【上 田泰記念感染症 ・化学療法研究奨励賞受賞者論文】
MorphologicalNaoyukiMiyashita 1),Akira
Masamitsu

DivisionofKawasaki

MedicalDivisionof

Nakajima 1),Yoshihito

Respiratory

Diseases,1) Department

recent

years

acute

respiratorytheseclinicalisolates

andtrachomatisalocation

dimensionthemember

of

of

the

of

hexagonallymembrane,werethatcompared18

present

their

L2/434/Bu

the"pear-shape"ofrecognizing

ed

March

isolatingthestrainsofthenasopharynx

Chlamydia

that

of

in

C.trachomatis,C.psittaci
those

of

Key

words:Chlamydiaofarrayedmorphology

frompecorum

body

is

(EB)

all

members

of

the

and

C.pecorum

C.pneumoniae

projections

regularsurface

on

the

the

Chlamydia.It

theorganisms,and

surfaceofwasround-shaped

EB,Button

ilar

intracellularparasites,

to a single genus,Chlamydia,

which

four

the

to
are

species,Chlamydia

toof

C.trachomatis

investigations

ciationof C.pneumoniae

ic

trachomatis 2),Chlamydia

thatrecentofprojec-cardiovascularsuggested

andC.pneumoniae

ele-

KKpn-1811,14,15),from

the

with

tract

are two morphologicallyforms:oneis the infectious

mentary

body (EB) and the other is the reproductive

reticulate

body (RB).TheEB penetrates

host cell by being phagocytized

the

last

four

years,werespiratory

a susceptible

study,weexamined

isolates(KKpn-1•`1(Kpn-18)

an RB,which

multiplies

by binarythen undrgoes

maturation

throughfission.The

RB

an intermediatemorphology

progeny EB 4)
ChlamydialThepneumonia,bronchitis,sinusitis,andpharyngitis,aswell as being responsible for up to 10% of cases ofpneumoniae

community-acquired

KKpn-15141),KKpn-1611)

of

sim-

of

and

comparedstrainsofpatiertts

thepresent

theclinicalitwithof C.pneumoniC.pecorum.

Methods

strains

ratory pathogen 5).The organism is a common cause of

infections has a worldwide status

morphology

and

18

KKpn-1•`andMaterials

pneumonia 6,7).The prevalence

isolating

infections.In

ae,C.trachomatis,C.psittaci

is well recognized as a major respi-

innasopharynx

of establishedofdisease10,11),and

succeeded

the

asso-

atherosclerot-strains,designatedacuteaddition,exacerbations

pulmonaryand is converted into

the

with

8,9),acutecycle in whichInIn
a possible

infection

disorders

psittaci 2) and Chlamydia pecoruma).These
organisms
multiply through a common,uniquedevelopmental

C.pneumoniae

thatdifferent

infections 6)

have

asthma12,13).

C.pneumoniae

outerstructure,surface

obstructive

body,to form a matureEB

profileschronic

distinctthetheidentical
EB

bronchial

functionallyforstrains.

have been assigned

there

criterion
properties,such

was,therefore,conclud-strainsthat

Introduction

pneumoniae 1),Chlamydia

10,
indicatedstructure

The chlamydiae,obligate
comprises

resultsmorphologicaloforganisms,and

pneumoniae,pear-ortion,hexagonal

genus

Frt-Hu/Cal

The

inner

of

TW-183,morphologicaltheKKpn-1-—KKpn-18

TWAR

valid

structures
of

on

a
basic

with

morphology

C.pneumoniae

Bo/E58.

notthepatients

thepsittaci

Chlamydia

C.pneumoniae,although

nucleus,presence

similartheelementary

pneumoniae
ofexaminedwithMicrobiology,

19,1997)

study,wemorphology

and

of

3)

(TWAR),IOL-207,Kajaani-6,YK-41,Chlamydia

Chlamydiaasand 2)Department

KKpn-1•`KKpn-18,from

infections.In

Matsushima 1)

Welfare18,1997•EAcceptedinToshiharu

701-01,Japan

February

succeeded

Medical

Kishimoto 1),

city,Okayama

of

have

and

Medicine

University

designatedtract

AR-39,AR-388

that

we

we

of

577,Kurashiki

Welfare,Kawasaki

pneumoniae

Soejime,Toshio

Niki 1)

School,Matsushima

strains,whichof Chlamydia

Matsumoto 2),Rinzo(ReceivedIn

analysis

followingchlamydial

strains

were

used:C.

TW-183,AR-39,AR-3887)(TWAR),

IOL-20716),Kajaani-617),YK-4118),KKpn-115),

psittaci

andFrt-Hu/Cal 1019);C.trachomatis

15

clinicalisolates15),C.
L2/434/Bu20)

andC.pecorum

AR-39

Bo/E5821)

andWashington
Strains
Saikku,were

Research

Foundation,
and

was

of

suppliedpurchased
Seattle,were

Helsinki,byY,Japan.

Instituteof Public

Health,KKpn-15,

KKpn-16

and

other

pneumoniae

laboratory.

These

in

HeLa

229

3

postinoculation.

Hirai

strains

K,

HL

of
L2

National
hasbeenmaintained

20

years.

C.

grown

tures

and
day

2

trachomatis
in

L929

HeLa

229

or,3

propagatedCultivationof229

pneumoniae
werestrains.

onto

then

incubated

and

were

incubated

at

37•Ž

in

Dulbecco-modified
with

10%

Light
To

tions

examine

the

HeLa

229

cells

inoculated

Afterincubation

air-dried,withiodineandTheethanol

and

cific

monoclonal(Washington
SCP-5323),

48•`72absolute

for

Cal

more

than

psittaciormonolayer
then

were

cul-

tion

E58
line.

gationat

experiments

remove

cell

with
all

room

C.

of

5%

Nissui,

CO2

at

Tokyo)

andofTokyo)

5%

precipitate
treatedstainedwithResearch
C.trachomatis

Foundation,

cells

above.

infected

cells
and

were

stainedwithabsolute
species-spe-(MAbs),
Seattle,species-specific

RR-402atroomtemperature
obtainedtowas(bottomtoplay-layer,

layer,

50%

30%

vol/vollayered

umn

(40
60

min

the

gradient7.3]);

at4•ŽJapan).
wereThe

12,000•~g

RNase

for

30

min

min
in

and

the

in

SPG

4•Ž.

The

and

then

37•Ž.

at

SPG

(finalatswing

together30

concentration;The

suspension

onto

a continuous

Urografin

gradient

52%

vol/vol)andcentrifuged

at

Two

distinct

bands

4•Ž.
column.

EBs

The

was

presence

confirmed

tron

microscopy.

After

were

suspended

in

SPG

EB

outer

the

formedofnumber

with

andstoredcol-

8,000•~g

were

lower

washing

30

centrifuged

RPS-25precipitateforsuspended

in

then
an

suspended

andatcomplete

and
with

was

to

for

[pH
min

DNase

each)

was

acid;
Germany]

obtainedwith20ƒÊg/ml

a

band

by

SPG,

the

in
of
elecEBs

at -70•Ž

until

required.
Preparation

staining
fixed

C. pneumoniae

antibodies

thediameter)

mm

described

60

at

of

centrifu-

supernatantbufferforcentrifuged

in

(14
as

Tris-HCl
8,000•~g

and

L929

homoge-briefBerlin/Bergkamen,

Cal

of

and

After

[3,5-diacetamido-2,4,6-triisobenzoic
AG,

cells

supplement-min

Na2HPO4

0.072%)

cushionTokyo,

in10

solution;

layer

CO2

cells
solu-

0.052%,

acid

two-layer

bottomthenstainabilitya(Hitachi,monolayerstrainC.tra-

KH2PO4theturbid

The
L929

for

debris,rotor

supplemented
serum.

mM

andinfectedhomogenizer.

sucroseSchering

were

C.pneumoniae

h postinoculation,

glutamic

900•~g

onto

their

method7.5%,

a Teflon

Urografin

temperacells

ered

i or

and

sucrose-phosphate-glutamate

0.1529%,wt/volinoculainsucrose

with

was

H20

72

Higashi24)

new

of
At

collected
(SPG:

nized

MDBK

also

EBs28).

2methanol,were

purification

harvested

cell

at

the

chomatis

10

Cal

and

May-Gruenwald-Giemsa
cells

strains

for

E58

the

of

Our

prepara-C.trachomatis

10

and

h,

and

EB

a

slip

in

C.pneumoniae

wereHigashi24).

thatestablished

on

for

of

and

indicated

therefore

onto

incubated

TamuraWe

a monolayera coveroffragments.

inclusions,

and

fixed

solutions.

morphology

methodexperiments

strainsS,

serum.

was

and

day

microscopy

intracytoplasmic
or

grown
on

the

C.pecorum

Yamasakicalf(Nissui,

by

and

by

inoculated

MEM

purified

C.psittaciof RBsandatmosphereownof the

and

(MEM,

an

EBs

Tamuraatmospheretheheat-shock

a number

infectionwithspecies-spe-

contained

Similarly,

inoculatedthemethod

the

fetal
were

EBe

and

KDa

the

cycloheximide

strainsof

to60

al.25)

Infectedandirectedetminmediumcentrifugation.

cells

ed

in

pneumoniaebysonication,60

heat-inactivatedL2

withoutfor

35t

1ƒÊg/ml

10%

10

for

of

preliminary

C. psittacimonolayers.

essential

containing
with

cell
at

minimuminbrief(900•~g

the

cells

MAb

C.

and

laboratory

Caldwell

C.

used

an

of

our

purifieda MDBK

After

centrifuged

ture)

in

were

in

be

Preparation

supplied

C.pecorum

infected

cells

to

epitopeobtained

Inc.)

C.

cultures

in

was

MAb,

Systems,byandpostinoculation.

confirmed

genus-specific

University.

suspension
cell

continuously

HeLa

was

which

cific

Co.),
Diagnostics

Gifu

laboratory
L2

P.

(Syva
(Ortho1997

protein14).

our

were

E58

Japan.

our

by

in

harvested

suppliedin

XC-60,

isolatesstrains

Health,USA.

KKpn-1,andAgriculture,

of

WA,

Hiroshima

isolated

cultureswas

Institute

were

were

C.pecorum

Facultytrachomatis

on

cell

the

Strain,clinicalC.pneumoniae

or

from

Cultureset

Finland.

PrefectualC.

MicroTrak

supplied

Kanamoto15

TW-183,

Strains

Kajaani-6

University

YK-41

strains.

AR-388

IOL-207

theMAY

日本化 学 療 法 学 会雑 誌

256

Four
bufferofmillilitersofpurified

was

with

6g

diameter;andUSA)22)

Biihler,

MAb,

After

membranes

mixedM

shakenandcentrifugationEBsuspension

glass

beads

S Instruments

vigorously
TiThingen,ofon

a CellGermany)
at10,000•~g

Inc.,

in
(0.1

Osaka,Mill

shaker

at

70

CPS

for

60

min

Tris

mm

in

Japan)(Edmund
for
at

4

min.

4•Ž

on

VOL.45

NO.5

a sucrose

Morphologicalcushion(sucroseanalysis

15% wt/vol) ,theof Chlamydia

precipitate was recovered in 0 .2 M Tris-HC1 buffer (pH 7.4)
containing MgC12 5 mg/ml and treated with a mixture(LPS)

of DNase and RNase (final concentration;20 ug/ml
each) at 37C for 60 min .This was followed by trypsin

coveredtreatment

Agriculture,Gifu(50 ug/ml) at 37C for 60 min and finallywith 0.125% sodium dodecylsulfate(SDS) at 37C for

10 min to remove the inner membranes.Aftering twice,theinsoluble outer membranes

pended in distilled waterrequired.
Electronand stored

washwere sus-

at-70C

until

microscopy

To examine

the morphology

situ,infectedof the organisms

cells were collected at 24,48

inon thelabeling

for 60 min.Thin

sectionsO

asreportedor outermembranes

werecoatedplat-

For shadow-casting,EBs
mountedon a specimen

inum-palladium
tive

grid

staining,outerwas examinedelectron

Determinationof thetransformation

by Fourier

To determine

phologicalfreezing

damagea Balzers
described
The11-5003AF

wascalculateddiffraction

by

40% glycerol

by ice crystal

301

pelleted

to preventmor-

formation.After
were preparedfreeze-replica

fractionpatternobtaina clearapparatusasor JEM-2000

examinedwithtransmission

a Hitachi

electronmicro-

scope at 75 or 80 kV.

To observe projections

on the surface of C.pneumo-

purified EBs were mounted on a nickel

grid without a supporting
fixed with the glutaraldehyde

membraneand doubly
and OsO4 fixatives.The

specimens

treatedwere additionallywith 2% tannic

Fourier

original

theas a diffracof the

without

regularof thedis-(frequency

0)cases,thefrom the selectedimage

was cal-that

by measuring

by the inverse

systemintegralof the transform
some(Zeiss,of thespacingspots.In

imagesspots in the dif-

Fouriertransform

topresent

inthe "noise"

micrographs.
ResultsinInclusion
ties

were

from the origin

the original

previously29)

niae EBs,thewas displayedreconstituted

glutaraldehyde,and

memoryaveragewerenitrogen,replicas

specimensin the micrographto thewithin liquid

image

pre-

cells werein a micrographsystem,so7.3) solution.To

2.5%

information
thestructureinsystema camera,and

tance

with

programmed

analysisin the computer

on areplicas,infected

regularmembrane,diffraction

culated

of 1%

H-500

of the hexagonal

throughpattern.The

washings,

struc-

image

mountedcentrifugation,fixed

(15nm

the

tion

into

were

acid (PTA,pH

impregnatedGermany)18).Briefly,theparticles

the Hitachi

by a formula

image

onto a droplet

phosphotungstic

then

For nega-

obtainedmixedof regularEB outer2000

and placed

grid

freezewithshadower28)

of

at 75 kV.

spacing

the spacingin thein puttothenseveralwithmicroscope

ture

were

exposed

K,Faculty

A-goldCo.).Aftertransmission

membranes

carbon-covered

pare

and

in a vacuumof protein

the specimen

IBAS

on a gridandby Hiraia suspension

imagesof Birkelund

University,Gifu,Japan),washedin diameter,Amersham

waspreviously27).

membrane(suppliedwithand 72 h

examineda collodionMAbmethodwas mounted

phosphate-buffered-saline,and

an

and

with

by thespecimenwith

0.1 M cacodylate buffer (pH 7.3) for 60 min and 2.5%
preparedof all strains,immunogold

was performed

anti-LPS

postinoculation
by brief centrifugation(300xg
for 3min) and doubly fixed with 2.5% glutaraldehydeinsO4 in the same buffer

EB surface

et al.31).Briefly,thestructure

were

257

pneumoniae

morphology

of clinicalisolates

The inclusions
other

and

staining

proper-of C.pneumoniae

of KKpn-1,KKpn-15,KKpn-16

15 clinicalisolates

positively

andstainedwithC.

pneumoniae-specific
MAb (Cultureset),but

MAbs and the genus-specificnot with C.trachomatis-spe-cific MAb (MicroTrak)or iodinestain.Theirmorphol-

ogy in HeLa

closely

resembledthat

229 cells

psittaciand C.pecorum,which
the early stages in the Giemsa

are oval and dense
staining preparations

reported

host nucleus.These properties of the 18 clinical isolates obtaineOk our laboratory were identical withthose of TWAR strains,indicating they are the members of the species C.pneumoniae.
Morphology of C.pneumoniae
EBs in thin sections and shadowcast preparations
The chlamydial bodies in in situ inclusions of theethanol series,dried
dryer,followed
a Hitachi

in an

in a Hitachi HCP-1 critical pointby coating with Pt-palladium

E-102

were examined

dehydrated

Ion Sputter
with a Hitachiunit.The

alloy in

specimens

S-570 scanning

elec-

tron microscope at 25 kV.
To determine

the locationof lipopolysaccharide

compression.

acid to stabilize the morphology of the projections,asand then

without

in

The inclusions

previously30)

expandedof C.

of the日本 化学 療 法 学 会雑 誌

258

C.

pneumoniae

shown
seen

in
in

strains

Fig.
the

in

the

RB

The

EBs

of

KKpn-1,

Kajaani-6,

trast,

andthepostinoculation
of

were
of

EBs
no

the

KKpn-15,
15

to

the

RBs

of

strainsother strainsdifference between

TWAR andwas seen when the purified

EBs were

isolates(Fig.

dried in air and shadowcast (Fig. 2). The EBs of C.
ecorum, C. psittaci and C. trachomatis strains had pa

their

1
mor-

round "fried egg" shapepecorum,C.

IOL-207,

EBs 11, 14, 18, 27, 32).
TWAR

similar morphologicalstrains.

and
C.

are

differ-different

clinicalthattrachomatisofandKKpn-16,

theperiplasmic space limited by a wavy outer membrane,
creating "pear-shaped"
profiles 14. 18, 32, (Fig.
35) 1b). A

aredistinctround 11, 14, 17, 18, 32•`34)

identical
C.

EBs

There

and

were

was

psittaci,hmorphology

YK-41,

however,phology60

1 11,14,18, 32). A number
inclusions.

ences

a),

at

MAY 1 9 9 7

had

from

the

EBs

that was indistinguishableof strains

IOL,-207,

KKpn-1,

In

con-

a

wide

clinical isolates 14, 18, 32,(Fig.
33) 2a). However, the EBs ofFig. 1. Thin sections of KKpn-15
and AR-39 strains
at 60 h postinoculation.
a, Chlamydia
pneumoniae

Kajaani-6,

YK-41, and the 15

in HeLa 299 cells
KKpn-15;b, C.

pneumoniae
AR-39 at higher
magnification.
KKpn-15
EBs have anarrow
periplasmic
space and are round in shape, whereas
AR-39
EBs
are
enclosedby a wavy
outermembraneandare"pear-shaped"
in profile. c, KKpn-15
inclusion.
d, AR-39 inclusion.
Arrowheads
point to mitochondria
locatedclose to inclusions,but
unassociatedwith the inclusion
membrane.
Bars; 500 nm.

Fig.

2.
(a)

Shadowcast
and

agar
methodBars;

TW-183plate,
with1 ƒÊm.

ofpurified

EBs

of

(b)

strains.

EBs

were

a

specimentransferredcollodion,

KKpn-15,

KKpn-16,

to
and

then

Chlamydia

pneumoniae

air-driedshadowcast

on

grid

by
with

KKpn-15asmooth-surfaced

thepseudoreplica

Pt-palladium

alloy

.

VOL. 45 NO. 5

Morphological

the TWAR strains

exhibited

observations

area even byon the distributionof mito-chondria in the host cells infected with all strains
C. pneumoniaeof Chlamydia

the "pear- shaped" mor-

phology with a wide outer-membrane
the air-drying method (Fig. 2b).
Furtheranalysisofrevealed the absence of any mitochon-

drial association with the inclusion membrane 11,14, 18,32)
27,(Fig. 1c, 1d arrowheads). This strongly suggested thatthe absence of the mitochondria-inclusion
associationis common in C. pneumoniae

RBs.
To directly confirm the presence of projections

the surface of C. pneumoniae EBs, purified EBs weredried by the critical point drying method and examined by scanningwere observed on a limited area of the EB surface of
the C. pneumoniae strains18,32) (Fig. 5). The EBs of
KKpn-15 were globular in shape (Fig. 5a), whereas
those of the TWAR strains

inclusions

mic space. The number of projections

is a natural

occurrence

duringRegularand related

the EB envelope

were preparedstructurein the outer

In a preliminary

structuresand inclusion

mem-of the inclusion-bearingcells

at 60 h postinoculation.

on the EBs ofeach of the strains was not determined.

the reproductive

stage27).
To examine(Fig. 5b) had irregularshapes as a result of the presence of a wide periplas-with C. psittacibrane, freeze-replicas

electron microscopy18,32) Projections

althoughassociation

projectionson

multiplication,

mitochondrial

Surface259

pneumoniae

experiment,

membranes
it was found that the

As shown in

Fig. 3, the cleaved faces of the EBs and RBs were
exposedas convex or concave

membrane.

Buttonfaces in the replica

structures(B structures)29)orcraters36) were clearly seen in a limitedarea of theconcave

When thefaces18, 32)(Fig. 3, arrowheads).

inclusion membrane

was replicated,cles grouped together

within several areas were fre-many fine parti-

quently observed on the convex faces18,.32)(Fig. 4,
arrowheads). The morphological properties of the B
structureson the concave faces of in situ chlamydial

bodies and the particles
sion membraneon the convex faces of inclu-

were identicalpreviously4). Therefore,

with those mentioned

the results

obtainedFig.

in the4.

present study strongly suggested the presence of projections on the surface of the C. pneumoniae EBs andstrainFine

3.

Freeze-replica

ies

and

60

h

imagesinclusion

postinoculation.

into

convexseen(arrowheads).

orofmembranes

in

situ
of

Chlamydial
concavefaces.
Bar;

1 ƒÊm.

Many

chlamydial

strainKKpn-15

bodiesareBstructures

imagesinclusion
at
in
The
Bar;

of

membrane

KKpn-15
particles

arrowheads.
outlines.

Fig.

Freeze-replica

chlamydial

60

groups
face

h

in

situ

containing

postinoculation.
are

is

an

ruggedindicated
over

by
the

RB

1 ƒÊm.

bodat
cleaved
are

Fig.

5.
Scanning
electronmicrographof EBs ofChlamydia pneumoniae KKpn-15 (a) and AR-39 (b).
Both micrographs
show hexagonally arrayed projections in a limited area of the surface. Bars; 300 nm.

日 本化 学 療 法 学 会雑 誌

260

EB outer membranesof C. pneumoniae

strains

brokein thepresent

fragments

when theirenveloped

were

The

with 0.5% Sl)

to removethe innermem-brane, whereas those of C. psittaci and C. trachoma-

periodicity
difference

tis strains

pear-shaped

into small
treated

did not. This suggested

membraneof C. pneumoniae'

solution.

Therefore,

reducedfrom 0.5% to 0.125%

membranes
When

that

the outer

ERs is fragile in SDS

the SDS concentration
to isolatewasthe outerof C. pneumoniae.

the

outer

the hexagonally

clearly

seen despite

werearrayednegatively

structures

the difference

were

in chlamydial

species18,32)(Fig. 6). The dimensionsof the structuresappearedexamined.

regularto be similar in all strains
structures

preparations
disintegration

only on the inner surfaceof the outer membrane

shown). These results
those reported

Suchwere also seen in the shadowcast
exposed by(data notwere in good agreement

withpreviously37), indicating that the innersurface of the EB outer membraneof C. pneumoniae

strains is also composed of hexagonallyarrayed struc-

study

are

was measured

summarized

in thisin Tablemanner.

1. The averageof all strains
was very similar despite
in morphology,
i. e., the roundindicate

inner

layer

phologicalbranesmembranes

stained,results

results
the

MAY 1997

profile,in thinthat

the regularstructurecomposingof the EB outermembrane

is a mor-

propertyof themembranes

sections.

theorcommongenus

to theChlamydia,of the TWARThese

ER outeralthoughEBs showmem-

the outer

peculiar

profilesin thin sections.
Immunolabelingty of theof LPS

I.PS

When

on

EBs andimmunogold-labelingwith

was carriedout, the surface

dial

was heavily

strainstheanti-LPS

the LPS was located
The

IDEIA

kit

MAb

of the Elis of all chlamy-

labeledwith protein

particles(Fig. 8). This finding
indicated
immunogold-labelingmethod was accurate

test

diversi-of C. pneumoniae

A-goldthat theand thaton the EB surface.

chlamydia

is designed(DAKO

to detect

Diagnostics

Co.)26.

C. trachomatis

LPS,tures.
A computer
determine

Fourierthe spacing

transformof the hexagonal

Fig. 7 shows an example.
transform,

the fragment

was carriedout18)tostructure.

As a result of the Fourierof the KKpn-15

outer mem-brane shown in Fig. 6a yielded the diffraction
Fig. 7a. The spots show threedifferent

spots instructuralunits of about 176, 90 and 50A, respectively. When
reconstruction by the inverse Fourier transform was
performed
hexagonal

for the innermost spots (176A), a clear
pattern was obtained (Fig. 7b), and theperiodicity in the outer membraneof all strains

usedFig. 7.

Image

processingof thehexagonal

structures

Table

1.

Comparisonof periodicity

in chlamydial

speciesFig.6.NegativelystainedEBoutermembraneof
Chlamydia
pneumoniae
KKpn‑15
strain .b,higher
magni fication of the micrographin a .The hexagonal
structures are seen throughout
the outer membrane.
Bar; 200nm.

inthe outermembraneof strain
KKpn-15
EB. a,
Diffraction
spots obtainedby Fouriertransformationfrom the original
micrographshown
in Fig. 6b; b,
Image
reconstituted
by inverse
Fouriertransformation from the six spots of the diffraction
showninFig. 7a.

Periodicity

measuredfrom centerto centerby Fouriertransform

VOL. 45 NO. 5

Morphological

Fig.

8.

analysis

Immunogold-labelingof Chlamydia

on thesurface261

pneumoniae

of EBsof theChlamydia

pneumoniea
KKpn-15(a) and TW-183(b) strains.
The samples
weretreatedwith monoclonal
antibody
directed
to Chlamydia
psittaci
LPS,
and thenlabeledwith protein
A-goldparticles. Intact
EBs and EB
envelopes
are heavily
labeledwith the protein
A-gold particles.
Bars;
500 nm.

Table

2.

Diversityof Chlamydia

intensity

to the IDEIA

pneumoniae
test

strains

in reaction

the basis of their properties,

kita)

C. pneumoniae-specificiodine,were foundincluding reactivity

withMAbs and stainability

withto have a narrow

periplasmic

space in sections and shadowcast preparations11,14,18,32)
Carter et al.34)demonstrated

that the EB morphologyof C. pneumoniae IOL-207 strain was also round in
thin sections and indistinguishable
from that of C.
trachomatis

and C. psittaci EBs. Similarly,

al.17) demonstrateda)

The numbers

which

is

antigen,C.

of EBs are shown

a

major

and

of

the

C.

correlation

and

EB

moniaestrainsorquantity

structurehas

proposed.

been

results

EBs

IDEIA

kit26).

aparameter,theintensities.

reaction

intensity

However,itthedifferent

resultedfromdifferences

in

the

EB

although

on
of

the

reactivities

wassurface,antigenic

notepitopequalithe
in

LPS

was

morphology

ofcompared

psittaci
"pear

with

Cal

10.

-shaped"sections35)

The, withwhereasC.

thatpneumoniaeoftogetherstudy,

gest that the "pear-shape" profiles of EBs in thin sections are not common among C. pneumoniae strains,
is not valid as a mor-

phological criterion for identifying C. pneumoniae
species.
Based on a series of electron-microscopic studies on
C. psittaci

EBs, Matsumoto

proposeda possible dia-structures4). In a scanning
Gregoryelectron microscopy study ,et al.39) found theet al.38) and Matsumoto

C.

TWAR

TWAR

trachomatis
EBs

were

a largeperiplasmic

those

of

C.

ning electron microscopy showed projections

strains
L2

and

C.

shown

to

be

space,

trachomatis

in

thin

and

C.

on TWAR EBs but on KKpn-1,

KKpn-15,

cal

were

difference

study.

However,strains,which

observedtowasconfirmed

the
were

EBs

be

round.

This

again
of

recognizedinstructures 29)or craters36) on the concave face of EBs in
freeze replicas

strongly suggests that the projections

KKpn-1•`18
asC.

of the genus Chlamydia ,thereby supporting the proposition by Gregory et al .38)
that the projections are a phenotypic marker for rec-morphologithepresent

and

YK-41

pneumoniae

not only
KKpn-16 ,and YK-41 EBs as well. Moreover, the presence of B

are common to all members

psittaciwith thestrongly sug-presence of projections on the EBs of many strains of
C. trachomatis and C. psittaci, and in our study , scan-

Discussion
The

These results,
in the present

gram showing the projections grouped together within a limited surface area and their related internalof C. pneuthe

strain.

obtained

Popov etEBs of C. pneumoniae

and thus that the pear-shapenumberdifferent

2).

and

particleasbetween(TableC. psittaci

the

with

found

of LPSchemicalofwithtestedatwaswhether

LPS

positively

morphology33)

clarifiedty

waspneumoniae

occurredNo

reactskit

Kajaani-6(genus-common)

the

also
IDEIA

purifiedreactionorder

genus-specificconsequently

pneumoniae

Whenin increasing

round

on

ognizing members of the genus Chlamydia.

MAY

日本 化 学 療 法 学 会 雑 誌

262

Matsumotoisolated

examined

C.psittaci

from infected

freeze-replica

techniques

hexagonallyarrayedthe inclusions

Cal 10 inclusions

cells by thin sectioning
and concluded

particles

reported

were projections

the presence

directlyof identicalconvex face of in situ inclusions
and C.psittaci

straine.Inor vesicles

the

would

that

on the convex face ofconnecting

the RBs to the host cytoplasm.Matsumoto

alsoparticles

membrane.If

andon theof C.trachomatis

L2

the present study,thepar-this were not so,aderivedfrombe regularly

conversionof the RB outer

duringmaturationthestrains,because

their

outer

the otherstrains

also possess projections

connection

betweenis,there-

and that the direct

RBs and inclusion

membranes

with the projections is a common phenomenon
multiplication

duringstructure

RB outermembraneMatsumoto examined the outer membranes of C.
psittaci EBs and reported that the dimensions of thehexagonal structures had a periodicity of 167A by an
optical transformation method using laser beams.
However,in the present study, the periodicity in the
Cal 10 outer membranes was estimated to be 175A by
the Fourier transform in the computer system.This
difference (approximately 5%) appeared to be due tothe difference in methods of measurement
andseemed unavoidable.It was noteworthy that the EBsof the C.pneumoniae strains examined possessed
hexagonal structures in the inner surface of the outermembrane and that no marked difference in periodicity was recognized between the round-shaped EBs(KKpn-1,KKpn-15,KKpn-16,IOL-207,Kajaani-6,
YK-14) and the pear-shaped EBs (TW-183,AR-39
and AR-388).Based on these findings,it is very likely that the hexagonal structure composing the inner
layer of the EB outer membrane is a morphologicalThisof thebetweenthe presence

rigidityof themembranes
formedandobtained

defective

observations
al strainsin the

from

with

round

be diminisheda correlation

structures

bodies.In

and theaddition,outerabnormal,giant

hexagonalRBs

structure42).Routineof manychlamydi-

EBs,suchas C.psittacithat

RB outera largein sizeinto

a smallwithphenomenon

EB outerthe results
that

the

associationisin C.pneumoniae

species.
C.pneumoniae
specieswas

eralclassified

in 1989as a newon the basis of EB morphology,serology,andDNA analysisof the TWAR

strains1).Since

then,sev-

C.pneumoniae

strains,including

our isolates,

have been establishedfrom patientswith respiratory

diseases.It

is noteworthy

differencein EB morphology,only

been

that

in spite of the striking
one serotype

identified1).Althoughhumansthought

to be the natural

reservor

similarchlamydiae

been isolated

and

koala

varietyhave

bear44).Theseof reactivityof many

pneumoniae

serotypes

that

Microscope

Y,Department
M,Division

Departmentof Medicine,Kawasaki

ed by Project Research#6-506

GrantsJ T, Kuo

Chlamydia

pneumoniae

S

Diseases,

Medical School#3-507,#4-504,#
Medical School

Grant (04771177) from theand Culture,Japan.

Literature
Grayston

T andwork was support-

from Kawasaki

Ministry of Education,Science

TWAR.

C.

of Microbiology,andof Respiratory

and a Science ResearchstraindifferentCenter,Ohmori

Nakabayashi

1)isolateswish to thank Uehira K,Suda

K,Electron

andwith theexist.

and Tanaka

5-503

been

from a horse43)

C.pneumoniae

kit led us to speculate

The authors

have

has

of C.pneumoniae,

findings,together

in the IDEIA

and C.

membranedetected.

is likely

for their technical assistance.Thisof penicillin41) were also fragile

on the multiplication

trachomatis,suggest
mayof hexagonalchlamydial

in the presence

suggestedweremitochondria/inclusion

YamaneManire37)

within size of theusedagreement

absencepear-shaped
profiles.On
the basisstudy of EB and RB of C.psittaci
andstrainswith the inclusion-con-strains

is in good

Acknowledgments

Matsumotoassociateda commonwith ain size of the RB

in thereduction

previously27).Therefore,it

propertycommon to the genus Chlamydia,although
the outermembranesof the TWAR EBs showof a comparative
Cal 10 strain,

and thereportedalso

tightly

as C.pneumoniae
TWAR.
between the formationof theC.pneumoniae
findingoccur

is still unclear.

No mitochondria
tainingof the genus Chlamydia.

reductionis incomplete

hexagonal

shouldled us to speculate,on

thispear-shapedEBs,suchThus,therelationshipstrains.It

the RBs of C.pneumoniae

the
simi-

KKpn-1—KKpn-18

EBs are enclosed

hand,that

membraneduringmaturation.A

membranemembrane.This

cated inclusions of C.pneumoniaethat

membranesof theouter

likelyof fragments

seen in the inclusion

lar reduction

round

numberRB outerfrom RB to EB during

ticles were again seen on the convex face of the replifore,very

the

1997

Cited
C-C,

Campbell

L A, et Ed.:

sp. nov. for Chlamydia

Int J Syst Bacteriol

39: 88-90,

sp.
1989

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Evidence
origin.

の形態学的解析明2)・ 副 島

林 造3)・ 岸 本

芳 人1)・ 松 島

寿 男1)

敏 春1)
微 生物 学教 室

か らKKpn‑18とに 新 種 と な っ た ク ラ ミ ジ ア で あ り,従性 呼 吸 器 感 染 症 患 者 の 咽 頭 ス ワ ブ か ら 計18株

命 名)。 今 回 こ れ ら 臨 床 分 離18株

TW‑183,AR‑39,AR‑388(以比 較 検 討 し た 。TWAR株

を 中 心 に 超 微 構 造 を,既traehomatisL2/434/Bu株の 基 本 小 体(elementary

梨 状 を 呈 し,C.pneumoniaeを

spaceをた こ と か ら,EBの広 いperiplasmic

spaceばC.pneumoniaeの

ラ ミ ジ ア 独 特 の 表 面 突 起(surface

様SPを

らHSに

*

projection;SP)およ びB構

interactionの

のHSと

24th

Ave.N‑E.#334,

形 成 さ れ,脆音 波 処 理 ・界 面 活 性 剤(SDS)処Seattle,造,封Washington

入 体 膜 上 のSP

外 膜 内 面 はhexagonal

弱 な 網 様 体(reticulate

剛 性 の 問 の 相 関 性 が 強 く示 唆 さ れ て い る 。TWAR株

相 違 が み ら れ る と 予 想 さ れ た が,い絡 先):4717

円形 であっあ る こ と が 示 唆 さ れ た 。EBず れ の 株 に お い て もHSが98105,USA

body;RB)外

膜 に

のEBが

西洋梨

理 に 対 す る 脆 弱 性 ・感 受 性 が 異 な る こ と か
認 め ら れ,そ

岡山県倉敷市松島577
現 住 所(連様形 態 学 的特 徴 とは い え な い こ とが判 明

株 間 で有 意 差 は な か っ た。

**

回対

基 本 形 態 に 差 が な い こ と が 判 明 し た 。 し た が っ てC.

介 す るhost‑Chlamydia

な 規 則 的 構 造(hexagonalstructure;HS)で状 を 呈 し て い る こ と や,超ともつ 西 洋と し て 挙 げ ら れ て い る 。 し か し,今の 剛 性 は 外 膜 に 依 存 し て い る と 考 え ら れ て お り,C.psittaci,C.trachomatisのな い こ と か ら,こBo/E58株
spaceを有 し て い た 。 ま た 同 様 に10L‑207,Kajaani‑6,YK‑41株EBも集 団 が 用 い た ク ラ ミ ジ ア 全 株 に 認 め ら れ,EBの
psittaci,C.trachomatis同pmrum

いperiplasmic

円 形 でC.psittaci,C.traehomatis,C.pecorumのEB同に 狭 いperiplasmic

はHSが存 のC.pneumeniae
psittaci

な ら び にCんlmツdia

body;EB)は,広新 種 と し た ク ラ イ テ リ ア の1つ

象 と し たKKpn‑1〜KKpn‑18株EBはし た 。 し か し,ク来その分離培 養は困難 とさの 分 離 に 成 功 し た(KKpn‑1

上TWAR株),10L‑207,Kajaani‑6,YK‑41株,Chlamydia

Frt‑Hu/Cal10株,Chlamydia

forJ

Kaltenboeck B, Kousoulas K G, Storz J: Structures
and allelic diversity and relationships among the
major outer membrane protein (ompA) genes of thefour chlamydial species. J Bacteriol 175: 487-502,
1993

川 崎 医 科 大 学 呼 吸 器 内 科*,2)同pneumoniaeは1989年

theorganisms

3)川崎 医療福祉 大学 医療福 祉学科

れ て い た 。 最 近 我 々 は,急

T,

1986

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