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Jourrtal of General Microbiology (1987), 133, 2233-2236.
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Amino Acid Sequence and Antigenicity of the Amino-terminus of the
168 kDa Adherence Protein of Mycoplasma pneumoniae
By E . J A C O B S , K . F U C H T E A N D W . B R E D T *
Department of Microbiology and Hygiene, Institute for Medical Microbiology and Hygiene,
University of’Freiburg, 0-7800 Freiburg, FRG
(Received I I December I986 ;revised I I March 1987)
The amino-terminal end of the 168 kDa adherence protein from the membrane of Mycoplasma
pneumoniae was sequenced up to 12 amino acids. A synthetic peptide containing nine amino
acids of this sequence was used to study the antigenicity of the amino-terminus of the 168 kDa
protein and the involvement of the homologous sequence of the protein in the adherence
process. The synthetic peptide when coupled to ovalbumin was immunogenic in rabbits.
Antibodies against this peptide epitope could be demonstrated in sera taken during natural M .
pneumoniae infection in humans. The structural domain of the 168 kDa protein homologous with
the synthetic peptide did not appear to be involved in adherence, as the synthetic peptide or its
homologous antibody failed to inhibit adherence of M . pneumoniae.
INTRODUCTION
Adherence of Mycoplasma pneurnoniae to erythrocytes or epithelial cells is mediated by a
168 kDa membrane protein (Hu et al., 1982; Feldner et al., 1982). Besides its function as an
adhesin the 168 kDa protein is a major immunogen of M . pneumoniae. In early stages of human
infection it is so far the only protein antigen known against which antibodies are developed by
all patients (Leith et al., 1983; Hu et al., 1983; Jacobs et al., 1986~).Using the 168 kDa protein
isolated from SDS-PAGE it was possible to establish an ELISA with this antigen for a sensitive
and specific serological diagnosis of M . pneumoniae infection (Jacobs & Clad, 1986; Jacobs et al.,
19866). However, the considerable costs entailed in growing M . pneumoniae, and the low yield of
M . pneumoniae cells and accordingly of the 168 kDa antigen, strongly suggested the need for
alternative methods of antigen production. One possibility seemed to be the use of synthetic
peptides representing relevant antigenic determinants of the 168 kDa protein. However, neither
the amino acid sequence of the 168 kDa protein nor the base sequence of its gene is yet known,
although some cloned DNA sequences of the M . pneumoniae chromosome have been shown to
encode large sections of this antigen (Trevino et al., 1986).
The development of a high-yield electroelution method (Jacobs & Clad, 1986) provided
sufficient amounts of the 168 kDa protein for further analytical studies. The amino-terminal end
of the 168 kDa adhesin has now been sequenced and investigated for antigenic and functional
properties using a homologous synthetic peptide.
METHODS
Isolation andpurification of the 168 kDa protein. M . pneumoniae strain FH was grown on glass and harvested as
described previously (Jacobs et al.. 1980). The 168 kDa protein was electroeluted from SDS-PAGE gels as
described by Jacobs & Clad (1986). Approximately 65-75 pg of the protein could be obtained from 5 mg of total M.
pneumoniae cell protein processed per gel. The amino-terminal end of three different preparations of the purified
protein was sequenced by Edman degradation (Edman & Henschen, 1975) in a gas-phase protein sequencer 470A
(Applied Biosystems). Phenylthiohydantoin derivatives of amino acids were identified by reversed-phase
chromatography as described by Lottspeich ( 1980).
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E . JACOBS, K . F U C H T E A N D W . B R E D T
Synthesis of peptide. An oligopeptide containing nine amino acids (positions 4-1 2) of the amino-terminal
was synthesized commercially by the solid-phase procedure
sequence (Asn-Pro-Arg-Leu-Thr-Pro-Phe-Thr-Tyr)
according to the Merryfield method by Orpegen, Heidelberg, FRG. For analytical reasons the tryptophan in
position 10 was replaced by the similarly charged and structurally related phenylalanine.
Zmmunizatwn of rabbits. For immunization the tyrosine end of the peptide was coupled to ovalbumin (Sigma
A2512, grade VI) as carrier molecule using the bisdiazotized benzidine method of Bassiri et al. (1979) with a molar
ratio of albumin to peptide of 1 :5 . Rabbits were immunized intradermally with 10 mg peptide conjugate or
ovalbumin in complete Freund’s adjuvant. Intravenous booster injections ( 5 mg each) were given after 6 and 4
weeks respectively and the animals were bled 14 d after the last injection. Immunization with purified 168 kDa
protein was performed following the same schedule.
Patients’sera. Paired sera were obtained from six children with clinical symptoms of M . pneumoniae pneumonia
and with a titre increase (at least twofold) in the complement-fixation test. Sera were taken on days 8 ( f 3 d) and 16
( & 4 d) on average after onset of symptoms. Control sera were obtained from children with no respiratory disease,
whose sera had been sent for other serological tests.
ELZSA. An ELISA with the 168 kDa protein as antigen (1 pg per well) or with 10 ng peptide per well was carried
out as described previously (Jacobs et al., 1986 b) using polystyrene microtitre plates (Greiner, FRG) instead of a
nitrocellulose filtration plate. Measurements (A4os)were performed on 1 : 100 dilutions of the respective antisera,
if not otherwise indicated.
Zmrnunoblotting. This was done as described previously (Jacobs et al., 1986 a ) with 80 pg mycoplasma cell
protein or 10 pg 168 kDa protein loaded per track of the SDS-PAGE gel.
Adherence inhibition test. This was done as described by Jacobs et ul. (1985). Briefly, M . pneumoniue strain FH
was grown in flat-bottomed microtitre plates; the mycoplasma layers were washed, treated with the respective
antiserum for 1 h and then overlaid with a suspension of sheep erythrocytes. The plates were sealed, incubated for
another hour and then inverted for 15 min to allow non-attached erythrocytes to detach from the mycoplasma
layer. The sealing tape was removed, the wells were washed once and the attached erythrocytes were lysed by
addition of 100 pl distilled water per well. After 10 min the A414 of the lysate was measured. The A414 of lysates
from wells without serum treatment was taken as maximum adherence (100%).
RESULTS A N D DISCUSSION
Amino acid sequence of the 168 kDa adhesin
Analysis of the amino-terminal region of the_ _168 kDa protein showed the following sequence
lle
for the first 12 amino acids : N-( 1)Asn-Ala- -Asn-Pro-Arg-Leu-Thr-Pro-Trp-Thr-Tyr(12).
GlY
Position 3 showed a possible microheterogeneity .Besides hydrophobic amino acids like alanine,
leucine, tyrosine and tryptophan the amino-terminus contained hydrophilic components such as
asparagine, arginine and threonine. Comparison with sequences stored in the European
Molecular Biology Laboratory (EMBL, Heidelberg, FRG) and Protein Identification Resource
(PIR, Washington, DC, USA) data banks revealed no homologous sequences.
Immunogenicity of synthetic peptide homologous with the amino-terminus of the 168 kDa protein
Antigenically active regions of proteins usually contained polar residues which are located on
the surface of the native molecule (Walter et al., 1980). The sequence described here contained a
combination of hydrophobic and hydrophilic amino acids and its immunogenicity could not be
predicted. Therefore an oligopeptide of nine amino acids (positions 4-1 2) was synthesized,
coupled to ovalbumin and used for immunization of rabbits. The resulting antiserum was
immunoblotted against M . pneumoniae proteins (Fig. 1). The weak but distinct reaction of the
anti-peptide serum with the 168 kDa protein is apparent (lane 5) when compared with the
preimmunization serum (lane 4). The more intense band resulting from the reaction with
antiserum raised by immunization with purified 168 kDa protein is shown in lane 3.
For quantification the anti-peptide serum was tested in an ELISA against the 168 kDa
antigen. The test showed an increase in antibody activity from A405 0.04 (preimmunization
serum) to A405 0.93 (anti-peptide serum) (serum dilutions 1 :loo). When the ELISA was
performed with peptide as antigen (serum dilutions 1 :800) the activity increased from A405 0.05
(background, preimmunization serum) to A405 0.58. An anti-168 kDa serum showed a
comparable increase from A405 0.08 (preimmunization serum) to A405 0.9. The control antiDownloaded from www.microbiologyresearch.org by
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N-terminus of M . pneumoniae adhesin
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Fig. 1. Reaction of anti-peptide serum with M. pneumoniue protein antigens. Lanes: 1, separated M.
pneumoniue proteins, stained with Coomassie blue; 2, purified 168 kDa protein, stained with Coomassie
blue; 3-5, immunoblots against separated M. pneumoniue proteins (antisera: lane 3 anti-168 kDa
serum; lane 4, preimmunization serum of peptide-immunized rabbit; lane 5 , anti-peptide serum).
Table 1. IgM antibodies in paired sera of six children with M . pneumoniae infection and of six
healthy controls in ELISA using 168 kDa protein or synthetic peptide as antigen
ELISA titres (A40s)*
I
Antigen tested
Controls
1st serumt
2nd serum:
168 kDa protein
Oligopeptide
0.08 f 0.03
0.1 1 f 0.05
0.15 f 0.06
0.24 f 0.08
0.66 f 0.1 8
0.48 f 0.15
The results are means f standard deviation of six sera in each case (serum dilutions 1 :loo).
t Sera taken on day 8 ( f3 d) after onset of symptoms.
$. Sera taken on day 16 ( f4 d) after onset of symptoms.
ovalbumin serum reacted neither with the 168 kDa protein (A4050.06; 1 :loo) nor with the
peptide antigen (Ado50-04; 1 :800).
The results indicated that the amino-terminal sequence functioned as an immunologically
active epitope of the 168 kDa protein.
Immunogenicity of the amino terminus of the 168 kDa protein during natural infection with
M . pneumoniae
Despite the immunological reactivity of the synthetic peptide corresponding to the amino
terminal sequence of the 168 kDa protein, the amino-terminus of the native protein need not
necessarily be immunogenic in the intact organism. It could be situated within or on the inner
side of the membrane, eliciting no or only a late immune response during natural infection.
Therefore paired sera from six children suffering from M. pneumoniae infection were tested for
IgM antibodies in an ELISA using in parallel the synthetic peptide and the 168 kDa protein as
antigens. The results (Table 1) showed comparable antibody titres against both antigens. The
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E. JACOBS, K . FUCHTE A N D W . B R E D T
differences between controls and first sera or controls and second sera were significant
(P= 0.005 and 0.001 respectively, by Student’s t-test). Apparently the epitope of the 168 kDa
protein corresponding to the synthetic peptide sequence is sufficiently exposed during natural
infection to stimulate an early immune response.
Adherence inhibition tests with synthetic peptide and anti-peptide serum
The possible involvement of the amino-terminal end of the 168 kDa protein in the adherence
of M. pneumoniae was examined by testing the anti-peptide serum in the quantitative adherence
inhibition assay (Jacobs et al., 1985). Pretreatment of M. pneumoniae layers with the anti-peptide
serum did not inhibit the attachment of sheep erythrocytes. Furthermore, pretreatment of the
sheep erythrocytes with 100 pg synthetic peptide ml-l for 30 min did not inhibit binding. Thus,
this structural domain of the amino-terminus of the 168 kDa protein appeared to have little or no
role in the binding function.
The results suggest that the amino-terminal sequence of the 168 kDa protein is possibly
exposed on the surface of the M. pneumoniae cell, but does not substantially contribute to the
adherence process. The knowledge of the sequence of the amino-terminal end of the 168 kDa
adhesin should help to identify the corresponding DNA sequence within the M . pneumoniae
chromosome. Furthermore the results indicate the potential value of synthetic epitopes as
antigens for the serological diagnosis of M. pneumoniae infections.
This work was supported by a grant from the Bundesministerium fur Forschung und Technologie. The technical
help of Karin Oberle and the secretarial skills of Ruth Thomas are gratefully acknowledged. We are indebted to
Emile Schiltz (Institut fur Oranische Chemie und Biochemie) for advice and practical help with the amino acid
sequencing procedure, and to Roland Friedrich for help with the data bank search.
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