A Structural and Immunological Comparison of Rickettsial HSP60 Antigens with Those of Other Species,...

A Structural and Immunological Comparison of Rickettsial HSP60

Antigens with Those of Other Speciesayb GREGORY A. DASCH,',d WEI-ME1 CHING,'

PETER Y. KIM,' HUONG PHAM,' CHARLES K. STOVER,'.' EDWIN V. OAKS,' MICHAEL E. DOBSON,' AND

EMILIO WEISS' 'Rickettsia1 Diseases Division

Naval Medical Research Institute Bethesda, Maryland 20814-5055

and 'Department of Enteric Infections

Walter Reed Army Institute of Research Washington, D.C. 20307

INTRODUCTION

At a cellular level, the physiological responses of plants, animals, and both eukaryotic and prokaryotic microorganisms to a rapid increase in temperature (heat shock) are remarkably universal.' Generally, a select subset of proteins is produced in greatly increased amounts while the majority of other proteins de- cline in content. The proteins which are hyperinduced are known as heat-shock proteins (HSPS).~ Heat-shock proteins are thought to have an important role in protection of the cell against heat as well as the physiological stresses, including exposure to heavy metal ions or other toxins, ultraviolet light, or ethanol, which also induce synthesis of heat-shock proteins. Seventeen HSP have been identified in Escherichia cok3 Under heat-shock conditions, a number of very different bacteria have been shown to synthesize a set of proteins with molecular weights remarkably similar to those of the HSP initially found in E. ~ o l i . ~ - * Strikingly, by DNA sequence analysis of genes encoding HSP, by comparison of physical properties of the proteins themselves, and, indirectly, by demonstration of conserved antigenic domains with monoclonal and polyclonal antibodies, a number of these proteins have also been shown to be highly conserved between eukaryotes and prokaryotes. Such conservation has most lnotably been observed for proteins related to the E. coli DnaK protein {equivalent to the eukaryotic HSP70 family of

'This work was supported in part by the Naval Medical Research and Develop- ment Command, Department of the Navy, work units 3M161102.BS13.AK.lll and 3M162770.A870.AN. 121.

$he opinions and statements contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.

dAddress correspondence to Dr. Gregory A. Dasch, Naval Medical Research Institute, Bethesda, Maryland 20814-5055.

fPresent address: Molecular Vaccines, Inc., 19 Firstfield Road, Gaithersburg, MD 20878. 352

DASCH el al.: RICKETTSIAL HSP60 ANTIGENS 353

70-78-kDa proteins), the GroEL protein (equivalent to the eukaryotic HSP60 family of 58-65-kDa proteins), and the HtpG (or C62.5) protein (equivalent to the eukaryotic HSP90 family of 83-108-kDa protein^).^,^ In addition, although a related E. coli protein has not been described, Mycobacterium leprae was shown recently to have a 19-kDa protein homologous to the small HSP family (16-40 kDa) of e u k a r y ~ t e s . ~ Finally, although related eukaryotic sequences have not yet been described, genes with significant DNA sequence homology to that of the E. coli GroES HSP have been described for prokaryotes as diverse as Mycobacte- rium tuberculosis, Chlamydia psittaci, Rickettsia tsutsugamushi, and Coxiella burnetii. It may be surmised that some of the other 13 E. coli HSPs will prove to he highly conserved among most prokaryotes, if not also in eukaryotic organ- isms.

Although the role of the HSP in responses to stressful environmental conditions has stimulated their investigation, it has become clear that many HSP also have important roles in normal cell physiology. A number of the E. coli HSP were originally discovered b‘ecause mutations in those genes of the E. coli host inter- fered with the growth or morphogenesis of bacteriophage.14 The finding that both HSP60 and HSP70 molecules participated in a wide range of assembly processes in both prokaryotic and eukaryotic cells lead to their description as “molecular chaperones.”15 Molecular chaperones interact with other proteins to prevent their aggregation and precipitation until their proper assembly can be effected but are not components of the final structure. This concept has been both broadened and extended to include for molecular chaperones a catalytic role as specific “protein unfoldases” that hydrolyze ATP during the assembly of oligomeric proteins and a role in maintaining an unfolded transition state in proteins, allowing them to be translocated across membranes. 16-18 In this context, the increase in synthesis of these proteins under conditions of heat shock or other stress may be seen as a mechanism to facilitate proper assembly or translocation of proteins under conditions which would otherwise lead to a great increase in the rate of protein dena- turation and a resulting loss of cell function.

A number of HSP have been shown to be essential (GroEL, GroES, rpoD) or conditionally essential (DnaK, DnaJ) for growth of E. coli under a variety of condition^.'^ The central role of the E. coli GroEL (HSP60) and GroES proteins is apparent in the pleiotropic effects of mutations in their genes on translocation of some proteins,20 on phage morphogenesi~,’~ on folding of both oligomeric and monomeric proteins ?1*22 and on proteolysis .23 The strong conservation of functions of the HSP60s is obvious in the heat inducibility and oligomeric structure of the Tetrahymena mitochondrial 58-kDa protein,24 the essential role of the yeast HSP60 protein in assembly of proteins imported into the m i t o c h ~ n d r i o n , ~ ~ ~ ~ ~ and the selective mutagenesis of the mammalian mitochondrial protein P1 in response to microtubule inhibitor^.^^,^^ In chloroplasts, HSP60 functions as a binding protein which catalyzes the assembly of the oligomeric enzyme ribulose bisphosphate carboxylaseloxygenase, the key enzyme in the photosynthetic fixation of COz in plant^.^^,^^ No cytoplasmic homologues of HSP60s have been described in plants, animals, or lower eukaryotes. DNA sequence homologies of these proteins are in agreement with the endosymbiotic theory that mitochondria originated from purple bacteria and chloroplasts from ~yanobacteria.~’

Although the existence of cross-reactive polysaccharide antigens has been known for many years, it was only in 1975 that K a i j ~ e r ~ ~ and H ~ i b y ~ ~ (antigen 10 of Pseudomonas) each described the widespread existence of a common major protein antigen in numerous bacterial genera. By means of crossed immunoelectrophoretic techniques, Hoiby and his c o - ~ o r k e r s ~ ~ , ~ ~ extended these findings to

354 ANNALS NEW YORK ACADEMY OF SCIENCES

many other genera, including some gram-positive bacteria. In 1980, Sompolinsky et ~ 1 . ~ ~ 9 ~ ~ isolated the antigen from Pseudomonas aeruginosa and demonstrated that it had an apparent monomeric molecular weight of 62 kDa by SDS-polyacrylamide gel electrophoresis but that it formed aggregates of 600-900 kDa. How- ever, it was not until 1987 that three groups of investigator^^*-^^ characterizing the genes encoding the 65-kDa protein antigens of Mycobacterium species realized that this 65-kDa protein was related to the E. coli GroEL protein and that each protein belonged to the ubiquitous common protein antigen family. The two proteins were highly immunoreactive molecules; they contained broadly reactive epitopes, as well as genus and species-specific epitopes, had similar amino acid compositions, formed oligomers of greater than 240 kDa; and their genes had very extensive regions of sequence homology.

The strong sequence similarity of bacterial HSP60s to the mitochondria1 HSP60s has stimulated considerable speculation about the potential of these antigens to trigger autoimmune responses, particularly rheumatoid a r th r i t i~ .~ I -~~ Al- ternatively, host cell HSPs may have important roles in immune surveillance and

Both T cell recognition of and antibody binding to epitopes on the 65-kDa protein of Mycobacterium leprae and M. tuberculosis have been in- vestigated extensively .47-50 The great antibody cross-reactivity among the bacterial common antigens has long stifled interest in these proteins for diagnostic purposes and has complicated contemporary efforts to employ them as protective vaccine antigens. However, monoclonal antibody and DNA sequence analyses of the HSP60s of bacteria have suggested that genus-specific, and occasionally species-specific, monoclonal antibodies can be obtained which recognize the variant regions of these protein^.^'-'^

The HSP60s of two Rickettsiaceae, Rickettsia tsutsugamushi and Coxiella burnetii, have been cloned and sequenced. 12~13 In addition, a number of rickettsial HSP60s have been purified and partially ~haracter ized.~~ Immune responses to these proteins have also been examined with both polyclonal and monoclonal a n t i b o d i e ~ . ' ~ . ~ ~ , ~ ~ Here we review these findings and speculate on the importance of the host immune responses to the rickettsial HSP60s during rickettsial infection and the function of these proteins in rickettsial physiology.

HSP60 ISOLATION AND CHEMICAL CHARACTERIZATION

Since all Rickettsiaceae except Rochalimaea are obligate intracellular para- sites, it is difficult and expensive to produce sufficient mass to isolate and chemi- cally characterize individual proteins from these species. For investigating the properties of the rickettsial HSP60s, we felt that prior experience in characterizing the Rochalimaea proteins would be valuable, particularly since they are the closest free-living relatives known for the rickettsiae. Moreover, we had previously noted the presence of a major protein band of 60 kDa on SDS-polyacrylamide gels that was present in high concentration in the wash fluid of cell enve- lopes prepared from Rochalimaea quintana and R . vinsonii following French pressure-cell disruption of the organism^.^* Rochalimaea whole cells analyzed by Western blotting with polyclonal sera obtained from rabbits immunized with typhus group rickettsiae, Proteus, or Legionella often exhibited strong reactivity with a protein of this molecular weight. However, occasionally even normal rabbit or guinea pig sera also reacted with this band. In addition, similar molecular weight proteins were prominent in the SDS-polyacrylamide gel protein profiles of

DASCH et af.: RICKElTSIAL HSP60 ANTIGENS 355

both typhus and spotted fever group rickettsiae, and similar less abundant antigens could also be detected by Western blotting in three Proteus and eight Le- gionella strains. These observations strongly suggested that the 60-kDa protein of each of these species was its common bacterial antigen or HSP60.

To prove this hypothesis, we then applied the procedure described by Pau et aLS6 for the Legionella pneumophila common antigen to the purification of the Rochalimaea 60-kDa proteins (FIG. 1) and to similar proteins from a number of control species. The fractionation of the cells was monitored by dot blotting of crude fractions and by Coomassie blue staining and Western blotting of proteins analyzed by polyacrylamide gel electrophoresis. Yields of 60-kDa protein in each fraction and for each bacterium varied greatly. The wash of the cell envelope fraction generally contained the greatest portion of the 60-kDa antigen, although a significant portion was also present in the cytoplasmic and washed envelope fractions. A similar 60-kDa protein was also obtained in the envelope wash from Escherichia, Shigella, Proteus, Plesiomonas, Aeromonas, Pseudomonas, Rick- ettsia typhi, and R . prowazekii. Each of these proteins was further purified by gel filtration on a Pharmacia Superose-12 column. Some proteins were further purified by ion exchange on a Mono Q column. The native molecular weights of proteins obtained in sufficient quantity and purity were determined on a Dupont GF-450 gel filtration column.

The native GroEL protein of E. coli was shown previously by electron micros- copy to consist of two rings of seven monomers each, one ring stacked above the other, with a nominal molecular mass of 840 kDa.57 A similar symmetry had also been observed for the pea chloroplast protein, which had a molecular mass of 900-950 kDa by gel filtration and equilibrium ~edimentat ion.~~ Values of 665 kDa were obtained for the Pseudomonas HSP60 by gel filtration, while higher values were obtained by electrophoresis and ultracentrifugation (800-900 kDa).37 By HPLC gel filtration, we observed native values of 680-750 kDa for the Profeus, Pseudomonas, Plesiomonas, Shigella, and Escherichia 60-kDa proteins. By HPLC gel filtration, Pau et ~ 1 . ~ ~ observed that the Legionella 60-kDa protein eluted at 650 kDa. However, we found that Aeromonas hydrophila 60-kDa protein eluted in two peaks of 620 and 840 kDa, respectively, while both Rochalimaea proteins had values of only 450 kDa, a value consistent with oligomers of 8 rather than 14 monomers. The Mycobacteriurn bovis HSP60 protein has a molecular weight of 246 kDa by gel filtration suggesting it is a tetramer.58 All of the bacterial species with 60-kDa proteins whose native sizes are compatible with the presence of tetradecamers are gamma-subdivision purple bacteria, while Rochalimaea be- longs to the alpha s u b d i ~ i s i o n . ~ ' , ~ ~ Consequently, the aggregation behavior of HSP6O proteins does not appear to be completely conserved.

To confirm the identity of the Rochaiimaea 60-kDa protein antigens as members of the HSP60 family, the amino acid compositions of R . quintana and R . uinsonii were compared to the compositions deduced from DNA sequences of HSP60 genes (TABLE 1). The compositions of the Rochalimaea proteins were very similar, even though consistent differences existed in the pattern of HSP60 fragments (presumably derived from protease activity during growth or purification) detected in each species by Western blotting. The amino acid compositions of the Rochalimaea 60-kDa proteins were entirely comparable to those of the HSP60 standards, with the exception of the amounts of tyrosine, phenylalanine, and arginine, which were somewhat higher. To provide more definitive identification of the 60-kDa proteins we have purified, automated amino-terminal amino acid sequence analyses were also done (TABLE 2). Three conclusions are apparent from these data. First, each of the proteins had sufficient homology to the E. coli

b

m e 0 * 0 E b

FIGURE 1. A

naly

sis

of s

ubce

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r fra

ctio

ns o

f R

ocha

limae

a vi

nson

ii (V) a

nd R

. qui

ntan

a (Q

) by

SDS-

poly

acry

lam

ide

gel e

lect

roph

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nd

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tern

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tting

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rest

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ular

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ght s

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ards

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ne 1

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ee F

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h pr

essu

re-c

ell

extr

act,

(lane

2)

200,

000

x g

supe

rnat

ant

of

extr

act,

(lane

3)

200,

000

x g

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t of

ext

ract

(ce

ll en

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ract

ion)

, (la

ne 4

) 20

0,00

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ope

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tion,

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) was

hed

cell

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frac

tion.

20

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f pr

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n ex

cept

the

cell

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lope

was

h fr

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ns (1

0 pg)

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e se

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ted

on 1

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els.

(Pa

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e bl

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ile. (

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l B)

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tern

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t de

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ith

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l C) W

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se m

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l ant

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rote

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nd 4

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tain

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rn '

TABLE 1.

Com

paris

on o

f C

ompo

sitio

ns o

f 60

-kD

a H

eat-S

hock

Pro

tein

s Spec

ies"

Roc

ha'.

Sacc

. C

HO

Tr

it.

Myc

ob

a~

t.~

~ C

hlam

. R

icke

tt.

Cox

. Pr

ooer

tv

rsut

su.'2

bu

rnet

ii"

E. c

0P9

leor

. tb

lbo.

D

sitta

ci"

QU

. wi

n.

cere

.26

Cells

28

H. s

aDien

s2'

aest.

29

Am

ino

acid

com

posi

tion

(num

ber o

f re

sidu

es)

Ala

52

Ile

41

Leu

32

Val

66

CY

S 3

Met

10

AS

P 37

G

lu

44

Asn

29

G

ln

20

His

8

Arg

25

LY

S 50

Ph

e 12

TY

7

Trp

0

Pro

12

Ser

26

Thr

32

Tota

l res

idue

s 55

5 M

olec

ular

mas

s 59

.7

GlY

49

(kD

a)

63

56

36

41

61 2 23

34

47

22

14 4 21

46 7 7 0 14

28

26

552

58.3

76

58

33

40

58 3 22

35

46

19

16 1 22

40 7 7 0 15

17

33

548

57.2

73

52

29

54

52 0 9 33

53

16

13 2 21

45 8 7 1 14

21

38

54 1

56.9

71

56

29

55

54 0 7 32

55

16

14 2 22

43 8 7 1 16

18

34

540

56.7

64

44

42

46

44 6 13

31

53

16

15 6 20

49 8 9 0 16

30

32

544

58.1

65

77

32

45

41

N 21

45

64

-

- 5 30

38

13

13

N 20

31

32

57 1

60

69

65

33

45

40

N 13

46

65

-

- 6 33

39

13

14

N 21

27

37

565

60

56

56

42

49

49 5 11

32

51

17

13 2 28

44

14 7 0 23

38

35

572

60.7

60

56

43

51

54 3 17

36

44

21

17 3 22

54 9 7 1 18

25

32

573

61.0

57

56

43

48

57 3 18

37

43

20

17 3 22

53

10 7 1 19

25

34

573

61 .O

69

42

50

44

48 3 6 30

51

20

18 3 21

38 7 8 1 18

30

36

543

57.5

a C

ompl

ete

open

rea

ding

fra

me

of t

he D

NA

seq

uenc

e us

ed f

or a

ll de

term

inat

ions

exc

ept

Roc

halim

aea,

whi

ch w

as b

ased

on

anal

ysis

of

the

prot

ein

and

a m

onom

eric

pro

tein

siz

e of

60

kDa.

For

Roc

halim

aea,

the

Asp

and

Asn

val

ues,

and

Gln

and

Glu

val

ues,

are

com

bine

d as

Asp

and

Glu

, re

spec

tivel

y. S

ourc

es o

f da

ta a

re in

dica

ted

by re

fere

nce

num

bers

. Ric

kett.

tsut

su.,

Ric

ketts

ia t

suts

ugam

ushi

; C

ox. b

urne

tii, C

oxie

lla b

urne

tii; E

. co

li, E

sche

rich

ia c

oli;

Myc

obac

t. le

pr.,

Myc

obar

teri

um le

prae

; M

ycob

act.

tb.l

bo.,

Myc

obac

teri

um tu

berc

ulos

islb

ouis

; C

hlam

. psi

ttaci

, C

hla-

m

ydia

psi

ttaci

; R

ocha

l. q

u.,

Roc

halim

aea

quin

tana

; R

ocha

l. ui

n., R

ocha

limae

a vi

nson

ii; S

acc.

cer

e.,

Sacc

haro

myc

es c

ereu

isia

e; C

HO

cel

ls,

Chi

nese

ham

ster

ova

ry c

ells

; H. s

apie

ns, H

omo

sapi

ens;

Tri

t. ae

st.,

Triti

cum

aes

tiuur

n. N

, no

data

ava

ilabl

e.

TABL

E 2.

Com

paris

on o

f th

e A

min

o-T

erm

inal

Am

ino

Aci

d Se

quen

ces

of P

roka

ryot

ic 6

0-kD

a H

eat-

Shoc

k Pr

otei

ns

Bac

teriu

m

Sour

ce

Sequ

ence

" D

iff.

Esc

heri

chia

col

lz9

DN

A

MA

AK

DV

KF

GN

DA

RV

KM

LR

GV

NV

L

Ples

iom

onas

shi

gello

ides

Pr

otei

n ??

?DV

KF

GN

DA

RV

KM

LR

GV

NV

L

0 Sh

igel

la Jl

exne

ri

Prot

ein

???T

VK

FG

ND

AR

VK

ML

RG

VN

VL

1

Pseu

dom

onas

aer

ugin

osa

Prot

ein

AA

KE

VK

FG

MA

R&

KM

LV

?V

NV

L

5 C

hine

se h

amst

er o

vary

cell

s28

DN

A

-

YA

KD

VK

FG

AD

AR

UM

LQ

GV

DL

L

I H

omo

sapi

ensz

7 D

NA

Y

AK

DV

KF

GA

DA

RU

ML

QG

VD

LL

I

Roc

halim

aea

quin

tana

Pr

otei

n A

AK

EV

KF

Gm

AR

mL

RG

VW

L

8 R

ocha

limae

a ui

nson

ii Pr

otei

n A

AK

EV

KF

Gm

AR

mL

RG

VU

L

8

Ric

ketts

ia t

yphi

Pr

otei

n ?

? K

UK

HQ

?_K

? R

NM

>

I C

hlam

ydia

psi

ttaci

" D

NA

M

AA

KU

KY

NE

DA

R&

KE

KG

VK

TL

11

C

oxie

lla b

urne

tii"

DN

A

MA

AK

VL

KF

SH

EV

LH

AM

SR

GV

BV

L

11

Pisu

m s

ativ

um a

lpha

sub

unit6

1 Pr

otei

n A

AK

DU

FD

QH

SR

UM

QA

GD

12

Pi

sum

sat

ivum

bet

a su

buni

t61

Prot

ein

AK

UF

NK

DA

R.

KL

QN

GV

NE

L

12

Ric

ketts

ia p

row

azek

ii Pr

otei

n ?

? K

UK

HQ

? &A

R _E

? M

L

>6

Myc

obac

teri

um le

prae

" D

NA

M

.AK

TIA

YD

EE

AR

RG

LE

RG

LN

SL

13

M

ycob

acte

rium

tb.

lbov

iss9

D

NA

M

.AK

TIA

YD

EE

AR

RG

LE

RG

LN

AL

13

Sa

ccha

rom

yces

cer

euisi

ae26

D

NA

S

HK

BK

FG

VE

GR

AS

LL

KG

VX

L

13

Ric

ketts

ia t

suts

ugam

ushi

'2

DN

A

M.S

KQ

IVH

GD

QC

RK

K~

GIN

V~

14

Tr

iticu

m a

estiu

uma

DN

A

DA

KE

IAF

DQ

KS

RA

AL

QA

GV

EK

L

15

Syne

choc

occu

s 63

Ol6O

D

NA

M

TA

KR

IIY

NE

NA

RR

AL

EK

GU

L

15

Aer

orno

nas

hydr

ophi

la

Prot

ein

??

GA

GS

GK

VT

FN

GE

IIN

AP

?E

19

A

min

o ac

ids

diff

erin

g fr

om th

ose

foun

d at

the

corr

espo

ndin

g po

sitio

n in

the

E. c

oli

Gro

EL p

rote

in a

re u

nder

lined

and

enu

mer

ated

in

the

diff

eren

ce (

Diff

.) co

lum

n. T

hree

am

ino

acid

s re

mov

ed f

rom

the

P. s

ativ

um b

eta

subu

nit

sequ

ence

wer

e co

unte

d as

mis

mat

ches

. Th

e le

ader

se

quen

ces o

f se

vera

l euk

aryo

tic p

rote

ins

are

not d

ispl

ayed

.

.c 0 E

DASCH ef al.: RICKE’ITSIAL HSPQ ANTIGENS 359

HSP60 to establish its identity as an HSP60, with the exception of the Aeromonas hydrophila 60-kDa protein. The conservation of certain residues, such as the lysines at positions 4 and 7, the phenylalanine at 8, the arginine at 13, the glycine- valine pair at 19-20 and the leucine at 23, are particularly helpful in aligning and comparing sequences. Second, although HSP60s are believed to be among the most conserved proteins known, the amino terminus has undergone considerable alteration in evolution. Although the limited sequence available from this ap- proach only comprises about 4% of the protein, it is interesting that certain clusterings can be deduced from this information. A number of bacteria clustered by 16s rRNA sequence analysis in the gamma and alpha branches of purple b a ~ t e r i a ~ ~ ~ ~ ~ have very similar HSP60 amino-terminal sequences that diverge little from the mitochondrial HSP60 sequences. On the other hand, Coxiella burnetii is a gamma group member, but its HSP60 appears to have diverged to the same extent as Chlamydia HSP60, approaching the divergence seen in the Mycobacte- rium, cyanobacteria (Synechococcus), yeast mitochondrial, and chloroplast HSP60s. The close relationship of Rochalimaea quintana and R . uinsonii is confirmed by the identity and uniqueness of their HSP60 sequences, as was also observed for the R . typhi and R . prowazekii HSP60s, although these data were less complete. Finally, the Rickettsia tsutsugamushi HSP60 sequence is quite divergent from that of all the other sequences, supporting its exclusion from the genus Rickettsia and the likelihood that it represents a distinct lineage of bacteria. These conclusions will be reexamined below by means of epitope analysis with monoclonal antibodies and the very limited set of DNA sequences of cloned HSP genes.

IMMUNOLOGICAL RELATIONSHIPS AMONG HSP60 ANTIGENS

Hindersson et summarized the results of crossed immunoelectrophoretic detection of the bacterial common antigen in 25 different genera of gram-negative, gram-positive, and spirochete bacteria. They suggested that the’ antigen might contain a number of phylogenetically stable epitopes and that normal human immune responses to bacterial flora might elicit antibodies and immune responses against this antigen that could comprise a type of “natural immunity” against pathogens sharing this antigen. The very extensive analyses conducted on both T cell- and antibody-mediated immune responses to the 65-kDa antigens of Myco- bacterium leprae and M . tuberculosis provide some support for at least the first of these ~iews.~’-~O At least 14 spatially separate epitopes have been defined by competition assays among monoclonal antibodies recognizing the HSP6O of M . l e p r ~ e . ~ ’ The reactivity of these antibodies for 23 species of mycobacteria was further determined. One antibody recognized only M. leprae, three recognized all species to varying degrees, while the others recognized at least eight of the species. A number of these antibodies have further been shown to react with a number of genera of Archaebacteria and both gram-positive and gram-negative genera, including Rickettsia r i~ke t t s i i .~~-~O Monospecific serum prepared against the Chlamydia psittaci HSP60 also cross-reacted with 57-60-kDa proteins from E. coli, Salmonella, Neisseria, Coxiella, Rickettsia rickettsii, Borrelia burgdorferi, and Mycobacterium tuberculosis.” More than 80% of the sera from culture- positive or serologically confirmed human infections with Legionella pneumophila contained antibodies against the Legionella HSP60.* The Le- gionella HSP60 was shown to contain Legionella-specific and non-specific epi-


topes recognized by both human and rabbit antisera.65 Helsel et al.S2 found a monoclonal antibody against Legionella HSP60 that recognized 22 species of Legionella but only Acinetobacter IwofJii and Bordetella among 27 other bacterial species. Both conserved and specific epitopes on the HSP60 of Borrelia burgdorferi were recognized by human serum from cases of acrodermatitis chronica atrophicans.%

Monoclonal antibodies against Mycobacterium and those prepared against several species of Rickettsiales were used to demonstrate the existence of both cross-reactive and specific epitopes on the HSP60s of several genera (TABLE 3, FIG. 2).'2353 Although all typhus and spotted fever group rickettsiae appeared to react very similarly with polyclonal sera and most monoclonal antibodies, R . Canada HSP60 and Rochalimaea appeared to lack the particular conserved epitope recognized by Mycobacterium antibody Y 1.2 (FIG. 2A), while a second, more conserved epitope was recognized by an antibody against R . typhi HSP60 (FIG. 2B). Other epitopes that were Ehrlichia-specific and typhus and spotted fever group rickettsiae-specific were also detected. Most interesting, however, were antibodies which detected Ehrlichia, Rickettsia, and Rochalimaea, or did not react with these genera but did detect Wolbachia and other gamma-subdivision purple bacteria such as Escherichia, Legionella, and Proteus. These group- ings were consistent with phylogenetic relationships deduced from 16s rRNA sequencing.62 On the other hand, neither the polyclonal sera nor broadly reactive typhus monoclonal antibodies exhibited much cross-reactivity against the HSP60 of Rickettsia tsutsugamushi.12 The 16s rRNA sequence of scrub typhus is not known, but these data suggest that scrub typhus may be a distinct eubacterial lineage, lying outside the purple bacteria, possibly diverging from this group more than do the Chlamydia, Mycobacterium, or even the spirochete branches.63

STRUCTURAL CONSERVATION OF HSP60 GENES

The complete sequences of the HSP60 genes of only two rickettsiae, Rickett- sia tsutsugamushi and Coxiella burnetii, are known at present.I2J3 These genes share with E. coli, Chlamydia psittaci, and Synechococcus 6301 genes11p29,60 a common organization in an operon consisting of a heat-shock-like promoter region followed by two open reading frames (ORFs), the first ORF with homology to the GroES gene of E. coli and the second with the GroEL gene. The two genes appear to be cotranscribed. The cloned genes of Coxiella appeared to be heat- regulated in E. coli, while those of R . tsutsugamushi were not. In Mycobacte- rium, the two genes are not present in the same operon.I0 Gly-Gly-Met motifs in the carboxyl-terminal region have been observed for each of the known HSP60 sequences except for Chlamydia and for wheat, Triticum aestivum. This hydro- philic domain may function as a membrane anchor, as it appears to follow a hydrophobic, putative transmembrane domain. Hydrophobicity plots of the individual HSP60s are remarkably superimposable. j2 Pairwise comparisons of deduced amino acid sequences indicated homologies of 40-50%. Since many of the non-identical amino acids were conservative replacements, overall HSP60 sequence similarities were approximately 70%. No one region of the protein con- tains the majority of the conserved amino acids; rather, regions of no more than ten amino acids of identical sequence among the various HSP60 are scattered throughout the protein. Similarly, regions showing low similarity in sequence were no greater than ten amino acids in length and were also scattered through the

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2 a

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N

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N

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N

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N

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N

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N

o. 3

N

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N

o. 5

N

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f

2 f

3 -

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* 3

1 1

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limae

a 4

4 4

4 4

4 Ty

phus

, SF

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etts

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3 2

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utsu

gam

ushi

N

N

N

E

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., se

nn.

2 N

N

1

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-

N

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-

-

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0 N

, not

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ome o

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from

Ref

s. 1

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The

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(4).

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s No.

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: G

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.6, V

55-8

D10

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s ric

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s No.

1-5

: TC

30-2

F2.2

, T5-

3H10

.1, T

C30

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8.1,

T5-

1F1.

5,

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. Ehr

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o. 1

): m

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No.

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DASCH el al.: RICKETTSIAL HSP60 ANTIGENS 363

protein sequence, notably at both the carboxyl and amino termini. As might be guessed from the differences among HSP60s in amino acid composition, cysteine and histidine appear rather variable in location, while a number of tyrosine, phenylalanine, proline, arginine, and lysine residues may have important structural roles, as deduced from their invariance in location. As anticipated from the amino-terminal sequence comparisons (TABLE 2), the sequence data suggests that Coxiella burnetii and E. coli HSP60s are relatively closely related, while the Rickettsia tsutsugamushi sequence is rather different from the other known sequences.

In order to compare structural aspects of different HSP60s, Garnier-Os- guthorpe-R~bson~~ predictions of the secondary structure of the five known complete bacterial proteins were plotted (shown in FIG. 3 for R. tsutsugamushi HSP60). Those regions predicted to have a high likelihood of antigenicity were also determined by the algorithm of Jameson and Wolf.68 None of the secondary structure plots was very similar. Several regions of high antigenic index appeared to be conserved (residues 25-60,190-220,350-380,460-480) among the proteins, while the scrub typhus HSP60 had relatively unusual antigenic domains at both the amino and carboxyl ends of the protein. Except for domain 25-60, none of these regions appeared to be much more strongly conserved than other domains. Although two of these domains are in regions of the M . leprae protein sequence where monoclonal antibodies are known to bind, the other predicted antigenic domains do not apparently bind murine an t ibodie~ .~~

DISCUSSION AND CONCLUSIONS

The analysis of stress proteins in pathogenic bacteria is most advanced for the agents of leprosy and tuberculosis. At least three mycobacterial antigens are known to be homologues of well-characterized stress proteins from E. coli or euka ryo te~ .~~ The implications of this observation for other pathogenic bacteria are obvious. In retrospect, it appears logical to expect that immune responses would cause some stress in pathogenic microorganisms and that elevations in the amounts of such stress proteins would be reflected in antibody- and T cell-mediated responses to such proteins. However, as regards the HSP60s of C. burnetii, Rochalimaea, Chlamydia, Ehrlichia, and all three groups in the genus Rickettsia, the apparent constitutive levels of these proteins appear remarkably high. In the absence of any direct information on the temperature regulation of any of these proteins, it may be asked whether the process of growth at their usual temperature of cultivation (34°C) constitutes sufficient inductive stress relative to the ambient environment they would encounter in a host arthropod. Alternatively, intracellu-

4 FIGURE 2. Western blot analysis of bacterial antigens with monoclonal antibodies against the HSP60 proteins. Antigens were separated on 8-16% SDS-polyacrylamide gradient gels. Protein loading: (lanes 1, 2) 5 pg, (lanes 3-10) 40 pg, (lanes 11-20) 20 pg. (Lanes 1, 2) Mycobacterium bouis, M. phlei cell walls; (lanes 3-5) Proteus 0x19, 0x2, and OXK cells; (lanes 6-8) Legionella pneumophila, L. micdadei, and L. bozernanii cells; (lanes 9-10) Rochalimaea quintana, R . uinsonii cells; (lanes 11-20) rickettsiae: R. typhi, R. prowazekii, R. Canada, R. rickettsii, R. conorii, Israeli tick typhus, R . sibirica, R . akari, R . australis, and Thai tick typhus. Blots were developed as in FIGURE 1 but with (Panel A) monoclonal antibody Y1.2, against Mycobacterium leprae 65-kDa protein or (Panel B) monoclonal antibody T5-1F1.5, which recognizes R. typhi HSP60.

DASCH et af.: RICKE’ITSIAL HSP60 ANTIGENS 365

lar growth may constitute significant stress in itself to induce synthesis of these proteins. Synthesis of stress proteins may be particularly essential for adaptive growth in professional phagocytes such as macrophages. Cross-reactive 70-kDa antigens are present in Rochalirnaea and both the typhus and spotted fever group rickettsiae (Dasch, unpublished observations), but their relationship to the DnaK proteins of E. coli and Mycobacteriurn has not been proven.

Structural, immunologic, and DNA sequence information is now available about the HSP60 proteins in Rickettsiaceae and a few other bacteria. Although the broad immunological cross-reactivity of this common bacterial antigen was initially discouraging for its use as a diagnostic antigen, the existence of at least genus-specific epitopes and the distribution of sequence substitutions suggest that this protein has undergone considerable evolution that may be exploited. For example, even with the limited sequences available, there are remarkable paral- lels that can be drawn to phylogenetic relationships deduced previously by 16s rRNA sequencing. If monoclonal antibodies can be selected for binding to phylogenetically important specific amino acid sequence changes, a more rapid method of typing novel bacteria for phylogenetic purposes may be possible than can be achieved by nucleic acid sequencing. At least such HSP60 studies should indepen- dently validate conclusions about phylogeny based on the ribosomal RNA sequences. The relatively large amount of the HSP60 antigen present in pathogenic bacteria may permit new approaches to rapid detection of bacteremia utilizing the cross-reactive epitopes and antigen-detection technologies. Alternatively, poly- merase chain reaction amplification of conserved sequences may also permit development of simple broad methods for enumeration of slowly growing or non- cultivable bacteria.

Although the 65-kDa antigen of Mycobacteriurn has been proposed as a vaccine candidate, this seems inappropriate due to the evidence for its involvement in rheumatoid arthritis and the recent demonstration that the Chlamydia HSP60 antigen may be the cause of ocular hypersensitivity and blindness in trachoma infections.” Similarly, the presence of immune responses to the HSP6O of Bor- relia burgdorferi suggests that autoimmune responses to specific bacterial HSP60 antigens may be significant in the pathology of certain diseases. At present, autoimmunity appears not to be a significant aspect of disease caused by the genus Rickettsia, but it should not be ruled out for Coxiella, particularly in cases of chronic endocarditis. Indeed, even if the rickettsial HSP60 antigens do not elicit autoimmune problems following vaccination, evidence for a protective response against these highly conserved proteins may be difficult to obtain. Despite their conservation, the proteins are clearly immunogenic. Many individuals have a variety of preexisting antibodies to HSP60 proteins and may eliminate vaccines containing these proteins rapidly due to their “natural immunity.” Furthermore, with the rickettsiae, long-lasting protection seems to be more dependent on species-specific antigens. Following infection, these antigens elicit stronger antibody- and cell-mediated responses in man than do the HSP60 antigens.”

The most important aspect of the HSP60 antigens will probably relate to their normal physiological role in the rickettsiae. Discovery of therapeutic agents which affect the function of these proteins might be used in inhibiting rickettsial replication. On the other hand, the likely role of these proteins in promoting proper translocation, folding, and assembly of proteins suggests that cloned rickettsial antigens may be produced more efficiently in E. coli expression systems if, rather than having these processes rely solely on constitutive levels of the E. coli GroEL protein, the appropriate overexpressed rickettsial HSP60 is also present.


ACKNOWLEDGMENTS

We thank David Marana, Walter G. Sewell, Rick Jaffe, Dzung Pham, Janet Miller, and Richard Grays for their technical assistance on different phases of this project.

1. 2.

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A Structural and Immunological Comparison of Rickettsial HSP60 Antigens with Those of Other Species,...

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