Mycoplasma pneumoniae Cytadherence Phase-Variable Protein ...

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Vol. 174, No. 13

JOURNAL OF BACTERIOLOGY, JUIY 1992, p. 4265-4274

0021-9193/92/134265-10$02.00/0 Copyright © 1992, American Society for Microbiology

Mycoplasma pneumoniae Cytadherence Phase-Variable Protein HMW3 Is a Component of the Attachment Organelle MARLA K. STEVENSt AND DUNCAN C. KRAUSE* Department of Microbiology, University of Georgia, Athens, Georgia 30602 Received 8 January 1992/Accepted 18 April 1992

The subcellular location of the phase-variable cytadherence-accessory protein HMW3 in Mycoplasma pneumoniae has been examined by biochemical and immunoelectron microscopic techniques. Analysis by Western blot (immunoblot) with HMW3-specific antiserum established the presence of this protein within the M. pneumoniae Triton X-100-insoluble fraction or triton shell. Immunogold labeling of Triton-extracted mycoplasmas with affinity-purified antibodies localized HMW3 to the terminal knob on the rodlike extensions of the triton shell, a location that would correspond to the adherence organelle in whole mycoplasmas. Treatment of triton shells with KI resulted in the selective removal of the adherence-accessory proteins HMW1 to HMW4. Analysis of these triton shells by transmission electron microscopy revealed dramatic ultrastructural changes in the filamentous network and core structure. Immunogold labeling of KI-extracted shells reflected the removal of HMW3 from the disrupted tip structure. An examination of ultrathin sections of wild-type cells by transmission electron microscopy following labeling with HMW3-specific antibodies provided further evidence for the nonrandom distribution of HMW3 and its localization to the terminal portion of filamentous cell extensions. Most colloidal gold molecules were associated with the cell interior, but limited peripheral labeling of the terminal region was also observed. Postfixation antibody labeling of whole cells suggested limited exposure of HMW3 on the mycoplasma surface at the tip structure. However, prefixation antibody labeling failed to indicate surface exposure, raising some uncertainty regarding the relationship of HMW3 with the mycoplasma membrane.

extending from the opposing base into the matrix of the cell body (3, 33, 34), which is similar in appearance and organization to the triton shell (22). Molecular and genetic studies have begun to reveal the complexity of M. pneumoniae cytadherence through the identification and characterization of accessory components to this process. Wild-type, virulent M. pneumoniae exhibits a high-frequency, reversible switching of the cytadherence phenotype (16, 17). These spontaneously arising noncytadhering variants have been grouped into classes on the basis of differences in their protein profiles (17). The most common class of variants (class I) lacks five proteins designated HMW1 to HMW5 (Mr, 140,000 to >340,000) (17, 29). The attachment organelle in class I variants lacks the characteristic truncation and nap observed with wild-type M. pneumoniae (1). The class I variants also fail to cluster the cytadhesin P1 at the tip of the attachment organelle, despite the presence of P1 in significant quantities elsewhere along the cell surface (1). These data suggest that one or more of the cytadherence-accessory proteins HMW1 to HMW5 may have a structural role in maintaining the truncated architecture of the attachment organelle and the appropriate distribution and/or disposition of the cytadhesin P1 on the mycoplasma surface. An analysis of the triton shell component proteins of wild-type M. pneumoniae cells reveals at least 20 highmolecular-weight proteins including the adhesin P1 (14), HMW1 and HMW4 (30), and HMW5 (29), which appear to form an interconnected protein complex (30). HMW1 and HMW4 are exposed on the M. pneumoniae cell surface, primarily along the filamentous cell extensions, and are associated with the rodlike structure of the triton shell (30), which is consistent with a structural role for these proteins as elements of this intracellular scaffolding. In the studies described here, we have likewise utilized immunogold elec-

Adherence to host epithelial cells (cytadherence) is a pivotal step in colonization of the human respiratory epithelium by the bacterial pathogen Mycoplasma pneumoniae (7, 8, 13, 27). This organism possesses a differentiated terminal extension (the attachment organelle) which is positioned in close association with the host cell surface during cytadherence (8, 13, 34). In negatively stained preparations, the tip of this terminal extension appears truncated, having a bulblike end (1). In addition, this region of the cell envelope possesses a surface layer or nap that exhibits differential staining compared with that of the rest of the cell (1). Two putative mycoplasma adhesins, proteins P1 (1, 9, 12, 13, 15) and P30 (2), both of which cluster specifically at the attachment organelle (1, 9, 12), have been identified. The adhesin P1 also remains scattered elsewhere across the surface of the mycoplasma cell at a much lower density (1). Extraction of virulent M. pneumoniae cells with the nonionic detergent Triton X-100 solubilizes the cell membrane, leaving insoluble networks of filamentous proteins (triton shells) (14) that retain the same orientation and general shape as unextracted cells (10, 22). Individual triton shells exhibit a rodlike structure that terminates in a knob formation at one end and a meshwork of fibers at the other end (10, 22). Examination of thin sections of filamentous M. pneumoniae by transmission electron microscopy likewise reveals an electron-dense core extending the length of the attachment organelle (8, 13, 33, 34). The core structure has a striated appearance or periodicity, suggesting that it contains repeating subunits (22, 33). Furthermore, the core structure terminates in a knob formation at one end and has fibers * Corresponding author. t Present address: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235.

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tron microscopy and biochemical analysis to evaluate the subcellular location of HMW3 in order to clarify its role as an accessory component in mycoplasma adherence to host cells.

MATERUILS AND METHODS Organisms and culture conditions. These studies were carried out with virulent M. pneumoniae M129 B18 (20) and a cytadherence-deficient variant lacking HMW1 to HMW5 (class I isolate 2) (17). Mycoplasma cultures were grown in 150-cm2 tissue culture flasks containing approximately 50 ml of Hayflick medium at 37°C (11). 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 (6), 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 adheres poorly to plastic and was therefore collected by centrifugation of the culture medium at 9,500 x g for 20 min; this was followed by three washes with PBS. The protein content was determined by a modified Lowry assay (21) or the BCA assay (Pierce, Rockford, Ill). Anti-HMW3 serum. All protocols with vertebrate animals were reviewed and approved by the institutional animal care and use committee. Sera were collected from rabbits immunized with HMW3 which had been purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as described by Tjian et al. (31). The immunoglobulin G (IgG) fractions were purified as previously described (30). HMW3-specific IgG antibodies were affinity purified as described elsewhere (18). Briefly, total protein from wildtype M. pneumoniae was separated by SDS-PAGE (19) and electrophoretically transferred to nitrocellulose (32). HMW3 was identified by Ponceau S red staining (25) on the basis of its characteristic electrophoretic mobility, and nitrocellulose strips containing HMW3 were carefully cut from the blot and blocked in a 20 mM Tris-HCI (pH 7.5)-500 mM NaCl (TBS) containing 4% calf serum. HMW3-specific antiserum was reacted with nitrocellulose strips containing bound HMW3. The strips were washed in TBS containing 0.5% Tween 20, and antibodies were eluted with 100 mM glycine (pH 2.5)-0.2 M NaCl containing 0.1% bovine serum albumin (BSA). The pH was brought to 7.5 by the addition of 1 M Tris-HCI (pH 8.0). The resulting antibody preparation was dialyzed (molecular weight cutoff, 12,000 to 14,000) and concentrated by adsorption of H20 with polyethylene glycol (molecular weight, 15,000 to 20,000) until the final volume was approximately 1 ml. The antibody specificity was examined by Western blotting (immunoblotting) (32). Triton X-100 extraction and SDS-PAGE. Mycoplasmas were suspended at approximately 1 mg of protein per ml in 20 mM Tris-HCl (pH 7.5)-150 mM NaCl (Tris-NaCI) containing 1 mM phenylmethylsulfonyl fluoride and extracted with 2% Triton X-100 (Sigma Chemical Co., St. Louis, Mo.) as previously described (30). Proteins were subjected to SDS-PAGE (19) with 3 and 4.5% acrylamide in the stacking and separating gels, respectively. The resulting gels were either visualized with silver stain (24) or examined by Western blotting (32). For the latter, proteins were transferred electrophoretically to nitrocellulose, blocked in TBS containing 4% calf serum overnight at 4°C, and incubated with HMW3-specific antiserum overnight at 4°C. The next day, the blot was washed four times in TBS containing 0.5% Tween 20 at room temperature, reacted with HRP-conju-

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gated goat anti-rabbit-IgG antibody (Bio-Rad Laboratories, Richmond, Calif.) for 4 h at room temperature, washed well in TBS-Tween, and then developed by using HRP color development reagent (Bio-Rad). Immunogold labeling of whole cells. Log phase wild-type and class I M. pneumoniae cells were suspended in fresh Hayflick medium and passed through a 25-gauge needle and a 1.2-p,m-pore-size Acrodisc filter (Gelman Sciences, Ann Arbor, Mich.) to disperse cell aggregates. The cell suspensions were placed in wells of a tissue culture dish containing UV-sterilized, Formvar-carbon-coated, 400-mesh nickel grids. For the class I variant M. pneumoniae, the grids were coated with poly-L-lysine to promote attachment. After a 1to 2-h incubation at 37°C, the grids were removed, washed gently with HEPES-NaCl (25 mM N-2-hydroxyethylpiperazine-N-2'-ethanesulfonic acid [pH 7.4], 0.8% NaCI), and fixed in 1% glutaraldehyde-1% paraformaldehyde-0.1% picric acid in 0.2 M sodium cacodylate (pH 7.2) for 30 min at 4°C. Immunogold labeling was performed as previously described with minor modifications (30). Briefly, grids coated with poly-L-lysine were blocked in 5% nonfat dry milk in 0.2 M Tris-HCl (pH 8.2)-0.8% NaCl-1% BSA for 30 min at room temperature. All samples were incubated in a 1:5 dilution of the HMW3-specific, affinity-purified IgG antibodies overnight at 4°C. After a 1-h incubation at room temperature with a 1:10 dilution of the goat anti-rabbit IgG conjugated to 10-nm colloidal gold (Amersham, Arlington Heights, Ill), 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. Transmission electron microscopy of Triton X-100-extracted mycoplasmas. Wild-type M. pneumoniae cells were grown on electron microscope grids as described above, washed with HEPES-NaCl, and extracted with TrisNaCl-2% Triton X-100 with gentle mixing. The tissue culture dish containing the grids was placed in a water bath for 30 min at 37°C, after which the grids were washed with HEPES-NaCl and fixed in 1.5% glutaraldehyde in HEPESNaCl for 30 min at 4°C. The grids containing the detergentextracted mycoplasmas were either immunolabeled, postfixed, and examined as described above for whole cells or directly postfixed in 0.1% osmium tetroxide in H20 for 10 min at room temperature, stained in 1% filtered aqueous uranyl acetate for 10 min at room temperature, dehydrated in alcohol, critical-point dried, and shadowed with a thin layer of platinum. Immunogold labeling of thin sections. Thin sections of wild-type and class I variant M. pneumoniae cells were prepared and processed as described in detail previously (30). Immunogold labeling was performed as described above with whole cells. KI extraction of mycoplasma Triton shells. Wild-type M. pneumoniae cells were grown and extracted with 2% Triton X-100 as described above. The resulting Triton X-100insoluble pellet was washed in Tris-NaCl and resuspended in either 0.6 or 1.8 M KI. The suspensions were incubated in a water bath for 1 h at 37°C. The triton shell was isolated by centrifugation at 15,000 x g for 30 min at 4°C. The supernatant was carefully removed and saved, and the pellet was washed with Tris-NaCl. The supernatants and pellets were mixed with Laemmli sample buffer (19), analyzed by SDSPAGE (19), and visualized by silver staining (24). Transmission electron microscopy of the KI-treated triton shell. Wild-type M. pneumoniae were grown on electron microscope grids and extracted with Tris-NaCl-2% Triton

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able in the Triton-insoluble fraction of the variant (lane e). Detection of HMW3 in the variant profiles was probably due to the presence of revertants in the variant population, enrichment by Triton fractionation, and the sensitivity of the technique. No HMW3 was detectable in the Triton-soluble profile of the variant (lane g). These observations confirmed the partitioning of HMW3 in the triton shell of M. pneumo-

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niae. Antiserum specificity. Immunogold electron microscopy was performed to localize HMW3 within the M. pneumoniae cell. Unfortunately, significant reactivity to other M. pneumoniae proteins was observed in the HMW3-specific IgG at the antibody concentrations necessary for immunoelectron microscopy. This prompted us to affinity purify the HMW3-

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specific IgG antibodies for such studies. The affinity-purified antibodies (diluted 1:5) reacted exclusively with a protein band, corresponding to HMW3 in relative mobility, in the wild-type M. pneumoniae protein profile, while no reactivity was observed with the class I variant protein profile (data not shown). HMW3-specific IgG affinity-purified antibodies were used at the same concentration (1:5 dilution) for all FIG. 1. Western blot analysis of Triton X-100-fractionated wildtype and class I variant M. pneumoniae with anti-HMW3 serum. Pellets of wild-type and class I variant mycoplasmas with similar amounts of total cell protein were solubilized in Triton X-100 and separated into detergent-soluble and -insoluble fractions by centrif-

ugation (30). Mycoplasma proteins were subsequently resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antiHMW3 serum at a dilution of 1:5,000, as detailed in the text. Lanes: a, protein standards as indicated in the left margin in kilodaltons (k); b and c, wild-type and class I variant M. pneumoniae, respectively, prior to extraction (ca. 25 ,ug of protein); d and e, wild-type and class I variant detergent-insoluble fractions, respectively (ca. 15 pg of protein); f and g, wild-type and class I variant detergent-soluble fractions, respectively (ca. 35 ±g of protein). X-100 as described above. The Triton X-100-extracted sam-

ples

were

then incubated in Tris-NaCl, 0.6 M KI,

or

1.8 M

KI in a water bath for 30 min or 1 h at 37°C. The grids were then washed in HEPES-NaCl and postprocessed as described above. For some studies, the 0.6 M KI-extracted

sample was immunolabeled with a 1:5 dilution of the HMW3specific, affinity-purified rabbit IgG as described above. RESULTS Localization of HMW3 to the Triton X-100-insoluble fraction. A previous study employing SDS-PAGE and Western blot analysis of the M. pneumoniae triton shell clearly demonstrated that HMW1 and HMW4 are components of this insoluble network (30). This study also suggested that HMW3 is likewise a component of the triton shell. Our first goal was to confirm immunologically the presence of HMW3 in the Triton-insoluble fraction. M. pneumoniae cells were extracted with Triton X-100, and the resulting soluble and insoluble fractions were analyzed by Western blotting with antiserum specific for HMW3 at a 1:5,000 dilution (Fig. 1). A single protein band, corresponding to HMW3 in relative mobility, was present in the total protein profile of wild-type M. pneumoniae and absent from the profile of the class I variant deficient in HMW1 to HMW5 (Fig. 1, lanes b and c, respectively). HMW3 was enriched in the Triton-insoluble fraction of wild-type M. pneumoniae (lane d) and was barely detectable in the Tritonsoluble fraction (lane f). HMW3 was likewise barely detect-

immunoelectron microscopy. Immunogold labeling of Triton X-100-extracted whole M. pneumoniae cells. In order to evaluate the distribution of HMW3 in the triton shell of M. pneumoniae, mycoplasmas grown on electron microscope grids were extracted with Triton X-100 and examined by immunoelectron microscopy with HMW3-specific, affinity-purified IgG antibodies. Gold particles concentrated specifically at the terminal knob of the rodlike extensions of the triton shell (Fig. 2A and B), indicating localization of HMW3 to this region of the mycoplasma cell. Interestingly, in some cases the gold particles appeared to be oriented in a linear pattern (Fig. 2A and B, arrows). As a control for labeling specificity, HMW1-to-HMW5deficient variant M. pneumoniae cells were examined in a similar manner. However, because these variants did not attach well to electron microscope grids, it was necessary to first coat the grids with the adhesive poly-L-lysine. As expected, Triton-extracted variant cells showed very little labeling (Fig. 2D). Furthermore, the triton shell of the variants did not contain well-defined rodlike extensions and lacked the knoblike terminus present in wild-type cells. As a control, triton shells from wild-type cells grown on poly-Llysine-coated grids were also immunolabeled. Identical labeling patterns were observed with these triton shells (Fig. 2C) and with those from wild-type mycoplasmas grown on grids not coated with poly-L-lysine. Immunogold labeling of thin sections. As an alternative approach to evaluate the subcellular location of HMW3, thin sections of wild-type and class I variant cells were immunolabeled with HMW3-specific, affinity-purified IgG antibodies and examined by electron microscopy. Gold particles were consistently confined to the terminal portion of the filamentous extensions of wild-type M. pneumoniae cell sections (Fig. 3A, B, and C, arrowheads). Gold particles were also seen in association with small spherical regions, which may represent glancing or cross-sections of the filamentous extension (data not shown). Only a few cell sections in any field retained the gold label, reflecting the apparently nonrandom and limited distribution of HMW3 within the mycoplasma cell. Finally, most of the gold particles appeared to localize within the interior of the filamentous extension. However, some particles were also present on the cell periphery, suggesting that HMW3 may associate with the cell membrane, perhaps with limited surface-exposed epitopes. The

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FIG. 2. Immunoelectron microscopy of wild-type and class I variant M. pneumoniae cells extracted with Triton X-100. Mycoplasmas were grown on electron microscope grids, extracted with Triton X-100, and incubated with affinity-purified anti-HMW3 IgG at 1:5 and then with a goat anti-rabbit IgG-10-nm colloidal gold secondary antibody. (A and B) Wild-type M. pneumoniae; (C) wild-type M. pneumoniae grown on poly-L-lysine-coated grids; (D) class I variant M. pneumoniae grown on poly-L-lysine-coated grids. Arrows indicate linear patterns of labeling.

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FIG. 3. Immunoelectron microscopy of thin sections of wild-type and class I variant M. pneumoniae cells. Mycoplasmas were fixed, dehydrated, and embedded in LR White resin (Bio-Rad Microscience Division, Cambridge, Mass.). Thin sections were placed on electron microscope grids and incubated with affinity-purified anti-HMW3 IgG (1:5 dilution) and then with a goat anti-rabbit IgG-10-nm colloidal gold secondary antibody. (A, B, and C) Wild-type M. pneumoniae; (D) class I variant.

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FIG. 4. Immunoelectron microscopy of M. pneumoniae whole cells. Wild-type and class I variant mycoplasmas were grown on electron microscope grids for 1 or 2 h, fixed, and incubated with affinity-purified anti-HMW3 IgG at 1:5 and then with a goat anti-rabbit IgG-10-nm colloidal gold secondary antibody. (A and B) Wild-type M. pneumoniae; (C) wild-type M. pneumoniae grown on grids coated with poly-L-lysine; (D) class I variant M. pneumoniae grown on grids coated with poly-L-lysine. Arrowheads indicate labeling of the tip region with affinity-purified anti-HMW3 IgG. Arrows indicate cells that lack labeling with the antibodies.

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i4

-1

-2

-3 116-

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FIG. 5. SDS-PAGE profiles following KI treatment of the Triton-insoluble fraction of wild-type M. pneumoniae. Triton shells were treated with 0.6 or 1.8 M KI and then centrifuged to separate dissociated proteins from the triton shell. Samples (ca. 20 ,ug of protein each) were subjected to SDS-PAGE (23) and visualized with silver stain (28). Lanes: a, Triton-insoluble fraction; b, proteins removed from triton shells with 0.6 M KI treatment; c, proteins removed from triton shells with 1.8 M KI treatment. Protein standards are indicated in the left margin in kilodaltons (k). HMW proteins are denoted in the right margin.

HMW1-to-HMW5-deficient class I variant cell sections did not retain any gold particles (Fig. 3D).

Immunogold labeling of whole cells. To examine whether epitopes of HMW3 were accessible to antibodies at the cell surface, wild-type M. pneumoniae cells were grown on electron microscope grids, fixed as described above, and incubated with HMW3-specific, affinity-purified IgG antibodies. Cell labeling was possible only with an extended incubation (overnight at 4°C) and high-titered anti-HMW3 antibodies. Under these conditions, gold particles clustered on the cell surface at the differentiated tip of the filamentous extensions (Fig. 4A, arrowheads). This labeling pattern was generally confined to only one end of the M. pneumoniae cell, specifically the bulblike terminal region, which was the same subcellular distribution of HMW3 as was observed with triton shells and thin sections. Interestingly, however, not every cell was labeled (Fig. 4A, arrow). Fig. 4B shows a higher magnification of a wild-type M. pneumoniae cell with two heavily labeled bulbous tip regions. Formation of a new, differentiated tip structure, commonly seen in M. pneumoniae cells undergoing a modified form of binary fission (28), could account for the HMW3-specific labeling of the two extensions on the wildtype cell in Fig. 4B. Wild-type cells grown on poly-L-lysinecoated grids exhibited the same surface labeling at the tip (Fig. 4C). As expected, no labeling was observed on the surface of the class I variant cells (Fig. 4D), confirming the specificity of the labeling pattern. The variant cells lacked the bulblike tip on the filamentous extensions, consistent with both the observation reported previously (1) as well as the appearance of the variant triton shells (Fig. 2). Effects of KI treatment on Triton X-100-extracted cells. Gobel et al. (10) reported that incubation of Triton X-100extracted wild-type M. pneumoniae cells with 0.6 M KI disrupts the integrity of the detergent-insoluble filamentous

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network. We have expanded upon this work in an attempt to identify protein components essential in maintaining the integrity of the attachment organelle. The triton shells of wild-type M. pneumoniae were incubated with increasing concentrations of KI, and the resulting dissociated proteins and remaining triton shell component proteins were analyzed by SDS-PAGE (Fig. 5). Figure 5, lane a, shows the typical profile for the Triton-insoluble fraction. Treatment of triton shells with 0.6 M KI resulted in the partial dissociation of HMW1 to HMW4 from the Triton-insoluble fraction (lane b). On the basis of the staining intensity, a greater portion of HMW3 relative to the other HMW proteins appeared to be removed. Treatment with 1.8 M KI resulted in a significantly greater dissociation of HMW1 to HMW4 from the triton shell, as was apparent by their increased staining intensity in the dissociated fraction (lane c). However, dissociation was not complete, as these proteins were clearly visible in the 1.8 M KI-treated insoluble fraction (data not shown). To visualize the effects of KI treatment and to attempt to correlate the partial dissociation of the HMW proteins with changes in the appearance of the triton shell, KI-treated triton shells were examined by transmission electron microscopy (Fig. 6). The appearance of triton shells incubated in the buffer control was consistent with previous descriptions (10, 22, 30), as shown by networks of filaments and individual filaments with rodlike extensions having terminal knobs (Fig. 6A, arrowhead). KI treatment of M. pneumoniae triton shells resulted in dramatic ultrastructural changes. Some networks of filaments were still present in samples treated with 0.6 M KI (Fig. 6B). Overall, however, the network of filaments appeared less organized than in the control samples, and more isolated rodlike core structures were observed. Many of the core structures lacked the terminal knob (Fig. 6B, arrow). Treatment with 1.8 M KI fragmented the triton shell. Individual core structures with terminal knobs and filaments associated with large aggregates of cells were completely absent (Fig. 6C). In order to evaluate ultrastructurally the fate of HMW3 after KI extraction, triton shells treated with 0.6 M KI were examined by electron microscopy following immunogold labeling with HMW3-specific antibodies. Gold particles associated with small thin fibrils extending from the disrupted knoblike terminus and with small particulate matter that covered the background were observed (Fig. 6D). These fibrils appeared to be connected to the tip of the core structure. The increase in gold associated with the background particulate matter in the 0.6 M KI-treated triton shell appeared to result from fragmentation of the terminal knob and the release of HMW3, suggesting that HMW3 is a major component of the terminal knob of triton shells.

DISCUSSION Scanning and transmission electron microscopy of M. pneumoniae cells reveal a Triton X-100-insoluble residue that forms a filamentous multiprotein network (10, 22, 30), similar to eukaryotic cytoskeletal structures (23). This network may serve as a scaffold controlling changes in cell shape, cell division, and motility (4, 5, 28). Furthermore, this structure may be directly involved in the adherence of M. pneumoniae to host cells (cytadherence) through the interaction with the adhesins P1 (1, 26) and possibly P30. The cytadherence-accessory protein HMW3, along with HMW1 and HMW4 (30), partitioned with the Triton-insoluble fraction (Fig. 1). However, rather than clustering in distinct regions along the rodlike extensions of the triton

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FIG. 6. Electron microscopic analysis of KI-treated triton shells of wild-type M. pneumoniae. Triton shells were incubated in Tris-NaCl buffer (control) (A), 0.6 M KI (B), or 1.8 M KI (C) and processed for electron microscopy. Also shown are triton shells following treatment with 0.6 M KI and immunogold labeling with HMW3-specific antibodies (D). Arrowheads indicate isolated core structures with terminal knobs; arrows indicate core structures lacking the terminal knob.

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shell as seen with HMW1 and HMW4, HMW3 concentrated specifically at the terminal knobs (Fig. 2). The linear pattern of gold labeling (Fig. 2) may contribute to the periodicity observed by others in the core structure and terminal knobs (22, 33). Immunogold labeling of thin-sectioned (Fig. 3) and whole (Fig. 4) M. pneumoniae cells confirmed this localization of HMW3 to the tip structure. Finally, double-antibody labeling experiments with HMW3- and adhesin P1-specific antibodies provided additional evidence to underscore the identification of this location as the adherence organelle (data not shown). Disruption of the knob and core structure of the M. pneumoniae triton shell with increasing concentrations of KI (Fig. 6) correlated with increased removal of the HMW1 to HMW4 proteins from the triton shell (Fig. 5). Immunogold labeling of the 0.6 M KI-treated triton shells indicated fragmentation of HMW3 specifically from the tip structure. Lower-molecular-weight proteins not resolved by the conditions used for SDS-PAGE in Fig. 5 may also be extracted by KI treatment and contribute to the observed ultrastructural disruption of the triton shells. However, the absence of the knob formation at the tip of the core structure of the variant deficient in HMW1 to HMW5 suggests that one or more of the HMW proteins account for the observed changes, consistent with previous speculation that these proteins contribute to the maintenance of the proper architecture of the attachment organelle (1). Whole-cell labeling studies, while reinforcing the limited subcellular distribution of HMW3, were somewhat problematic with regard to possible exposure of this protein at the cell surface. Immunogold labeling was observed only after an overnight incubation of fixed cells with concentrated HMW3-specific antibodies. These conditions suggest (i) low antigenicity of surface-exposed epitopes of HMW3, (ii) very limited exposure of the surface epitopes, and/or (iii) artifactual labeling as a result of sample processing under these conditions. Repeated attempts at labeling unfixed cells with anti-HMW3 antibodies under a variety of conditions were unsuccessful (data not shown), raising the possibility that fixation may expose epitopes which are not normally antibody accessible. Additional studies addressing the relationship between HMW3 and the mycoplasma membrane, perhaps also employing monoclonal antibodies to HMW3, may be required to resolve this issue. HMW3 could serve at least two roles in the terminal knob of the attachment organelle. As a component of the triton shell, HMW3 could function to stabilize the attachment organelle. The absence of the knob formation in both the class I variant whole cells and triton shells, the disruption of the knob structure by KI (corresponding to removal of HMW3 along with the other HMW proteins), and the localization of HMW3 to this region of the triton shell suggest that HMW3 may participate in the formation of and/or be a component of the terminal knob of the M. pneumoniae cytoskeleton. In addition, HMW3 may directly anchor the adhesin P1 at the tip of the adherence organelle through a linkage to the cytoskeleton. The gene for HMW3 has been cloned (25) and sequenced (26). The deduced amino acid sequence of HMW3 indicates a high proline content, and secondary structure algorithms predict an extended, probably rigid conformation (26), consistent with a structural role for this protein within the triton shell. The absence of HMW3 in the class I variants, and consequently the linkage to P1, could account for (i) the inability of these cells to cluster P1 at the tip of the adherence organelle, (ii) the presence of a significant number of P1 molecules randomly

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distributed across the cell surface, and (iii) their reduced adherence capacity. Recently, we described structural features of HMW3 that might promote such an interaction with P1 (26). Additional studies are under way to evaluate the possibility of a direct interaction between HMW3 and P1. ACKNOWLEDGMENTS This work was supported by Public Health Service research grant A123362 and research career development award AI00968 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 F. Gherardini, P. Wyrick, M. Farmer, J. Paulin, W. Steffens, C. Kelloes, and M. Ard for advice and technical assistance with the immunoelectron microscopy.

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