Pathogenic bacteria have evolved in close association with their
hosts and have developed sophisticated mechanisms to increase their
chances of survival. Some of these mechanisms exploit normal host
processes and signaling systems thus illustrating the powerful
adaptation of pathogens to their environment. Some of the better
examples of bacterial mimicry relate to mechanisms involved in
colonization of the host. Pathogens such as Bordetella
pertussis, Salmonella typhimurium, enteropathogenic Escherichia coli, and Yersinia species subvert
different host systems to aid in their
colonization(1, 2, 3) .
Staphylococcus
aureus is an important pathogen possessing the potential to
express a variety of different virulence determinants. These include
exotoxins, exoenzymes, and a family of specific protein adhesins
(MSCRAMMs) that mediate the adherence of the organism to host tissues
and extracellular matrix
components(4, 5, 6) . Some of these adhesins
have been characterized in molecular detail, including the fibronectin
adhesins (FnBPA and FnBPB), the collagen adhesin (Cna), and the
fibrinogen adhesin (clumping factor, ClfA) (reviewed in Refs. 5 and 6).
Site-specific mutants have been isolated and compared with parental
strains in both in vitro and in vivo models of
adherence and infection, and there is significant evidence that each of
these adhesins is an important determinant for colonization and
virulence(5, 6) . These proteins have similar
features, including a signal peptide at the NH
terminus
(which is cleaved during secretion across the plasma membrane) and at
the COOH terminus, an LPXTG motif preceding a hydrophobic
membrane spanning region, and a positively charged tail, which are
involved in anchoring the proteins in the cell wall.
S. aureus also expresses several proteins which interact with the immune
system. Protein A is cell wall-associated protein that comprises five
(in some isolates four) repeated units of 58 amino acids, each of which
can bind to the Fc region of IgG(7) . Protein A interferes with
opsonophagocytosis (8) and is an important virulence
factor(9, 10) . Some of the expressed toxins
(enterotoxins A-E, epidermolytic toxin A, and TSST-1) act as
superantigens (11) by binding to human and mouse class II major
histocompatibility complex (MHC) (
)proteins and stimulating
T cells to proliferate nonspecifically(12, 13) . These
superantigens can act as important mediators of toxic shock and other
acute reactions.
Recently, we described a novel 72-kDa surface
protein of S. aureus strain FDA 574 that is capable of binding
to several extracellular matrix proteins, including fibronectin,
fibrinogen, vitronectin, bone sialoprotein, and
thrombospondin(14) . In this paper, we report on the molecular
cloning and the complete nucleotide sequence of the gene encoding this
protein. The deduced protein contains repeated subdomains that share
striking sequence homology with a segment of the peptide binding groove
of the
chain of MHC class II mammalian proteins. We have
designated the protein as Map (MHC class II analogous
protein) and demonstrated that the purified recombinant bacterial
protein specifically recognizes a 15-amino acid residue synthetic
peptide derived from vitronectin.
EXPERIMENTAL PROCEDURES
Materials
E. coli strain JM101 was used
as the bacterial host for plasmids and pBluescript SK(+)
(Stratagene, La Jolla, CA) was used as the cloning vector. S.
aureus strain FDA 574 was obtained from the United States Food and
Drug Administration. Luria agar and broth (LA and LB; Difco) were used
for growth of E. coli and S. aureus strains.
Ampicillin (100 µg/ml) (Sigma) was added when appropriate.
Restriction and modification enzymes were purchased from U. S.
Biochemical Corp. or Life Technologies, Inc.
Isopropyl-
-D-thiogalactopyranoside was purchased from
Life Technologies, Inc. Purified oligonucleotides were obtained from
Advanced DNA Technologies Laboratory, Texas A & M University,
College Station, TX. All other chemicals were molecular biology grade
from Sigma or U. S. Biochemical Corp.
DNA Manipulation and Sequencing
DNA manipulations
were performed using standard procedures(15, 16) . DNA
was sequenced with the Sequenase 2.0 DNA sequencing Kit (U. S.
Biochemical Corp.) and with the thermocycling sequencing method using
the Circum Vent Thermal Cycle Dideoxy DNA Sequencing Kit (New England
Biolabs). Oligonucleotides corresponding to sequences from the
Bluescript clones were used as DNA sequencing primers. The final
sequence was determined from both strands.
Cloning of the map Gene from S. aureus FDA
574
Polyclonal antibodies raised against the purified native Map (14) were used to screen a
gt11 library constructed from
genomic DNA of S. aureus FDA 574. One clone with a
5.7-kilobase pair insert was detected and called pMAP4. The
NH
-terminal amino acid sequences of trypsin generated
fragments of the native Map protein from S. aureus strain
Newman were determined. These sequences were found to be almost
identical to a partial sequence from the S. aureus strain ATCC
25923 OMP-70 (EMBL, accession number 13404; SAHLB.PIR; S04522).
Oligonucleotides were used to PCR amplify a 360 nucleotide fragment
from pMAP4. Additional
gt11 clones were isolated using the random
prime-labeled 200-base pair fragment corresponding to the 3` end of the
369-base pair fragment as a probe in a plaque hybridization assay. DNA
from the
clones was isolated and cloned into the Bluescript
vector.
Expression and Purification of Native and Recombinant
Map
Native Map was extracted from S. aureus FDA 574
cells with 1 M LiCl and purified as described
previously(14) . The map gene was amplified from
chromosomal DNA of S. aureus FDA 574 by PCR, using the primers
5`-CGGGATCCGCAGCTAAGCAAATAGATA-3` and
5`-GCGTCGACGCGGCAAATCACTTCAAGT-3`, containing the restriction cleavage
sites BamHI and SalI, respectively (underlined). The
reaction mixture contained 10 ng of target DNA, 200 pM of
forward and reverse primers, 1.5 mM MgCl
, 2
µM of each dNTP, 10 mM Tris-HCl, pH 9, 50 mM KCl, 0.1% Triton X-100, and 2.5 units of Taq DNA
polymerase (Life Technologies, Inc.). The reaction mixtures were
overlaid with 100 µl of mineral oil and amplified for 30 cycles
consisting of a 1-min denaturation period at 94 °C, a 1-min anneal
temperature at 55 °C, and a 1-min extension period at 72 °C.
After amplification, 10 µl was analyzed by agarose gel
electrophoresis (1% agarose). The PCR product was treated with SDS and
proteinase K prior to digestion and ligation(17) . The product
was digested with BamHI and SalI and ligated with
plasmid pQE30 (Qiagen Inc.) to form pMAP1. This plasmid was transformed
into E. coli strain M15 (Qiagen Inc.). A culture harboring
pMAP1 was grown in LB until it reached an A
of
0.6. Isopropyl-
-D-thiogalactopyranoside was added to a
final concentration of 0.5 mM, and cells were incubated at 37
°C for another 4 h. Cells were harvested, lysed by passage through
a French press (twice at 20,000 pounds/inch
), and the
lysate was centrifuged at 50,000
g for 20 min. The Map
fusion protein, which contained six histidines at the amino terminus,
was localized to the insoluble pellet. This pellet was solubilized in 6 M guanidine hydrochloride, and the Map fusion protein was
purified by metal chelating chromatography (18) under
denaturing conditions. The recombinant Map protein migrated slightly
slower than the native map in SDS-polyacrylamide gel electrophoresis.
Some His
-tagged proteins have been shown to run more slowly
on SDS gels than equivalent untagged proteins(18) .
SDS-Polyacrylamide Gel Electrophoresis and Western Ligand
Blotting
Proteins were fractionated on a 10% polyacrylamide gel
and transferred to a nitrocellulose membrane (Schleicher & Scheull)
for 1 h at 100 V in 20 mM Tris, 150 mM glycine, 20%
(v/v) methanol. Remaining binding sites were blocked for 2 h in 5%
(w/v) non-fat dry milk in TBS. The filters were probed with vitronectin
or with recombinant osteopontin(19) , which had been
I-labeled using the chloramine-T method (20) or
with fibrinogen that had been conjugated to horseradish
peroxidase(21) .
Binding of Recombinant Map to Synthetic
Peptides
Detachable Immulon 1 microtiter wells were coated with
an increasing amount of each peptide overnight at 4 °C in 50 µl
of phosphate-buffered saline (PBS). The plates were washed three times
with PBS containing 0.1% Tween 20 (PBST). 100 µl of a 0.1% bovine
serum albumin solution was added to the wells to block any remaining
binding sites. After 1 h at room temperature, approximately 50,000 cpm
I-labeled recombinant Map in 100 mM CAPS, 20
mM piperazine, pH 9.4, was added to the wells and allowed to
bind for 4 h at room temperature. Following the incubation period, the
wells were washed three times in PBST and then counted in a
counter. The assays were performed in triplicate. Bovine serum
albumin-coated wells served as a control. Results are expressed as mean
± S.D.
Peptides
I-Labeled recombinant Map
was tested for binding to the following peptides: 1) vitronectin
peptide, AKKQRFRHRNRKGYR, charge +8, M
2001.14(22) ; 2) influenza virus peptide, PKYVKQNTLKLAT,
charge +3, M
1503.62(23) ; 3)
cystatin-C peptide, DAYHSRAIQVVRARKQ, charge +4, M
1899.03 (24) ; 4) thrombomodulin peptide,
GTLGGPAQDVDFPEDRIAR, charge +2, M
2014.07, 5)
three thrombospondin peptides ((a) ELTGAARKGSGRRLVKGPD, charge
+5, M
1968.06, (b)
ASLRQMKKTRGTLLALERKDHS, charge +6, M
2539.73, (c) TRDLASIARLRIAKGGVNDN, charge +4, M
2140.26); and 6) syndecan 4 peptide,
RMKKKDEGSYDLGKKPIYKKAPTNEFYA, charge +8, M
3306.39.
RESULTS AND DISCUSSION
Cloning and Nucleotide Sequence of the map Gene from S.
aureus FDA 574
DNA fragments containing overlapping segments of
the map gene were cloned as described under
``Experimental Procedures.'' DNA sequencing of these clones
revealed a single open reading frame of 2067 nucleotides which began
with an ATG at position 71 and ended with a TAA at position 2138 (Fig. 1A). The putative Map protein is composed of 689
amino acids with a predicted molecular mass of 77,040 daltons,
including the signal peptide. The deduced primary structure sequence
revealed a highly unusual protein that is dominated by a 110-amino
acid-long domain that is repeated six times (Fig. 1B).
A 30-amino acid-long signal peptide has been identified at the NH
terminus of the protein. The sequence coding for the 10 amino
acids after the putative signal sequence corresponded exactly to the
amino acid sequence obtained from the NH
-terminal sequence
of the 1 M LiCl extracted and high performance liquid
chromatography-purified native protein from S. aureus FDA 574.
This sequence suggested that the signal peptide cleavage site is
located after the alanine at residue 30. Following the signal peptide,
there is a spacer region of 19 residues followed by the repeated
domains. Typical motifs, associated with cell wall-anchored proteins
and found in most Gram-positive bacterial surface proteins, were not
present in the COOH terminus of the Map protein. This is in agreement
with our previous study(14) , which demonstrated that the Map
protein can be released from the bacterial cell surface by extraction
with 1 M LiCl, suggesting that Map is not covalently anchored
to the cell wall.
Figure 1:
Nucleotide and deduced amino acid
sequence of the map gene of S. aureus FDA 574. A, a single open reading frame of 2067 nucleotides beginning
at position 71 and ending at position 2138 is indicated. The signal
peptide cleavage site was determined experimentally and is indicated by
a vertical arrow. The beginning of each of the 110-amino acid
repeats is indicated by a horizontal arrow. B, Schematic
representation of the 72-kDa Map protein of S. aureus FDA 574.
The protein consists of a signal sequence (S) of 30 amino
acids followed by a unique sequence (U) of 19 amino acids and
six repeated domains (1-6) of approximately 110 amino acids.
Within each domain there is a subdomain (shaded area) of 31
amino acids that is highly homologous to part of the peptide binding
region in MHC class II DR
molecules. The nucleotide sequence has
been deposited in GeneBank(TM) (accession number
U20503).
S. aureus FDA 574 Map Is Homologous to Eukaryotic MHC
Class II Proteins
Within each of the six repeated 110-amino acid
domains, there is a subdomain of 31 residues (Fig. 1B).
These subdomains are highly homologous with the amino-terminal end of
the
chain of many MHC class II proteins from different mammalian
species (Fig. 2). For example, when compared with the
amino-terminal end of the
chain of HLA-DR-9a, the respective
subdomains were 48, 48, 29, 28, 39, and 10% identical. However, if
conservative amino acid substitutions are included, the respective
subdomains were 61, 65, 52, 59, 52, and 45% similar. The
three-dimensional structure of the human MHC antigen HLA-DR1 has been
determined(25) . The binding site for peptides has been located
to a groove composed of 8
sheet strands flanked by two
antiparallel helical walls at the amino-terminal end of the 
heterodimer. It was concluded that conserved as well as highly
polymorphic amino acids participate in peptide binding within the
groove(25, 26) . The region in the
chain of MHC
class II molecules, which is homologous to the subdomains of Map,
contains residues involved in peptide binding.
Figure 2:
Sequence comparison of the six deduced 31
amino acid subdomains from S. aureus FDA 574 Map with part of
the peptide binding region of various MHC class II DR
chain
molecules from human, mouse, and monkey. The amino acid sequences of
the six homologous subdomains were aligned with the NH
terminus of the
chain of MHC class II DR molecules from
human, mouse, and monkey. The identical amino acids are shown in bold.
Western Ligand Blotting Analysis of Purified Native and
Recombinant Map
The remarkable sequence similarity between the S. aureus FDA 574 Map protein and the eukaryotic MHC class II
molecules suggested (a) that the reported binding of Map to
several matrix proteins may be due to the recognition of specific and
possibly similar peptide sequences in these matrix proteins rather than
to other common structural features such as carbohydrates and (b) that Map might be capable of binding to small peptides in
an analogous fashion to MHC class II molecules. To examine these
hypotheses, recombinant Map was expressed in E. coli as a
fusion protein with a polyhistidine tail and purified by
metal-chelating chromatography (Fig. 3, lane 2). Both
native and purified recombinant Map protein bound to labeled
vitronectin, fibrinogen, recombinant osteopontin (Fig. 3, lanes 3-8), and fibronectin (data not shown). The
recombinant osteopontin was analyzed by electrospray mass spectroscopy
and shown to have a mass that corresponds to the predicted amino acid
sequence. Hence, the protein has not been subjected to any
post-translational modifications. Therefore, the binding of
I-labeled recombinant osteopontin, which does not contain
any additional moieties, indicates that Map binding to the matrix
protein involves a specific protein-protein interaction.
Figure 3:
Western affinity blot analysis of native
and purified recombinant S. aureus FDA 574 Map protein. Lanes 1, 3, 5, and 7, purified native Map from S.
aureus FDA 574; lanes 2, 4, 6, and 8,
recombinant affinity-purified Map. Samples were stained with Coomassie
Brilliant Blue R-250 (lanes 1 and 2) or probed with
I-labeled vitronectin (lanes 3 and 4),
horseradish peroxidase-labeled fibrinogen (lanes 5 and 6), and
I-labeled recombinant osteopontin (lanes 7 and 8). Migration distances and molecular
masses (kDa) of the standard proteins are indicated by the numbers on the left.
Binding of Map to a Synthetic Peptide
To
investigate whether Map is capable of binding to short peptides,
purified recombinant Map was labeled with
I and studied
for binding to a panel of eight immobilized synthetic peptides in an
enzyme-linked immunosorbent assay type assay. Radiolabeled Map bound to
a 15-amino acid peptide (residues 347-361 of vitronectin) (Fig. 4). This sequence contains the heparin binding domain of
vitronectin, and this peptide was previously found to partially inhibit
the adherence of S. aureus to immobilized
vitronectin(22) . The binding of
I-labeled Map to
immobilized peptide increased when increasing amounts of peptide were
used to coat the microtiter wells (Fig. 4). In a Western
affinity blot assay, binding of the
I-labeled vitronectin
peptide to both native and recombinant Map could also be detected (data
not shown). The Map protein failed to bind to the other seven peptides
tested. Some of these peptides were of similar size and charge as the
vitronectin peptide, suggesting a specific interaction between Map and
the vitronectin peptide.
Figure 4:
Binding of
I-labeled
purified recombinant S. aureus FDA 574 Map protein to
synthetic peptides. Purified recombinant S. aureus FDA 574 Map
bound to a vitronectin peptide (AKKQRFRHRNRKGYR) (
) but failed to
bind to seven other peptides (listed under ``Experimental
Procedures''), including an influenza virus peptide
(PKYVKQNTLKLAT) (
) and a rat cystatin-C peptide
(DAYHSRAIQVVRARKQ) (
).
Previous studies have shown that S.
aureus can bind a number of different host proteins (reviewed in (5) ). In some cases, the bacterial proteins have been
characterized and shown to be unique proteins which are different from
the Map protein. However, in many cases the bacterial proteins have not
been identified and the Map protein could be responsible for some of
the observed binding activities. The Map protein is, to our knowledge,
the first identified structural and functional bacterial
``analog'' to mammalian MHC class II molecules and may
potentially interfere with the immune defense system of the host. The
mechanism and consequence of such interference is at this point
speculative and is the topic of future investigations.