Localization of the Mycoplasma pneumoniae Cytadherence ...

Vol. 173, No. 3

JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1041-1050

0021-9193/91/031041-10$02.00/0 Copyright © 1991, American Society for Microbiology

Localization of the Mycoplasma pneumoniae CytadherenceAccessory Proteins HMW1 and HMW4 in the Cytoskeletonlike Triton Shell MARLA K. STEVENS AND DUNCAN C. KRAUSE* Department of Microbiology, University of Georgia, Athens, Georgia 30602 Received 24 July 1990/Accepted 19 November 1990

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the ifiamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae ceUs incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the ifiamentous extensions of the mycoplasma cells.

variant lacks proteins A, B, C, and P1. Reacquisition of those proteins missing in the variants results in spontaneous reversion to the cytadherence-positive phenotype (25). The cytadhesin P1 localizes in high concentrations at the terminal attachment organelle of wild-type M. pneumoniae (1, 5, 13, 20) and is also detected less densely elsewhere on the mycoplasma surface (1). The cytadherence-negative variants described above which lack HMW1 through 4 (class I; 24) possess P1 scattered across the cell surface but lack the ability to cluster P1 in the tip and lack the characteristic tip morphology observed in wild-type M. pneumoniae (1). These observations imply a role for HMW1 through 4 in maintaining the architecture of the tip organelle necessary for P1 clustering. Ultrastructural studies with scanning and transmission electron microscopy have revealed that treatment of M. pneumoniae cells grown on grids with the nonionic detergent Triton X-100 dissolves the cell membrane, leaving a rodlike tip structure and a network of filamentous strands (14, 31) analogous to cytoskeletons in a variety of eukaryotic cells (12, 32). The anchoring of P1 might involve the interaction of one or more of HMW1 through 4 with this cytoskeletonlike structure. Recently, Kahane et al. initiated a biochemical characterization of the M. pneumoniae cytoskeletal network, comparing the protein profiles of the Triton-soluble and -insoluble fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting (22). That work revealed that a portion of the cytadhesin P1 population localizes in the detergent-insoluble fraction, termed the triton shell (22). In the present study, we expand upon this finding and methodology, combining biochemical techniques and electron microscopy to examine the cellular location of HMW1 and HMW4 and their function in cytadherence.

The cell wall-less bacterium Mycoplasma pneumoniae is a pathogen of the human respiratory tract that causes tracheobronchitis and primary atypical pneumonia (10). Attachment of viable mycoplasmas to the respiratory epithelium (cytadherence) is a prerequisite for tissue colonization and subsequent disease production (10, 11, 21, 34). M. pneumoniae cytadherence results from the interaction of trypsinsensitive moieties, including the 170-kDa surface protein P1 on mycoplasmas (21), with neuraminidase-sensitive and -insensitive sites on host cells (21, 34-36). Brief trypsinization of mycoplasmas results in the simultaneous removal of P1 and a decrease in the adherence of virulent M. pneumoniae to hamster tracheal rings (21). Furthermore, in vitro binding studies with detergent-solubilized mycoplasma preparations and glutaraldehyde-fixed hamster tracheal epithelial cells have demonstrated specific binding of P1 to host cells (23), and P1-specific antibodies block hemadsorption and adherence to tracheal epithelium (13, 20, 24). M. pneumoniae cytadherence is a complex, multifactorial process, however, and the surface protein P1 alone is not sufficient for mycoplasma attachment to host cells (2, 16-18, 23, 26). Characterization of the protein profiles of spontaneously arising cytadherence-negative variants by one- and two-dimensional gel electrophoreses established four variant classes lacking specific proteins that were present in the cytadherent parent strain (17, 26). Class I variants lack high-molecular-weight proteins 1, 2, 3, and 4 (HMW1, HMW2, HMW3, and HMW4, respectively), ranging from 140 to 215 kDa; class II variants lack a 32-kDa surface protein which, like P1, appears to function as an adhesin (2); class III variants lack three proteins designated A, B, and C (72, 85, and 37 kDa, respectively); and a single class IV *

Corresponding author. 1041

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STEVENS AND KRAUSE

MATERIALS AND METHODS

Organisms and culture conditions. These studies were carried out with virulent M. pneumoniae M129 B18 (28) and a cytadherence-deficient variant lacking HMW1 through 4 (class I isolate 2; 26). Mycoplasma cultures were grown in 150-cm tissue culture flasks containing approximately 50 ml of Hayflick medium at 37°C (19). Organisms were harvested in the log phase when the phenol red indicator in the growth medium became orange (48 to 72 h after inoculation). Since wild-type organisms attach to plastic (8), the cell, "monolayer" was washed three times with phosphate-buffered saline (PBS [pH 7.2]), scraped, and collected by centrifugation at 9,500 x g for 20 min. The class I variant lacks the ability to adhere to plastic; it was collected by centrifugation of the culture medium at 9,500 x g for 20 min and washed three times with PBS. Protein content was determined with a modified Lowry assay (30). Peptide mapping analysis of mycoplasma HMW1 and HMW4. The structural relatedness of HMW1 and HMW4 was studied by peptide mapping as described previously (9, 25) with a slight modification. Total M. pneumoniae protein was separated in the first dimension by SDS-PAGE, and individual lanes containing a complete mycoplasma protein profile were sliced immediately from the first-dimension slab gel without staining and equilibrated for 30 min in PM buffer (0.125 M Tris hydrochloride [pH 6.8], 0.1% SDS, 1 mM EDTA). The gel slices were applied directly to a second slab gel (3% stacking and 12% separating gels), overlaid with 50 jig of ot-chymotrypsin (Sigma Chemical Co., St. Louis, Mo.), and electrophoresed until samples were 0.5 cm from the top of the separating gel. The current was turned off for 30 min to allow proteolysis, and electrophoresis was resumed. Peptides were visualized by silver staining (33) or blotted to nitrocellulose paper (38) and probed with rabbit anti-HMW1 serum at a 1:100 dilution as described below. Triton X-100 extraction. Mycoplasmas were suspended at approximately 1 mg of protein per ml in 20 mM Tris hydrochloride (pH 7.5)-150 mM NaCl-1 mM phenylmethylsulfonyl fluoride, and Triton X-100 (Sigma) was added to 2% (vol/vol). After 30 min of incubation at 37°C, the samples were centrifuged for 30 min at 15,000 x g and 4°C to separate the soluble and insoluble fractions. The insoluble fraction was resuspended in fresh Tris hydrochloride-NaCl-phenylmethylsulfonyl fluoride-Triton X-100 and recentrifuged. Each fraction was subjected to SDS-PAGE (27) with slab gels composed of 3% stacking and 5% separating gels. Proteins were visualized by silver staining. Sucrose density gradient centrifugation. Wild-type M. pneumoniae cells were solubilized in Triton X-100 as described above. After 30 min of incubation at 37°C, the samples were either loaded directly onto a 25 to 50% sucrose gradient or centrifuged to isolate the detergent-insoluble fraction, which was subsequently loaded onto the gradient. The samples were centrifuged for 50 min at 5,000 rpm (3,086 x g) and 4°C. Fractions were removed sequentially from the top of the gradient, dialyzed in 10 mM Tris hydrochloride0.02% SDS (pH 6.8), and dried in a speed vacuum. The samples were resuspended in 50 ,ul of Laemmli sample buffer (27) modified to contain only 10 mM Tris hydrochloride. Proteins present in each fraction were analyzed by SDSPAGE and visualized by silver staining. Anti-HMW1 serum. All protocols with vertebrate animals were reviewed and approved by the institutional animal care and use committee. Serum was collected from rabbits immunized with HMW1 purified by preparative SDS-PAGE as

J. BACTERIOL.

described by Tjian et al. (37). The immunoglobulins (Ig) were purified on an affinity column containing Staphylococcus protein A agarose beads (Bio-Rad Laboratories, Richmond, Calif.), Bound antibodies were eluted with 100 mM glycine (pH 3.0), after which the pH was neutralized with Tris hydrochloride (pH 7.5)-1% bovine serum albumin. Serum specificity was examined by Western immunoblotting. Immunogold labeling of thin sections. Wild-type and class I variant M. pneumoniae cells were grown to the log phase and harvested as described above. The pellets were washed once in 0.1 M sodium cacodylate buffer (pH 7.2) and fixed in 1% glutaraldehyde-1% paraformaldehyde-0.1% picric acid in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at 4°C. The cells were washed in sodium cacodylate buffer to remove excess fixative, dehydrated in alcohol, and embedded in LR White resin. Thin sections (silver-gold) were cut with a diamond knife on a Sorvall. microtome and placed on carbon-coated, Formvar-coated 400 mesh nickel grids. Grids were etched for 30 min with saturated sodium meta-periodate, blocked in goat serum diluted 1:10 in 0.2 M Tris hydrochloride (pH 8.2)-saline-1% bovine serum albumin (TBS-BSA) for 30 min, washed with TBS-BSA, and incubated overnight at 4°C in the appropriate primary antibody diluted in TBS-BSA. Grids were washed with TBS-BSA to remove excess antibody and were incubated in a 1:20 dilution of goat antirabbit IgG conjugated to 5-nm colloidal gold (Janssen Life Sciences, Piscataway, N.J.) for 1 h at room temperature. The grids were counterstained in 2% aqueous uranyl acetate-4.4% lead citrate and examined in a JEOL Cx 100 electron microscope at 80 keV. Immunogold labeling of whole cells. Wild-type M. pneumoniae cells were grown to the log phase as described above. The cells were scraped into fresh Hayflick medium and passed through a 25-gauge needle and a 1.2-,um-pore Acrodisc filter (Gelman Sciences, Ann Arbor, Mich.) to disperse cell aggregates. The cell suspensions were placed in the wells of a tissue culture dish containing UV-sterilized, carbon-coated, Formvar-coated 400 mesh nickel grids. The cells were incubated for 4 h at 37°C to allow attachment to the grids. The grids were removed, washed gently with PBS, fixed for 30 mim, and probed with antibodies as described above for thin sections, with the omission of etching with sodium meta-periodate. Immunogold labeling of Triton X-100-extracted whole cells. Wild-type M. pneumoniae cells were grown on electron microscope grids as described above, washed with PBS, and extracted with 20 mM Tris hydrochloride (pH 7.5)-150 mM NaCl-2% Triton X-100 with gentle mixing. The tissue culture dish containing the grids was partially submerged in a water bath for 30 min at 37°C, after which the grids were washed with PBS, fixed for 15 to 30 min, and immunolabeled as described above for the whole mycoplasmas. Scanning electron microscopy. Wild-type M. pneumoniae cells grown on sterile glass coverslips were either fixed immediately as described above or extracted with 2% Triton X-100 as described above and then fixed. After fixation and dehydration (in alcohol), the coverslips containing the cells were critical point dried in liquid C02, gold sputter coated, and examined with a Philips 505 electron microscope at 20 keV. RESULTS Peptide mapping analysis of HMW1 and HMW4. Peptide mapping studies have demonstrated that HMW1 through 3 are structurally distinct polypeptides (25). However, suffi-

CYTOSKELETAL ELEMENTS AND M. PNEUMONIAE ADHERENCE

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FIG. 1. Peptide mapping analysis of HMW1/4. Mycoplasma proteins were separated in the first dimension by SDS-PAGE, and an individual lane containing a complete protein profile (unfixed and unstained) from the first-dimension gel was placed along the top of a second slab gel. Chymotrypsin was added, and digestions were carried out as detailed in Materials and Methods. The peptides generated by proteolysis were separated by SDS-PAGE in the second slab gel and visualized by silver staining (A) or electroblotted to nitrocellulose and probed with anti-HMW1 serum at a dilution of 1:100 (B). Peptide profiles of HMW1 through 4 are indicated, as are the directions of electrophoresis in the first and second dimensions. The solid dark bands near the bottom of the silver-stained gel (A) correspond to the protease.

cient quantities of HMW4 were not available to permit analysis with that protocol. When antiserum raised against HMW1 was evaluated for its specificity by radioimmunoprecipitation, reactivity was observed with both HMW1 and HMW4 (25). It was not clear at the time whether this reactivity resulted from a failure to completely separate HMW1 and HMW4 by SDS-PAGE when attempting to purify the former for immunization in rabbits or whether HMW1 and HMW4 are immunologically related (25). To explore this second possibility, we modified the peptide mapping protocol to eliminate the need to stain or excise the protein bands from the first-dimension gel. An M. pneumoniae protein profile generated by SDS-PAGE in one dimension was subjected to proteolytic digestion and then analyzed by SDS-PAGE in the second dimension, perpendicular to the first. When visualized by silver staining (Fig. 1A), a characteristic peptide profile was generated for each protein band that had been resolved in the first-dimension gel. The profiles for HMW1 through 3 shown here are comparable to

those reported previously (25). The profile for HMW4 was only barely visible by silver staining (Fig. 1A); however, when a corresponding Western blot was probed with antiHMW1 serum (Fig. 1B), the HMW1 and HMW4 profiles were clearly detectable and appeared nearly identical. While the patterns generated did not appear to be the result of complete proteolysis, on the basis of the fragment sizes, we were unable to increase the extent of digestion significantly by extending the digestion time or increasing the amount of protease used. Nevertheless, the virtually identical peptide patterns observed with HMW1 and HMW4 suggest that these proteins are structurally related. Antiserum specificity. The Ig used for immunoelectron microscopy were purified from polyclonal preimmune or immune serum made against HMW1. One consequence of using polyclonal sera is the presence of antibodies reactive to proteins other than HMW1 and HMW4 (hereafter referred to as HMW1/4). However, these antibodies could be effectively diluted out, yielding a specific probe for further studies. Figure 2 shows immunoblot profiles of wild-type and class I variant M. pneumoniae total proteins probed with preimmune or anti-HMW1/4 Ig. A 1:7,500 dilution of the anti-HMW1/4 Ig reacted specifically with only two proteins (215 and 210 kDa) in the wild-type profile (Fig. 2, lane e); these were absent in the class I variant profile (Fig. 2, lane f). Antibodies which reacted to proteins other than HMW1/4 at a 1:100 dilution (Fig. 2, lane c) were effectively diluted out at a 1:7,500 dilution. The faint reactivity of the Ig with proteins in the class I variant profile (Fig. 2, lanes d and f) was probably due to the presence of revertant organisms. The preimmune Ig possessed only trace reactivity to M. pneumoniae proteins (Fig. 2, lanes a and b). Immunogold labeling of thin sections. Figure 3 is a composite of electron micrographs of immunogold-labeled sections of wild-type and class I variant M. pneumoniae cells. Four features were consistently observed when wild-type mycoplasmas were incubated with a 1:100 dilution of antiHMW1/4 Ig (Fig. 3A). (i) Gold particles appeared densely

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VOL. 173, 1991


clustered along the filamentous extension of the cell; (ii) clusters of gold particles were also observed in the smaller spherical cell sections and may represent cross-sections or glancing sections of (i), depending upon the orientation of the cell during sectioning; (iii) most gold particles appeared to label within the cell interior, although some appeared around the cell periphery, suggesting a membrane location; and (iv) labeling was in many cases confined to only a limited number of cells in a given field (Fig. 3A). This same labeling pattern was observed at a 1:1,000 dilution of anti-HMW1/4 Ig (Fig. 3B), although with a lesser degree of labeling. No specific labeling was visualized when a 1:100 dilution of anti-HMW1/4 Ig was incubated with sections of a class I variant (Fig. 3C) or when preimmune Ig at a 1:100 dilution was incubated with sections of wild-type M. pneumoniae (Fig. 3D), confirming the specificity of anti-HMW1/4 Ig. In addition, sections of wild-type mycoplasmas incubated with only the secondary antibody as a control failed to show any retention of the label (data not shown). These data suggest a predominantly intracellular location for HMW1/4 in wildtype M. pneumoniae. Immunogold labeling of whole cells. Figure 4A shows M. pneumoniae whole cells grown on grids and labeled with anti-HMW1/4 Ig at a 1:100 dilution. The gold particles clustered in specific regions on the surface of the cells, predominately along the filamentous extensions and near the tips of these filaments. Some labeling was also apparent on the body of the cells. Wild-type whole cells incubated with preimmune Ig at a 1:100 dilution showed some labeling (Fig. 4B), although to a much lesser extent than did cells incubated with anti-HMW1/4 Ig. Labeling was absent on wildtype whole cells probed with a 1:500 dilution of preimmune Ig (Fig. 4C), while a 1:500 dilution of anti-HMW1/4 Ig still labeled predominately along the filamentous extensions of the cells (Fig. 4D). Wild-type cells incubated with only the secondary antibody retained no label (data not shown). Unfortunately, class I variant cells would not freely attach to electron microscope grids and therefore could not be used as a parallel control. However, the absence of specific labeling of sections of the class I variant with anti-HMW1/4 Ig (Fig. 3C) and the lack of significant detection of HMW1/4 on immunoblots probed with anti-HMW1/4 Ig (Fig. 2, lanes d and f) seemed to minimize the need for this control. Localization of HMW1/4 to the Triton X-100-insoluble fraction of M. pneumoniae. The nonionic detergent Triton X-100 dissolves eukaryotic cell membranes, leaving an insoluble mass of protein filaments (32). These residual filaments retain the same general shape as do the original cells and usually constitute an intracellular cytoskeleton. Figure 5A and B are scanning electron micrographs of wild-type M. pneumoniae cells grown on glass coverslips before and after extraction with Triton X-100. Unextracted M. pneumoniae cells exhibited typical filamentous and coccoid morphologies (Fig. 5A). When the cells were extracted with Triton X-100, the insoluble residue remained in the same orientation as in the unextracted M. pneumoniae cells (Fig. 5B). Extracted cells had a reduced appearance. Membrane removal exposed aggregates of filaments or individual filaments, consistent with results described previously by others (14). When a suspension of mycoplasmas is extracted with Triton X-100, two fractions that can be separated and analyzed by SDS-PAGE result: a supernatant containing the solubilized portions of the cell, such as individual proteins and lipids, and a pelletable material containing insoluble components of the cell, a mass of interconnecting proteins that form the intracellular scaffolding visible by scanning and

1045

transmission electron microscopy (14, 31). Approximately 20 major proteins appeared to constitute the detergentinsoluble fraction of wild-type and class I variant M. pneumoniae cells examined by SDS-PAGE (Fig. 6, lanes c and d, respectively). As reported by Kahane et al. (22), the major adhesin P1 partitioned in both the detergent-soluble and -insoluble fractions of the wild-type and class I variant M. pneumoniae cells (Fig. 6, lanes c to f). The detergentinsoluble fraction from wild-type mycoplasmas was enriched for protein bands comigrating with the adherence-accessory proteins HMW1 through 4 (Fig. 6, lane c). These bands were absent or present only in very reduced amounts in the corresponding fraction from a class I variant (Fig. 6, lane d). Because of the reversible nature of M. pneumoniae cytadherence phase variation (25), the presence of revertant organisms often allowed the detection of small quantities of the high-molecular-weight proteins in variant profiles. The detergent-soluble fraction of wild-type M. pneumoniae contained small quantities of HMW1 and HMW2 (Fig. 6, lane e), which were absent in the corresponding detergent-soluble fraction of the class I variant (Fig. 6, lane f). While HMW4 was only barely detectable in the silver stains in Fig. 6, the more sensitive Western immunoblot analysis with antiHMW1/4 Ig confirmed the partitioning of this protein in the detergent-insoluble fraction (data not shown). These data suggest that HMW1/4 constitute part of the cytoskeletonlike matrix of M. pneumoniae. The partitioning of HMW1 to HMW4 in the detergentinsoluble fraction of M. pneumoniae could be due to their individual insolubilities or to their participation in a multiprotein complex that could serve as an intracellular scaffolding. To distinguish between these two possibilities, we subjected detergent-solubilized mycoplasmas and the detergent-insoluble fractions of these to sucrose density gradient centrifugation (Fig. 7). After each sample was centrifuged on the sucrose gradients, fractions were removed from the top to the bottom and processed for SDS-PAGE. When mycoplasmas were solubilized with Triton X-100 but not separated into soluble and insoluble fractions and subsequently analyzed in this manner (Fig. 7, left half), the profile of proteins at the top of the gradient (T) appeared identical to that of the detergent-soluble fraction shown in Fig. 6, lane e. Likewise, the profile of proteins at the bottom of the gradient (B) appeared identical to that of the detergent-insoluble fraction shown in Fig. 6, lane c. When the detergentinsoluble fraction was isolated and analyzed by sucrose density gradient centrifugation (Fig. 7, right halft), most of the proteins, including HMW1 through 4 and P1, cosedimented to the bottom of the gradient (B). This profile was likewise almost identical to that shown in Fig. 6, lane e. Some of the proteins appeared at both the top and the bottom of the gradients, suggesting partial removal as a result of centrifugation. Identical results were obtained when Triton X-100 was included in the sucrose gradients or when samples were centrifuged under a variety of conditions. These results suggest that HMW1 through 4 are components of a large complex of interconnected proteins. Immunogold labeling of Triton X-100-extracted cells. To visualize HMW1/4 in the triton shell, we grew M. pneumoniae cells on grids, extracted them with Triton X-100, and examined the remaining residues by immunoelectron microscopy. Wild-type M. pneumoniae cells incubated with anti-HMW1/4 Ig diluted 1:5,000 (Fig. 8A) or 1:10,000 (data not shown) bound gold in the same regions as did whole cells (Fig. 4A and D). Distinct clusters of gold particles were present along the filamentous extensions of the cells. Fur-

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FIG. 5. Scanning electron microscopy of wild-type M. pneumoniae cells before and after Triton X-100 extraction. Wild-type M. pneumoniae cells were grown on glass coverslips, fixed as intact cells (A) or extracted with 2% Triton X-100 (B), and prepared for election microscopy.

thermore, membrane dissolution with Triton X-100 appeared to expose many more epitopes than were accessible on whole cells, on the basis of the labeling intensity with much more dilute anti-HMW1/4 Ig. Wild-type cells incubated with a 1:1,000 dilution of preimmune Ig failed to show any significant labeling (Fig. 8B). Furthermore, cells incubated with only the secondary antibody retained no label (data not shown). These data confirm the biochemical localization of HMW1/4 to the detergent-insoluble complex. DISCUSSION The M. pneumoniae cytadhesin protein P1 is necessary but not sufficient for adherence to host cells (17, 18, 25). Cytadherence-deficient variants lacking HMW1 through 4 fail to cluster P1 at the attachment organelle, although P1 is still present along the surface of these cells. One can speculate that one or more of HMW1 through 4 may play a role in controlling the distribution of P1 on the cell surface. To fulfill such a role, these proteins may be components of a mycoplasma cytoskeleton responsible not only for P1 orientation but also for changes in cell shape, division, and movement. We have demonstrated that HMW1 and HMW4 were structurally closely related, on the basis of similarities in their peptide profiles. Whether HMW1 and HMW4 are the products of duplicated genes or share a precursor-product relationship is not clear. Pulse-chase experiments to test the latter have proven inconclusive (data not shown). We have cloned the gene for HMW1 (22a), and nucleotide sequence

analysis of this gene should clarify the relationship between HMW1 and HMW4. Two conclusions are apparent from the labeling pattern on thin sections of wild-type M. pneumoniae with anti-HMW1/4 Ig. First of all, the distribution of the label was nonrandom. In any given field there were some cell sections that labeled intensely, while in other cell sections absolutely no labeling was observed. When there was labeling, it was confined to filamentous portions of the mycoplasmas or what appeared to be cross-sections thereof. Unlabeled cells may be a consequence of (i) nonrandom distribution of HMW1/4 in M. pneumoniae; (ii) inaccessible antigenic sites within the resin; (iii) variability in the degree of fixation within the samples, resulting in loss of antigenicity; or (iv) cell growth stage, reflecting potential variability in expression or distribution of HMW1/4. Antibody labeling data obtained with whole cells and triton shells, however, supported the first option, i.e., that HMW1/4 are not randomly distributed in the cells. In addition, most of the gold particles appeared to localize in the cell interior, with only limited surface exposure. Taken together, these observations suggest that HMW1/4 are membrane proteins with predominately an intracellular exposure and are located primarily along the filamentous extensions of mycoplasma cells. Antibody labeling of whole cells was likewise confined primarily to the filamentous extensions of the cells, notably along the filamentous extensions of the cells and near the tip structures. Morphological studies have indicated that M. pneumoniae cells possess a leading filamentous tip structure (used for attachment to animal cells), a cell body, and a

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FIG. 6. SDS-PAGE analysis of Triton X-100-fractionated wildtype and class I variant M. pneumoniae cells. Pellets of wild-type and class I variant M. pneumoniae cells were solubilized with Triton X-100, separated into detergent-soluble and -insoluble fractions by centrifugation, and analyzed by SDS-PAGE. Protein profiles were visualized by silver staining. Lanes: a, wild-type M. pneumoniae prior to extraction; b, class I variant M. pneumoniae prior to extraction; c, wild-type detergent-insoluble fraction; d, class I variant detergent-insoluble fraction; e, wild-type detergent-soluble fraction; f, class I variant detergent-soluble fraction. Protein standards are shown on the right. Arrows on the left denote HMW1 through 4 and P1.

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filamentous tail (6, 7). It is not clear for the cells in Fig. 4 which ends are attachment filaments and which ends are tails. For some cells, both filaments were labeled with anti-HMW1/4 Ig (Fig. 4A and D). The possibility exists that these cells were undergoing binary fission, giving rise to two cells that were attached by their tails and had their attachment filaments extending in opposite directions. HMW1/4 partitioned in the cytoskeletonlike detergentinsoluble fraction, which consisted of at least 20 different proteins, including a portion of the P1 population. The cosedimentation of these proteins during sucrose density gradient centrifugation suggests that they form an interconnected, high-molecular-weight network. These biochemical findings were confirmed by transmission electron microscopy, as HMW1/4 were visualized by immunogold labeling along the filamentous extensions of detergent-extracted cells (Fig. 8A). Antiserum prepared against HMW1 possessed reactivity with several other mycoplasma proteins. This reactivity was easily diluted out, yielding a reagent specific for HMW1/4. However, because of differences in the sensitivities of Western immunoblot analysis and immunoelectron microscopy, it is difficult to establish correlations between the two procedures. By using the highest possible dilution of antibodies that yields interpretable labeling, we have attempted to circumvent this complication. It is important to note that the antigenic and structural relatedness of the two proteins precludes our making a distinction between them in immunoelectron micrographs. The cytadhesin P1 is densely clustered at the attachment organelle (1, 13, 20) and scattered less densely elsewhere along the mycoplasma surface (1). A peripheral location of

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FIG. 7. SDS-PAGE profiles following sucrose density gradient centrifugation of Triton X-100-solubilized M. pneumoniae or the detergent-insoluble fraction of M. pneumoniae. Mycoplasmas were solubilized with Triton X-100 and either loaded directly onto a 25 to 50% sucrose gradient or first separated into the soluble and insoluble fractions. The insoluble fraction was loaded onto a separate gradient. After centrifugation, aliquots were removed from the top to the bottom of the gradient, dialyzed, dried in a speed vacuum, and analyzed by SDS-PAGE. Proteins were visualized by silver staining. SOLUBILIZED denotes M. pneumoniae cells that were solubilized with Triton X-100 at 37°C for 30 min and loaded directly onto the gradient. INSOLUBLE denotes M. pneumoniae cells that were solubilized with Triton X-100 and separated into fractions by centrifugation as in Fig. 6 before the insoluble fraction was loaded onto the gradient. T, Top fraction of each gradient. B, Bottom fraction of each gradient. Protein standards are shown on the left. Arrows on the right denote HMW1 through 4 and P1.


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FIG. 8. Immunoelectron microscopy of wild-type M. pneumoniae extracted with Triton X-100. Wild-type M. pneumoniae cells were grown on Formvar-carbon-coated nickel grids, extracted with Triton X-100, and incubated with either anti-HMW1/4 Ig at 1:5,000 (A) or preimmune Ig at 1:1,000 (B) and with 5-nm goat antirabbit IgG-colloidal gold. Arrowheads indicate regions of labeling along the filamentous cell extensions.

HMW1/4 in the mycoplasma filaments and transmembrane orientation within the cell would permit their interaction, either directly or indirectly, with P1, possibly contributing to the polar clustering of P1 necessary for attachment. An analogy can be drawn between the cytoskeletonlike structure in M. pneumoniae cells and that found in eukaryotic cells. For example, the membrane skeleton of an erythrocyte is defined as the set of proteins that remains after extraction with Triton X-100 (3, 4, 29). These proteins appear as "irregularly oriented filaments dotted with globular protrusions" (29), a description extremely similar to that given for the M. pneumoniae intracellular network (14, 31). In the erythrocyte cytoskeleton, the integral membrane protein band 3 spans the phopholipid bilayer in such a way that its cytoplasmic domain attaches to ankyrin, which is linked to spectrin and the cytoskeleton (3, 4, 15, 29). Likewise, in M. pneumoniae, HMW1/4 may contribute to the anchoring of P1 to the cytoskeleton. Further biochemical and electron microscopic analyses will be necessary to develop a greater understanding of the nature of this interaction. ACKNOWLEDGMENTS

This work was supported by Public Health Service research grant

A123362 and research career development award A100968 from the National Institute of Allergy and Infectious Diseases and by a Faculty Research Grant from the University of Georgia Research Foundation to D.C.K. We thank P. Wyrick, M. Farmer, J. Paulin, and W. Steffens for

advice and technical assistance with the immunoelectron microscopy and Pat Bates for expert word-processing skills. REFERENCES 1. Baseman, J. B., R. M. Cole, D. C. Krause, and D. K. Leith. 1982. Molecular basis for cytadsorption of Mycoplasma pneumoniae. J. Bacteriol. 151:1514-1522. 2. Baseman, J. B., J. Morrison-Plummer, D. Drouillard, B. PuleoScheppke, V. V. Tryon, and S. C. Holt. 1987. Identification of a 32 kilodalton protein of Mycoplasma pneumoniae associated with hemadsorption. Isr. J. Med. Sci. 23:474-479. 3. Bennett, V. 1985. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu. Rev. Biochem. 54:273-304. 4. Bennett, V., and P. J. Stenbuck. 1979. The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes. Nature (London) 280:468-473. 5. Biberfeld, G., and P. Biberfeld. 1970. Ultrastructural features of Mycoplasma pneumoniae. J. Bacteriol. 102:855-861. 6. Boatman, E. S. 1979. Morphology and ultrastructure of the Mycoplasmatales, p. 63-102. In M. F. Barile and S. Razin (ed.), The mycoplasmas, vol. 1. Academic Press, Inc., New York. 7. Bredt, W. 1979. Motility, p. 141-155. In M. F. Barile and S. Razin (ed.), The mycoplasmas, vol. 1. Academic Press, Inc., New York. 8. Bredt, W., J. Feldner, and I. Kahane. 1981. Adherence of mycoplasma to cells and inert surfaces: phenomena, experimental model, and possible mechanisms. Isr. J. Med. Sci. 17:586588. 9. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in so-

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STEVENS AND KRAUSE

dium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. 10. Colier, A. M., and W. A. Clyde, Jr. 1971. Relationships between Mycoplasma pneumoniae and human respiratory epithelium. Infect. Immun. 3:694-701. 11. Collier, A. M., and W. A. Clyde, Jr. 1974. Appearance of Mycoplasma pneumoniae in lungs of experimentally infected hamsters and sputum from patients with natural disease. Am. Rev. Respir. Dis. 110:765-773. 12. Crossley, R., and D. V. Holberton. 1983. Characterization of proteins from the cytoskeleton of Giardia lamblia. J. Cell Sci. 59:81-103. 13. Feldner, J., U. Gobel, and W. Bredt. 1982. Mycoplasma pneumoniae adhesin is localized to the tip structure by monoclonal antibody. Nature (London) 298:765-767. 14. Gobel, U., V. Speth, and W. Bredt. 1981. Filamentous structures in adherent Mycoplasma pneumoniae cells treated with nonionic detergents. J. Cell Biol. 91:537-543. 15. Haest, C. W. M. 1982. Interactions between membrane skeleton proteins and the intrinsic domain of the erythrocyte membrane. Biochim. Biophys. Acta 694:331-352. 16. Hansen, E. J., R. M. Wilson, and J. B. Baseman. 1979. Isolation of mutants of Mycoplasma pneumoniae defective in hemadsorption. Infect. Immun. 23:903-906. 17. Hansen, E. J., R. M. Wilson, and J. B. Baseman. 1979. Twodimensional gel electrophoresis comparison of proteins from virulent and avirulent strains of Mycoplasma pneumoniae. Infect. Immun. 24:468-475. 18. Hansen, E. J., R. M. Wilson, W. A. Clyde, Jr., and J. B. Baseman. 1981. Characterization of hemadsorption-negative mutants of Mycoplasma pneumoniae. 32:127-136. 19. Hayflick, L. 1965. Tissue cultures and mycoplasmas. Tex. Rep. Biol. Med. 23(Suppl. 1):285-303. 20. Hu, P. C., R. M. Cole, Y. S. Huang, J. A. Graham, D. E. Gardner, A. M. Collier, and W. A. Clyde, Jr. 1982. Mycoplasma pneumoniae infection: role of a surface protein in the attachment organelle. Science 216:313-316. 21. Hu, P. C., A. M. Collier, and J. B. Baseman. 1977. Surface parasitism by Mycoplasma pneumoniae of the respiratory epithelium. J. Exp. Med. 145:1328-1343. 22. Kahane, I., S. Tucker, D. K. Leith, J. Morrison-Plummer, and J. B. Baseman. 1985. Detection of the major adhesin P1 in the Triton shells of virulent Mycoplasma pneumoniae. Infect. Immun. 50:944-946. 22a.Krause, D. C. Unpublished data. 23. Krause, D. C., and J. B. Baseman. 1982. Mycoplasma pneumoniae proteins that selectively bind to host cells. Infect. Immun. 37:382-386. 24. Krause, D. C., and J. B. Baseman. 1983. Inhibition of Myco-

J. BACTERIOL.

plasma pneumoniae hemadsorption and adherence to respiratory epithelium by antibodies to a membrane protein. Infect. Immun. 39:1180-1186. 25. Krause, D. C., D. K. Leith, and J. B. Baseman. 1983. Reacquisition of specific proteins confers vitulence in Mycoplasma pneumoniae. Infect. Immun. 39:830-839. 26. Krause, D. C., D. K. Leith, R. M. Wilson, and J. B. Baseman. 1982. Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence. Infect. Immun. 35: 809-817. 27. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 28. Lipman, R. P., W. A. Clyde, Jr., and F. W. Denny. 1969. Characteristics of virulent attenuated and avirulent Mycoplasma pneumoniae strains. J. Bacteriol. 100:1037-1043. 29. Lux, S. E. 1979. Dissecting the red cell membrane skeleton. Nature (London) 281:426-429. 30. Markwell, M. A. K., S. M. Haas, L. L. Bieber, and N. E. Tolbert. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206-210. 31. Meng, K. E., and R. M. Pfister. 1980. Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal. J. Bacteriol. 144:390-399. 32. Niggli, V., and M. M. Burger. 1987. Interaction of the cytoskeleton with the plasma membrane. J. Membr. Biol. 100:97-121. 33. Oakley, B. R., D. R. Kirsh, and N. R. Norris. 1980. A simnplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105:361-363. 34. Powell, D. A., P. C. Hu, M. Wilson, A. M. Collier, and J. B. Baseman. 1976. Attachment of Mycoplasma pneumoniae to respiratory epithelium. Infect. Immun. 13:959-966. 35. Roberts, D. D., L. D. Olson, M. F. Barile, V. Ginsburg, and H. C. Krivan. 1989. Sialic acid-dependent adhesion of Mycoplasma pneumoniae to purified glycoproteins. J. Biol. Chem. 264:9289-9293. 36. Sobeslawsky, O., B. Prescott, and R. M. Chanock. 1968. Adsorption of Mycoplasma pneumoniae to neuramic acid receptors of various cells and possible role in virulence. J. Bacteriol. 96:695705. 37. Tjian, R., D. Stinchcomb, and R. Losick. 1975. Antibody directed against Bacillus subtilis a factor purified by sodium dodecyl sulfate slab gel electrophoresis. J. Biol. Chem. 250: 8824-8828. 38. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.