Journal of Bacteriology, April 2001, p. 2686-2690, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2686-2690.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of Outer Membrane Proteins in
Chlamydia trachomatis LGV Serovar L2
Regina J.
Tanzer and
Thomas P.
Hatch*
Department of Molecular Sciences, University
of Tennessee Center for Health Sciences, Memphis, Tennessee 38163
Received 3 November 2000/Accepted 22 January 2001
 |
ABSTRACT |
We used a photoactivatable, lipophilic reagent,
3'-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine,
to label proteins in the outer membrane of elementary bodies of
Chlamydia trachomatis LGV serovar L2 and mass
spectrometry to identify the labeled proteins. The identified proteins
were polymorphic outer membrane proteins E, G, and H, which were made
late in the developmental cycle, the major outer membrane protein, and
a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.
| |
TEXT |
The cell envelope of
Chlamydia species resembles that of other gram-negative
bacteria, consisting of an outer membrane (OM), an inner membrane (IM),
and a periplasmic space. Two unusual features of the chlamydial
envelope are a deficient and perhaps novel peptidoglycan structure
(reviewed in references 6, 10, and
21) and the presence of a disulfide-bond-cross-linked
major outer membrane protein (MOMP) in the OM and cross-linked
cysteine-rich proteins (CRPs) in the periplasm (reviewed in
reference 12).
The MOMP is the only chlamydial OM protein that has been well
characterized. It is a porin (2, 30), is surface exposed (7, 28), and may play a role in the attachment of
Chlamydia trachomatis to host cells (27, 28).
Genes that potentially encode a family of proteins, referred to as
polymorphic outer membrane proteins (Pomps), have been identified in
the genomes of Chlamydia species: nine Pomp genes
(pmpA to pmpH) are present in the genomes of
C. trachomatis serovars D and L2, 21 genes (pmp1 to pmp21) are in C. pneumoniae, and at least six
genes (Pomp90 and Pomp98 families) are encoded by the genomes of ovine
strains of C. psittaci (15, 20, 26). However,
only two Pomps have been shown to be produced in C. trachomatis and C. pneumoniae, and only four Pomps have
been identified in C. psittaci (11, 16, 20, 22,
29). The functions of the Pomps are not known, nor is it known
when C. trachomatis Pomps are made during the developmental
cycle. Several other OM proteins can be predicted from chlamydial
genomic sequences (15, 26); however, none of these
proteins have been experimentally identified in OMs.
The OM of chlamydiae is poorly characterized, in part because of the
difficulty of growing large quantities of chlamydiae but mainly because
chlamydial OMs cannot be separated from IMs by density gradient
centrifugation. Criteria that have been used to identify chlamydial OM
proteins include surface exposure, as detected by susceptibility to
trypsin or reaction with antibodies following treatment of infectious
elementary bodies (EBs) with these reagents, and insolubility in the
weak anionic detergent sodium lauryl sarcosinate (Sarkosyl). Although
these methodologies have been useful, they can yield deceptive results.
For example, damage to EBs or contamination of EBs with osmotically
fragile reticulate bodies during harvesting and purification can
expose proteins that are not on the surface of EBs, and the failure to observe positive reactions will result if a surface-exposed protein lacks a trypsin-sensitive site or an immunodominant epitope.
Insolubility in Sarkosyl is also subject to misinterpretation.
The technique was originally developed by Filip et al.
(9) to remove cytoplasmic membrane proteins from
well-characterized integral OM proteins of Escherichia coli
and was adapted to chlamydial studies by Caldwell et al.
(4) and Hatch et al. (14). However, the
reason for the differential solubility of IM and OM proteins in
Sarkosyl is not known, and it is likely that some non-OM proteins
fractionate in the Sarkosyl-insoluble fraction and that some OM
proteins are released from OMs by Sarkosyl. For these reasons we chose
to identify C. trachomatis LGV serovar L2 OM proteins on the
basis of their reaction with
3'-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine
([125I]TID), a photoactivatable lipophilic
reagent developed by Brunner and Semenza (3) to label
amino acid side chains and other ligands in the lipophilic environment
of cytoplasmic membranes of eukaryotic cells. Because TID is
lipophilic, it penetrates the OM of gram-negative bacteria, where it
becomes trapped and incapable of passing through the hydrophilic
periplasm and beyond (8).
Identification of OM proteins in C. trachomatis
L2.
EBs were harvested at 48 h after infection of L929 cells,
treated with 1% Nonidet P-40 (Sigma Chemical Co., St. Louis, Mo.) to
eliminate osmotically fragile reticulate bodies, and purified by
centrifugation for 30 min at 80,000 × g on a
three-step gradient of 29, 34, and 40% Hypaque-76 (Nycomed Inc.,
Princeton, N.J.). Purified EBs from 2 × 108
cells were reacted with 25 µCi of [125I]TID,
as previously described (8), and incubated for 30 min at
37°C in the presence or absence of 12 µg of trypsin (type III from
bovine pancreas; Sigma Chemical Co.) in 200 µl of phosphate-buffered saline (pH 7.4), followed by the addition of 24 µg of trypsin inhibitor (type II-O from chicken egg white; Sigma). After the EBs were
washed once in trypsin inhibitor, one half of the EB preparation was
solubilized by heating to 90°C in Laemmli buffer (18)
containing 5% 
-mercaptoethanol and 10 mM dithiothreitol, and the
other half was extracted with 500 µl of 0.5% Sarkosyl (Sigma) in
phosphate-buffered saline for 30 min at 37°C. The Sarkosyl-insoluble fraction was collected by centrifugation (14,500 × g
for 20 min) and washed twice in 500 µl of 0.5% Sarkosyl before it
was solubilized in Laemmli buffer with reducing agents. Proteins in
whole EBs and the Sarkosyl-insoluble fraction of EBs were fractionated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5 to15% gradient gels, and incorporation of
125I into proteins was detected by
phosphorimaging of the dried gels.
The protein profiles of whole EBs and the Sarkosyl-insoluble fraction
of EBs are shown in Fig. 1. A number of
proteins were seen in the Coomassie brilliant blue-stained
Sarkosyl-insoluble fraction (Fig. 1A, lane 3), including the 40-kDa
MOMP and its likely degradation products (28), the 60-kDa
CRP doublet, and several unknown proteins. The unknown proteins
included one of about 120 kDa (protein 1), a poorly resolved doublet of
about 100 kDa (proteins 2 and 3), and a protein of approximately 46 kDa
(protein 4). Only the MOMP, likely degradation products of the MOMP,
and proteins 2 to 4 were labeled extensively with TID; the periplasmic
CRPs, notably, were not labeled (Fig. 1B, lane 3). The TID-labeled,
Sarkosyl-insoluble profile was similar to that of the TID-labeled whole
EB protein profile (Fig. 1B, lanes 1 and 3). Treatment of EBs with
trypsin prior to the preparation of the Sarkosyl-insoluble fraction
reduced the intensity of the protein doublet 2-3, with the appearance
of new peptides, designated tryptic fragments T1 to T3.
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FIG. 1.
Identification of proteins labeled with
[125I]TID by SDS-PAGE. (A) Coomassie-stained 7.5 to 15%
polyacrylamide gel, with prestained protein standard sizes shown in
kilodaltons at each side; (B) phosphorimage of the gel. Lanes: 1, whole
EBs; 2, whole EBs following treatment with trypsin; 3, the
Sarkosyl-insoluble fraction; 4, the Sarkosyl-insoluble fraction of EBs
treated with trypsin. Arrows point to proteins discussed in the text.
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In order to better resolve the high-molecular-weight proteins, the
Sarkosyl-insoluble fraction of EBs was fractionated on a 5.0 to 7.5%
gel and proteins were visualized with the reversible, negative
zinc-imidazole stain described by Castellanos-Serra et al.
(5). Under this condition of analysis, the intensity of protein 2 was reduced when the EBs were treated with trypsin, suggesting that the band may consist of a mixture of two or more proteins (Fig. 2). Gel slices containing
proteins 1 to 3 and trypsin fragment T1 were excised from the gel shown
in Fig. 2 and prepared for analysis by matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry
after in-gel digestion with trypsin, as described by Shevchenko
et al. (25). Protein 4, the MOMP, and trypsin fragments T2
and T3 were similarly analyzed following electrophoresis of the
Sarkosyl-insoluble fraction on 7.5 to 15% gels. Because the C. trachomatis L2 genome has not been completely sequenced and
annotated, we identified proteins with the C. trachomatis D
database located on the Protein Prospector website
(http://prospector.ucsf.edu). Our findings are summarized in Table
1.
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FIG. 2.
Zinc-imidazole-stained 5 to 7.5% polyacrylamide gel of
high-molecular-weight proteins present in the Sarkosyl-insoluble
fraction of EBs. Lanes: 1, Sarkosyl-insoluble fraction of EBs; 2, Sarkosyl-insoluble fraction of EBs treated with trypsin. Sizes of
prestained protein standards in kilodaltons are shown on the left.
Arrows point to bands (numbered the same as in Fig. 1) that were
analyzed by mass spectrometry.
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When EBs were not first treated with trypsin, protein bands 2 and 3 contained a mixture of Pomps E, G, and H. After treatment of EBs with
trypsin, these bands were resolved to a mixture Pomps E and H, with
PompE being the predominant protein in band 2 and PompH being the
predominant protein in band 3; fragment T1 was identified as a
degradation product of PompG. We concluded that the trypsin-sensitive
PompG and the trypsin-insensitive PompE comigrate as band 2, with
contamination with Pomp H (protein 3) due to smearing during
electrophoresis. Pomps G and H were previously identified by Mygind et
al. (22) in the Sarkosyl-insoluble fraction of C. trachomatis L2.
The best identification of band 1 was C. trachomatis D open
reading frame (ORF) 664 (26). The predicted peptide of ORF
664 possesses a potential FHA (forkhead-associated) domain, thought to
be important in protein-protein interactions, and is weakly homologous
to several proteins found in eukaryotes and prokaryotes; however, its
function in chlamydiae is not clear. PSORT analysis (http://psort.nibb.ac.jp) predicted an uncleavable signal sequence and
an IM location for the protein encoded by ORF 664, which is consistent
with the lack of labeling of protein 1 by
[125I]TID. The predicted molecular weight of
the mature protein encoded by ORF 664 is 89,649, considerably less than
the relative molecular weight (120,000) determined by SDS-PAGE. The
predicted pI of the mature protein is 4.32, and the acidic nature of
the protein, including several stretches of high-density negative
charge, may be responsible for its anomalous migration rate.
Interestingly, ORF 664 is weakly paralogous to PompC (E value = 5e-06); however MALDI-TOF analysis failed to identify PompC of either
C. trachomatis D or C. trachomatis L2 (only a
partial sequence is available) in band 1.
Protein band 4 (relative molecular weight of about 46,000) was found to
be a mixture of the MOMP and ORF 623 (26) by MALDI-TOF analysis, with the tryptic peptides with the highest counts being assigned to the MOMP. N-terminal amino acid analysis confirmed the
presence of the MOMP and ORF 623 in protein band 4, with the MOMP being
present in a ratio of about 2 to 1. ORF 623 encodes a predicted
chlamydia-specific protein of unknown function; it possesses a
predicted signal sequence, which was confirmed by the N-terminal
sequence analysis, and the characteristics of an OM protein as
determined by PSORT analysis. The predicted molecular weight of the
mature form is 45,669. The presence of the MOMP in protein band 4 may
be the result of smearing of the abundant 40-kDa MOMP during
electrophoresis or may be due to the presence of a posttranslationally
modified form of the MOMP in the band. Kuo and colleagues
(17) have presented evidence that the 40-kDa MOMP of
C. trachomatis L2 is N-linked glycosylated with a
high-mannose oligosaccharide. It is possible the carbohydrate
identified by Kuo et al. (17) was the result of
contamination of unmodified 40-kDa MOMP with modified 46-kDa MOMP.
However, in preliminary studies, we failed to detect alteration in the
migration rate of any protein in the Sarkosyl-insoluble fraction when
EBs were treated with calf intestinal alkaline phosphatase
(Boehringer-Mannheim; 5 U in 200 µl of phosphatase buffer for 1 h at 37°C), N-glycosylase F (Boehringer-Mannheim; 2 U in
200 µl of phosphate-buffered saline for 1 h at room temperature,
followed by the addition of Triton X-100 to 0.25% and incubation at
room temperature for 30 min and then overnight at 4°C), and
endoglycosylase H (Boehringer-Mannheim; 2 mU, same incubation
conditions as for N-glycosylase F) (data not shown).
In a further attempt to identify the MOMP in band 4, an immunoblot was
treated with monoclonal antibody L2I-45, which recognizes the
serovar-specific epitope in the MOMP of C. trachomatis L2 (1). Only the abundant 40-kDa MOMP band and degradation
products of the MOMP were recognized by the antibody (Fig.
3).
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FIG. 3.
SDS-PAGE (A) and immunoblot (B) analysis of the
Sarkosyl-insoluble fraction of EBs incubated in the presence (lane 2)
and absence (lane 1) of trypsin. The blot was treated with monoclonal
antibody L2I-45, which reacts with the serovar-specific epitope in the
MOMP of C. trachomatis L2 (1). Trypsin
fragment T4 was not analyzed by mass spectrometry; however, the
intensity of the band suggests that it was derived from the MOMP.
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Stage-specific synthesis of Pomps.
We recently demonstrated
that three Pomps of the ovine C. psittaci Pomp90 family and
three other potential Pomps are synthesized cotemporally with the
late-stage-specific CRPs in C. psittaci 6BC
(29). We examined stage-specific synthesis of C. trachomatis L2 Pomps by adding 275 µCi of
[35S]Cys-[35S]Met
(Protein Labeling Mix; NEN, Boston, Mass.) to
150-cm2 monolayers of infected cells (2 × 107) at 14 h postinfection (p.i.) and
preparing a Sarkosyl-insoluble fraction directly from the infected
cells at 16, 18.5, and 23 h p.i. and from the harvested medium
supernatant fluid and cells remaining attached to the flasks at 48 h p.i. Cycloheximide (0.5 µg/ml) was present during the entire
infection to inhibit incorporation of label into host cells.
Incorporation of label into chlamydial proteins in the
Sarkosyl-insoluble fraction was assessed by autoradiography of a dried
polyacrylamide gel, with the amount of material loaded onto the gel
adjusted so that the amount of MOMP (present in relatively constant
amounts at all times p.i.) for each sample was the same (Fig.
4). The results indicated that the Pomps
are made at 18.5 h and later times, whereas synthesis of the
late-stage-specific CRPs (13, 23) is not obvious until
23 h. The 16 h p.i. sample was difficult to interpret; the
large amount of material loaded resulted in a high background and
included proteins which comigrated with the CRPs that were not present
at late times p.i. Nonetheless, it appears that the Pomps, at least
relative to the MOMP, are not made in significant amounts between 14 and 16 h p.i.
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FIG. 4.
Autoradiograph of a 7.5 to 15% polyacrylamide gel
showing incorporation of [35S]Cys-[35S]Met
into the Sarkosyl-insoluble fraction from 14 to 48 h p.i. The
Pomps appear to be made in greater amounts late in the cycle, relative
to the MOMP, but at detectable levels sooner than the
late-stage-specific 60-kDa CRPs.
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Conclusions.
[125I]TID labeling of
C. trachomatis L2 EBs identified three Pomps (E, G, and H),
the MOMP, and another protein or proteins that migrate during SDS-PAGE
with a relative molecular mass of about 46 kDa. It is likely that
additional proteins are present in the OM but in insufficient amounts
to be detected by the amount of TID label that we used. This is almost
certainly the case for PompF, which is encoded immediately upstream of
PompE on the same operon, with the UAA stop codon of PompF located
only 2 bp upstream of the AUG start codon of PompE (26).
Also, in a preliminary, study Lindquist and Stephens (19)
detected transcripts of the genes encoding Pomps B, D, I, and G by
reverse transcription-PCR in C. trachomatis L2. The Pomps
that we detected are encoded by genes that are clustered in the same
region of the genome: the operon encoding Pomps F and E
(pmpFE) and three divergently transcribed genes encoding
Pomps G, H, and I. It is interesting that the divergently transcribed
Pomp genes are cotemporally expressed.
Mass spectrometric and N-terminal amino acid analysis indicated that
the MOMP was a major constituent of the TID-labeled, 46-kDa protein
band, suggesting that the protein may be a posttranscriptionally modified form of the MOMP, perhaps identical to the modified form described by Kuo et al. (17). However, the presence of the
MOMP was not confirmed by immunoblot analysis and we found no evidence of glycosylated or phosphorylated protein in either the 46- or the
40-kDa protein. Alternatively, the TID-labeled protein may be encoded
by ORF 623, a predicted OM protein of unknown function. Definitive
identification of the proteins in the 46-kDa band will require
additional investigation. Whatever the identity of the TID-labeled
46-kDa protein is, there is evidence that it may be antigenic in that
Newhall et al. (24) identified a band of similar size that
reacted on immunoblots of multiple serovars of C. trachomatis with the sera of infected patients. Although the
proteins in the 46-kDa band did not possess a surface-exposed
trypsin-sensitive site, their presence in the OM suggests they may be
useful as diagnostic tools or in the development of a subunit vaccine.
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ACKNOWLEDGMENTS |
We thank Harlan Caldwell for the provision of monoclonal antibody
L2I-45.
This work was supported by Public Health Service grant AI-19570 from
the National Institute of Allergy and Infectious Diseases. Mass
spectrometric studies were carried out in the Stout Neuroscience Laboratory at the University of Tennessee, which is supported by Public
Health Service grant RR 105222 and National Science Foundation grant
DBI 9604633.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Sciences, University of Tennessee Center for Health Sciences, 858 Madison Ave., Memphis, TN 38163. Phone: (901) 448-4664. E-mail: thatch{at}utmem.edu.
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D. Longbottom, and A. J. Herring.
1998.
The major outer membrane protein of Chlamydia psittaci functions as a porin-like ion channel.
Infect. Immun.
66:5202-5207[Abstract/Free Full Text].
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Journal of Bacteriology, April 2001, p. 2686-2690, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2686-2690.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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