FEMS Microbiology Letters 83 (1991) 247-254
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
ADONIS 037810979100444G
247
FEMSLE 04637
Analysis of the molecular mass heterogeneity of the transferrin
receptor in Neisseria meningitidis and commensal Neisseria
C.M. Ferreirds, M.T. Criado, M. Pi nt or and L. F e r r d n
Departamento de Microbiolog[a, Facultad de Farmacia, Uniuersidad de Santiago de Compostela, Santiago de Compostela, Spain
Received 30 May 1991
Revision received 12 July 1991
Accepted 15 July 1991
Key words: Neisseria spp.; Transferrin; Receptor; Iron
1. SUMMARY
Peroxidase-conjugated transferrin was used to
detect transferrin receptors both in intact outer
membrane vesicles (OMVs) from Neisseria
species in a dot blot assay, and in SDS-PAGEseparated OMV proteins after transferring to nitrocellulose membranes. All N. meningitidis
strains produced transferrin receptors after culturing in either iron sufficiency or iron restriction
although expression was higher in the latter case,
whereas only six N. lactamica and two N. sicca
(among 20 commensal species) were able to bind
transferrin. Molecular mass (MM) of the receptors were mainly between 78 kDa and 85 kDa
(87.5% of strains), 12.5% had receptors with MM
close to 70 kDa, and 5% showed receptors with
MM over 85 kDa. Our results confirm the molecular mass heterogeneity of the transferrin receptors in N. meningitidis, completely disagree with
Correspondence to: C.M. Ferreir6s, Departarnento Microbiologla y Parasitologla, Facultad de Farmacia, Universidad de
Santiago de Compostela, 15706 Santiago de Cornpostela,
Spain.
the 'universal' 98 kDa receptor proposed by some
authors, and show a low expression of the receptor in commensal Neisseria.
2. INTRODUCTION
Human biological fluids and mucosa possess
macromolecules such as transferrin and lactoferrin able to avidly bind iron (affinity constant over
1036), leaving free iron reduced to very low concentrations (10-18 M) hampering bacterial growth
[1]. To ensure survival into the human host, microorganisms have developed high-affinity iron
uptake systems to compete for iron with the
above-mentioned macromolecules. The most
common iron-catching mechanism is based on the
synthesis of low molecular mass iron chelators
named siderophores [2]. Nevertheless, in the
genus Neisseria iron acquisition is achieved by a
different approach in which direct bacteria-toprotein contact through specific surface receptors
is involved. Proteins (transferrin or lactoferrin)
are deprived of iron and left outside the microorganism, whereas the iron is internalized [3].
248
Neisserial outer membrane proteins, including
receptors for transferrin and lactoferrin, are induced under iron restriction (FeRP) [3-6], and
the inability to synthesize some of these proteins
is related to deficiencies in the iron-uptake mechanisms. Schryvers and Morris [7] identified a 71kDa outer membrane protein as the transferrin
receptor in one N. meningitidis strain, and in
additional studies demonstrated the existence of
a transferrin receptor complex that included a
98-kDa outer membrane protein, this 98-kDa receptor being present in all the Neisseria strains
they studied [8].
Ala'Aldeen et al. [9] using different experimental approaches and monospecific anti FeRP70 serum (i.e. to the 71-kDa protein described by
Schryvers and Morris [7]), showed the lack of
transferrin binding by this protein, the binding
activity being localized in proteins ranging from
60 kDa to 90 kDa in different strains. Also,
Griffiths et al. [10] and Ferreirds et al. [6] found
molecular masses of the transferrin receptor in
N. meningitidis to be variable between 60 kDa
and 90 kDa. Despite this, a recent review [11]
considered the 98-kDa iron-restricted protein to
be "in all strains and under all forms of iron
limitation" the sole transferrin receptor.
Our aim in this study was to clarify the situation evaluating the molecular heterogeneity of
the transferrin receptor using a high number of
Neisseria strains obtained from different sources
(carrier and invasive).
3. M A T E R I A L S AND M E T H O D S
3.1. Bacterial strains and growth conditions
The strains of the genus Neisseria used in this
study were taken from our collection. Neisseria
meningitidis (carrier strains) and 20 commensal
strains (N. lactamica and N. sicca) were isolated
from the oropharynx of healthy individuals.
Twenty invasive N. meningitidis strains were isolated from either cerebrospinal fluid or blood of
patients, all in our region. Serogrouping and
serotyping was done in the Centro Nacional de
Microbiolog~a, Virologla e Inmunologia in Majadahonda (Spain). All strains were identified by
standard methods [12] and stored in 5% sodium
g l u t a m a t e / 5 % bovine albumin [13] at - 8 0 °C in
50 /xl until used. The strains were cultured on
Choc-Iso agar plates [14] for 24 h at 3 7 ° C in a
5% atmosphere and then one isolated colony
subcultured for 8 h in the same conditions before
inoculation.
3.2. Chemicals
Peroxidase-conjugated human transferrin was
obtained from Jackson Immunoresearch Laboratories, West Grove, PA. 4-Chloro-l-napthol,
ethylenediamine dihydroxyphenyl acetic acid
(EDDA) and speciality chemicals for SDS-PAGE
were from Sigma Chemical Co., St. Louis, MO.
3.3. Induction of IRPs
Erlenmeyer flasks with 100 ml of Mueller-Hinton broth (MH; normal iron) or Mueller-Hinton
broth with 39 /xM ethylene-diamine-dihydroxyphenyl acetic acid (MH-EDDA, iron restriction)
were incubated overnight at 3 7 ° C in a water
bath. with shaking at 100 rpm.
3.4. Extraction of outer membrane cesicles (OMVs)
Both excreted outer m e m b r a n e vesicles
(OMVs, blebs) and bacterial outer membranes
were obtained from the cultures as described
previously [6]. The cultures were centrifuged at
48000 x g, the pellets were resuspended in 0.1 M
acetate buffer, pH 5.8 with 0.2 M lithium chloride
with ultrasonic pulses, incubated at 4 5 ° C in a
shaking water bath and then passed through a 21
gauge needle. The suspensions were then centrifuged at 1 0 0 0 0 x g and the pellet was discarded. Membrane fragments were obtained from
the supernatant by centrifugation at 50000 x g
and suspended in 0.5 ml of distilled water. Protein content was determined by the Coomassie
Blue dye method [15]. Aliquots were then stored
frozen at - 2 0 °C until analysed by SDS-PAGE.
3.5. Transferrin binding assay
The dot enzyme assay was developed essentially as described by Schryvers and Morris [7]. To
determine the level of expression of transferrin
receptor activity serial two-fold dilutions were
prepared from outer membrane protein extracts
249
containing 5 0 0 / z g / m l of protein and aliquots of
2 /xl were directly applied onto nitrocellulose
membranes (0.45/xm, BioRad Laboratories). The
membranes were then incubated in blocking solution (0.5% skim milk and 0.001% antifoam A in
buffer) and washed with Tris-buffered saline
(TBS) prior to addition of blocking solution containing the conjugated transferrin. After allowing
binding, the membranes were washed with TBS
and developed with 4-chloro-l-napthol and 0.01%
hydrogen peroxide [16].
3. 6. SDS-PA GE and electroblotting
SDS-PAGE was performed as described previously [6]. The OMVs were subject to 10% sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with the discontinuous
buffer system of [17]. Aliquots containing 20 /zg
of sample were either boiled at 100 ° C for 5 min,
heated in sample buffer at 37 °C for 20 rain or
maintained overnight at room temperature.
After electrophoresis, proteins were transferred from the gels to nitrocellulose membranes
using a Milliblot SDE-Electroblotting System
(Millipore Ib4rica S.A.) and a low ionic discontinuous buffer system composed of two anode buffers
(0.3 M Tris, 10% methanol pH 10.4/25 mM Tris,
10% methanol pH 10.4) and one cathode buffer
(25 mM Tris, 40 mM 6-aminohexanoic acid, 20%
methanol pH 9.4). Electrotransfer was run for 30
min at 2.5 m A / c m 2 of gel area. Transferred
proteins were blocked, incubated with peroxidase-conjugated human transferrin and developed with the chloronaphthol/hydrogen peroxide substrate mixture as described in the transferrin-binding assay.
4. RESULTS AND DISCUSSION
Most Neisseria meningitidis strains used in this
study were characterized as serogroup B (40% of
Table 1
Characteristics of Neisseria meningitidis strains and molecular mass and thermal stability of their transferrin receptors
Carrier strains
Invasive strains
Strain
Sg/St ~
MM
(kDa)
Method b
Strain
Sg/St
MM
(kDa)
Method
P000
P009
P095
P097
P124
P136
P139
P148
P164
P192
P214
P224
P230
P242
P346
P361
P391
P536
P608
P636
B/15
AA/I
B/15
B/12
AA/NT
B/2
NG/1,8
B/1
B/2
B/15,8
AA/NT
AA/2
AA/2
AA/15
Y/15
AA/5
AA/15
B/1
Y/NT
AA/8,15
83.8
83.4
81.9
84.0
81.2
81.0
81.4
79.5
80.1
80.9
80.0
80.1
72.9
80.7
82.9
84.7
84.9
88.3
72.2
86.5
1
1
2
1
1
2
2
2
1
2
3
1
3
1
1
1
1
1
2
1
V00I
V002
V003
V006
V009
V010
V011
V012
V013
V014
V015
V019
V021
V022
V026
V028
V029
V036
C019
L341
C/2
B/15
B/15
B/15
B/12
B/NT
B/4
B/NT
B/NT
B/NT
B/15
B/1
B/NT
B/15
B/NT
C/2
B/NT
C/2
B/8,15
B/2
84.0
71.4
79.1
78.5
83.4
80.2
80.2
81.1
81.1
79.6
78.7
78.0
80.7
83.5
78.3
67.1
81.4
68.2
81.7
78.0
3
3
1
1
3
1
1
1
1
1
1
1
1
3
1
1
1
1
1
1
a Serogroup/Serotype. NG, not groupable; NT, not typable.
b Method of sample processing for electrophoresis: (1) heating to 100 o C for 10 min; (2) heating to 37 o C for 20 min; (3) overnight
incubation at room temperature.
250
the carrier and 85% of the invasive isolates), their
serotype being more variable (Table 1). This is in
agreement with the fact that in our region
(Galicia, Spain) most bacterial meningitis is
caused by serogroup B N. meningitidis, this illness
being endemic in this area.
In order to investigate the effect of iron restriction on the outer m e m b r a n e protein composition of the bacteria, cultures were grown at 100
rpm constant shaking in Mueller-Hinton broth
with added E D D A . O M V s were obtained by
lithium chloride extraction as described in MATERIAL AND METHODS and proteins were analysed
by S D S - P A G E and laser densitometry. Iron restriction produced either a 'de novo' synthesis or
an expression increase of outer m e m b r a n e proteins when compared with the iron-sufficiency
situation (Fig. 1); the transferrin receptor being
Table 2
Transferrin receptor expression level in Neisseria meningitidis
strains determined by dot blot enzyme assay
Carrier strains
Strain
PO00
PO09
P095
P097
P124
P136
P139
P148
P164
P192
P214
P224
P230
P242
P346
P361
P391
P536
P608
P636
Invasive strains
Fe discharge
Strain
Normal ~l
Low b
1/4
1/4
1/4
0
1/4
1/4
1/4
1/2
1/4
1/2
1/2
1/16
1/16
1/8
1/8
1/4
1/8
1/16
1/8
1/32
1/128
1/64
1/64
1,/32
1/64
1/64
1/32
1/32
1/32
1/64
1/32
1/32
1/32
1/32
1/64
I/64
1/64
1/64
1/64
1/64
VO01
VO02
VO03
VO06
VO09
V{}IO
V{}ll
VO12
V{}I3
VO14
VO15
VO19
V(I21
V(122
V026
V028
V029
Vl)36
C019
L341
Fe discharge
Normal
Low
1/8
1
1}
1/ 16
1
1/4
1/4
1/8
1
1.8
1
1/2
1/4
1
1/4
I,/2
1/8
1
1/4
1/4
1/32
I/8
l/32
1/32
1/16
1/32
1/32
1/64
1/16
1/64
1/32
1/64
1/32
1/16
1/64
1/32
1/512
1/32
1/32
1/64
~' Growth in Mueller-Hinton broth.
h Growth in Mueller-Hinton broth with 39 /xM ethylene-diamino-dihydroxy-phenyl acetic acid.
Fig. 1. Laser scanning densitograms of the outer membrane
proteins of strains N. meningitidis P000 (upper graphs) and
V002 (lower graphs) growth in normal iron (A) and iron
restriction (B) conditions. OMVs were obtained by lithium
chloride extraction and proteins analysed by SDS-PAGE (10%
polyacrylamide).
included in these new or enhanced proteins. Recent studies by other authors [4,7] are in agreement with this, showing that the expression of
transferrin binding activity is regulated by iron
levels in the culture media.
The level of expression of the Tf receptor was
estimated by a dot blot enzyme assay onto nitrocellulose p a p e r using peroxidase-conjugated
transferrin and testing the OMVs obtained from
40 (20 carrier and 20 invasive) N. meningitidis
strains. Receptor activity was detected in all
strains,, in samples from cultures grown both in
iron sufficiency (with only two exceptions, strains
P097 and V003, which were negative) or iron
restriction. Nevertheless, as can be seen in Table
2, transferrin receptor expression was higher in
the strains cultured in iron restriction although
both the expression level and the expression increase were highly variable among the strains:
iron restriction expression levels ranged from a
251
Table 3
Molecular mass and thermal stability of the transferrin receptors of commensal Neisseria
Strain a
N.
N.
N.
N.
N.
lactamica
lactamica
lactamica
lactamica
lactamica
P343
P544
P576
P580
P671
MM
Method b
83.0
78.1
79.0
79.1
79.3
3
3
3
3
3
a Other two N. lactamica, 11 N. sicca and one Neisseria sp.
did not produce transferrin receptor in the experimental
conditions tested.
b Methods of sample processing for electrophoresis: (1) heating to 100 ° C for 10 min; (2) heating to 37 ° C for 20 rain; (3)
overnight incubation at room temperature.
m i n i m u m o f 1 / 8 in strain V002 to a m a x i m u m of
1 / 5 1 2 in strain V029, a n d e x p r e s s i o n i n c r e a s e s
v a r i e d f r o m 1 / 1 6 - 1 / 3 2 (two-fold; m i n i m u m in-
c r e a s e ) in strain V006 to 1 / 8 - 1 / 5 1 2 (64-fold) in
strain V029 or 0 - 1 / 3 2 ( t h e o r e t i c a l l y oo; m a x i m u m
i n c r e a s e ) in strains P097 a n d V003 (Fig. 2). T h e
S D S - P A G E analysis of the O M V p r o t e i n s (Fig. 3)
shows t h a t the t r a n s f e r r i n r e c e p t o r in strain V006
is c o i n c i d e n t with o n e of t h e m a i n o u t e r m e m b r a n e p r o t e i n s , also e x p r e s s e d u n d e r iron sufficiency c o n d i t i o n s , w h e r e a s t h a t o f strain P097
c o r r e s p o n d s to a ' d e novo' s y n t h e s i z e d p r o t e i n .
Griffiths et al. [10] using e l e c t r o b l o t t i n g techn i q u e s r e p o r t e d t h e d e t e c t i o n of t r a n s f e r r i n rec e p t o r s in m o s t o f t h e i r strains, a l t h o u g h d e t e c tion was not possible w h e n t h e y grew in iron
sufficiency. T h e lack o f a g r e e m e n t in this p o i n t
with o u r results m a y be d u e to t h e d i f f e r e n t
m e t h o d s u s e d for t h e t r a n s f e r r i n r e c e p t o r d e t e c tion; as t h e s e a u t h o r p o i n t out, s a m p l e p r e p a r a tion for S D S - P A G E a n d e l e c t r o b l o t t i n g can inactive the b i n d i n g ability of t h e r e c e p t o r . It is
OMV protein (ng)
1000
500
250
125
62.5
31.3
15.6
7.8
Strain
V006
P097
Fig. 2. Quantification of the transferrin receptor expression in Neisseria meningitidis. The strains were cultured in Mueller-Hinton
broth with (upper lanes) or without (lower lanes) 39 ~M EDDA. After overnight incubation outer membrane vesicles (OMVs) were
extracted, adjusted to 500 p~g ml -I and 2-/xl aliquots of serial two-fold dilutions of these suspensions were blotted onto
nitrocellulose membranes, probed with transferrin-peroxidase and revealed for peroxidase activity with 4-chloro-l-naphtol.
252
Fig. 3. Laser scanning densitograms of the outer membrane
proteins of strains IV. meningitidis P097 (upper graphs) and
V006 (lower graphs) growth in normal iron (A) and iron
restriction (B) conditions. OMVs were obtained by lithium
chloride extraction and proteins analysed by SDS-PAGE (10%
polyacrylamyde). Asterisks indicate the localization of the
transferrin receptor as detected after electrotransfer to nitrocellulose paper and incubation with peroxidase-conjugated
transferrin.
evident that in our case, using dot-blot with intact
OMVs, the chances for a correct activity of the
receptors are much higher.
Griffiths et al. [10] reported that only one of
their six serogroup A strains showed a 'strong
reaction' with transferrin and two of the strains
were negative for binding. In our case the nine
serogroup A A strains tested (all of them from
carriers) showed binding activity with titers >
1 / 3 2 in the dot blot assay and in all of them the
receptor was detected after iron sufficiency
growth although, evidently, at lower titers.
For the characterization of the transferrin receptor proteins in the 40 N. meningitidis strains
and 20 other commensal Neisseria strains, O M V s
were subject to SDS-PAGE, proteins electro-
transferred to nitrocellulose m e m b r a n e s and
probed with peroxidase-conjugated transferrin. In
some strains in which transferrin receptor activity
was detected by dot blot, this was not detectable
after S D S - P A G E and electrotransfer, this being
in agreement with the above-mentioned results
obtained by Griffiths et al. [10]. Consequently, in
order to test if this lack of detection was due to
experimental problems (of receptor inactivation)
three different sample preparation protocols at
different temperatures and incubation times in
sample buffer were used for electrophoresis of
the OMVs. Twenty-eight strains showed transferrin receptors demonstrable after 'normal' sample
preparation (100 ° C / 5 min). Six of the other 12
samples (15%) could be shown by treatment at
37 ° C / 2 0 min, which indicates a slight thermolability, and the last six samples (15%) were only
detected if overnight incubation (14 h) at ambient
temperature (20 ° C) was used, indicating a relatively high thermolability. Although all slightly
labile strains were obtained from carriers, four of
the 20 invasive strains were highly labile, which
indicates that receptor thermolability cannot be
completely associated with the origin of strains.
Nevertheless, the number of either slightly or
strongly labile carrier strains is twice that of the
invasive ones.
In this study, transferrin receptors of the N.
meningitidis strains showed variable molecular
masses (Table 1). Most of the strains (87.5%) had
receptors with molecular masses close to 80 kDa
(from 78 kDa to 85 kDa). Five strains (12.5%)
had receptors with molecular masses near to 70
kDa (from 67 kDa to 73 kDa) and only two
showed receptors with higher molecular masses,
86.5 kDa and 88.3 kDa, respectively. These results completely agree with those of Griffiths et
al. [10] which found that 11.5% of their strains
had receptors with molecular masses neighbouring 68 kDa and all other strains with molecular
masses from 78 kDa to 84 kDa. Although these
authors did not find higher molecular mass receptors, this does not constitute a real discrepancy because of the low percentage (5%) found
by us within that range. These results are also in
agreement with those of Ala'Aldeen et al. [9]
which showed that in 12 N. meningitidis strains
253
studied transferrin receptors were always present
and with molecular masses ranging from 60 kDa
to 90 kDa. All these results along with ours
completely disagree with those of Schryvers and
Lee [8] which maintain that all Neisseria strains
possess similar transferrin receptors with molecular masses about 98 kDa.
The method routinely used by these authors
[8,18], based on affinity techniques, does not test
if the purified material binds transferrin and it is
probable that errors can arise. We have applied
this method (data not shown) to our strains, trying to detect transferrin binding activity after
electroblotting without any success even with our
most active (by our method) strains. Perhaps the
amount of receptor purified is not enough to
detect binding of transferrin but able to be detected by silver staining.
Schryvers and Lee [8] found lactoferrin binding activity in all comensal Neisseria strains tested
(using the above mentioned affinity method).
Transferrin binding activity was less evident in
commensal strains (five of the eight N. lactamica
strains). In all five cases, the receptors were highly
thermolabile with molecular masses close to 80
kDa. It has to be mentioned that one N. lactamica (P281) and two N. sicca (P189 and P314)
strains showed receptor activity in the dot blot
assays, but not after SDS-PAGE and electrotransfer. Perhaps this inability to bind transferrin
in the commensal species and the lability of the
N. lactamica receptors could explain their low
pathogenicity when compared to N. meningitidis
and N. gonorrhoeae, as has already been proposed for the strains lacking transferrin receptors
[19].
ACKNOWLEDGEMENTS
This work has been supported by Grants
PM88-0209 of the Direcci6n General de Investi-
gaci6n Cientifica y T6cnica (DGICYT, Spanish
Government) and XUGA81503988 of the Direcci6n Xeral de Ordenaci6n Universitaria e Polftica
Cientifica (Autonomic Government).
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