FEMS MicrobiologyLetters 112 (1993) 141-146
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
141
FEMSLE 05586
Brucella group 3 outer membrane proteins contain
a heat-modifiable protein
C a r l o s G a m a z o a, A n a I. V i t a s a, I g n a c i o M o r i y 6 n ~, I g n a e i o L 6 p e z - G o f i i a a n d R a m 6 n D i a z b
a Departamento de Microbiologla, Facultad de Medieina, and b Departamento de Microbiologfa, Cllnica Universitaria,
Universidad de Navarra, Pamplona, Spain
(Received 10 May 1993; revision received 28 May 1993; accepted 2 June 1993)
Abstract: Brucella melitensis and B. ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular
mass upshift from 25 kDa to 30 kDa. A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which
was also blocked by exposure of the protein to Zwittergent 314. The B. melitensis heat-modifiable protein and Escherichia coli
OmpA shared antigenic determinants. These data indicate that the Brucella group 3 outer membrane proteins belonged to the
OmpA-familyof proteins.
Key words: Brucella; OmpA; Outer membrane blebs
Introduction
It has been shown by sequential SarkosylZwittergent extraction that the outer m e m b r a n e
(OM) of Brucella contains three major groups of
O M proteins, each one appearing in S D S - P A G E
as a cluster of polypeptides of close apparent
molecular mass [1,2]. The characterization of the
Brucella O M proteins of groups 2 and 3 has been
achieved in part. G r o u p 2 has been shown to
have porin activity [3], and the aminoacid composition of the cluster of polypeptides included in
group 3 [2] suggests that they could be the Bru-
Correspondence to: C. Gamazo, Departamento de Microbiologla, Facultad de Medicina, Universidad de Navarra, Aptdo.
273, 31080, Pamplona, Spain.
counterparts of Escherichia coli OmpA.
However, O m p A is characterized by the fact that,
in contrast to most proteins, its electrophoretic
mobility decreases upon incubation at 100°C in
SDS, and such an upshift in apparent molecular
mass has not been observed previously in Brucelia group 3.
It has been shown that exponentially growing
B. melitensis and B. ovis spontaneously release
O M blebs enriched in group 3 O M proteins [4,5].
We report here that a fraction of the main
polypeptide in O M blebs is a heat-modifiable
protein sharing antigenic determinants with
O m p A . We also report that the fraction not displaying the apparent molecular mass upshift is
tightly bound to LPS and that the use of Zwittergents in the extraction of Brucella O M proteins
precludes the observation of the heat modifiability of group 3.
celia
142
Materials and Methods
Bacterial strains and growth conditions
B. melitensis 16M (smooth, virulent), B.
melitensis 115 (rough, avirulent), B. ovis (naturally
rough species) strain REO 198, and E. coli K12
W 1485, were grown in flasks containing tryptic
soy broth on a rotary shaker at 37°C. When strain
16M was used, the ceils were killed after growth
by addition of phenol to the broth (0.5% final
concentration) and overnight incubation at room
temperature.
Preparation of OmpA and OM blebs
E. coli K12 OmpA was obtained and purified
by the method of Hindennach and Henning [6].
To obtain the OM blebs of B. melitensis and B.
ovis, the cells of exponentially growing cultures
were removed by centrifugation (15 min at 8 000
× g), and the supernates concentrated by dialysis
under vacuum at 4°C and clarified by filtration
through 0.2 /zm filters. The OM blebs in the
clarified fluid were collected by ultracentrifugation (2 h at 100000 × g , 4°C), resuspended in
distilled water and lyophilized [4,5].
SDS-PAGE
SDS-PAGE was performed in 12.5 or 13.5%
acrylamide slabs with the discontinuous buffer
system of Laemmli [7] and gels stained either
with Coomassie blue [8] or alkaline-silver for proteins [9], or periodate-alkaline-silver for lipopolysaccharide (LPS) [10]. Molecular mass standards
were phosphorylase b (94 kDa), bovine serumalbumin (66.6 kDa), E. coli OmpF (38 kDa) prepared by the method of Rosenbusch [11], carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14.3 kDa). Densitometry of Coomassie blue stained gels was
performed at 450 nm in a Beckman R 115 gel
scanner.
Antisera and immunoblotting procedure
Antibodies to B. melitensis 115 OM blebs were
raised in rabbits by injection of five intramuscular
doses of 600/zg in Freund's incomplete adjuvant,
administered at weekly intervals. Immunoblotting
was carried out as described previously [5] with
peroxidase-conjugated goat anti-rabbit IgG
(Nordic) and 4-chloro,l-naphthol as substrate.
Proteolytic treatment
B. melitensis 16M OM blebs at t0 mg/ml in
Tris. HCI 40 mM, pH 7.5 were digested with
Tritirachium album proteinase K (MerckSchuchardt, F.R.G.) (0.5 mg/ml) for 6 h at 37°C,
and the products analyzed by SDS-PAGE. As a
control, blebs were disrupted and denatured by
boiling in the same buffer supplemented with
0.5% SDS and digested and analyzed as above.
Results
Figure 1 summarizes the experiments designed
to test the temperature-dependent molecular
mass shift of group 3 proteins. It can be seen
that, when the samples were incubated in the
SDS sample buffer at room temperature (Fig. 1,
lane 2) only a single major band (25 kDa) followed by a smear was observed in 13. melitensis
st
m
1
2
3
~ qllmWmllll ~
94K-i
eTK-g
38K30K-
•..
21 K 14K-
Mm
Fig. 1. S D S - P A G E analysis of B. melitensis 16M O M blebs
after incubation in SDS at 100°C (1), at room temperature (2)
or in 0.3% Zwittergent 314 first and then in SDS at 100°C (3).
Coomassie blue stain was used.
143
16M O M blebs. In contrast, when this material
was analyzed after brief incubation in the SDS
sample buffer at 100°C (Fig. 1, lane 1) two distinct bands of apparent molecular mass 25 k D a
and 30 k D a were observed. Moreover, densitometric scanning showed that the decrease in protein content of the 25 k D a band correlated with
the amount of protein in the 30 kDa band (not
shown)• On the other hand, when the O M blebs
from B. melitensis were preincubated in Zwittergent 314 (10 mg b l e b s / m l of Zwittergent 0.3% in
Tris • HC1 10 mM, p H 7.5, during 20 min at room
temperature) before treatment in SDS at 100°C, a
smear or group of three fuzzy bands clustering
around 25 kDa was observed (Fig. 1, lane 3).
1
2
i~ii!!!!~iiiiiil
!iii!iiiiiii~iiiiili
25K-
(Jmp A
1 2
1
Q
Fig. 3. S D S - P A G E analysis of B. melitensis 115 (lane 1) and
16M (lane 2) O M blebs stained with periodate-silver stain to
reveal LPS. The smear in lane 2 is due to the presence of
S-LPS. Lipid A is stained in the bottom of 1 and 2.
k
38 K
30 K -
--
A
B
Fig. 2. (A) SDS-PAGE analysis of E. coli OmpA after incubation in SDS at 100°C (1), or at room temperature (2).
Coomassie blue stain was used• (B) Western blot analysis of
OmpA (as in A1) revealed with rabbit antiserum to B.
melitensis 115 OM blebs. The band at the top of the gel
corresponds to a marker included to show the position of the
lane in the nitrocellulose paper.
Similar results were observed with B. melitensis
115 and B. ovis R E O 198 O M blebs (not shown)•
Purified O m p A from E. coli was analyzed
with O M blebs antiserum from B. melitensis 115
by immunoblotting. The result (Fig. 2B) showed
the reaction of the O m p A with these antibodies.
A reaction of the R-LPS in such O m p A preparation was also observed in the running front of the
gel (Fig. 2B).
The preceding results showed that, in contrast
to the O m p A controls (Fig. 2A), only a fraction of
the protein appearing in the 25 kDa band underwent the 30 k D a shift after incubation at 100°C
(Fig. 1, lane 2 vs. 1). Schewitzer e t al. [!2] have
shown that denatured O m p A shifts back to a
lower apparent ('native') molecular mass upon
incubation with LPS; for this reason the presence
on the Brucella group 3 proteins of tightly bound
LPS was examined. With blebs of the rough and
smooth strains, the periodate silver method
144
1
2 3
30 K25 K-
Fig. 4. SDS-PAGE analysis and protein silver stain of B.
melitensis 16M OM blebs (lane 1), of the same material
treated with proteinase K before (lane 2) and after (lane 3)
boiling in SDS. The band of the proteinase K is marked with
an asterisk.
stained the smear characteristic of the rough and
smooth LPS and the 25 kDa band (Fig. 3). Since,
the 30 kDa band was not stained (Fig. 3), this
result strongly suggests that LPS remained tightly
bound to the 25 kDa band. This interpretation is
consistent with the result of the proteolysis experiments in which it was observed that the 25 kDa
band but not the 30 kDa band was resistant to
the hydrophobic proteinase K in intact blebs (Fig.
4, lane 2). The resistance of the 25 kDa band to
proteinase K was not intrinsic, since disruption of
the OM blebs in SDS at 100°C made the proteolytic treatment fully effective (Fig. 4, lane 3).
Discussion
The results presented here demonstrate that
Brucella OM contains a protein sharing with E.
coli OmpA heat modifiability. The upshifl in apparent molecular mass of OmpA is due to the
unusually high 3-structure content of this protein
that makes the native OmpA bind amounts of
SDS larger than those bound by the SDS-denatured form [13]. Due to the overall similarity in
amino acid composition between Brucella group
3 and OmpA [2], this same explanation can be
advanced for the heat modifiability of the
polypeptide present in Brucella OM blebs. This is
also supported by the cross-reactivity detected by
immunoblot between OmpA and the Brucella
OM blebs protein. Despite this, the apparent
molecular mass in SDS-PAGE of the Brucella
polypeptide was clearly lower than that of OmpA.
Based on the aminoacid composition presented
by Verstreate et al. [2], a minimal molecular mass
of 37.7 kDa can be calculated, which is very close
to the 37.0 kDa of OmpA. The simplest explanation for the higher SDS-PAGE mobility of the
Brucella polypeptide group 3 with respect to
OmpA is that it binds more SDS in its not fully
denatured form. This increased binding of SDS is
consistent with the comparatively higher hydrophobicity of the Brucella cell envelopes previously postulated [1].
The fact that the molecular mass upshift could
not be observed after exposure of the protein to
Zwittergent 314 explains why this feature had not
been observed before. It has to be emphasized
that methods milder than the Sarkosyl-Zwittergent extraction of OM proteins from cell envelopes (i.e. spheroplasting and use of non-ionic
detergents) are of no use in BruceUa [1].
Only part of the protein in the 25 kDa band
exhibited the molecular mass upshift upon boiling. This could be due to the presence of two
different polypeptides with the same electrophoretic mobility before exposure to SDS at
100°C, or to a single polypeptide, a fraction of
which is bound to a hydrophobic molecule that
would prevent the binding of SDS and the subsequent molecular mass upshift. The detection of
tightly bound LPS on the 25 kDa band but not on
the 30 kDa band and the respective resistance
and sensitivity to the hydrophobic proteinase K in
intact blebs supports the second hypothesis.
Moreover, both features stress the similarity with
145
OmpA, since this protein interacts strongly with
LPS and is partially resistant to proteases in
intact membranes [12].
OmpA contributes to the maintenance of OM
integrity [14], although it has been found that it
also produces apparently nonspecific diffusion
channels [15]. In all likelihood, because of this
important role, heat-modifiable proteins are conserved in many Gram-negative species, including
several E n t e r o b a c t e r i a c e a e , Moraxella, Neisseria,
P s e u d o m o n a s , Vibrio , Pasteurella , A c tinobacillus ,
and H a e m o p h i l u s , recently described [16-20]. Be-
her et al. [16] have proposed that heat-modifiable
proteins have been highly conserved in the evolution of Gram-negative bacteria. Because Brucella
is not taxonomically related to the genera mentioned above, the results of Verstreate et al. [2]
and those reported here give further support to
such a hypothesis.
Acknowledgements
This research was supported in part by the
Spanish CICYT (PA86-0376-C02-02), and DGICYT (PM92-0140-C02-02). A.I. Vitas is a Fellow
of the 'Gobierno de Navarra'.
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