iScience
Article
Morphological remodeling of
Coxiella burnetii
during its biphasic developmental cycle revealed
by cryo-electron tomography
Doulin C.
Shepherd,
Mohammed
Kaplan, Naveen
Vankadari, ...,
Robert A. Heinzen,
Grant J. Jensen,
Debnath Ghosal
mohammedk@uchicago.edu
(M.K.)
grant_jensen@byu.edu (G.J.J.)
debnath.ghosal@unimelb.
edu.au (D.G.)
Highlights
Imaging developmental
stages of
Coxiella burnetii
cells using cryo-electron
tomography
Revealing the
macromolecular structure
of
C. burnetii
type IV
secretion system
The ultrastructural
features of the
C. burnetii
small and large cell
variants
Shepherd et al., iScience
26
,
107210
July 21, 2023
ª
2023 The
Authors.
https://doi.org/10.1016/
j.isci.2023.107210
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iScience
Article
Morphological remodeling of
Coxiella burnetii
during its biphasic developmental cycle
revealed by cryo-electron tomography
Doulin C. Shepherd,
1
,
9
Mohammed Kaplan,
2
,
9
,
*
Naveen Vankadari,
1
Ki Woo Kim,
2
,
3
Charles L. Larson,
4
Przemysław Dutka,
2
,
5
Paul A. Beare,
4
Edward Krzymowski,
6
Robert A. Heinzen,
4
Grant J. Jensen,
2
,
7
,
*
and Debnath Ghosal
1
,
8
,
10
,
*
SUMMARY
Coxiella burnetii
is an obligate zoonotic bacterium that targets macrophages
causing a disease called Q fever. It has a biphasic developmental life cycle where
the extracellular and metabolically inactive small cell variant (SCV) transforms in-
side the host into the vegetative large cell variant (LCV). However, details about
the morphological and structural changes of this transition are still lacking. Here,
we used cryo-electron tomography to image both SCV and LCV variants grown
either under axenic conditions or purified directly from host cells. We show
that SCVs are characterized by equidistant stacks of inner membrane that pre-
sumably facilitate the transition to LCV, a transition coupled with the expression
of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were
associated with extracellular densities possibly involved in host infection. Also,
SCVs contained spherical multilayered membrane structures of different sizes
and locations suggesting no connection to sporulation as once assumed.
INTRODUCTION
Coxiella burnetii
is a highly infectious wide-ranging zoonotic pathogenic bacterium and the causative
agent of human Q fever, a severe and debilitating form of influenza-like illness.
1
,
2
C. burnetii
exhibits a
biphasic developmental cycle of metabolically active and replicating (exponential phase) large cell variant
(LCV) present inside the host, to nonreplicating (stationary phase) dormant small cell variant (SCV) that can
survive outside the host cell.
3–6
Each variant has distinct morphological features; while SCVs are character-
ized by their rod-shaped, shorter length (0.2–0.6
m
m), dense periplasmic space and condensed DNA, LCVs
are pleomorphic in shape, with length exceeding 1
m
m, and exhibit relatively sp
arse periplasmic space and
dispersed DNA.
3–5
The SCVs are highly stable in external environments (outside the host cell) and recalci-
trant to physical and chemical stresses.
3
,
7
,
8
The primary mode of human infection is inhalatio
n of mainly SCVs through contaminated aerosol.
9
C. burnetii
SCVs predominantly target primary mononuclear phagocytes (e.g., alveolar macrophages) to
begin the intracellular biphasic life cycle.
10
,
11
Following internalization,
C. burnetii
establishes a specialized
membrane-bound replicative niche known as the
Coxiella
containing vacuole (CCV) which matures into an
autophagolysosome-like compa
rtmentwithanacidifiedlumen(pH
4.75), acid hydrolases, cationic pep-
tides, and lysosomal markers suc
h as LAMP-1 and CD63 (LAMP-3).
12
,
13
The acidification of the CCV upre-
gulates metabolic activity and gene expression in SCVs
leading to a transition to
the metabolically active
LCVs.
5
,
14
,
15
In addition, these developmental transitions can be mimicked in host cell-free conditions by
growing
C. burnetii
in an axenic medium known as second generation acidified citrate cysteine medium
(ACCM-2).
16
The maintenance and maturation of the CCV is largely mediated by the activity of the
C. burnetii
Dot/Icm
(defective in organelle trafficking/intracellular multiplication) type IV secretion system (T4SS) that delivers
more than 130 unique effector proteins into the host cytosol.
17
,
18
The bacterial T4SSs a
re multimegadalton
molecular machines that span through the bacterial cell envelope and are involved in transport of nucleo-
protein complexes as well as proteins across the cellular envelope.
19
Based on genetic composition and
1
Department of Biochemistry
and Pharmacology, Bio21
Molecular Science and
Biotechnology Institute,
The University of
Melbourne, Melbourne, VIC,
Australia
2
Division of Biology and
Biological Engineering,
California Institute of
Technology, Pasadena, CA
91125, USA
3
School of Ecology and
Environmental System,
Kyungpook National
University, Sangju, Korea
4
Coxiella Pathogenesis
Section, Laboratory of
Bacteriology, Rocky
Mountain Laboratories,
National Institute of Allergy
and Infectious Diseases,
National Institutes of Health,
Hamilton, MT, USA
5
Division od Chemistry and
Chemical Engineering,
California Institute of
Technology, 1200 California
Boulevard, Pasadena, CA
91125, USA
6
Department of Physics and
Astronomy, Brigham Young
University, Provo, UT 84604,
USA
7
Department of Chemistry
and Biochemistry, Brigham
Young University, Provo, UT
84604, USA
8
ARC Centre for Cryo-
electron Microscopy of
Membrane Proteins, Bio21
Molecular Science and
Biotechnology Institute,
University of Melbourne,
Parkville, VIC, Australia
9
These author contributed
equally
10
Lead contact
*Correspondence:
mohammedk@uchicago.edu
(M.K.),
grant_jensen@byu.edu
(G.J.J.),
Continued
iScience
26
, 107210, July 21, 2023
ª
2023 The Authors.
This is an open access article under the CC BY-NC-ND license (
http://creativecommons.org/licenses/by-nc-nd/4.0/
).
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phylogenetic analysis, they are classified
into two major classes, T4ASS and T4BSS.
20
The
C. burnetii
T4SS
belongs to the T4BSS class and is phylogenetically very closely related to the
Legionella pneumophila
T4BSS.
21
,
22
C. burnetii
T4BSS effector proteins subvert multiple host cellular pathways such as autophagic, secretory,
and endolysosomal trafficking and aid biogenesis of the CCV to facilitate intracellular replication of
C. burnetii
.
23
,
24
Earlier studies suggested that the transition from SCV to LCV inside the CCV is correlated
with the upregulation of T4BSS expression and activity.
5
,
25–27
Intriguingly, under
in vitro
and axenic (host
cell-free) growth conditions,
C. burnetii
slowly differentiates from LCVs to SCVs; this transition is evident
after 10 days, and the majority of LCVs convert to SCVs after 21 days.
16
Despite decades of research, the structur
al changes and remodeling that occur in
C. burnetii
during its
biphasic cycle from LCV to SCV and vice versa remain elusive in part because of the lack of high-resolution
in situ
imaging of this obligate pathogen. While recent advances in single particle cryo-electron micro-
scopy (cryo-EM) and cryo-electron tomography (cryo-ET) methods have enabled the investigation of
T4SSs in great detail in various bacterial species,
28–32
the macromolecular architecture, localization and
developmental regulation of the
C. burnetii
T4BSS with respect to its biphasic cycle remain poorly under-
stood. Moreover, previous studies on the ultrastructure of
C. burnetii
, including their ability to form ‘‘spore-
like’’ structures’’, used conventional transmission electron microscopy (TEM) where sample preparation
(fixation and dehydration) and sta
ining may disrupt membranes.
33–35
Here, we used cryo-ET to visualize
C. burnetii
LCVs and SCVs in axenic (host cell-free) growth conditions as
well as host-derived
C. burnetii
variants at macromolecular resolution. Our comprehensive cryo-ET analyses
captured changes in cellular morphology and intricate membrane dynamics associated with LCV to SCV
transition. In addition,
in situ
structural analyses of the
C. burnetii
Dot/Icm T4BSS under different develop-
mental conditions provided insights into the regulation of its expression and activity.
RESULTS
Cryo-ET imaging of
C. burnetii
cells grown under axenic conditions
To reveal the morphological features of
C. burnetii
cells and the macromolecular architecture of their
T4BSS and its regulation during different developmental stages, we used cryo-ET to image frozen hydrated
C. burnetii
cells (both SCVs and LCVs) grown in host cell-free second-generation acidified citrate cysteine
media (ACCM-2). Earlier studies showed that the biphasic developmental transition and the growth ki-
netics and viability of
C. burnetii
can be recapitulated in ACCM-2 media.
16
These transition and growth ki-
netic of
C. burnetii
are very similar to those occurring inside a host cell such as Vero (African green monkey
kidney fibroblasts) cells.
5
,
16
Hence, we decided to image
C. burnetii
grown in ACCM-2 medium for 5 and
14 days to capture the morphological and structural signatures of SCV to LCV transition and vice versa.
While we cannot exclude that axenic growth might induce modifications not naturally occurring in the phys-
iological biphasic growth, this approach is advantageous for cryo-ET imaging where the small size of
C. burnetii
makes them suitable for direct imaging.
Our cryo-ET imaging of the day 5 axenic culture showed that
62% of the cells are LCVs (cell size >800 nm),
35% cells are in their transition state (transition state cell variant (TCV), 600–800 nm) and only 3% cells were
SCVs (<600 nm) (
Figure S1
). We based our classification on the morphological and ultrastructural features
of the cells like cell length and compact chromatin (for SCVs), as previously described.
36
Similar to what is
recently reported,
37
the ribosomes appeared to be excluded from the compact nucleoid region in SCVs.
Compared to SCVs, LCVs exhibited relatively sparse periplasmic space, dispersed DNA, and contained
many ‘‘Wi-Fi’’ shaped structures spanning across the cell envelope (
Figures 1
A and 1B). These structures,
with numbers ranging between 1 and 8 per cell (
4 on average per cell), were composed of two major den-
sities - an outer membrane (OM) associated layer and a lower periplasmic layer (
Figure 1
C). In partially lysed
cells, we occasionally observed clear top views of these particles that appeared to have two concentric
rings with the outer ring diameter being
40 nm (
Figure 1
D). Owing to their similarity to the T4BSS particles
in
L. pneumophila
,
38
we hypothesized that these particles are the T4BSS of
C. burnetii
. Accordingly, these
particles were absent in 17 cryotomograms of a
C. burnetii
strain with a complete
dot/icm
knockout, con-
firming that they are T4BSS particles (
Figure S2
A). In contrast, cryo-ET imaging of the day 15 axenic culture
contained a mixed population of SCVs (19%), LCVs (
37%) and TCVs (44%), and here also T4BSS particles
were present only in LCVs and TCVs but not in SCVs (
Figure S2
B). Our observation that the assembly
debnath.ghosal@unimelb.
edu.au
(D.G.)
https://doi.org/10.1016/j.isci.
2023.107210
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and maintenance of the T4BSS apparatus is coupled
to the developmental transition from SCV to LCV is
consistent with previous studies.
5
,
25–27
While the majority of T4BSS particles of
C. burnetii
were located at the cell poles, there were also a few
(7 from 825 particles in 138 tomograms) that were positioned away from the cell poles (
Figure S3
A). In
our previous work on
L. pneumophila
, we observed only a few T4BSS particles located away from the
cell pole in >3,500 cryotomograms.
39
,
40
Similar to our previous observation in
L. pneumophila,
we identi-
fied T4BSS particles at the division plane in dividing
C. burnetii
, suggesting that they start assembling at the
future new poles during the septation process (
Figure S3
B).
Macromolecular architecture of
C. burnetii
T4BSS
To decipher the macromolecular architecture of the
C. burnetii
T4BSS, we averaged 414 particles and
generated a subtomogram average at a resolution of 2.5–4.5 nm (
Figures 1
Eand
S4
A). This average was
generated from 69 cryotomograms of cells grown in ACCM-2 for 5 days and resuspended in phosphate
buffer saline pH 7.2. Our initial average nicely resolved densities associated with the OM and the periplas-
mic complex; however, the inner membrane (IM) associated densities were missing. A closer look at the
individual particles revealed that the distance betw
een the OM and IM varied significantly in individual par-
ticles with most of them lacking clear densities associated with the IM (
Figure 1
Candlaterin
Figure 2
).
Consequently, we generated two separate averages with different masks, one with a mask on the OM
and one on the IM and juxtaposed them together to produce a composite average with a local resolution
between 2.5 and 4.5 nm (
Figures 1
Eand
S4
A). At this resolution, this composite average showed marked
structural similarity to the T4BSS of
L. pneumophila
, where both consisted of an OM complex, a periplasmic
complex and a stem-like structure connecting the two (
supplemental information Figures S5
A and S5B).
ABCD
G
F
E
Figure 1.
In situ
structure of the
C. burnetii
T4SS
(A and B) Slices through electron cryotomograms of LCV
C. burnetii
cells highlighting the presence of T4SS particles
(black arrows). Scale bar 100 nm.
(C) Enlargements of the T4SS particles shown in A (top panel) and B (lower panel). Scale bar 10 nm.
(D) A slice thorough an electron cryotomogram of a lysed
C. burnetii
cell illustrating the pres
ence of multiple top views of
T4SS particles (white arrows). Scale bar 100 nm. An enlarge
ment of a top view of a T4SS particle is shown in the white-
boxed area.
(E and F) Slices through the subtomogram averages of T4SS of
C. burnetii
at pH 7.2 (left) and pH 4.75 (right). Dashed
yellow line indicates a composite of two averages obtained with different masks (see
STAR Methods
) concatenated
together. Scale bar 10 nm.
(G) A cross section through the T4SS OM complex (at the lev
el indicated with the orange arrow in E) showing 13-fold
symmetry. OM = outer membrane, IM = inner membrane.
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The OM complex showed a 13-fold sy
mmetry and a maximum diameter of
40 nm (
Figure 1
G), and formed
together with the periplasmic complex what appears to be a secretion chamber similar to the
L. pneumophila
T4BSS system. This structural similarity between the two systems is consistent with the
homology between at least 23 genes of the 30 T4BSS genes in
L. pneumophila
.
22
Subsequently, we docked the recently reported near-atomic structure of the
L. pneumophila
T4BSS core
complex
41
into our subtomogram average which allowed us to tentatively assign components that
contribute to different densities in our structure. This fitting suggested that the OM core complex
comprised DotC, DotD, DotF, DotH, DotG and DotK proteins while the periplasmic ring complex contains
parts of DotG and DotH (
supplemental information Figures S5C
). Despite several data processing trials, we
could not resolve the cytoplasmic ATPase complex. Th
is is consistent with a contemporaneous study on
C. burnetii
T4BSS which used more than 7,000 particles to generate an
in situ
average,
37
butitalsolacked
the cytoplasmic complex suggesting that it is either flexible or rarely associated.
Earlier studies have shown that acidification of the CCV inside the host triggers the differentiation of SCVs into
LCVs. This developmental transition has been linked to enhanced metabolic activities, higher gene expression
and biogenesis and secretion of the
C. burnetii
T4BSS.
5
,
25–27
Based on this, we hypothesized that under au-
tolysosomal pH conditions (pH
4.75), we might capture actively secreting T4BSSs in our cryotomograms.
Therefore, we cultured
C. burnetii
cells in ACCM-2 media for 5 days and then resuspended them in a low
pH media (citrate buffer saline pH
4.75). Unlike the particles observed in the previous tomograms of cells
at pH 7.2, individual particles in these tomograms of cells at pH 4.75 showed less variability in the distance be-
tween the OM and IM. We extracted 411 T4BSS particles to produce a subtomogram average (with a single
mask) at 2.5–5 nm resolution (
Figures 1
F and
S4
B) which revealed a few structural differences (as suggested at
the resolution of our structures) compared to the average at pH 7.2 including the existence of what could be a
secretion channel and density for the wing structures (as seen before in the
L. pneumophila
T4BSS,
Figures S5
and
S6
). These observations are consistent with the fact that the T4BSS is active in the acidic autolysosome
environment and therefore acidic pH might have ‘primed’ these complexes.
Of interest,
17% of individual T4BSS particles showed extracellular densities associated with them in day 5
(both pH 4.75 and 7.2) and day 15 cultures. We classified these extracellular densities into 4 major
A
B
C
Figure 2. T4SS-associated extracellular densities
(A) Slices through electron cryotomograms of
C. burnetii
cells indicating the presence of T4SS particles without any
extracellular densities associated with them.
(B) Slices through electron cryotomograms of
C. burnetii
cells showing the presence of unstructured extracellular
densities above the T4SS particles.
(C) Slices through electron cryotomograms of
C. burnetii
cells illustrating different types of extracellular densities
associated with T4SS such as: short filament-like density (black a
rrow), crown-like density (yellow arrows), tubular densities
(orange arrows). Green arrow point to tubular densities where no T4SS could be identified at their bases. Scale bar is
20 nm. OM = outer membrane, IM = inner membrane.
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categories:(1)thosewithanamorphousshape,(2)thinfi
lament-like, (3) crown-like structures, and (4) those
with a rod or tubular-like form (
Figures 2
A–2C). However, some of these extracellular densities were also
occasionally localized on the cell surfaces without an obvious T4BSS particle underneath them (
Figure 2
C,
right most). This could be either because they are relics after the T4BSSs are disassembled or they
were secreted and then drifted away from T4BSS. Alternatively, it is also possible that they were secreted
in a T4BSS-independent manner. These extracellular densities were absent in 17 cryotomograms of a
C. burnetii
strain with a complete
dot/icm
knockout. The low number of particles with extracellular den-
sities precluded the possibility of producing a decent subtomogram average to see if there are any struc-
tural changes in T4BSS associated with the presence of these extracellular densities.
Of the different types, the ‘tube-like’ densities were the most interesting as they are reminiscent of a T4ASS
pilus filament. These tube-like filaments were
8nmwide,
25 nm in length and showed flexibility. No
extracellular features associated with T4
SS were reported in previous studies of
C. burnetii
,
37
or other spe-
cies such as
L. pneumophila
.
39
,
40
Furthermore, no pilin-like candidates have been identified for any of the
T4BSSs to date. Therefore, what these different extracellular densities are and if they are related to
released/secreted proteins (such as DotA or IcmX
37
) remain unknown.
Inner membrane stacking, a characteristic feature observed only in SCVs
In the day 15 axenic culture, 19% of cells were SCVs,
37% were LCVs and 44% are in their transition state
(
Figure S1
). The SCVs, which are 600 nm or shorter, exhibited relatively dense periplasmic space and
condensed DNA compared to LCVs (
Figure S2
B). Another interesting feature in the cryotomograms of
SCVs was the presence of a stack of tightly packed membranes in the cytoplasm (
Figures 3
A–3E,
supple-
mental information Video S1
). In 58 cryotomograms, we always observed a single membrane stack per SCV
with each stack having 2–6 layers consistently spread
10 nm apart as revealed by density profile analysis
AB
C
D
EF
Figure 3. Multilayered cytoplasmic membrane invaginations in
C. burnetii
SCVs
(A–E) Slices through electron cryotomograms of
C. burnetii
cells (purified from infected vero cells 28 dpi) highlighting the
presence of multilayered cytoplasmic membrane invaginati
ons (orange arrows) with zoom-ins of these invaginations
shown in the enlargements at the lower right corner of each panel. The white arrows in panel C point to a membrane of
presumably the host vacuole still attached to the OM of
C. burnetii
cell. Scale bar is 100 nm.
(F) Lower panel: average density profile taken along the ar
ea indicated between the yellow lines in the upper panel
highlighting the equidistant spacing of
10 nm between the different layers of the inner membrane invaginations.
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(
Figure 3
Fand
supplemental information Video S1
). This membrane stacking was generated by invagi-
nating the IM toward the cytoplasm and was initiated in LCVs, but densely packed mature stacks were
exclusively found in SCVs which suggests that maturation of these stacks is developmentally linked to
the LCV to SCV differentiation.
To investigate if the membrane stacking changes during an infection condition, we imaged the 28 days
post-infection host (Vero cells) -derived
C. burnetii
variants using cryo-ET. In this sample,
35% of the cells
were SCVs, 41% were in transition state and 24% were LC
Vs. Similar to the day 15 axenic culture, only host-
cell derived SCVs had densely packed membrane stacks
while LCVs and transition state cells only revealed
early stages of these membrane invaginations. Intriguingly, only in the host-cell-derived SCVs we also
observed a dark linear density with an average length of few hundred nanometers (
100–300 nm) nm
and located
10 nm from the IM (
Figure S7
). This linear density appeared morphologically rather different
from the IM stacks suggesting that it is of a different nature.
Cytoplasmic spore-like structures in SCVs and transitional cells
Another prominent feature in
C. burnetii
SCVs of the day 15 axenic culture is the presence of IM invagina-
tions that extended and curved to form a multilayered spherical structure in the cytoplasm. Initially, the
IM invaginated into the cytoplasm forming a septum with a protein-like layer visible at one side (
Figure 4
and
supplemental information Video S2
). This protein-like layer was located
7–8 nm from the invaginated
membrane. Subsequently, this septum elongated and curled with the protein-like layer still visible at one
side to ultimately form a concentric multilamellar spherical cytoplasmic structure with an average diameter
of
120–150 nm (
Figures 4
and
S8
,
Video S2
). In total, we identified 16 such structures in 58 cryo-tomograms.
It was difficult to determine how many layers this structure has at the resolution of our cryo-tomograms but
the number of membrane layers in these structures appeared to be variable in different cells as they had
ABC
Figure 4. Different stages of membrane invaginations in
C. burnetii
cells
(A–C) Top panels: Slices through electron cryotomograms of
C. burnetii
cells (grown in ACCM-2 for 15 days) illustrating
different stages of inner membrane invaginations (orange arrows) with 3D segmentations of these stages shown in the
lower panels. An early stage of IM invagination is shown in panel A where a protein-like density can be seen at one side of
this invagination (red density in the segmentation). Panel B s
hows a long and curved IM invagination where a protein-like
density can be seen between this invagination and the IM (red density in the segmentation, green arrow in inset). A whole
multilayered vesicular structure can be seen in panel C. Note that it was difficult to trace the different layers in the
tomogram hence it is shown as one large blob in the segmentation in the lower panel (See
supplemental information
Video S2
). Scale bar is 100 nm.
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