unit, Research Technology Section, Research
Technology Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved January 22, 2009 (received for review November 26, 2008)
The inability to propagate obligate intracellular pathogens under
axenic (host cell-free) culture conditions imposes severe experi-
mental constraints that have negatively impacted progress in
understanding pathogen virulence and disease mechanisms. Cox-
iella burnetii, the causative agent of human Q (Query) fever, is an
obligate intracellular bacterial pathogen that replicates exclusively
in an acidified, lysosome-like vacuole. To define conditions that
support C. burnetii growth, we systematically evaluated the or-
ganism’s metabolic requirements using expression microarrays,
genomic reconstruction, and metabolite typing. This led to devel-
opment of a complex nutrient medium that supported substantial
growth (approximately 3 log10) of C. burnetii in a 2.5% oxygen
environment. Importantly, axenically grown C. burnetii were
highly infectious for Vero cells and exhibited developmental forms
characteristic of in vivo grown organisms. Axenic cultivation of C.
burnetii will facilitate studies of the organism’s pathogenesis and
genetics and aid development of Q fever preventatives such as an
effective subunit vaccine. Furthermore, the systematic approach
used here may be broadly applicable to development of axenic
media that support growth of other medically important obligate
axenic growth ? metabolism ? microaerophile ?
obligate intracellular pathogen
like illness (1). Shortly after the discovery of Q fever as a clinical
axenic (host cell-free) conditions (3). However, despite over 6
limited to colonization of a viable eukaryotic host cell.
Early studies showed minimal C. burnetii metabolic capacity in
buffers adjusted to neutral pH (4). The organism’s intracellular
growth compartment was subsequently described as ‘‘phagoly-
sosomal-like’’ (5) which led to the discovery by Hackstadt and
Williams (6) that significant metabolic activity by C. burnetii only
occurs in buffers that mimic the moderately acidic (approxi-
mately pH 4–5) conditions of this vacuole. Building on this work,
we recently developed a nutrient medium termed Complex
Coxiella Medium (CCM) that supports axenic metabolic activity
by C. burnetii for at least 24 h (7). Critical components of CCM
include 3 complex nutrient sources (neopeptone, FBS, and
RPMI cell culture medium), a high concentration of chloride
(140 mM), and citrate buffer (pH approximately 4.75) (7).
The obligate intracellular nature of C. burnetii imposes con-
siderable experimental limitations that impede progress in un-
derstanding the organism’s physiology and virulence. Indeed,
systems to genetically manipulate Coxiella are lacking and
lipopolysaccharide is the only defined virulence factor of the
in CCM (unpublished data), the pathogen’s sustained axenic
metabolic activity in this medium suggested a modified formu-
lation might support replication. To this end, we evaluated the
organism’s transcriptome during metabolism in CCM and iden-
oxiella burnetii is the causative agent of human Q fever, a
disease that typically manifests as a debilitating influenzae-
tified a potential nutritional deficiency of this medium. More-
over, using genomic reconstruction and metabolite typing, we
defined C. burnetii as a microaerophile. These data allowed
development of a medium that supports axenic growth of
infectious C. burnetii under microaerobic conditions.
initial step to identify nutritional deficiencies of CCM that could
preclude C. burnetii cell division, a comparison of genome wide
transcript profiles between organisms replicating in Vero cells
and incubated in CCM for 24 h was conducted. This analysis
showed substantially reduced expression of ribosomal genes
during incubation in CCM (supporting information (SI) Table
S1), suggesting that protein synthesis was insufficient to support
C. burnetii replication in this axenic medium. Supplementation
of CCM with pyruvate, succinate, or glutamate, efficiently
oxidized energy sources of C. burnetii (9), did not improve C.
burnetii de novo protein synthesis in CCM (7), suggesting energy
starvation was not the reason for reduced ribosomal gene
Supplementation of CCM with Protein Precursors Improves C. burnetii
Catabolic Activity. Amino acid deficiencies in CCM could also
explain reduced ribosomal gene expression. C. burnetii has
multiple amino acid auxotrophies that appear compensated for
by amino acid and peptide transporters (10). Moreover, intra-
cellular bacteria frequently use amino acids as carbon sources
(11), with an exceptionally high concentration of L-cysteine
required for axenic growth by some (12). To evaluate whether
supplementation of CCM with amino acids and peptides im-
proves C. burnetii metabolic activity, casamino acids (a mixture
medium (Table 1). Following 24 h preincubations in media, C.
burnetii was subjected to a 3 h [35S]Cys/Met pulse and the fold
increase in radiolabel incorporation over the negative control
(i.e., organisms labeled in labeling buffer at pH 7) used to assess
the catabolic capacity (7) of the organism. CCM supplemented
with casamino acids or L-cysteine supported statistically signif-
icant increases in C. burnetii radiolabel incorporation of (39.1 ?
5.1)-fold and (134.5 ? 23.4)-fold, respectively (Fig. 1A). The
effect of supplementing CCM with both casamino acids and
L-cysteine was additive, resulting in a (232.7 ? 33.5)-fold
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
March 17, 2009 ?
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no. 11 www.pnas.org?cgi?doi?10.1073?pnas.0812074106
Acidified Citrate Cysteine Medium (ACCM) (Table 1) sup-
ported an approximately 13-fold increase in protein synthesis
compared to CCM. SDS/PAGE and autoradiography confirmed
that radiolabel was incorporated into C. burnetii de novo syn-
thesized protein (Fig. 1B).
C. burnetii Substrate Oxidation Increases Under Microaerobic Condi-
tions. Terminal oxidases containing either cytochrome o or
cytochrome bd (associated with aerobic and microaerobic res-
piration, respectively) are encoded by the C. burnetii genome
(Fig. 2A). This observation suggested that C. burnetii responds to
alterations in oxygen tension during intracellular growth. There-
fore, we assessed the effect of oxygen tension on C. burnetii
metabolism of a wide variety of metabolites, including amino
acids and carbohydrates, using Phenotype Microarrays (PM). C.
burnetii oxidation of 95 substrates in PM-1 arrays was tested
under oxygen tensions of 20%, 5%, and 2.5%. Incubation in 20%
oxygen resulted in efficient oxidation of only succinate (Fig. 2B).
However, incubations in 5% or 2.5% oxygen showed oxidation
of 10 and 17 substrates, respectively (Fig. 2B). Intermediates of
major metabolic pathways including the tricarboxylic acid cycle
and glycolysis were most efficiently oxidized (Fig. 2C). Several
substrates efficiently oxidized by C. burnetii in PMs have also
been identified as substrates for the organism in independent
studies (e.g., succinate, glutamate, and proline) (9, 13).
ACCM Supports C. burnetii Replication in a Microaerobic Environment.
The characterization of C. burnetii as a potential microaerophile
suggested that incubation in ACCM in an optimal oxygen
environment might support growth. Therefore, ACCM was
inoculated with C. burnetii and the cultures incubated in a 20%
or 2.5% oxygen environment. Cultures were monitored for C.
burnetii replication by measuring bacterial genome equivalents
(GE) by quantitative Taqman PCR (QPCR) every 24 h over 6
days. Incubation of C. burnetii in ACCM in 20% oxygen did not
result in an increase in C. burnetii GE (Fig. 3A). However,
substantial replication of C. burnetii occurred in 2.5% oxygen
in ACCM in 1% or 5% oxygen was similar to that observed in
2.5% while no replication was observed in 10% oxygen (data not
shown). The growth cycle of organisms incubated in ACCM in
Table 1. Components of Acidified Citrate Cysteine Medium
Ions Final concentrationa, b
Nutrients Final concentration
aConcentrations are in mM unless otherwise specified.
bExcluding contribution from neopeptone and FBS.
cAbsent in CCM.
determined by incubating organisms in CCM or CCM supplemented with 2.5
mg/ml casamino acids, 1.5 mM L-cysteine or both (ACCM). Bacteria were
preincubated in the respective media for 24 h, then labeled with [35S]Cys/Met
in labeling buffer (pH 4.5) for 3 h. (A) De novo protein synthesis by C. burnetii
was measured by quantification of radiolabel incorporation by scintillation
counting. Results are expressed as fold increase in incorporation when com-
pared with incorporation of bacteria preincubated in CCM, then labeled in
labeling buffer (pH 7.0) (negative control). Casamino acids and L-cysteine
significantly improved C. burnetii metabolic activity. (B) SDS/PAGE and auto-
radiography confirmed incorporation of radiolabel into bacterial proteins.
Values are mean ? SEM (n ? 3). The level of radiolabel incorporation in CCM
(pH 7.0) is normalized to 1.
decreasing oxygen availability. The ability of C. burnetii to oxidize substrates
encoding terminal oxidases associated with aerobic (cytochrome o) and mi-
croaerobic (cytochrome bd) metabolism suggested C. burnetii can respire
under microaerophilic conditions. (B) Purified C. burnetii was added to PM-1
plates and incubated for 24 h in 20%, 5%, and 2.5% oxygen. The number of
metabolites oxidized increased with decreasing oxygen tension, consistent
(C) Seventeen substrates were efficiently oxidized by C. burnetii in 2.5%
and expressed as relative OmniLog units (OLU). Quantitative analysis is rep-
resentative of at least 3 independent experiments. Substrate key (rows A-H,
columns 1–12): A1, no substrate control; A5, succinate; A8, L-proline; A11,
D-mannose; B12, L-glutamate; C2, D-galactonic acid-?-lactone; C9, ?-D-
glucose; D6, ?-ketoglutarate; E1, L-glutamine; E12, adenosine; F5, fumarate;
F6, bromo succinate; G4, L-threonine; G5, L-alanine; G9, mono methyl succi-
nate; H8, pyruvate; H9, L-galactonic acid-?-lactone; H11, phenylethylamine.
The number of substrates oxidized by C. burnetii increases with
Omsland et al.PNAS ?
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2.5% oxygen consisted of a lag phase of approximately 1 day,
followed by 3 days of exponential growth and the onset of
stationary phase thereafter. The generation time of C. burnetii
during exponential growth in ACCM was 9.1 h which is 1–2 h less
than the generation time in Vero cells (14). C. burnetii also
formed colonies (approximately 0.05–0.1 mm in diameter) after
a 14 day incubation in an ACCM-based solid agarose medium
To confirm that the increase in C. burnetii genome equivalents
corresponded to an increase in C. burnetii infective units,
organisms incubated in ACCM in 2.5% oxygen were assayed for
Vero cell infectivity using a fluorescent infectious focus-forming
unit (FFU) assay that employs a Coxiella-specific antibody.
ACCM culture aliquots taken every 24 h for 6 days postinocu-
lation showed an increase in FFUs of 2.19 logs (Fig. 3 B-C) with
the kinetics of FFU development directly corresponding to
increases in GE (Fig. 3A). Coxiella containing vacuoles showed
typical staining for the lysosomal marker CD63 (data not
shown). Ten to fifteen genomes were required for development
of each vacuole, equivalent to published GE/FFU ratios (14).
Collectively, these data show that ACCM supports robust cell
division of infectious C. burnetii that are antigenically similar to
cell culture-cultivated organisms.
C. burnetii Developmental Transitions Occur in ACCM. During infec-
tion of eucaryotic host cells, C. burnetii undergoes a biphasic
developmental cycle characterized by transition of metabolically
dormant, nonreplicative small cell variants (SCV) to metaboli-
cally active, replicative large cell variants (LCV) (14). To deter-
mine whether this developmental program occurs during repli-
cation in ACCM, medium was inoculated with purified SCVs
and morphological differentiation assessed by transmission elec-
tron microscopy (TEM). Ultrastructural features of the inocu-
lum were characteristic of the SCV, most notably small cell size
(? 0.2 ?m) and electron-dense chromatin (Fig. 4 A-B) (14).
Organisms incubated in ACCM for 3 days and in exponential
phase displayed ultrastructural characteristics of the LCV in-
cluding increased cell size (? 0.2 ?m) and dispersed chromatin
(Fig. 4 A-B) (14). Organisms incubated in ACCM for 6 days and
in early stationary phase were a mixture of SCVs and LCVs (Fig.
4 A-B). ACCM cultures initiated with purified SCVs also showed
approximately 3 logs of growth after 6 days of incubation (Fig.
S2). Developmental transitions were confirmed by immunoblot-
ting for ScvA, a DNA-binding protein specific to the SCV (14,
15). ScvA was detected only in the SCV inoculum and the
LCV/SCV mixture present at early stationary phase (Fig. 4C).
Thus, C. burnetii undergoes a developmental program in ACCM
that is similar to in vivo propagated organisms.
The systematic approach described herein allowed identification
of nutritional and biophysical conditions that support C. burnetii
host cell-free growth. Based on a 2.5–3 log increase in C. burnetii
GE after 6 days of culture in ACCM in 2.5% oxygen, we estimate
that 100 ml of ACCM can yield the same number of organisms
as 7 ? 108infected Vero cells. Growth of C. burnetii in ACCM
was also established with an inoculum as low as 100 GE/ml (data
not shown), suggesting ACCM can be used to isolate C. burnetii
from the small number of organisms typically contained in
clinical samples. Axenic culture of C. burnetii in ACCM will
improve our ability to define factors required for intracellular
growth and pathogenesis. Moreover, growth of C. burnetii on
solid ACCM medium will facilitate clonal isolation and devel-
opment of genetic tools for this organism.
microaerobic conditions. (A) C. burnetii GE were assessed by QPCR daily for 6
days. Incubation in 20% (f) oxygen did not support C. burnetii replication
while incubation in 2.5% (?) oxygen resulted in considerable C. burnetii
replication. (B) Increases in C. burnetii GE during incubation in ACCM corre-
lated with production of infectious bacteria as determined by a quantitative
FFU assay. Values are mean ? SEM (n ? 3). (C) Representative staining of FFUs
contained in equal aliquots of ACCM harvested at 2, 4, and 6 days post
in also shown. (Scale bars, 30 ?m.)
ACCM supports axenic cell division of infectious C. burnetii under
whether C. burnetii transitions between nonreplicative SCV and replicative
LCV developmental forms during incubation in ACCM, medium was inocu-
chromatin. Organisms incubated in ACCM for 3 days exhibited ultrastructural
characteristics of the LCV including increased cell size (average diameter:
0.456 ? 0.0078 ?m) and dispersed chromatin. Following 6 days of incubation,
a mixed population of SCVs and LCVs was observed, resulting in an overall
reduction in cell size (average diameter: 0.290 ? 0.0087 ?m). Values are
SCV inoculum and the LCV/SCV mixture present in stationary phase.
C. burnetii SCV to LCV development occurs in ACCM. To determine
www.pnas.org?cgi?doi?10.1073?pnas.0812074106Omsland et al.
The substantial increase in C. burnetii metabolic activity in
ACCM compared to CCM is largely an effect of L-cysteine. An
elevated level of L-cysteine is also required for in vitro culture
of Legionella pneumophila (16, 17) where the amino acid may
serve as an antioxidant in scavenging hydrogen peroxide (18).
While L-cysteine is bio-accessible to L. pneumophila, the oxi-
dized form L-cystine is not, making it necessary to supplement
nutrient media with L-cysteine in great excess of what is con-
sumed by the organism (12). ACCM containing the alternative
antioxidant L-glutathione instead of L-cysteine did not support
C. burnetii growth (data not shown), suggesting that C. burnetii’s
requirement for L-cysteine in ACCM is nutritional. In addition
to serving as a precursor in protein synthesis, L-cysteine may also
be a source of sulfur.
C. burnetii replication in ACCM is optimal in a 2.5% oxygen
environment and the presence of genes encoding cytochrome bd
physiological explanation for the observed growth phenotype.
Interestingly, the intracellular bacteria Mycobacterium tubercu-
losis (19), Chlamydia trachomatis (20), and Rickettsia rickettsii all
encode cydAB, suggesting adaptation to microaerobic metabo-
lism in intracellular bacteria may be underappreciated. Indeed,
transcriptional analysis of M. tuberculosis during infection of
macrophages indicates the organism adapts to a reduced oxygen
environment (21), and improved growth of Chlamydia pneu-
moniae is observed under low oxygen conditions (22). Like M.
tuberculosis, C. burnetii can occupy tissue granulomas (23), a
defined low oxygen environment (24). Mammalian cell intracel-
lular oxygen tension can be significantly lower than the extra-
cellular oxygen tension (25). Moreover, the membrane of the C.
burnetii replicative vacuole is enriched in cholesterol (26), and
cholesterol-rich membranes are known to impede oxygen diffu-
sion (27). Collectively, these observations suggest the vacuolar
compartment of C. burnetii is a low oxygen environment. In
addition to potentially serving as a terminal oxidase in low
oxygen conditions, CydAB may also protect C. burnetii against
oxidative agents of the phagolysosome. In support of this
hypothesis, cydB mutants of Brucella abortus and Escherichia coli
are hypersensitive to hydrogen peroxide (28, 29).
The debilitating nature of acute Q fever, along with C.
burnetii’s environmental stability and aerosol route of transmis-
sion, have raised concerns over potential illegitimate use of this
microorganism (30). In this regard, axenic cultivation of C.
burnetii will aid molecular characterization of the organism to
enable development of protective measures against Q fever,
including improved diagnostic tools and efficacious vaccines.
Moreover, the strategy used here to establish culture conditions
for C. burnetii may be broadly applicable to identifying media
formulations and biophysical conditions that support growth of
other currently obligate intracellular bacterial pathogens of
humans within the genera Anaplasma, Ehrlichia, Treponema,
Chlamydia and Rickettsia.
Materials and Methods
Cultivation and Purification of C. burnetii from Vero Cells. C. burnetii Nine Mile
phase II (RSA439, clone 4) was propagated in African green monkey kidney
(Vero) fibroblasts (CCL-81; American Type Culture Collection) grown in RPMI
medium (Invitrogen Corp.) supplemented with 2% FBS. At 7 days post infec-
tion, host cells were disrupted by sonication and C. burnetii purified by
differential centrifugation as described (31, 32). At this time point postinfec-
tion, infected Vero cells contain roughly equal numbers of SCV and LCV
morphological forms (14). C. burnetii SCVs were generated by prolonged
culture in Vero cells as previously reported (14) and purified as described
use. With the exception of experiments examining C. burnetii biphasic devel-
opment (Figs. 4 and S2), all experiments used C. burnetii purified from Vero
cells at 7 days post infection.
Transcription Microarray Analysis. For analysis of C. burnetii transcript profiles
during intracellular growth, Vero cells at 90–95% confluence in T-75 cell
to remove noninternalized bacteria, then 10 ml RPMI supplemented with 2%
washed with 10 ml of Hank’s buffered salt solution, then lysed with 3 ml of
TRIzol reagent (Invitrogen). For analysis of C. burnetii transcript profiles in
CCM, 2.5 ? 109GE of C. burnetii were incubated in triplicate in 0.5 ml CCM in
24-well plates for 24 h. Bacteria were transferred to a 1.5 ml Microfuge tube,
pelleted by centrifugation, then TRIzol reagent (1.0 ml) added to each tube.
Total RNA in TRIzol samples was purified and processed as previously de-
A MicrobEnrich kit (Applied Biosystems) was used to increase the relative
level of C. burnetii RNA derived from Vero cell-propagated organisms. En-
riched RNA (approximately 1 ?g) was amplified using a MessageAmp II
Bacteria kit (Applied Biosystem). Briefly, double stranded (ds) cDNA was
synthesized, product was purified using a QiaQuick 96-well system, and
All cRNA originating from Vero cell-propagated C. burnetii and 3 ?g cRNA
from CCM-cultivated organisms were hybridized to a custom Affymetrix
GeneChip designed as previously described (35). Microarray data were ana-
lyzed using Partek Genomics Suite software (Partek Inc.) essentially as de-
Radiolabeling with [35S] Cysteine/Methionine. Radiolabeling of C. burnetii
proteins was conducted using 2.5 ? 109GE of freshly thawed organisms.
Following preincubations in 6-well plates containing 2.0 ml medium per well,
buffer (7) supplemented with 1.0 mM glutamate (labeling buffer) to remove
excess nutrients. Bacteria were then resuspended in 500 ?l labeling buffer
containing 25–50 ?Ci [35S]Cys/Met protein labeling mix (Perkin-Elmer) and
incubated for 3 h in a screw-cap tube to allow incorporation of the radionu-
NaH2PO4, 150 mM NaCl, pH 7.8) to remove unincorporated [35S]Cys/Met.
Bacterial pellets were lysed in equal volumes of a SDS polyacrylamide gel
electrophoresis (SDS/PAGE) sample buffer and boiled for 10 min. Equal vol-
umes of each sample were analyzed by scintillation counting to determine
cpm. Twelve percent SDS/PAGE followed by autoradiography using CL-
Xposure (Pierce) film was used to visualize radiolabeled proteins (3 h expo-
sure). Precision Plus Protein Dual Color Standards (Bio-Rad) were used as
molecular mass markers.
Phenotype Microarrays. C. burnetii oxidation of substrates under different
oxygen concentrations was tested using Phenotype Microarrays (PM-1) (Bi-
olog Inc.) containing 95 substrates including amino acids and carbohydrates.
Purified C. burnetii were suspended in CCM (7) (5 ? 109GE/ml) supplemented
to promote oxidation of individual PM substrates. Bacterial suspension (100
?l) was added to each well of the 96-well PM plate and plates incubated for
to 5% CO2and 20%, 5%, or 2.5% O2. Atmospheric oxygen was displaced by
nitrogen gas. At the end of the incubation period, reduction of reporter dye
as OmniLog Units (OLU).
Preparation of ACCM Medium and Incubation Conditions. The ingredients of
ACCM are listed in Table 1. Casamino acids were prepared fresh at the time of
medium preparation while other components were kept as refrigerated
(citrate buffer, salt solution, RPMI cell culture medium) or frozen (neopep-
tone, FBS, L-cysteine, FeSO4) stocks. The pH of ACCM was adjusted to 4.75
using 6 N NaOH and the medium filtered through a 0.22 ?m filter to sterilize.
C. burnetii cultures were established in T-25 and T-75 polystyrene cell culture
with C. burnetii (1.0 ? 106GE/ml) purified from Vero host cells. Cultures were
incubated in an Innova CO-48 incubator as described for PM analysis. Growth
of C. burnetii on solid medium was conducted using a soft agarose overlay
method. A 2? solution of ACCM nutrients was adjusted to pH 4.75, sterilized
by filtration and 7.5 ml added to an equal volume of 2% (wt/vol in water)
molten UltraPure Agarose (Invitrogen). The 1% ACCM-agarose was poured
into 100 ? 20 mm Petri dishes to create a solid medium base. Purified C.
Omsland et al.PNAS ?
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no. 11 ?
burnetii was spread on the ACCM-agarose base and 10 ml 0.25% ACCM- Download full-text
agarose added as top agarose. Plates were incubated for 14 days in an Innova
CO-48 incubator adjusted to 5% CO2and 2.5% O2. Colonies were imaged
an AxioCam ICc 1 digital camera (Carl Zeiss).
Quantification of C. burnetii Cell Division and Infectivity. C. burnetii replication
during incubation in ACCM was quantified by QPCR of C. burnetii GE using a
(50 ?l) were diluted in 150 ?l sterile PBS, diluted 5-fold further with sterile
water, then mechanically disrupted to release bacterial DNA using a FastPrep
homogenizer (Q-Biogene Inc., CA) and 0.1 mm zerconia/silica beads (Biospec
Products Inc., Bartlesville, OK) as lysing matrix. Samples were centrifuged for
1 min at 20,000 ? g to pellet the lysing matrix, and equal volumes of
supernatant containing chromosomal DNA were used as template DNA for
PCR reactions. QPCRs were performed using TaqMan Universal PCR Master
Mix and a Prism 7000 sequence detection system (Applied Biosystems).
Infectivity of C. burnetii cultivated in ACCM was determined by a fluores-
cent focus forming unit (FFU) assay (14). Ten microliter aliquots of C. burnetii
cell culture medium which was then frozen at ?80 °C until needed. Equal
volumes of thawed culture aliquots were used to infect confluent Vero cell
cultures in 24-well plates. Following a 1 h incubation at room temperature
with rocking, 1 ml of fresh RPMI medium supplemented with 2% FBS was
added to each well. After a 5 day incubation, infected cells were fixed with
100% cold methanol and FFUs stained by indirect immunofluorescence em-
ploying polyclonal guinea pig antiserum generated against formalin-killed C.
burnetii and Alexa Fluor 448-conjugated goat anti-guinea pig IgG serum
(Molecular Probes). FFUs were enumerated by fluorescence microscopy using
for all images using Adobe PhotoShop.
Transmission Electron Microscopy. Specimens were fixed as previously de-
scribed (14). Thin sections were cut with an RMC MT-7000 ultramicrotome
(Ventana), stained with 1% uranyl acetate and Reynold’s lead citrate before
viewing at 80 kV on a Philips CM-10 transmission electron microscope (FEI).
sured with the AMT software measuring tool and data analyzed using Prism
software (GraphPad Software Inc.). Final images were processed with Adobe
PhotoShop (Adobe Systems, Inc.).
Immunoblotting. C. burnetii was pelleted by centrifugation and lysed by
determined using a DC Protein Assay kit (Bio-Rad). Samples were diluted in
SDS/PAGE sample buffer and 10 ?g total protein separated by SDS/PAGE on a
10–20% Tris-HCl Ready Gel (Bio-Rad). Proteins were transferred to an Immo-
bilon-P membrane (Millipore) that was blocked overnight at 4 °C in PBS
incubated for 1 h at room temperature in PBST containing anti-ScvA rabbit
polyclonal antibody (15). Membranes were washed, then incubated for 1 h at
room temperature in PBST containing anti-mouse IgG secondary antibody
conjugated to horseradish peroxidase (Pierce). Reacting proteins were de-
tected via enhanced chemiluminescence using ECL Pico reagent (Pierce) and
CL-XPosure film (Pierce).
Statistical Analysis. Statistical analyses were performed by unpaired Student’s
t test using Prism software (GraphPad Software Inc.). Differences between
data sets where P ? 0.05 were considered statistically significant.
ertson, and Frank Gherardini for critical review of this manuscript; Gary
Hettrick for graphic illustrations; and Kent Barbian for initial help with the
OmniLog detection system. This work was supported by the Intramural Re-
search Program of the National Institutes of Health, National Institute of
Allergy and Infectious Diseases.
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