Download The CS2 fimbrial antigen from escherichia coli, purification

FEMS Microbiology Letters 26 (1985) 207-210
Published by Elsevier
207
FEM 01995
The CS2 fimbrial antigen from Escherichia coli, purification,
characterization and partial covalent structure
(CS2; fimbriae; N-terminal sequence)
Per Klemm, Wim Gaastra, Moyra M. McConnell * and Henry R. Smith
*
Department of Microbiology, The Technical University of Denmark, Building 221, DK-2800 Lyngby, Denmark, and * Division of Enteric
Pathogens, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, U.K.
Received 19 October 1984
Accepted 2 November 1984
1. SUMMARY
The CS2 fimbrial antigen was isolated by salt
and isoelectric precipitation and by column chromatography. The purified antigen was free Of other
fimbrial proteins present on the same bacterial
strain. Analysis of the N-terminal amino acid sequence indicated extensive homology with the
CFA1 fimbrial antigen, which was surprising since
the two proteins do not show any immunological
cross reactivity. It could therefore be concluded
that the N-terminal parts of these fimbrial proteins
are not located on the surfaces of the proteins and
might be involved in conservation of the structural
integrity of these proteins:
2. INTRODUCTION
Fimbriae are known to adhere to various hosttissue surfaces. In enterotoxigenic Escherichia coli
strains, causing diarrhoea in man, a variety of
surface antigens have been characterized e.g. the
CFA1 and CFA2 antigens [1-3]. These fimbriae
are known to mediate adhesion to human intestinal epithelium [1-4], thereby permitting coloniza-
tion by enterotoxigenic E. coli strains of the upper
intestinal tract, and must therefore be regarded as
important virulence factors.
The entity originally referred to as CFA2 was
later found to consist of three distinct antigens,
viz. CS1, CS2 and CS3 [5,6]. The CFA2 antigens
are, like the CFA1, plasmid-encoded [7,8]. However, the expression of the CS1 and CS2 is highly
dependent on the serotype and biotype of the host
strain. The same plasmid codes for CS1 and CS2
in an O6:H16 biotype A strain and for the CS2
and CS3 in 06 : H16 strains of biotypes B, C, and
F [7,8]. A few CS2 only strains have been identified [5]. CS1 and CS2 are very similar rigid fimbriae
of 6-7 nm [9,10]. Though morphologically indistinguishable, they are immunologically distinct.
CS3 was originally thought to be non-fimbrial but
has recently been shown to consist of thin (2 nm)
flexibly wiry fimbriae [11]. CS1 and CS2 fimbriae
resemble those of CFA1 and must be assumed,
like the latter, to be homopolymers consisting of
approx. 1000 identical subunits.
In this context we have purified and characterized CS2 fimbriae and report here both immunological and protein chemical aspects of this
virulence factor.
0378-1097/85/$03.30 © 1985 Federation of European Microbiological Societies
208
3. MATERIALS A N D M E T H O D S
3.1. Bacterial strain and culture
E. coli 58R297 of serotype 0 6 : H16 containing
plasmid NTP156 [9] was used for the isolation of
the CS2 fimbriae. Cells were grown on 50 large (14
cm) L-broth agar plates.
3.2. Crude extract of fimbriae
The bacteria were harvested and suspended in
200 ml of 0.1 M sodium phosphate pH 7.0. The
suspension was blended in a Sorval omnimixer at
maximum setting for 3 x 3 rain under ice-cooling.
Bacterial cells and large debris were removed by
centrifugation at 27000 X g for 15 min, and the
supernatant was filtered through a 0.80-/xm pore
size filter (Millipore Corp., Bedford, MA).
3. 3. lsoelectric and salt precipitation
To a crude extract of fimbriae 1 M acetic acid
was added to pH 4 under continuous stirring. The
solution was left for 30 min at 4°C and the precipitate collected by centrifugation (10 000 x g for
20 min). The pellet was resuspended in 0.05 M
Tris-HC1 pH 7 and 1 M MgCI 2 was added to a
final concentration of 0.1 M. The solution was left
overnight at 0°C and the precipitate collected by
centrifugation.
3. 4. Gel filtration
Gel filtration of partially pure fimbriae was
performed on a Sepharose CL6B column (1.5 x 90
cm) eluted with 6 M guanidinium chloride.
3.5. Polyacrylamide gel electrophoresis
The purity and M r of the CS2 protein were
assessed by electrophoresis on polyacrylamide slab
gels in the presence of 0.1% SDS as previously
described [9].
3.6. Amino acid analysis
Amino acid analysis was performed from
duplicate samples, hydrolyzed for 24 h, 48 h and
72 h in 6 M HC1 containing 0.1% phenol. Cysteine
was determined as cysteic acid from performic
acid-oxidized samples. Values for serine and
threonine were extrapolated to zero time, those for
leucine and isoleucine were determined from the
72 h hydrolysis values. The tryptophan content
was determined after hydrolysis in methane
sulfonic acid containing 0.2% 3-(2-aminoethyl)-indole [13].
3. 7. Sequence analysis
N-terminal amino acid sequence was determined by manual Edman degradation in the
presence of sodium dodecyl sulfate (SDS) [11].
Conversion into phenylthiohydantoin derivatives
and conversion into parent amino acids as well as
identification were performed as previously described [14,15].
3.8. Immunological techniques
Specific IgGs for CS2, CS3 and CFA1 were
prepared as previously described [9]. These IgGs
were labelled with horse-radish peroxidase to make
conjugates for use in the enzyme-linked immunosorbent assays (ELISAs). The assays have been
described in detail [9]. The antigens were suspensions of fimbriae (1 mg/ml). In the CS2 assays
doubling dilutions of antigens were used.
4. RESULTS A N D DISCUSSION
To suppress any production of type 1 fimbriae
strain 58R297 was grown on solid medium [16].
After harvesting the cells, the CS2 fimbriae were
liberated by blending. Initial purification was obtained by isoelectric- and magnesium chloride precipitation and the protein was thereby isolated to
approx. 80% purity in the form of intact fimbriae.
Testing with ELISA showed that this preparation
contained a small amount of material which gave a
positive reaction with anti-CS3 conjugate. The CS2
was detected at a dilution of 212 and CS3 at 25. As
a final purification step the fimbriae were disrupted in 6 M guanidinium chloride and the
liberated subunits were purified by gel chromatography as depicted in Fig. 1. The purification was
monitored by SDS gel electrophoresis (Fig. 2). No
CS3 was detected in this preparation by ELISA
while it gave a strongly positive reaction with
anti-CS2 conjugate.
The apparent M r of the CS2 fimbrial subunit as
determined by SDS electrophoresis was 17000. In
209
i----a
0.8
0./,
20
/.0
60
f i-act ion no
Fig. 1. Gel chromatography of partially purified CS2 protein
on a Sepharose CL6B column (1.5 x90 cm) eluted with 6M
guanidinium chloride in 0.2 M ammonium hydrogen carbonate.
Fractions of 1.8 ml were collected. Bar indicates pooled fractions containing the CS2 protein.
g o o d a g r e e m e n t with this result is the M r of 16 440
calculated on the basis of the a m i n o acid c o m p o s i tion (Table 1). A m i n o acid analysis showed the
CS2 p r o t e i n to c o n t a i n very few a r o m a t i c a m i n o
acid residues a n d for e x a m p l e no t r y p t o p h a n s .
C o n s e q u e n t l y the p r o t e i n would be expected to
have a very small a b s o r b a n c e at 280 n m which was
exactly what was o b s e r v e d during the purification
(Fig. 1). T h e CS2 fimbrial p r o t e i n does n o t c o n t a i n
a n y cysteine residues, a n d the subunits must therefore be held together b y n o n c o v a l e n t forces.
The N - t e r m i n a l a m i n o acid sequence of the CS2
p r o t e i n was elucidated for the residues 1 through
20. C o m p a r i s o n of the sequence with the N - t e r m i nal sequence of the C F A 1 fimbrial p r o t e i n [14] is
shown in Fig. 3. It is evident that the two sequences are nearly identical, the only observed
difference being a V a l - A l a shift in the N - t e r m i n a l
position. This homology, covering at least some
13% of the sequences, is quite surprising since the
two p r o t e i n s show no i m m u n o l o g i c a l cross-reactivity, as tested b y i m m u n o d i f f u s i o n a n d E L I S A .
However, since the a m i n o acid c o m p o s i t i o n s of
Table 1
Amino acid composition of the CS2 subunit protein
In parentheses are given the experimental values. The amino
acid composition of the CFA1 subunit protein has been included for comparison.
Amino acid
Fig. 2. SDS-polyacrylamide gel electrophoresis of CS2 preparations. Lane A crude extract, lane B, after isoelectric- and salt
precipitation, lane C, marker proteins and lane D, CS2 after
final purification on Sepharose CL 6B.
Asx
Thr
Ser
Glx
Pro
Gly
Ala
Cys
Val
Met
Ile
Leu
Tyr
Phe
His
Lys
Arg
Trp
Total
CS2
18 (17.8)
18 (16.8)
12 (12.2)
16 (16.1)
7 (6.9)
12 (12.1)
15 (14.9)
0 (0.1)
12 (11.6)
3 (2.7)
9 (8.9)
12 (11.9)
3 (2.9)
4 (3.9)
2 (2.0)
9 (9.0)
4 (3.9)
0 (trace)
155
CFA1
12
15
17
11
7
10
19
0
19
3
5
12
4
2
1
8
1
1
147
210
l
CS2
5
lO
15
20
: Al a-Gl u-Lys-Asn- I I e-Thr-Val -Thr-Al a-Ser-Val -Asp-Pro-Val - I I e-Asp-Leu-Leu-Gl n-Al a
CFAI : Val -GI u-Lys-Asn- I I e-Thr-Val -Thr-Al a-Ser-Val -Asp-Pro-Val - I I e-Asp-Leu-Leu-Gl n-A] a
Fig. 3. N-terminal amino acid sequence of the CS2 fimbrial subunit. The N-terminal sequence of the CFA1 protein is given for
comparison.
two proteins show substantial differences extensive
sequence differences accounting for the immunological variations must be located further inside
the peptide chains.
As the N-terminal region does not appear to
constitute an antigenic determinant (if it did the
two proteins would show immunological cross-reactivity) large parts or perhaps all of this segment
of the molecule might not be situated on the
surface of the molecule. Furthermore, since the
N-terminal sequence is conserved in both molecules it could fulfill a vital function in maintaining
the structural integrity of the CS2 and CFA1
fimbriae.
ACKNOWLEDGEMENTS
We are indebted to Marianne Hoff Moller for
excellent technical assistance. This work was supported by the Danish Technical and Medical Research Councils.
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