of 14
Discovery of chlamydial peptidoglycan reveals bacteria with
murein sacculi but without FtsZ
Martin Pilhofer
#3,4
,
Karin Aistleitner
#5
,
Jacob Biboy
6
,
Joe Gray
7
,
Erkin Kuru
8
,
Edward Hall
8
,
Yves V. Brun
8
,
Michael S. VanNieuwenhze
8
,
Waldemar Vollmer
6
,
Matthias Horn
2,5
, and
Grant J. Jensen
2,3,4
3
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
4
Howard Hughes Medical Institute
5
Division of Microbial Ecology, University of Vienna, Vienna, A-1090, Austria
6
Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Newcastle
University, Newcastle upon Tyne, NE2 4AX, United Kingdom
7
Institute for Cell and Molecular Biosciences, Pinnacle Laboratory, Newcastle University,
Newcastle upon Tyne, NE2 4AX, United Kingdom
8
Indiana University, Bloomington, IN, 47405, USA
#
These authors contributed equally to this work.
Abstract
Chlamydiae are important pathogens and symbionts, with unique cell biology features. They lack
the cell-division protein FtsZ, which functions in maintaining cell shape and orchestrating cell
division in almost all other bacteria. In addition, the existence of peptidoglycan (PG) in
chlamydial cell envelopes has been highly controversial. Using electron cryotomography, mass
spectrometry and fluorescent labeling dyes, here we show that some environmental chlamydiae
have cell-wall sacculi consisting of an unusual PG type. Treatment with fosfomycin (a PG
synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG
synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the
presence of PG in a member of the
Chlamydiae
. They also present a unique example of a
bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the
absence of a cell wall that renders FtsZ non-essential.
Introduction
Chlamydiae
are members of the
Planctomycetes-Verrucomicrobia-Chlamydiae
(PVC)
bacterial superphylum
1
. Like most other bacteria, some PVC bacteria are already known to
possess peptidoglycan, i.e. chains of alternating N-acetylglucosamine and N-acetylmuramic
acid sugars crosslinked by short peptides. PVC bacteria also display striking eukaryote-like
2
corresponding authors: Grant J. Jensen: jensen@caltech.edu, Matthias Horn: horn@microbial-ecology.net.
Author contributions
MP initiated the study. MP and KA performed all experiments except HPLC/MS analyses of sacculi, which were done by JB, JG, and
WV. EK, EH, YVB, and MSV provided the FDAA dyes and advice on the FDAA labeling experiments. MP, KA, WV, MH, and GJJ
designed the experiments and wrote the manuscript.
Competing financial interests
The authors declare no competing financial interests.
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Nat Commun
. 2013 December 2; 4: . doi:10.1038/ncomms3856.
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and archaea-like cell biological features, which have suggested intriguing hypotheses about
their role in cellular evolution
2, 3
.
In
Verrucomicrobia
and almost all other bacteria, septal PG synthesis is orchestrated by the
FtsZ cytoskeleton
4, 5
. In contrast,
Planctomycetes
lack both PG and FtsZ
5
. This fits to the
notion that PG-loss renders FtsZ dispensable, like in mycoplasmas
6
or L-form bacilli
7
.
While from genome sequences it is clear that chlamydiae do not possess FtsZ, the presence
or absence of chlamydial PG has been highly controversial
8, 9, 10, 11, 12, 13, 14, 15, 16
. One
early study reported the colorimetric detection of muramic acid in chlamydiae
14
, but more
reliable chromatographic methods subsequently failed to confirm this result
11, 15
. All
attempts to purify chlamydial sacculi have failed
10, 12
and no periplasmic density layers
have been detected between the inner and outer membranes of chlamydiae by electron
microscopy (including for instance
17, 18, 19
). The apparent absence of PG in chlamydiae is
surprising, however, since despite their highly reduced genomes, a nearly complete pathway
for the synthesis of PG is present in the genomes of all chlamydiae
13
. In addition, several of
the chlamydial PG biosynthetic enzymes have been characterized and shown to be
functional
in vitro
and in complementation assays
20, 21, 22, 23, 24, 25
.
Here we look for evidence of peptidoglycan cell walls in two diverse and deeply rooting
chlamydiae
26
,
Protochlamydia amoebophila
and
Simkania negevensis
. Through electron
cryotomography (ECT), biochemical purification, enzymatic digestion, mass spectrometry,
fluorescence microscopy, and antibiotic treatment, we show that
P. amoebophila
are indeed
surrounded by sacculi containing a new type of PG. In contrast, no evidence of PG is found
in
S. negevensis
. These results prove that some chlamydiae do in fact synthesize PG sacculi,
explaining the presence of PG-synthetic genes, but raising new questions about the identity
and purpose of the modifications and the mechanisms of cell division in the absence of FtsZ.
Results
Electron cryotomography of the chlamydial cell envelope
Two diverse and deeply rooting members of the chlamydial phylum,
Simkania negevensis
and
Protochlamydia amoebophila,
were imaged by electron cryotomography (ECT) in a
near-native state. Bacteria were purified from amoeba cultures, plunge-frozen, and 25 and
20 tomograms were collected of intact cells (Figure 1). Density profiles through the cell
envelopes of the two species were quite different. While four layers were resolved in
Simkania
envelopes (Figure 1 B, C), five layers were resolved in
Protochlamydia
(Figure 1
E, F). Because the individual leaflets of lipid bilayers can be resolved in some
cryotomograms, especially when the images are taken close to focus, in the case of
Simkania
, it is unclear whether the four layers represent the two leaflets of the outer and
inner membranes (“O” and “A” being the two leaflets of the outer membrane, and layers
“B” and “I” being the two leaflets of the inner membrane), or whether one or more of these
layers are non-membranous. The facts that layers O and A have fairly similar contrast and
are consistently spaced even through the undulations are consistent with them being two
leaflets of a single (outer) bilayer membrane. Their separation (~5 nm), however, is much
larger than typical phospholipid bilayer membranes, whose two density peaks (from the
phospholipid head groups) are only 3.7-4 nm apart
27
. Similarly,
Simkania
layers B and I
may be the two leaflets of a single (inner) membrane, since they have similar contrast and a
consistent spacing, but again they appear too far apart. In contrast to the
Simkania
envelope,
the profile of
Protochlamydia
surprisingly resembled those of other Gram-negative bacteria
with two membranes and a peptidoglycan cell wall
28, 29, 30
. Between the
Protochlamydia
outer and inner membranes (labeled “O” and “I”, respectively) there appeared to be three
additional layers (labeled “C”-“E”). The similar-looking three layers in
Treponema pallidum
(from the outside in) were identified as proteinaceous (lipoproteins), peptidoglycan, and
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again proteinaceous
29
. By analogy this suggests that layer C is composed of lipoproteins
(perhaps connecting the outer membrane to the cell wall) and other outer-membrane-
associated proteins, layer D is a cell wall, and layer E is composed of lipoproteins and inner-
membrane-associated proteins (perhaps including the penicillin binding proteins responsible
for cell wall synthesis, the lipoprotein OmcA, and the cysteine-rich protein OmcB). While
other interpretations remain possible (cysteine-rich disulphide-cross-linked envelope
proteins have been suggested to be the functional equivalent of PG in chlamydiae
31
), the
most important and clear observation was that
Protochlamydia
exhibit a distinct periplasmic
layer (D).
Purification and imaging of sacculi
In order to explore whether any of the observed periplasmic layers consisted of
peptidoglycan (PG), we attempted to purify sacculi by boiling chlamydial cells (obtained
from asynchronously infected amoeba cultures) in 4% sodium dodecyl sulfate. Strikingly, in
three independent experiments, we observed sacculus-like structures in preparations from
Protochlamydia
(Figure 2, A-D), but not from
Simkania
(two experiments).
Protochlamydia
sacculi diameters (679 nm +/−34 s.d. n=10) and morphologies matched the size and shape of
intact cells.
Protochlamydia
sacculi had one or two 5-7 nm thick layers (arrowheads in
Figure 2 E), plus mesh-like (up to 30 nm long) high-density aggregates attached to the
outside (arrows in Figure 2 D).
Digestion and biochemical analyses of sacculi
To check for the presence of peptidoglycan in the purified sacculi, we digested the samples
with cellosyl, a glycan strand-cleaving peptidoglycan muramidase. Cellosyl released soluble
material from insoluble sacculi, which was reduced with sodium borohydride and analyzed
by high pressure liquid chromatography (HPLC) using conditions for separating
muropeptides
32
. The chromatogram (Figure 2 F) showed three main peaks in the monomeric
region (20-50 min) and many peaks after 60 min that are poorly separated at higher retention
time (>75 min) forming a “hump”, which is typical for highly cross-linked and/or
incompletely digested PG material
33
. The retention times and overall pattern of cellosyl
digestion products were different, however, from those of muropeptide mixtures obtained
from other Gram-positive and Gram-negative bacteria.
To characterize this material, the three main cellosyl products in the monomeric region and
one well-separated main product at the beginning of the “hump” region were analyzed by
mass spectrometry (MS). The determined neutral masses of the earlier three products were
higher than what would be expected for monomeric muropeptides, but the masses of
products 1 and 2 and of products 2 and 3 both differed by 71 Da, a typical feature of
monomeric muropeptides with a tri-, tetra- and pentapeptide, respectively, due to the
presence of none, one or two D-alanine residues (Figure 2 F). In MS/MS analysis the three
products fragmented in a similar way, showing that they are related (Supplementary Fig. S1,
Table 1). For all three peaks, we observed mass differences to the parent ion corresponding
to the loss of Glc
N
Ac, Glc
N
AcMur
N
Ac(r) (r, indicates reduction to
N
-acetylmuramitol),
Glc
N
AcMur
N
Ac(r)-
L
-alanine and Glc
N
AcMur
N
Ac(r)-
L
-alanine-
D
-glutamate, confirming that
products 1-3 are all muropeptides. The neutral masses of the
Protochlamydia
products 1, 2
and 3 were all 314.12 Da larger than the masses of the reduced monomeric muropeptides
(with tri-, tetra- or pentapeptide) from Gram-negative bacteria
34
, however, suggesting the
presence of a common modification in the
Protochlamydia
muropeptides. The neutral mass
of product 4 was consistent with a peptide cross-linked dimer of product 2. Additional mass
differences that occurred in all fragmentation spectra indicated the presence of the same and
as yet unknown modifications with 129 and 203 Da, respectively, explaining the higher
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mass of
Protochlamydia
muropeptides 1-3 compared to the monomeric muropeptides of
E.
coli
.
To confirm that PG was a major component of
Protochlamydia
sacculi and check for the
presence of disulfide cross-linked protein components, purified sacculi were subjected to
lysozyme and DTT treatment, respectively, and imaged with negative stain electron
microscopy. Only the incubation with lysozyme degraded the sacculi (Supplementary Fig.
S2).
Fluorescence imaging of D-alanine incorporation
in vivo
To further confirm the presence of PG in
Protochlamydia
sacculi, we tested whether
fluorescently labeled amino acids (FDAA; labeled D-alanine in our experiments)
35
would be
incorporated into chlamydial cells
in vivo
. Incubation of amoeba cultures continuously
infected with
Protochlamydia
(including reticulate bodies, elementary bodies, and
transitional stages) with FDAA (HADA and BADA) resulted in multiple strong and
chlamydial cell-sized signals inside amoeba cells (Figure 3 A-D, Supplementary Fig. S3). In
many cases the FDAA labeling in infected amoebae overlapped with staining of the
chlamydial cells by DAPI or chlamydiae-specific fluorescence
in situ
hybridization (FISH).
Not all cells stained by FISH/DAPI showed a corresponding FDAA signal, at least in part
because chlamydial cells were in different developmental stages, including non-replicating
elementary bodies. No signals were detected when uninfected amoebae were incubated with
FDAA (Supplementary Fig. S3 D, E), or when infected amoebae were incubated with
DMSO only. Interestingly, purified
Protochlamydia
elementary bodies (which cannot
undergo cell division and are therefore probably not actively synthesizing new PG) also did
not show labeling upon incubation with FDAA (Supplementary Fig. S3 B, C), indicating
that PG synthesis takes place during
Protochlamydia
replication inside the host. Amoeba
cultures infected with
Simkania
, on the other hand, showed either no signals (using BADA)
or signals similar to the background level (using HADA) upon labeling with FDAAs
(Supplementary Fig. S4 A, B), consistent with the absence of purifiable sacculi. Purified
Simkania
cells were not labeled by either dye (Supplementary Fig. S4 C, D).
Protochlamydia
sensitivity to cell-wall-targeting antibiotics
Due to the high conservation of PG throughout the bacterial domain of life, many
antibacterial drugs target PG synthesis. The so-called “chlamydial anomaly”
16
is that despite
the fact that PG has not been detected in pathogenic chlamydiae, these organisms are
sensitive to cell wall-targeting
β
-lactam antibiotics. Penicillin, for instance, leads to the
formation of enlarged aberrant cells
36, 37
and blocks the conversion between developmental
stages
38
. Environmental chlamydiae, in contrast, are resistant to
β
-lactams
39, 40
- possibly
due to putative
β
-lactamases encoded in their genomes. To explore the role of the PG
sacculus in the
Protochlamydia
life cycle, we used an alternative PG synthesis-targeting
antibiotic (fosfomycin) to treat infected amoeba cells. The addition of 500
μ
g/ml fosfomycin
to
Protochlamydia
-infected amoeba cultures led to a significant decrease in infection rate
(20.2% ± 8 infected amoebae for fosfomycin-treated cultures vs. 95.8% ± 2.2 infected
amoebae for untreated cultures; p <0.0001, unpaired t-test).
Protochlamydia
cells within
treated cultures were also up to eight-times larger than normal (diameters of up to 6
μ
m)
(Figure 3 G, H). Lower fosfomycin concentrations (25
μ
g/ml and 100
μ
g/ml) induced the
formation of fewer aberrant forms and did not affect the infection rate (not shown).
Fosfomycin-treatment of
Simkania
-infected amoeba cultures led to only a slight decrease in
infection rate (59.9% ± 5.6 infected amoebae for fosfomycin-treated cultures vs. 67% ± 1.7
infected amoebae for untreated cultures) and no differences in cell size were detected
(Figure 3 E, F).
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Discussion
We conclude that
Protochlamydia
synthesize sacculi containing peptidoglycan that can be
hydrolyzed by cellosyl, contains monomeric and cross-linked muropeptides, and carries yet
unknown modifications at virtually every subunit. No evidence of peptidoglycan in
Simkania
was found. Fluorescence imaging of D-Ala incorporation
in vivo
and monitoring
of cell wall antibiotic sensitivity further suggested that the
Protochlamydia
PG sacculus
plays an important role in cell cycle and shape. This challenges previous speculations that
chlamydiae synthesize a small ring of PG only during cell division
41
. Because this might
still be true for
Simkania
and pathogenic chlamydiae, however, our data prompts a
reconsideration of whether these organisms lack PG entirely (and the effects of
β
-lactams
are pleiotropic) or if they synthesize novel PG structures that are not purified by standard
sacculus preparation protocols.
The presence of sacculi in
Protochlamydia
but not in
Simkania
matches the less complete
set of synthetic genes in the latter:
Simkania
, as well as pathogenic chlamydiae, lack an
undecaprenyl-diphosphate phosphatase (UppP) and alanine/glutamine racemases (Alr, MurI)
(Supplementary Table S1)
13, 42, 43
. Interestingly, transglycosylases have not been found in
any chlamydial genomes (Supplementary Table S1)
13
or in the genomes of a few other PG-
possessing bacteria
44, 45
, so some other enzyme(s) must be capable of synthesizing glycan
strands.
The presence of PG sacculi in
Protochlamydia
and in the
Chlamydiae
’s sister phylum
Verrucomicrobia
46
, together with the fact that the more basal chlamydial lineages have
more complete PG synthesis pathways make it likely that the last common chlamydial
ancestor synthesized a PG sacculus. The detection of a PG-containing sacculus in
Protochlamydia
challenges the view that FtsZ is essential in PG-possessing bacteria
4, 6, 7
,
however, because to our knowledge,
Protochlamydia
is the first example of a bacterium
with a PG cell wall, but without FtsZ. Studying cell division and septal development in this
organism could help clarify the role of FtsZ and the evolutionary transition to PG- and FtsZ-
independency.
Methods
Cultivation of organisms
Acanthamoeba castellanii
Neff infected with
Protochlamydia amoebophila
UWE25, or
A.
castellanii
UWC1 infected with
Simkania negevensis
, were cultivated in TSY medium (30
g/L trypticase soy broth, 10 g/L yeast extract, pH 7.3) at 20°C. Amoebal growth was
monitored by light microscopy and medium was exchanged every 3-6 days. The presence
and identity of the chlamydial symbionts was verified by isolation of DNA from cultures
followed by amplification and sequencing of the 16S rRNA genes. In addition, fluorescence
in situ
hybridization (FISH) using specific probes combined with 4
,6-diamidino-2-
phenylindole (DAPI) staining of infected cultures was performed using specific probes for
the respective symbiont
47
. Amoebae infected with chlamydiae were allowed to attach on
slides and were fixed with 4% formaldehyde at 20°C. Cells were hybridized for 1.5 hours at
46°C at a formamide concentration of 25% with the
Protochlamydia
-specific probe E25-454
(5
-GGATGT TAG CCA GCT CAT-3
) and the probe EUB338
48
. Subsequently, cells
were stained with DAPI (0.5
μ
g/ml in PBS) for 5 minutes, and slides were analyzed using an
epifluorescence microscope.
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Purification of chlamydiae
Infected
A. castellanii
cultures were harvested by centrifugation (7,197 × g, 10 min), washed
in Page’s Amoebic Saline (PAS)
49
, centrifuged and resuspended in PAS. Amoeba cells were
disrupted by vortexing with an equal volume of glass beads for 3 minutes. Glass beads and
cell debris were removed by centrifugation (5 min, 300 × g). The supernatant was filtered
through a 1.2
μ
m filter and centrifuged at maximum speed for 10 min. The obtained pellet
was resuspended in PAS.
Plunge-freezing
For plunge-freezing, copper/rhodium electron microscopy (EM) grids (R2/2 or R2/1,
Quantifoil) were glow-discharged for 1 min. A 20×-concentrated bovine serum albumin-
treated solution of 10 nm colloidal gold (Sigma) was added to purified chlamydiae or sacculi
(1:4 v/v) immediately before plunge freezing. A 4-
μ
l droplet of the mixture was applied to
the EM grid, then automatically blotted and plunge-frozen into a liquid ethane-propane
mixture
50
using a Vitrobot (FEI Company)
51
.
Electron cryotomography
Images were collected using a Polara 300 kV FEG transmission electron microscope (FEI
Company) equipped with an energy filter (slit width 20 eV; Gatan) on a lens-coupled 4 k×4
k UltraCam CCD (Gatan). Pixels on the CCD represented 0.95 nm (22,500×) or 0.63 nm
(34,000×) at the specimen level. Typically, tilt series were recorded from −60° to +60° with
an increment of 1° at 10
μ
m under-focus. The cumulative dose of a tilt-series was 180-220 e
−/Å
2
. UCSF Tomo
52
was used for automatic acquisition of tilt-series and 2D projection
images. Three-dimensional reconstructions were calculated using the IMOD software
package
53
or Raptor
54
. Tomograms were visualized using 3dMOD
53
. Density profiles were
generated using ImageJ.
Fluorescent labeling of peptidoglycan
Newly synthesized peptidoglycan was labeled using fluorescent D-amino acids
35
.
A.
castellanii
cells continuously infected with
Protochlamydia
or
Simkania
were harvested and
resuspended in a mixture of TSY and PAS (1:1). Cells were incubated with 1.5 mM HADA
(hydroxy coumarin-carbonyl-amino-D-alanine)
35
or BADA (4,4-Difluoro-5,7-Dimethyl-4-
Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid-3-amino-D-alanine) for 6 h with gentle
shaking. Cells were pelleted, washed three times and fixed with 4% formaldehyde followed
by FISH using the chlamydia-specific probe Chls-0523 or DAPI-staining. As a control,
uninfected amoebae and purified chlamydiae were treated in the same way.
Antibiotic treatment of infected amoebae cultures
A. castellanii
were seeded into the wells of a multi-well dish and infected with purified
Simkania
and
Protochlamydia
elementary bodies
55
. After centrifugation at 600 × g for 10
min, the medium was exchanged for TSY supplemented with fosfomycin (0, 25, 100 or 500
μ
g/ml, respectively). Medium was exchanged once at 48 hours post infection. Cells were
fixed with methanol at 96 hours post infection followed by immunofluorescence analysis
using either anti-PomS antibodies
55
or anti-
Simkania
antibodies raised against purified
chlamydiae. The number of infected amoebae was counted for each treatment.
Preparation and composition analysis of sacculi
Chlamydial cells were purified from 1.8 L (
Protochlamydia
) and 3 L (
Simkania
) of infected
amoeba culture and, depending on the amount of harvested cells, resuspended in 2.6-5.2 ml
4% SDS (w/v). After shipping (overnight, room temperature), the suspensions were dripped
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into 4% SDS (preheated to 90°C, 6 ml final volume) and stirred for 2.5 h at 90°C. Sacculi
were pelleted (30 min, 135,000 × g) in a TLA-100.3 rotor (Beckman Coulter), washed 4× in
3 ml water, resuspended in 150
μ
l water and supplemented with 0.02% (w/v) sodium azide.
Muropeptides were released from the PG by an overnight incubation with the muramidase
Cellosyl (Hoechst, Frankfurt am Main, Germany) on a thermal shaker at 37°C and 800 rpm.
The sample was boiled for 10 min and centrifuged for 10 min at 13,500 × g. The
muropeptides present in the supernatant were reduced with sodium borohydride and
separated on a 250×4.6 mm 3-
μ
m Prontosil 120-3-C18 AQ reversed phase column
(Bischoff, Germany) at 55°C using a 135 min gradient from 50 mM sodium phosphate pH
4.31 to 75 mM sodium phosphate pH 4.95, 15% methanol and a flow rate of 0.5 ml/min
32
.
Muropeptides were detected at 205 nm. Fractions were collected, concentrated in a
SpeedVac, acidified by 1% trifluoroacetic acid, and analyzed by offline electrospray mass
spectrometry on a Finnigan LTQ-FT mass spectrometer (ThermoElectron, Bremen,
Germany) in positive ion mode using mass scans over the mass range from
m
/
z
= 300 to
m
/
z
= 1900 at a typical spray voltage of 1.1-1.5 kV
34
. Parent ion scans were acquired with an FT
MS resolution setting of 100,000 (at
m
/
z
= 400) with a typical mass accuracy of 3 ppm. MS/
MS scans were performed in the ion trap, which has a typical mass accuracy for the
fragment ion of ± 0.3 Da. MS spectra were deconvoluted to generate uncharged masses
using the QualBrowser program (ThermoElectron, Bremen, Germany)
34
.
Negative-stain electron microscopy of treated sacculi preparations
Purified sacculi were incubated at 37°C for 12 h in 20
μ
l total volume with either lysozyme
(10 mg/ml), dithiothreitol (5 mM) or phosphate buffered saline. Samples were applied to a
Formvar-coated, carbon-coated, glow-discharged copper EM grid (Electron Microscopy
Sciences). Samples were aspirated, stained with 1.5% uranylacetate and imaged on an FEI
Tecnai T12 electron microscope.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was funded by the Austrian Science Fund FWF (Y277-B03 to MH), the European Research Council
(ERC StG “EvoChlamy” to MH), NIH grant GM094800B (to GJJ), the Caltech Center for Environmental
Microbiology Interactions (to GJJ, MP), a gift from the Gordon and Betty Moore Foundation to Caltech, the
BBSRC (BB/I020012/1 to WV), NIH grant AI059327 (to MSV) and NIH grant GM051986 (to YVB). We thank
Elitza Tocheva for discussions on the preparation of sacculi.
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Figure 1. Chlamydial cell envelopes are multi-layered
Simkania
(A-C) and
Protochlamydia
(D-F) cells were purified from asynchronously infected
amoeba cultures, plunge-frozen and imaged by ECT. Shown are tomographic slices through
reticulate bodies (A, D and B, E enlarged) and corresponding density profiles (C, F) of the
cell envelopes. Profiles are enlarged, aligned and cropped relative to the outer membrane.
Distances between peaks (in nm) are indicated. In contrast to the
Simkania
profile, the
Protochlamydia
profile resembles those of other bacteria with peptidoglycan cell walls (see
text for a full discussion of each profile and layer). Bar, 100 nm in A, D and 20 nm in B, C,
E, F.
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Figure 2.
Protochlamydia
synthesize purifiable sacculi that contain peptidoglycan
Cryoprojections (A) and tomographic slices (B-E) through sacculi-like structures purified
from
Protochlamydia
cells. We were unable to obtain similar structures from
Simkania
.
Sacculi had one or two layers (arrowheads) plus short high-density filaments (arrows) on the
outside (C enlarged in D, E). Sacculi were digested, reduced and separated by high-pressure
liquid chromatography (F). Mass spectrometry analysis of peaks 1-3 (neutral masses
indicated in Da) indicated the presence of modified peptidoglycan (see also Table 1 and
Supplementary Fig. S1). Bars, 100 nm.
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Figure 3.
Protochlamydia
incorporate D-alanine
in vivo
and are sensitive to fosfomycin
Protochlamydia
-infected amoebae (A-D), but not purified
Protochlamydia
cells
(Supplementary Fig. S2) or uninfected amoeba cells (Supplementary Fig. S2), stained
positively for new peptidoglycan synthesis with fluorescently labeled D-alanine dyes HADA
(A) and BADA (Supplementary Fig. S2), confirming the synthesis of peptidoglycan in
actively-growing
Protochlamydia
. Specific FISH staining of chlamydial cells (B, overlay
with A shown in C) and the eukaryotic host (D, shows overlay with C) are shown. The
treatment of
Protochlamydia
-infected amoeba cultures (amoeba cell outlines in white) with
cell wall synthesis-targeting fosfomycin (H, control shown in G) resulted in a dramatic
decrease in infection rate and aberrant or larger chlamydial cell shapes (shown are
immunofluorescent stainings of chlamydial outer membrane proteins), suggesting a crucial
role of PG in the
Protochlamydia
life cycle. Fosfomycin did not affect
Simkania
(F, control
shown in E). Bars, 5
μ
m in D, 10
μ
m in E-H.
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Pilhofer et al.
Page 14
Table 1
Fragmentation masses from fractions of reduced
P. amoebophila
muropeptides.
Fraction
number
or
muro-
peptide
Fragment
Mass of fragments,
H
+
form (m/z)
Proposed structure
determined
calculated
1
A
1167.3447
1167.4908
[M+H]
+
-H
2
O
B
1056.3231
1056.5014
[M+H]
+
-129.2
C
982.3848
982.4220
[M+H]
+
-GlcNAc
D
853.3392
853.4220
[M+H]
+
-129.0-GlcNAc
E
705.3032
705.3059
[M+H]
+
-Glc
N
AcMur
N
Ac(r)
F
634.3148
634.2688
[M+H]
+
-Glc
N
AcMur
N
Ac(r)Ala
G
505.2668
505.2262
[M+H]
+
-Glc
N
AcMur
N
Ac(r)AlaGlu
2
A
1238.3288
1238.5255
[M+H]
+
-H
2
O
B
1127.4075
1127.5361
[M+H]
+
-129.1
C
1053.3859
1053.4567
[M+H]
+
-GlcNAc
D
924.3934
924.4567
[M+H]
+
-129.0-GlcNAc
E
776.3376
776.3406
[M+H]
+
-Glc
N
AcMur
N
Ac(r)
F
705.3202
705.3035
[M+H]
+
-Glc
N
AcMur
N
Ac(r)Ala
G
576.3141
576.2609
[M+H]
+
-Glc
N
AcMur
N
Ac(r)AlaGlu
3
A
1309.4104
1309.5684
[M+H]
+
-H
2
O
B
1198.3875
1198.5790
[M+H]
+
-129.2
C
1124.3796
1124.4996
[M+H]
+
-GlcNAc
D
995.3871
995.4996
[M+H]
+
-129.0-GlcNAc
E
847.3485
847.3835
[M+H]
+
-Glc
N
AcMur
N
Ac(r)
F
776.3356
776.3464
[M+H]
+
-Glc
N
AcMur
N
Ac(r)Ala
G
647.3268
647.3038
[M+H]
+
-Glc
N
AcMur
N
Ac(r)AlaGlu
Tetra
from
C. jejuni
A
946.4128
946.3869
[M
+
Na]
+
-H
2
O
B
n.d.
835.4202
[M
+
Na]
+
-129
C
761.3908
761.3181
[M
+
Na]
+
-GlcNAc
D
n.d.
632.3181
[M
+
Na]
+
-129-GlcNAc
E
484.3317
484.2019
[M
+
Na]
+
-Glc
N
AcMur
N
Ac(r)
F
413.2619
413.1648
[M
+
Na]
+
-Glc
N
AcMur
N
Ac(r)Ala
G
284.3200
284.1222
[M
+
Na]
+
-Glc
N
AcMur
N
Ac(r)AlaGlu
Fraction numbers correspond to peak numbers in Figure 2 F. Fragments correspond to fragments in Supplementary Fig. S1. The parental ion ([M
+H]
+
or [M
+
Na]
+
) of each muropeptide fragments to ions lacking either water, an unknown modification with ~129 Da, a Glc
N
Ac residue, ~129
Da and Glc
N
Ac, a Glc
N
AcMur
N
Ac(r) disaccharide (r, indicates reduction to muramitol), Glc
N
AcMur
N
Ac(r)Ala or Glc
N
AcMur
N
Ac(r)AlaGlu (see
column ‘Proposed structure’). Tetra, reduced disaccharide tetrapeptide muropeptide fraction from
Campylobacter jejuni
56
. The Na
+
containing ion
with m/z 964.4202 was fragmented. n.d., not detected.
Nat Commun
. Author manuscript; available in PMC 2014 June 02.