JOURNAL OF BACTERIOLOGY, Apr. 2011, p. 1493–1503
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 7
The Coxiella burnetii Cryptic Plasmid Is Enriched in Genes Encoding
Type IV Secretion System Substrates?†
Daniel E. Voth,1* Paul A. Beare,2Dale Howe,2Uma M. Sharma,1Georgios Samoilis,3,4
Diane C. Cockrell,2Anders Omsland,2and Robert A. Heinzen2
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 722051;
Coxiella Pathogenesis Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Hamilton, Montana 598402; and Department of Chemistry3and
Department of Clinical Bacteriology,4University of Crete, Heraklion, Greece
Received 12 November 2010/Accepted 23 December 2010
The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a phagolysosome-like parasito-
phorous vacuole (PV), in which it replicates. The organism encodes a Dot/Icm type IV secretion system (T4SS)
predicted to deliver to the host cytosol effector proteins that mediate PV formation and other cellular events.
All C. burnetii isolates carry a large, autonomously replicating plasmid or have chromosomally integrated
plasmid-like sequences (IPS), suggesting that plasmid and IPS genes are critical for infection. Bioinformatic
analyses revealed two candidate Dot/Icm substrates with eukaryotic-like motifs uniquely encoded by the QpH1
plasmid from the Nine Mile reference isolate. CpeC, containing an F-box domain, and CpeD, possessing
kinesin-related and coiled-coil regions, were secreted by the closely related Legionella pneumophila Dot/Icm
T4SS. An additional QpH1-specific gene, cpeE, situated in a predicted operon with cpeD, also encoded a
secreted effector. Further screening revealed that three hypothetical proteins (CpeA, CpeB, and CpeF) encoded
by all C. burnetii plasmids and IPS are Dot/Icm substrates. By use of new genetic tools, secretion of plasmid
effectors by C. burnetii during host cell infection was confirmed using ?-lactamase and adenylate cyclase
translocation assays, and a C-terminal secretion signal was identified. When ectopically expressed in HeLa
cells, plasmid effectors trafficked to different subcellular sites, including autophagosomes (CpeB), ubiquitin-
rich compartments (CpeC), and the endoplasmic reticulum (CpeD). Collectively, these results suggest that C.
burnetii plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, providing
a plausible explanation for the absolute maintenance of plasmid genes by this pathogen.
Coxiella burnetii is a highly infectious intracellular bacterium
that causes Q fever, a zoonotic disease that typically presents
as an acute, influenza-like illness. Rare but serious chronic
disease can also occur and normally manifests as endocarditis.
C. burnetii displays an extensive array of animal reservoirs, with
humans exposed to the pathogen primarily via contact with
infected domestic livestock. Inhalation of contaminated aero-
sols is the main route of C. burnetii transmission to humans,
with alveolar mononuclear phagocytes considered the patho-
gen’s initial target cell (reviewed in reference 38).
Multiple in vitro studies indicate that C. burnetii replicates in
a parasitophorous vacuole (PV) with lysosomal characteristics
(34, 65). The early PV interacts with autophagosomes, which
may provide nutrients to activate pathogen metabolism (51).
Following an initial phagosome stall, the PV fuses with lyso-
somes and continually engages the endosomal pathway, as
indicated by delivery of fluid-phase markers (28). During early
stages of infection, C. burnetii differentiates from a nonrepli-
cating small-cell-variant morphological form into a replicating
large-cell-variant form (27). To accommodate pathogen
growth, the maturing PV expands by continual heterotypic
fusion with endolysosomal compartments to ultimately occupy
most of the host cell cytoplasm. PV expansion into a spacious
structure visible by phase-contrast light microscopy occurs co-
incident with entry of C. burnetii into the log phase of its
growth cycle (?1 to 3 days postinfection [dpi]) (17). The lys-
osomal character of the PV is shown by the presence of lyso-
somal membrane proteins, active acid hydrolases, and an acidic
lumen (?pH 5) (34, 65).
C. burnetii’s historical obligate intracellular nature has sty-
mied attempts to identify pathogen factors required for suc-
cessful infection. Lipopolysaccharide is the only confirmed vir-
ulence determinant of C. burnetii and is thought to shield the
bacterial cell surface from innate immune recognition (42, 59).
Predicted virulence factors include C. burnetii proteins that
modulate host cell processes. For example, C. burnetii protein
synthesis is required for PV maturation (33), autophagosome
interactions (51), apoptosis subversion (68), and activation of
the prosurvival kinases Akt and Erk1/2 (66). Maintenance of
host cell viability by induction of prosurvival responses is con-
sidered a pathogenic strategy that accommodates C. burnetii’s
slow growth rate.
C. burnetii protein effectors of host functions are likely de-
livered to the cytosol by a type IV secretion system (T4SS) with
homology to the Dot/Icm machinery of Legionella pneumophila
(57, 63). The refractory nature of C. burnetii to genetic manip-
ulation has necessitated using L. pneumophila as a surrogate
host to identify Dot/Icm substrates. These proteins often pos-
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, University of Arkansas for Medical Sciences,
4301 W. Markham Street, Little Rock, AR 72205. Phone: (501) 686-
8050. Fax: (501) 686-5359. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 7 January 2011.
sess eukaryotic motifs/domains predicted to functionally mimic
or antagonize the activity of host cell proteins (3, 14, 19, 46,
64). Indeed, multiple C. burnetii proteins with eukaryotic-like
ankyrin repeat domains (Anks) are secreted by L. pneumophila
in a Dot/Icm-dependent fashion (48, 67). An intriguing subset
of secreted bacterial proteins contains eukaryotic F-box do-
mains (2). F-box domain-containing proteins are components
of the mammalian E3 ubiquitin ligase enzyme complex, which
directs ubiquitination of target proteins, resulting in their pro-
teasome-dependent degradation or functional alteration (2).
C. burnetii isolates collectively contain three F-box-encoding
open reading frames (ORFs): CBUA0014, CBU0355, and
CBU0814 (2, 7).
CBUA0014 is present on the QpH1 plasmid, originally char-
acterized from the C. burnetii Nine Mile reference isolate (55).
All C. burnetii isolates examined to date maintain a related
autonomously replicating plasmid or have chromosomally in-
tegrated plasmid-like sequences (IPS) (6, 29, 44, 55, 56, 62).
Nucleotide sequences have been determined for QpH1, QpRS,
QpDG, QpDV, and IPS of the G and S isolates (7). Early
studies with a limited number of isolates demonstrated a cor-
relation between plasmid content and disease presentation,
i.e., isolates derived from ticks, infected animals, and patients
with acute Q fever harbored QpH1, while isolates from pa-
tients with chronic disease carried QpRS or IPS (56). However,
a recent genotyping study of 173 C. burnetii isolates revealed
that QpH1 is also carried by some human chronic Q fever
isolates, while showing correlations between QpDV and acute
infection and QpRS and chronic infection (24). Whether plas-
mid type confers human disease potential is unresolved. How-
ever, genetically distinct C. burnetii isolates can clearly be
grouped into pathotypes with different virulence in animal
models of acute Q fever (52). Aside from genes involved in
plasmid maintenance and segregation, plasmid genes primarily
encode hypothetical proteins. Nonetheless, the absolute main-
tenance of plasmid sequences by all C. burnetii isolates suggests
that they are critical for pathogen survival.
The presence of F-box-containing CBUA0014 on QpH1
prompted us to investigate whether this and other plasmid
ORFs encode Dot/Icm substrates. Using L. pneumophila as a
secretion model, we found that three QpH1-specific proteins
(including the CBUA0014 protein) and three of five hypothet-
ical proteins encoded by all C. burnetii plasmids and IPS are
translocated into the host cell cytosol by the Dot/Icm T4SS.
Importantly, plasmid effectors were also translocated into the
cytosol by C. burnetii, demonstrating secretion in a native set-
ting. Effector proteins contained a C-terminal region with sim-
ilarity to the predicted L. pneumophila Dot/Icm translocation
signal, and deletion analysis revealed that this region is neces-
sary for secretion by C. burnetii. All plasmid effector genes
were expressed during in vitro C. burnetii infection, and the
encoded proteins trafficked to different subcellular sites when
ectopically expressed. These results suggest that C. burnetii
plasmids play an important role in host cell modifications.
MATERIALS AND METHODS
Bacteria and mammalian cell culture. Bacteria used in this study are described
in Table 1. C. burnetii Nine Mile phase II, clone 4 (RSA439), was propagated in
Vero (African green monkey kidney) cells (CCL-81; ATCC, Manassas, VA) and
purified as previously described (16, 58). L. pneumophila strains were cultured on
charcoal yeast extract (CYE) agar plates. For plasmid selection, CYE plates
contained 10 ?g/ml chloramphenicol. For culture of DotA-deficient L. pneumo-
phila LELA3118, plates also contained 25 ?g/ml kanamycin. L. pneumophila
transformations were conducted as previously described (67). THP-1 human
monocytic cells (TIB-202; ATCC) and HeLa (human epithelioid carcinoma)
cells (CCL-2; ATCC) were maintained in RPMI 1640 medium (Invitrogen,
Carlsbad, CA) containing 10% fetal calf serum (Invitrogen) at 37°C and 5%
CO2. THP-1 cells were differentiated into macrophage-like cells by use of phor-
bol 12-myristate 13-acetate (PMA; EMD Biosciences, San Diego, CA) as previ-
ously described (68). Escherichia coli TOP10 (Invitrogen) was used for recom-
binant DNA procedures. For host cell-free growth of C. burnetii, 6-well plates,
T-75 flasks, or 0.2-?m-filter capped Erlenmeyer flasks containing ACCM (47)
were inoculated with organisms and incubated at 37°C in a 2.5% O2/5% CO2
Effector gene expression. mRNA quantification was performed using the
QuantiGene reagent system v.2.0 and custom-designed probes according to the
manufacturer’s directions (Panomics, Santa Clara, CA). THP-1 cells were incu-
bated with C. burnetii at a multiplicity of infection (MOI) of 25 for 2 h, and then
extracellular bacteria were washed from monolayers. Cells were lysed at various
time points with QuantiGene lysis buffer supplemented with 150 ng/ml protei-
nase K (Invitrogen) and solubilized by incubation for 30 min at 55°C, followed by
3 freeze-thaw cycles. Lysates were diluted in QuantiGene lysis buffer and com-
bined with blocking buffer and probes. Lysates were loaded into a 96-well capture
plate and incubated for 16 to 20 h at 55°C. mRNA was detected by luminescence
over 1,000 ms with a Safire2microplate reader (Tecan, Mannedorg, Switzerland).
mRNA from uninfected THP-1 cells was used to determine the background
signal, and this value was subtracted from each infected cell sample. Transcrip-
tional signals were normalized to C. burnetii genome equivalents established as
previously described (17).
Plasmid construction. The plasmid pJB2581 was used for expression of CyaA
fusion proteins in L. pneumophila (4). C. burnetii genes were amplified from
genomic DNA by PCR using Accuprime Taq polymerase (Invitrogen) and gene-
specific primers (Integrated DNA Technologies, Coralville, IA) where the 5?
primer incorporates a BamHI site and the 3? primer incorporates a SalI or PstI
site (see Table S1 in the supplemental material). Products were cloned into
pCR2.1-TOPO (Invitrogen), plasmids were digested with either BamHI/SalI or
BamHI/PstI (New England BioLabs, Ipswich, MA), and gene-containing frag-
ments were ligated to similarly digested pJB2581 by use of a Ligate-IT system
(U.S. Biologicals, Cleveland, OH).
Modified versions of pJB2581, designated pJB-CAT-BlaM and pJB-CAT-
CyaA, were constructed for expression of BlaM and CyaA fusion proteins,
respectively, in C. burnetii. The chloramphenicol acetyltransferase (CAT) gene
was amplified by PCR from pJB2581 using primers CAT-P1169F and CAT-
pJB2581-HindIIIrecR. The CBU1169 promoter (P1169) was amplified from C.
burnetii genomic DNA using primers P1169-pJB2581-Ab-HindIIIrecF and
P1169-R. The CAT gene was placed downstream from P1169 to create P1169-
CAT using overlapping PCR and the primers P1169-pJB2581-Ab-HindIIIrecF
and CAT-pJB2581-HindIIIrecR. P1169-CAT was cloned into HindIII-digested
pJB2581 by use of an In-fusion kit (BD Clontech, Mountain View, CA) to create
pJB-CAT. P1169 was then amplified by PCR from C. burnetii genomic DNA
using the primers P1169-pJB2581-F and P1169-R and fused by overlapping PCR
to either blaM, amplified by PCR from pXDC61 using the primers BlaM-p1169-F
and BlaM-pJB-CAT-R, or cyaA, amplified by PCR from pJB2581 using the
primers Cya-P1169-F and Cya-pJB-CAT-R. P1169-blaM and P1169-cyaA frag-
ments were cloned into EcoRI/PstI-digested pJB-CAT by use of the In-fusion kit
to create pJB-CAT-BlaM and pJB-CAT-CyaA, respectively. pJB-CAT-BlaM
and pJB-CAT-CyaA were then used to generate plasmids encoding BlaM or
CyaA fused to the N terminus of C. burnetii plasmid effector genes. C. burnetii
genes were amplified by PCR using gene-specific primers and cloned into a
unique SalI site in pJB-CAT-BlaM or pJB-CAT-CyaA by use of the In-fusion kit.
For green fluorescent protein (GFP) fusion constructs, C. burnetii genes were
amplified by PCR with gene-specific primers where the forward primer contains
CACC at the 5? end for directional cloning and a 5? Kozac sequence (ATGGGC)
for mammalian expression. Products were cloned into pENTR-D/TOPO (Invit-
rogen) and then subcloned into pcDNA6.2/N-GFP using LR Clonase II (Invit-
rogen). Plasmid constructions were confirmed by sequencing. All plasmids and
primers used in the study are listed in Table 1 and Table S1 in the supplemental
C. burnetii transformation. ACCM-cultured C. burnetii was collected,
washed with 10% glycerol, and then resuspended in 10% glycerol. Ten mi-
crograms of plasmid DNA was mixed with 50 ?l of C. burnetii in a 0.1-cm
cuvette, and organisms were electroporated as previously described (5). Fol-
lowing electroporation, bacteria were cultured in ACCM for 24 h, and then
1494 VOTH ET AL. J. BACTERIOL.
chloramphenicol (3 ?g/ml) was added to cultures for selection of transfor-
mants. The use of genes conferring resistance to chloramphenicol and am-
picillin for C. burnetii genetic transformation studies at the Rocky Mountain
Laboratories (RML) has been approved by the RML Institutional Biosafety
Committee and the Centers for Disease Control and Prevention, Division of
Select Agents and Toxins.
Immunoblotting. L. pneumophila transformant cultures were incubated with 1
mM IPTG (isopropyl-?-D-thiogalactopyranoside) (ICN Biomedicals, Costa
Mesa, CA) for 2 h to induce protein expression. Cultures were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting using a mouse monoclonal antibody directed against CyaA
(clone 3D1; Santa Cruz Biotechnology, Santa Cruz, CA). Reacting proteins were
detected using an anti-mouse IgG secondary antibody conjugated to horseradish
peroxidase (Pierce, Rockford, IL) and chemiluminescence using ECL Pico re-
agent (Pierce). To confirm fusion protein expression by C. burnetii transformants,
THP-1 cells (1 ? 106cells/well) were infected for 2 to 3 days and lysed with
SDS-PAGE sample buffer, and lysates were immunoblotted. CyaA expression
was detected as described above. BlaM expression was assessed using a mouse
TABLE 1. Bacteria and plasmids used in this study
Strain or plasmidGenotype or description
C. burnetii strains
RSA439 Phase II, clone 46
L. pneumophila strains
Salt-sensitive isolate of AM511
JR32 dotA::Tn903dIIlacZ3118, DotA?Kmr
Serogroup 1, clinical isolate
Serogroup 1, clinical isolate
E. coli strain TOP10F?mcrA ?(mrr-hsdRMS-mcrBC) ?80dlacZ?M15 ?lacX74 recA1 araD139
?(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG
cyaA fusion vector, Cmr
ankG in pJB2581
cbua0006 in pJB2581
cbua0007a in pJB2581
cbua0012 in pJB2581
cbua0013 in pJB2581
cbua0014 in pJB2581
cbua0015 in pJB2581
cbua0016 in pJB2581
cbua0023 in pJB2581
lpp1878 in pJB2581
lpl0189 in pJB2581
TA TOPO vector, Ampr
Gateway entry vector, Kmr
N-terminal GFP fusion vector, AmprCmr
pJB2581 containing P1169 driving cat
C. burnetii blaM fusion vector, Cmr
C. burnetii cyaA fusion vector, Cmr
cbua0006 in pJB-CAT-BlaM
cbua0006 in pJB-CAT-CyaA
cbua0007a in pJB-CAT-BlaM
cbua00012 in pJB-CAT-BlaM
cbua0013 in pJB-CAT-BlaM
cbua0013 in pJB-CAT-CyaA
cbua0014 in pJB-CAT-BlaM
cbua0014 in pJB-CAT-CyaA
cbua0015 in pJB-CAT-BlaM
cbua0015 in pJB-CAT-CyaA
cbua0016 in pJB-CAT-BlaM
cbua0016 in pJB-CAT-CyaA
cbua0023 in pJB-CAT-BlaM
cbua0023 in pJB-CAT-CyaA
cbua0015 lacking 2 aa in pJB-CAT-BlaM
cbua0015 lacking 5 aa in pJB-CAT-BlaM
cbua0015 lacking 7 aa in pJB-CAT-BlaM
cbua0015 lacking 10 aa in pJB-CAT-BlaM
pMMB207C containing blaM
VOL. 193, 2011COXIELLA PLASMID-ENCODED Dot/Icm SUBSTRATES 1495
monoclonal antibody directed against ?-lactamase (QED; Bioscience, San Di-
L. pneumophila and C. burnetii CyaA translocation assays. L. pneumophila
CyaA assays were performed as previously described, using a cyclic AMP
(cAMP) enzyme immunoassay (GE Healthcare, Piscataway, NJ) (67). For C.
burnetii CyaA assays, THP-1 cells (1 ? 106cells/well) were infected with C.
burnetii transformants (MOI of 100) for 3 days, cells were lysed, and then lysates
were processed as described above for CyaA assays. Positive secretion of CyaA-
effector fusion proteins was scored as ?2.5-fold more cytosolic cAMP then that
for cells infected with organisms expressing CyaA alone. In L. pneumophila
assays, CyaA fused to C. burnetii AnkG was used as a positive control (48, 67),
and confirmation of Dot/Icm-dependent secretion was conducted by repeating
the assay with the L. pneumophila DotA?mutant LELA3118.
C. burnetii BlaM translocation assay. For BlaM translocation assays, THP-1
cells were cultured in black, clear-bottomed, 96-well plates and infected with C.
burnetii transformants (MOI of 100) for 2 days. Monolayers were loaded with the
fluorescent substrate CCF4/AM (LiveBLAzer-FRET B/G loading kit; Invitro-
gen) in a solution containing 15 mM probenecid (Sigma). Cells were incubated
in the dark for 1 h at room temperature and then fluorescence quantified on a
Safire2microplate reader, with excitation detected at 405 nm and emission
detected at 450 nm. Average fluorescence from 8 uninfected wells was subtracted
from results from experimental wells. Results are presented as fold increase in
fluorescence above that for cells infected with C. burnetii expressing BlaM alone
(negative control). As with the CyaA assay, a fluorescence value ?2.5-fold above
that for the negative control was considered positive for effector translocation.
Infected cells processed for ?-lactamase activity were also visualized by epiflu-
orescence microscopy using an inverted Nikon TE2000 microscope. Represen-
tative images (?20 magnification) were captured with a ?-lactamase ratiometric
filter set (Chroma Technology, Rockingham, VT) and a Coolsnap HQ2 digital
camera (Photometrics, Tucson, AZ) controlled by Metamorph software (Molec-
ular Devices, Inc., Sunnyvale, CA). Images were processed using Adobe Photo-
shop (Adobe Systems, San Jose, CA).
HeLa cell transfections. Uninfected or infected HeLa cells cultured on 12-mm
glass coverslips were transfected with individual GFP fusion constructs by use of
Effectene reagent (Qiagen). At 18 h posttransfection, cells were processed for
fluorescence microscopy. Cells were incubated with 4?,6-diamidino-2-phenylin-
dole (DAPI; Invitrogen) to stain host and bacterial DNA. A rabbit polyclonal
anti-LC3B antibody (Cell Signaling Technology, Danvers, MA) was used to label
autophagosomes, mouse monoclonal antiubiquitin (clone FK2; Sigma) was used
to identify ubiquitinated proteins, and rabbit polyclonal anticalnexin (Stressgen,
Ann Arbor, MI) was used to detect the endoplasmic reticulum (ER). Antibodies
were detected with anti-rabbit or anti-mouse secondary antibodies conjugated to
Alexa Fluor 488 (Invitrogen). Fluorescence microscopy was performed using a
Nikon Ti-U microscope, and images were acquired with a 60? oil immersion
objective and a DS-Qi1Mc camera (Nikon, Melville, NY). Images were pro-
cessed using NIS-Elements software from Nikon.
Three QpH1-specific genes encode Dot/Icm substrates.
CBUA0014 is specific to QpH1 (Fig. 1A and Table 2) and
encodes a protein with a ubiquitination-related F-box domain
(2, 7), which comprises 47 amino acids (aa) of this small, 77-aa
protein. Immediately downstream of CBUA0014 is QpH1-spe-
cific CBUA0015, which encodes a protein with a predicted
55-aa coiled-coil domain (CCD). Common in eukaryotic pro-
teins and increasingly found in secreted bacterial effectors,
CCDs are ?-helical conformations that mediate protein-pro-
tein interactions (8, 20). The CBUA0015 protein also contains
a 25-aa region with similarity to kinesin-like protein 1 (KLP1)
of Giardia spp. Kinesin is a microtubule-associated motor pro-
tein involved in intracellular transport of vesicular cargo (30).
Because bacterial effector proteins often mimic host protein
activities, the eukaryotic domains in the CBUA0014 and
CBUA0015 proteins suggested that these proteins might be
Dot/Icm substrates. To test this hypothesis, Legionella pneu-
mophila was used as a surrogate model to assay secretion (48,
67). THP-1 cells were infected with L. pneumophila transfor-
mants harboring plasmids encoding the CBUA0014 or
CBUA0015 protein N-terminally fused to the C terminus of
Bordetella pertussis adenylate cyclase (CyaA) (60). The CyaA
assay relies on effector-mediated delivery of the fusion protein
to the cytosol, where the CyaA moiety is activated by binding
cytosolic calmodulin, resulting in supraphysiological levels of
FIG. 1. QpH1-specifc genes encode Dot/Icm substrates. (A) Circular map of QpH1, showing the locations of QpH1-specific CBUA0014,
CBUA0015, and CBUA0016 (red arrows) and five ORFs (CBUA0006, CBUA0007a, CBUA0012, CBUA0013, and CBUA0023) common to all C.
burnetii plasmids and IPS (green arrows). (B) CyaA translocation assays showing Dot/Icm-dependent secretion of CBUA0014 (CpeC), CBUA0015
(CpeD), and CBUA0016 (CpeE). Intracellular cAMP levels were determined following infection of THP-1 cells with L. pneumophila expressing
CyaA fused to individual C. burnetii proteins. Results are expressed as fold change over cAMP levels resulting from infection with L. pneumophila
expressing CyaA alone (negative control). L. pneumophila expressing C. burnetii AnkG fused to CyaA was used as the positive control (67).
Increased cAMP levels were observed when each fusion protein was expressed in wild-type L. pneumophila, and negative-control levels were
observed following expression in DotA-deficient L. pneumophila, indicating that secretion requires a functional Dot/Icm T4SS. Experiments were
performed in triplicate, and error bars indicate the standard deviations from the means.
1496 VOTH ET AL. J. BACTERIOL.
cAMP (60). Elevated cAMP levels were observed in lysates
from cells infected with wild-type L. pneumophila, but not a
DotA?strain (53), expressing either CyaA fusion protein (Fig.
1B). Thus, the CBUA0014 and CBUA0015 proteins, now de-
noted CpeC (Coxiella plasmid effector protein C) and CpeD,
respectively, are Dot/Icm substrates. CBUA0016 is QpH1 spe-
cific, encodes a hypothetical, hydrophilic protein, and resides
in a predicted operon with cpeD (39). This genetic arrange-
ment suggested that the CBUA0016 protein might also be a
T4SS substrate. Indeed, the CBUA0016 protein, now desig-
nated CpeE, was also translocated into host cells in a Dot/Icm-
dependent manner (Fig. 1B).
Conserved plasmid genes encode Dot/Icm substrates. Ge-
nus-specific hypothetical proteins lacking eukaryotic sequence
similarity are also common T4SS substrates (23). This is not
unexpected considering the broad repertoire of unique host-
pathogen interactions. Therefore, we investigated whether five
CBUA0013, and CBUA0023) encoding hypothetical proteins
that are conserved among all C. burnetii plasmids (Fig. 1A) and
IPS (6, 7) are Dot/Icm substrates. As shown in Fig. 2A, infec-
tion of THP-1 cells with wild-type, but not DotA?, L. pneu-
mophila expressing theCBUA0006,
CBUA0023 proteins fused to CyaA resulted in significantly
increased cAMP levels, indicating Dot/Icm-dependent translo-
cation into the host cytosol (Fig. 2A). These proteins are now
designated CpeA, CpeB, and CpeF, respectively (Fig. 2A and
Table 2). Conversely, the CBUA0007a and CBUA0012 pro-
teins were not translocated (Fig. 2A). By immunoblotting, all
CyaA fusion proteins were equally expressed (data not shown);
thus, negative results for CBUA0007a and CBUA0012 were
not due to lack of synthesis.
Homologs of C. burnetii T4SS effectors in other bacterial
species are unusual (67). While not a confirmed homolog, the
N-terminal 250 aa of CpeA show ?50% similarity to the N
termini of LPP1878 and LPL0189 from the L. pneumophila
Paris and Lens strains, respectively. Therefore, we tested
whether these proteins are secreted. As shown in Fig. 2B, both
TABLE 2. C. burnetii plasmid effector family
ORF or protein designation ina:
Nine Mile (QpH1)K (QpRS) G (IPS)
CpeA (CBUA0006) CBUKA0010CBUG0089CBUDA0032 Regions of L. pneumophila
lpp1878 and lpl0189
KLP1 and CCD
aGenBank accession numbers are as follows: for Nine Mile, AE016828; for K, CP001020; for G, CP001019; and for Dugway, CP000733. The plasmid type is shown
in parentheses beside the respective isolate.
FIG. 2. Conserved C. burnetii plasmid hypothetical proteins and L. pneumophila homologs are Dot/Icm substrates. Intracellular cAMP levels
were determined following infection of THP-1 cells with L. pneumophila expressing the indicated CyaA fusion proteins. Results are expressed as
fold change over cAMP levels resulting from infection with L. pneumophila expressing CyaA alone (negative control). C. burnetii AnkG fused to
CyaA served as the positive control. (A) Elevated cAMP levels were observed after infection of THP-1 cells with L. pneumophila expressing CyaA
fused to CBUA0006 (CpeA), CBUA0013 (CpeB), and CBUA0023 (CpeF), indicating secretion. Negative-control cAMP levels were observed
when these fusion proteins were expressed in DotA-deficient L. pneumophila, indicating that translocation is mediated by the Dot/Icm T4SS.
CBUA0007a and CBUA0012 were not secreted. (B) Elevated levels of cAMP were observed in THP-1 cells infected with L. pneumophila
expressing the CpeA homologs LPP1878 and LPL0189 fused to CyaA, indicating secretion. Experiments were performed in triplicate, and error
bars indicate the standard deviations from the means.
VOL. 193, 2011 COXIELLA PLASMID-ENCODED Dot/Icm SUBSTRATES1497
LPP1878 and LPL0189 caused increased levels of cAMP in
host cells when expressed in wild-type L. pneumophila, sug-
gesting a potential shared effector protein between these re-
Expression of Dot/Icm substrates is transcriptionally regu-
lated. L. pneumophila Dot/Icm substrate expression is tran-
scriptionally regulated to influence distinct infection events
(36). To confirm that the six plasmid effectors are expressed by
C. burnetii during infection and to resolve the kinetics of ex-
pression, we determined the transcriptional profile of each
effector gene over a 7-day time course of infection. This time
course represents the pathogen’s growth cycle in human mac-
rophages from initial infection through stationary phase (34).
As shown in Fig. 3, maximal expression of QpH1-specific and
conserved plasmid effector genes occurred at 2 and 3 days,
respectively, after infection of THP-1 cells. Thus, expression of
plasmid effector genes is temporally regulated, with highest
expression observed concomitant with early-log-phase growth
and rapid PV expansion.
Plasmid Dot/Icm substrates are translocated into the host
cytosol by C. burnetii. L. pneumophila has been invaluable in
identifying C. burnetii T4SS substrates (35, 48, 67). However,
direct demonstration of effector secretion by C. burnetii would
be ideal. Recent advances in C. burnetii genetic transformation
(5) and the discovery that RSF1010 ori-containing plasmids
autonomously replicate in the organism (15) allowed us to
develop both CyaA and ?-lactamase (BlaM) secretion assays
for use in C. burnetii. Like the CyaA assay, the BlaM assay is an
enzymatic reporter assay that relies on delivery of a BlaM-
effector chimera to the cytosol. Here, the BlaM moiety cleaves
the ?-lactam ring of a cell-loaded fluorescent compound, re-
sulting in blue cytosolic fluorescence (10, 18). The RSF1010
plasmid backbone used to create the C. burnetii expression
vectors pJB-CAT-CyaA and pJB-CAT-BlaM (see Fig. S1 in
the supplemental material) was derived from pJB2581 (4), the
same plasmid used in our L. pneumophila CyaA assays. Fusion
protein expression is driven by the C. burnetii CBU1169 pro-
moter, which encodes Hsp20 and is constitutively expressed by
C. burnetii (P. A. Beare, unpublished results).
Plasmid effector ORFs were cloned downstream and in
frame with cyaA or blaM. THP-1 cells were infected with C.
burnetii transformants cultured axenically under antibiotic se-
lection, and then CyaA and BlaM assays were conducted at 3
and 2 dpi, respectively. By immunoblotting, CyaA and BlaM
effector fusion proteins were all expressed in THP-1 cells (Fig.
4A and data not shown). THP-1 cells infected with C. burnetii
expressing CpeA, CpeB, CpeD, CpeE, and CpeF fused to
CyaA had ?2.5-fold-higher levels of cAMP than cells infected
with C. burnetii expressing CyaA alone, indicating transloca-
tion into the cytosol (67) (Fig. 4A). CyaA-CpeC induced sig-
nificantly more cAMP accumulation than CyaA alone; how-
ever, the level was slightly below the 2.5-fold cutoff. Based on
the same elevation in blue fluorescence relative to the level for
THP-1 cells infected with C. burnetii expressing BlaM alone,
CpeA, CpeB, CpeC, CpeD, and CpeE were positive for trans-
location in the BlaM translocation assay (Fig. 4B). Levels of
fluorescence with BlaM-CpeF were significantly higher than
levels with BlaM alone but fell below the 2.5-fold cutoff. Fur-
thermore, the CBU0007a and CBU0012 proteins were not
secreted using the BlaM assay (data not shown), which corre-
sponds with results from the L. pneumophila CyaA assay. Col-
lectively, as assessed by one or both secretion assays, all plas-
mid effectors were secreted by C. burnetii during intracellular
C. burnetii plasmid effectors contain a putative C-terminal
translocation signal. Deletion analysis indicates that an intact
C terminus is required for translocation of effectors by L.
pneumophila’s Dot/Icm T4SS (9, 43). Salient features of the
putative C-terminal secretion signal include depletion of neg-
ative amino acids in positions ?1 to ?6, enrichment of serine
and threonine in positions ?3 to ?11, and enrichment of
hydrophobic amino acids in positions ?1 to ?3 (9, 43). While
a lineup of the 20 C-terminal residues of each C. burnetii
plasmid effector protein showed little sequence identity, C
termini had the same general features of the putative L. pneu-
mophila translocation signal, including the lack of negatively
charged residues and enrichment of hydrophobic residues in
positions ?1 to ?6 (Fig. 5A). To test the importance of the C
FIG. 3. Plasmid effector genes are expressed during C. burnetii intracellular growth. THP-1 cells were infected with C. burnetii for the indicated
times, and then RNA was isolated. RNA was subjected to QuantiGene analysis to determine effector gene expression levels. Expression is shown
as relative light units (RLUs). QpH1-specific effector genes were maximally expressed at 2 dpi, while conserved plasmid effector genes were
maximally expressed at 3 dpi.
1498 VOTH ET AL. J. BACTERIOL.
terminus for C. burnetii secretion, BlaM translocation assays
were conducted with C. burnetii transformants expressing
BlaM-CpeD fusion proteins with C-terminal deletions of 2, 5,
7, or 10 aa. All fusion proteins were equally expressed by C.
burnetii following infection of THP-1 cells (data not shown).
Deletion of only 2 aa impaired secretion, while deletion of 7 aa
or more diminished secretion to near negative-control levels
(Fig. 5B). Thus, C. burnetii Dot/Icm substrates contain a C-ter-
minal region important for translocation.
Plasmid effectors traffic to distinct subcellular sites in mam-
malian cells. To probe effector function, we ectopically ex-
pressed plasmid T4SS substrates in HeLa cells as fusions to
green fluorescent protein (GFP) to visualize the subcellular
distribution. This commonly used approach provides clues
about bacterial virulence factor function, as effector proteins
typically modulate a host factor/process associated with the
targeted subcellular site (11, 18, 26, 45, 67). Specific localiza-
tion of GFP-CpeE and GFP-CpeF was not observed, while
GFP-CpeA and GFP-CpeC localized to small cytoplasmic
puncta, GFP-CpeB to vesicular structures, including the PV
membrane, and GFP-CpeD to filamentous structures (Fig. 6
and data not shown). Further analysis demonstrated that CpeB
trafficked to autophagosomes, as evidenced by colocalization
with LC3B (Fig. 6). CpeD partially localized to the endoplas-
mic reticulum (ER), as indicated by colocalization with
calnexin, and caused accumulation and eventual breakdown of
the ER network (Fig. 6). Finally, CpeC colocalized with ubiq-
uitin-rich structures (Fig. 6), consistent with the presence of an
F-box domain in this protein.
Here, we show that C. burnetii plasmids and IPS encode
Dot/Icm T4SS substrates. The functional relevance of C. bur-
netii plasmids has been elusive, despite description of the mol-
ecules over 25 years ago and subsequent nucleotide sequencing
(55, 56). An essential role in host cell modification provides the
first plausible explanation for absolute maintenance of plasmid
sequences by all C. burnetii isolates.
C. burnetii plasmid effectors were initially identified using L.
pneumophila as a surrogate model (48, 67). This screen indi-
cated that CpeA, CpeB, CpeC, CpeD, CpeE, and CpeF are
secreted in a Dot/Icm-dependent manner. Previous confirma-
tion of C. burnetii secretion of an effector originally identified
using L. pneumophila was achieved for AnkF by immunoblot-
ting the soluble cytosolic fraction of infected cells with specific
antibody (48). A major accomplishment of the current work is
direct demonstration of secretion by C. burnetii using genetic
methods. This screen was made possible using an RSF1010
ori-containing plasmid that autonomously replicates in C. bur-
FIG. 4. Plasmid effectors are translocated by C. burnetii. (A) THP-1 cells were infected for 3 days with C. burnetii expressing individual effectors
fused to CyaA, and then cell lysates were analyzed by immunoblotting and the cAMP assay. All CyaA-effector fusions were expressed by C. burnetii
(top), and CpeA, CpeB, CpeD, CpeE, and CpeF were translocated into the host cytosol (bottom), as scored by ?2.5-fold more cytosolic cAMP
than for cells infected with organisms expressing CyaA alone (negative control). Values at left in the top panel are molecular sizes in kilodaltons.
(B) THP-1 cells were infected for 2 days with C. burnetii expressing individual effectors fused to BlaM and then processed for ?-lactamase activity.
(Top) Micrographs showing blue-fluorescence-associated THP-1 cells infected with C. burnetii expressing BlaM-CpeD but not BlaM alone. Bar,
30 ?M. (Bottom) BlaM assay results indicating that CpeA, CpeB, CpeC, CpeD, and CpeE are translocated into the host cytosol, as scored by
?2.5-fold more blue fluorescence than for cells infected with organisms expressing BlaM alone (negative control). Experiments were performed
in triplicate, and error bars indicate the standard deviations from the means.
VOL. 193, 2011COXIELLA PLASMID-ENCODED Dot/Icm SUBSTRATES1499
netii, thereby providing a system for heterologous gene expres-
sion (15). When expressed in C. burnetii, five plasmid effectors
were positive in the CyaA assay and five were positive in the
BlaM assay. We are unsure why CpeC and CpeF were negative
in the CyaA and BlaM assays, respectively. Fusion proteins are
expressed at similar levels in C. burnetii; thus, negative results
are not associated with translational levels. However, native
CpeC is ?9 kDa in size, and attachment of CyaA (43 kDa) to
this small protein may alter folding and subsequent secretion.
In agreement with this hypothesis, CyaA-CpeC was also trans-
located at low levels in L. pneumophila compared to the other
plasmid effectors. The RSF1010 ori plasmid used in this study
has a copy number of 3 to 6 in C. burnetii (P. A. Beare and
R. A. Heinzen, unpublished data), and effectors are constitu-
tively expressed. Thus, overexpression of effector fusions might
inhibit the secretion apparatus, resulting in a negative readout.
However, all C. burnetii effector transformants productively
infect THP-1 cells to form a typical PV, suggesting that trans-
location of the effector repertoire required for intracellular
growth is unaltered. In the L. pneumophila CyaA assay, iso-
propyl-?-D-thiogalactopyranoside (IPTG) is added to cultures
before infection to induce fusion protein expression. A similar
system of temporally induced expression may be optimal for C.
burnetii translocation assays. Finally, fusion partners may affect
chaperone recognition of some effectors. Little is known about
chaperone function in C. burnetii type IV secretion, although
some, but not all, C. burnetii CyaA-Ank fusions require the
chaperone IcmS for translocation by L. pneumophila. None-
theless, combined results from both assays indicate that all six
plasmid effectors are secreted by C. burnetii.
The C termini of C. burnetii plasmid effectors show amino
acid enrichments similar to the proposed C-terminal secretion
signal of L. pneumophila Dot/Icm T4SS substrates (9, 43). We
previously demonstrated that deletion of 10 amino acids from
the C terminus of C. burnetii AnkI eliminates Dot/Icm-medi-
ated secretion by L. pneumophila (67). Here, we show that
deletion of only 2 residues from the C terminus of CpeD
adversely affects translocation by C. burnetii, with deletion of 7
or 10 aa lowering secretion to near negative-control levels.
Collectively, these data demonstrate a critical C-terminal se-
cretion signal in C. burnetii T4SS substrates.
Because Dot/Icm-translocated CpeC, CpeD, and CpeE are
specific to QpH1, their functions are strictly associated with
isolates that maintain this plasmid (24). Functional analysis of
specific and conserved plasmid effectors would be aided by
phenotyping respective gene knockouts. Unfortunately, meth-
ods of allelic exchange in C. burnetii are currently unavailable.
Therefore, to provide clues about effector protein activity, we
ectopically expressed plasmid effectors as GFP fusion proteins
in mammalian cells and monitored their subcellular localiza-
Consistent with the presence of an F-box domain (31), GFP-
CpeC traffics to ubiquitin-positive structures throughout the
cytoplasm. Ubiquitination mediated by bacterial F-box-con-
taining proteins can modify protein function or target proteins
for degradation by the proteasome (2). This process modulates
diverse host functions, including innate immune signaling, in-
flammation, and apoptosis (2). L. pneumophila produces five
F-box-containing proteins that are Dot/Icm substrates (22), the
most thoroughly characterized being AnkB (LegAU13). AnkB
forms a functional Skp–Cullin–F-box E3 ubiquitin ligase com-
plex (37) and is targeted to the cytosolic face of the L. pneu-
mophila-containing vacuole by a process requiring host cell
farnesylation (50). The substrate(s) (bacterial or host) targeted
by AnkB for ubiquitination is unknown, as is its role in L.
pneumophila parasitism, with some (1, 37, 49), but not all (22),
strains with inactivated AnkB showing strong infection defects.
Further characterization of CpeC-interacting proteins will pro-
vide insight into the specific function of this protein.
Ectopically expressed GFP-CpeD partially colocalizes with
the ER and disrupts the organelle’s dispersed architecture.
CpeD contains a region of homology to a kinesin-related
Giardia protein. Kinesins are well-characterized motor pro-
teins that direct plus-end vesicular cargo trafficking along mi-
crotubules (30) and mediate processes such as Golgi complex-
FIG. 5. C termini of plasmid effectors have features required for
efficient translocation. (A) The C-terminal 20 aa of the plasmid Dot/
Icm substrates were aligned, and residues having similar properties are
denoted. Each plasmid effector contained at least 2 hydrophobic res-
idues (L, V, I, and F) in the ?1 to ?6 positions relative to the C
terminus. Negatively charged residues (E and D) were absent in these
positions but present in the ?10 to ?20 region. Amino acids with
hydroxyl side chains (Ser and Thr) were randomly distributed across
each C-terminal segment. (B) A C-terminal deletion series of CpeD
fused to BlaM was tested for secretion using the BlaM assay conducted
as described in the legend for Fig. 4. Removal of only 2 aa impaired
secretion, while removal of 7 aa or more resulted in fluorescence near
negative-control levels. Experiments were performed in triplicate, and
error bars indicate the standard deviations from the means.
1500 VOTH ET AL.J. BACTERIOL.
directed secretory transport (69). CpeD also contains a CCD
that may mediate protein-protein interactions. When ectopi-
cally expressed, CCD-containing Dot/Icm effectors of L. pneu-
mophila can alter Saccharomyces cerevisiae secretory transport
and/or cause accumulation of ER-derived structures in CHO
cells (9, 18). Recent evidence suggests that C. burnetii manip-
ulates host secretory processes, with Rab1 recruitment to the
PV involved in proper vacuole formation (13). Additionally,
the ER protein Bip associates with a fractionated PV (32), and
the PV membrane decorates with calnexin (D. E. Voth, un-
published results). Thus, CpeD and potentially other Dot/Icm
substrates may influence ER function. It should be noted that
CpeD was also identified in a proteomic screen of C. burnetii
secreted proteins (54).
The third QpH1-specific Dot/Icm substrate, CpeE, showed
no specific localization when ectopically expressed. CpeE is a
hypothetical protein lacking any obvious homology to known
eukaryotic or prokaryotic proteins. CpeE was originally iden-
tified as a hydrophilic protein specific to acute Q fever isolates
and was termed CbhE? in reference to unique restriction map-
ping on QpH1 (40, 41). cpeE and cpeD reside in a putative
operon (39), a prediction supported by similar expression pro-
files. Like that for cpeC, maximal expression of both genes
occurs at 2 days postinfection, coincident with the onset of
rapid PV enlargement. Thus, CpeE and CpeD may act to-
gether to modulate PV formation or other host cell functions.
Of the five hypothetical proteins encoded by all C. burnetii
plasmids and IPS, three (CpeA, CpeB, and CpeF) are Dot/Icm
substrates. This effector group is maximally expressed at 3 days
postinfection, 1 day later than QpH1-specific effectors. CpeA is
similar to two hypothetical L. pneumophila proteins, LPL0189
and LPP1878, that we demonstrate are also Dot/Icm sub-
strates. The relatedness of these three secreted proteins may
correlate with a common biological function during intracellu-
lar growth. Only GFP-CpeB showed specific subcellular traf-
ficking in localizing to LC3B-positive autophagosome-derived
vesicles, including the PV membrane. Autophagosomes nor-
mally remove unwanted material, such as damaged organelles,
from the host cell cytoplasm and mature into autophagolyso-
somes, where this material is degraded (61). Numerous intra-
cellular pathogens subvert autophagic signaling to provide a
suitable growth environment (12). For example, Mycobacte-
FIG. 6. CpeB, CpeC, and CpeD traffic to distinct subcellular compartments. GFP-CpeB, GFP-CpeC, and GFP-CpeD were ectopically
expressed in HeLa cells, and cells were processed for fluorescence microscopy. GFP fusion proteins, antibody-labeled proteins, and DNA are
shown as green, red, and blue, respectively, in the merged images. Labeled proteins are indicated in the panels. CpeB trafficked to autophagosomes,
as evidenced by colocalization with LC3B. CpeC colocalized with ubiquitinated proteins. CpeD partially localized to the ER, as demonstrated by
calnexin staining, and caused disruption of the ER network. Bar, 10 ?m.
VOL. 193, 2011COXIELLA PLASMID-ENCODED Dot/Icm SUBSTRATES1501
rium tuberculosis inhibits autophagosome/lysosome fusion to
avoid lysosomal destruction (21). Conversely, exogenous in-
duction of autophagy benefits C. burnetii replication (25, 51),
and the organism actively engages autophagosomes a few min-
utes after infection (25, 51). It is intriguing to speculate that
CpeB benefits C. burnetii by acting alone, or in concert with
other effectors, to induce autophagy.
In summary, C. burnetii plasmids and IPS are enriched in
genes encoding Dot/Icm substrates. Functional characteriza-
tion of these proteins will provide needed insight into C. bur-
netii host cell parasitism. Host cell-free cultivation of C. bur-
netii (47) may provide a means to cure the pathogen of its
plasmid. Generation of an isogenic strain lacking plasmid se-
quences will help define the role of these molecules in host cell
infection and pathotype-specific virulence.
We thank Anita Mora for graphic illustrations, Katja Mertens and
James Samuel for unpublished information regarding the autonomous
replication of RSF1010 ori-containing plasmids in C. burnetii, and Seth
Winfree for BlaM microscopy. We thank Raphael Valdivia and Sergey
Konstantinov for helpful discussions and Xavier Charpentier for the
generous gift of L. pneumophila Paris and Lens genomic DNA.
This work was supported by funding from NIH NIAID grants
K22AI081753 (D.E.V.) and R01AI087669 (D.E.V.), the Arkansas Bio-
sciences Institute (D.E.V.), and the Intramural Research Program of
the National Institutes of Health, National Institute of Allergy and
Infectious Diseases (R.A.H.).
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