Morphological remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography

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

C. burnetii

type IV

secretion system

The ultrastructural

features of the

C. burnetii

small and large cell

variants

Shepherd et al., iScience

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,

Mohammed Kaplan,

Naveen Vankadari,

Ki Woo Kim,

Charles L. Larson,

Przemysław Dutka,

Paul A. Beare,

Edward Krzymowski,

Robert A. Heinzen,

Grant J. Jensen,

and Debnath Ghosal

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.

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), dense periplasmic space and condensed DNA, LCVs

are pleomorphic in shape, with length exceeding 1

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.

The primary mode of human infection is inhalatio

n of mainly SCVs through contaminated aerosol.

C. burnetii

SCVs predominantly target primary mononuclear phagocytes (e.g., alveolar macrophages) to

begin the intracellular biphasic life cycle.

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).

The acidification of the CCV upre-

gulates metabolic activity and gene expression in SCVs

leading to a transition to

the metabolically active

LCVs.

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).

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.

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.

Based on genetic composition and

Department of Biochemistry

and Pharmacology, Bio21

Molecular Science and

Biotechnology Institute,

The University of

Melbourne, Melbourne, VIC,

Australia

Division of Biology and

Biological Engineering,

California Institute of

Technology, Pasadena, CA

91125, USA

School of Ecology and

Environmental System,

Kyungpook National

University, Sangju, Korea

Coxiella Pathogenesis

Section, Laboratory of

Bacteriology, Rocky

Mountain Laboratories,

National Institute of Allergy

and Infectious Diseases,

National Institutes of Health,

Hamilton, MT, USA

Division od Chemistry and

Chemical Engineering,

California Institute of

Technology, 1200 California

Boulevard, Pasadena, CA

91125, USA

Department of Physics and

Astronomy, Brigham Young

University, Provo, UT 84604,

USA

Department of Chemistry

and Biochemistry, Brigham

Young University, Provo, UT

84604, USA

ARC Centre for Cryo-

electron Microscopy of

Membrane Proteins, Bio21

Molecular Science and

Biotechnology Institute,

University of Melbourne,

Parkville, VIC, Australia

These author contributed

equally

Lead contact

*Correspondence:

mohammedk@uchicago.edu

(M.K.),

grant_jensen@byu.edu

(G.J.J.),

Continued

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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.

The

C. burnetii

T4SS

belongs to the T4BSS class and is phylogenetically very closely related to the

Legionella pneumophila

T4BSS.

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

Earlier studies suggested that the transition from SCV to LCV inside the CCV is correlated

with the upregulation of T4BSS expression and activity.

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.

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.

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.

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.

Similar to what is

recently reported,

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

L. pneumophila

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.

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.

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

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

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

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.

(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

Subsequently, we docked the recently reported near-atomic structure of the

L. pneumophila

T4BSS core

complex

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,

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.

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

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

). 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

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. 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

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

or other spe-

cies such as

L. pneumophila

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

) 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

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

120–150 nm (

Figures 4

and

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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