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Infect Immun. Sep 2002; 70(9): 4925–4935.
PMCID: PMC128281

Identification and Functional Mapping of the Mycoplasma fermentans P29 Adhesin


Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

Mycoplasmal adherence to host cells is critical for colonization and maintenance of infection (34, 35). As a manifestation of the widely observed phenomenon of adaptive surface diversification (34, 51), some mycoplasmas display spontaneous loss and reacquisition of specific adhesins and corresponding adherence phenotypes (4, 21, 34). Identification and characterization of mycoplasma adhesins (19) and their role in virulence continue to be studied, including those of human mycoplasmal species. Perhaps most thoroughly examined are mycoplasma adhesins associated with the terminal tip organelle of some species, composed of multiple interacting proteins (4, 21, 23, 34, 37). The ligand binding components of tip structures, such as the P1 adhesin of Mycoplasma pneumoniae and MgPa adhesin of Mycoplasma genitalium, may in general show sequence homology and contain regions of sequence motifs common to many adhesins. However, the topology of the ligand binding interface appears to be complex and not fully understood, requiring either multiple interacting regions within a single translation product or the interaction of additional proteins. In contrast, tip organelle structures have not been observed on the majority of mycoplasma species (13), and yet individual surface protein adhesins have been defined and partially characterized, including, for example, the Vaa protein of Mycoplasma hominis (5, 20, 53, 54), the P97 ciliary adhesin of Mycoplasma hyopneumoniae (17, 29, 56), and the MAA1 antigen of Mycoplasma arthritidis (47, 48). These products reflect diverse sequences and structural motifs. In these cases as well, neither the topology of their ligand binding interface nor their precise ligand specificity has been fully determined.

We have studied the surface architecture of the human mycoplasma Mycoplasma fermentans as a prototype organism displaying features of adaptive surface variation possibly relevant to infection and pathogenicity, with the characteristics of mycoplasmas lacking specialized adherence structures. M. fermentans has received attention both for its potential roles as a primary or contributing pathogen in human disease (18, 25) and due to the recent discovery of potent macrophage-modulating activity associated with distinctively modified surface lipoproteins (7, 30-33, 41). M. fermentans displays several variable surface proteins, some lacking orthologous counterparts in other organisms and others with assignable specific functions (6, 10, 11, 45, 46, 49). Phase variation of some M. fermentans surface proteins has been shown to occur through frameshift mutation in tracts of redundant nucleotides (45), but other types of variation occur (11, 51), including the phase-variable differential display (or masking) of specific epitopes on the P29 lipoprotein (44, 46). Surface masking of specific membrane lipoproteins has also recently been observed for another human mycoplasma, M. hominis (55).

In previous studies (44), P29 was shown to be an abundant surface lipoprotein of 29 kDa. Anchored in the single, limiting membrane by diacylglyceryl moieties on the +1 Cys residue at the N terminus of the mature lipoprotein, the 219-amino-acid P29 polypeptide sequence resides entirely outside the membrane. It is hydrophilic and contains a 36-amino-acid disulfide loop in the central region of the protein and two short, nearly identical noncontiguous repeat units, Rep1 and Rep2 (44). No orthologs of the P29 sequence have been revealed through database searches (as of February 2002), and no function has been proposed for this protein. P29 undergoes an unusual mode of phase variation (44, 46). Whereas the P29 translation product is constitutively expressed by M. fermentans, accessibility of monoclonal antibody (MAb)-defined epitopes mapped to sequences at the N- and C-terminal ends of P29 was shown to phase vary independently in propagating populations of the organism (44). This raised the possibility that all or portions of the P29 molecule may be efficiently masked or unmasked as a heritable feature of phenotypic variants.

Because its distribution in host niches seems to be diverse (1, 2, 15, 16, 18, 22, 25), M. fermentans perhaps understandably is able to bind to a number of cell types. These include HeLa cells, a cell line used widely for mycoplasma adherence studies, and peripheral blood lymphocytes (8). Some studies suggest that M. fermentans can reside within (42, 43, 50) or fuse with (12, 14) some cultured cell lines including HeLa cells. The molecular components mediating these activities are becoming defined (50), but little is known about the mycoplasma components mediating initial binding to any host-derived cell. To identify M. fermentans surface proteins possibly involved in adherence, a set of recombinant fusion proteins representing known M. fermentans surface proteins was assessed for their ability to bind to HeLa cells. From this screening, a fusion protein representing the P29 lipoprotein of M. fermentans was identified and is further characterized in this report. We demonstrate that sequences within the P29 surface lipoprotein mediate specific, concentration-dependent binding of M. fermentans to a saturable ligand on HeLa cells that may be utilized by a divergent adhesin of at least one other human mycoplasma.


Bacterial strains and culture conditions.

M. fermentans PG18 clone 39, previously described in reference 49, and Mycoplasma capricolum ATCC 27343 were grown in modified Hayflick medium containing 20% heat-inactivated horse serum (Gibco BRL, Grand Island, N.Y.) as previously described (46). M. hominis 1620 was grown in modified Hayflick medium supplemented with 20% heat-inactivated horse serum and arginine as previously described (53). Escherichia coli DH10B (Gibco BRL) was used for both plasmid generation and fusion protein expression. E. coli cultures were grown in Luria-Bertani medium except during protein expression, when they were grown in Rich Medium (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 2 g of dextrose per liter of H2O). Both media were supplemented with 100 μg of ampicillin/ml.

Cell lines, culture conditions, and MAbs.

A suspension-adapted human epithelial HeLa S3 cell line (54) was used as the target in binding studies. Cells were grown in suspension to approximately 2 × 106/ml in RPMI 1640 medium (Gibco BRL) containing 10% fetal calf serum (Hazleton Research Products, Denver, Pa.), 2 mM l-glutamine, and 10 U of penicillin and 10 μg of streptomycin per ml. Cells were harvested by centrifugation at 400 × g, and viability was determined by trypan blue exclusion. Immunoglobulin G (IgG) MAbs C19 (F202C19A) and E12 (4437E12) recognizing the N-terminal and C-terminal regions of P29, respectively, were described previously (44, 49). The IgG MAb C15 (F203C15A) recognizing maltose-binding protein (MBP) was generated previously in this laboratory (M. F. Kim and K. S. Wise, unpublished data).

SDS-PAGE and immunoblotting.

Protein separation was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% gels as previously described (44, 46). Samples were heated in sample buffer under reducing conditions for 3 min prior to loading. Separated proteins were transferred electrophoretically to nitrocellulose membranes for immunostaining as described previously (44, 46). Immunodetection was performed with hybridoma culture supernatants containing MAb C15, C19, or E12 as primary Ab. Western blots were treated with a horseradish peroxidase-conjugated secondary goat Ab to mouse IgG (ICN/Cappel, Aurora, Ohio) diluted 1:2,000 in phosphate-buffered saline (PBS; 2.7 mM KCl, 1.2 mM KH2PO4, 138 mM NaCl, 8.1 mM Na2HPO4, pH 7.4) containing 10% heat-inactivated calf serum, and color was developed with the substrate 4-chloro-1-naphthol (Kirkegaard & Perry Laboratories, Gaithersburg, Md.).

Generation of recombinant protein expression plasmids.

The p29, p78, and vlpB genes were amplified by PCR from plasmids pMFPSEL1.2M (44), pMFPZ-4 (45), and pGEM7Z-17 (containing the isolated vlpB gene of Mycoplasma hyorhinis SK76) (24, 52), respectively. Forward and reverse primers (Table (Table1)1) were used to PCR amplify DNA fragments containing 5′ and 3′ restriction sites for directional cloning into the pMAL-c2 vector (New England Biolabs [NEB], Beverly, Mass.) (Fig. (Fig.1A).1A). PCR inserts were ligated into digested and dephosphorylated pMAL-c2 vector to generate protein expression plasmids that were then transformed into E. coli DH10B. The inserts of plasmids pFP29, pFP78, and pFPVlpB containing the p29, p78, and vlpB genes, respectively, were sequenced to confirm the anticipated fusion junctions. Truncated FP29 constructs were produced similarly. PCR products that contained specific regions of the p29 gene were generated by using the pFP29 plasmid as a template and mutagenic primers allowing in-frame cloning into pMAL-c2 (Table (Table1).1). For C-terminal truncations, these mutagenic primers also introduced a stop codon. One frameshift mutation was made in pFP29 by restriction at a unique MunI site, blunt filling the ends with the Klenow fragment of DNA polymerase I and deoxynucleoside triphosphates (28), and ligation. The frameshift resulted in replacement of the C-terminal nine amino acids of the authentic P29 sequence in the construct FP29t. MBP was expressed from the pMAL-c2 vector containing an in-frame stop codon at the XbaI site created by blunt filling the digested EcoRI site with the Klenow fragment of DNA polymerase I and deoxynucleoside triphosphates (R. Watson-McKown and K. S. Wise, unpublished data).

FIG. 1.
Generation of pFP29 and expression of fusion proteins. (A) The p29 gene, containing a UGA→UGG codon mutation, was cloned from plasmid pMFPSEL1.2M (44) into plasmid pMAL-c2 through a PCR product intermediate. The forward cloning primer Pr210 (Table ...
Oligonucleotide primer sequences

Expression, purification, and cleavage of fusion proteins.

Fusion protein expression and purification were performed as previously described (9, 53). Fusion proteins overexpressed in E. coli were purified by affinity chromatography with amylose resin (NEB) as directed by the manufacturer. Column fractions containing fusion protein were pooled, and protein concentrations were determined by the Folin assay of Lowry et al. (26) with a bovine serum albumin (BSA) protein standard. Purified fusion proteins were analyzed by SDS-PAGE to verify the degree of purity. Aliquots were stored at −80°C until use. For factor Xa cleavage of MBP from FP29t, the fusion protein was diluted 1:2 in 20 mM Tris (pH 8.0), and CaCl2 was added to a final concentration of 2 mM prior to addition of factor Xa protease (NEB). The cleavage reaction mixture was incubated at 37°C for 3 h. The fusion protein cleavage reaction mixture was immediately diluted in PBS containing 3% BSA (fraction V; Fisher Scientific) (PBS-BSA) to obtain the appropriate sample concentration for protein binding analysis and stored at −20°C until used.

Binding and inhibition assays.

Fusion protein binding assays were performed in 96-well round-bottomed microtiter plates (Nalge Nunc International, Naperville, Ill.). Freshly harvested, logarithmic-phase HeLa cells (105; of greater than 95% viability) and the fusion protein sample were added to each well in PBS-BSA to a final volume of 135 μl. Plates were covered with eight-well strip caps (Nalge Nunc International) and incubated for 60 min at 22°C while the plates were rotated on a 75° plane at 20 rpm (Roto-Torque; Cole-Parmer Instruments, Chicago, Ill.). Two intermediate washes were performed after each incubation step by centrifugation of plates at 400 × g followed by resuspension in PBS-BSA. Bound fusion protein was detected by an Ab sandwich technique. One hundred fifty microliters of MAb C15 (anti-MBP) hybridoma culture supernatant was incubated with HeLa cells for 60 min followed by 150 μl of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary Ab (ICN/Cappel) for an additional 60 min, both with rotation. Before flow cytometric assays, a final wash with PBS was performed. To identify proteins binding directly to HeLa cells, cells treated under the same conditions with the products of factor Xa cleavage of FP29t were rinsed with PBS, subjected to SDS-PAGE, and immunoblotted as described above. Bound proteins were detected with either MAb C19, recognizing a P29 epitope, or MAb C15, recognizing MBP.

For HeLa cell binding assays, mycoplasmas were grown in the specified medium as previously described (49, 54) in 10-ml broth cultures until medium color change occurred (red to orange for M. fermentans and M. capricolum or orange to red for M. hominis). Mycoplasma concentrations were normalized based on the absorbance (A) of broth cultures. A 1-ml sample of medium alone or 1 ml of mycoplasma culture diluted with medium in twofold serial dilutions was adjusted to pH 6.8 by the addition of 100 μl of 1.0 M sodium phosphate buffer (pH 6.8). ΔA600 (A600 of each serial dilution − A600 of the medium control) was then determined. A standard curve of average ΔA600 versus experimentally determined CFU was generated and used to standardize mycoplasma concentrations for subsequent experiments. ΔA600 of 0.115 was determined to equal approximately 3 × 107 CFU/ml. Mycoplasma cultures were concentrated by centrifugation at 9,000 × g for 10 min and resuspended to approximately 109 CFU/ml. CFU for each experiment were also determined experimentally by standard plate count. As an independent verification of consistency, normalized mycoplasma cultures prepared on different occasions were shown to contain equivalent amounts of mycoplasmal antigen by serial dilution and Western blotting, to determine endpoint staining with MAb to P29.

Mycoplasmas were labeled with the lipophilic fluorochrome DiIC18 (3) (Molecular Probes, Eugene, Oreg.) prior to mycoplasma binding assays, by a method modified from that of Baseman et al. (3). A 10-ml culture of mycoplasmas was centrifuged as described above, and the pellet was resuspended in 1 ml of PBS-BSA by trituration. Ten microliters of 1 mM DiIC18 in dimethyl sulfoxide was then added to the mycoplasma suspension, while the mixture was mixed by vortexing. The labeling suspension was incubated at 37°C for 60 min. Following labeling, mycoplasmas were washed twice by centrifugation at 9,000 × g for 3 min and resuspended in 1 ml of PBS-BSA containing 1% dimethyl sulfoxide, and once again in PBS-BSA, to a normalized concentration of 109 mycoplasmas per ml.

Assays to measure mycoplasma binding and inhibition of binding were performed in 96-well round-bottomed polystyrene plates (Nalge Nunc International). For binding assays, suspension cultures of HeLa cells of greater than 95% viability (determined by trypan blue exclusion) were harvested by centrifugation and suspended in PBS-BSA. HeLa cells (105) suspended in 135 μl of PBS-BSA were placed in wells prior to addition of mycoplasmas. Fifteen microliters of DiIC18-labeled mycoplasmas (approximately 1.5 × 107 mycoplasmas) were then added per well, and the wells were capped with eight-well strip caps (Nalge Nunc International). The plates were incubated for 120 min at 22°C while being rotated on a 75° plane at 20 rpm. After incubation, HeLa cells were washed twice by centrifugation at 400 × g and resuspended in PBS-BSA. Before flow cytometric assays, a final wash and resuspension in PBS were performed. For assays measuring inhibition of binding, HeLa cell preparations were suspended in 135 μl of PBS-BSA, alone (no-FP control) or containing purified fusion proteins, and incubated for 30 min prior to addition of labeled mycoplasmas as described above. Results for the binding inhibition assay were expressed as percents bound, by using the quotient of mycoplasma bound in a sample (average mean fluorescence) divided by the corresponding value for samples without added protein (no-FP control). In each case, the autofluorescence of HeLa cells alone was subtracted prior to the calculation.

Flow cytometry and fluorescence microscopy.

Flow cytometry was performed at the University of Missouri—Columbia Molecular Biology Program Cell and Immunobiology Core Facility on a FACS Vantage Turbosort flow cytometer (Becton Dickinson, San Jose, Calif.) with an excitation wavelength of 488 nm and detection with the 525-nm band-pass filter for FITC or a 575-nm band-pass filter for DiIC18. Gate voltages used were 300 for Ab C19, 450 for Ab C15, and 500 for DiIC18-labeled mycoplasmas. Samples were also observed with a Nikon Axioplan fluorescence microscope (Nikon Inc., Melville, N.Y.), and digital images were taken with a Spot2 digital camera (Diagnostic Instruments, Sterling Heights, Mich.). Within an experiment, all digital images were uniformly adjusted for brightness and contrast by using the accompanying Spot 32 software (Diagnostic Instruments).

DNA sequencing and computer sequence analysis.

Automated DNA sequencing was performed with an ABI model 377 sequencing apparatus (PE Applied Biosystems, Inc., Foster City, Calif.) with ABI BigDye terminator chemistry. Oligonucleotide primers were made with an ABI 3948 synthesizer. Both procedures were carried out by the University of Missouri—Columbia Molecular Biology Program DNA Core Facility. DNA and protein sequence analysis was performed with the Genetics Computer Group analysis software package through the Pittsburgh Supercomputing Center (http://www.psc.edu/biomed/). Coiled-coil predictions (27) were made with COILS version 2.2 (window = 28, weighted to exclude false positives in highly charged hydrophilic regions).


FP29 selectively binds to HeLa cells.

In order to identify M. fermentans surface proteins that might function as adhesins, recombinant fusion proteins representing a selection of characterized lipoproteins of M. fermentans and other mycoplasma species were screened for their ability to bind to HeLa cells. Fusion proteins (translational fusions with the MBP of E. coli) were constructed, expressed, and purified as described in Materials and Methods. Initial screening of binding by recombinant fusion proteins was performed by fluorescence microscopy, wherein binding was detected with the MBP-specific MAb C15 and an FITC-conjugated secondary Ab. FP29, representing the P29 lipoprotein of M. fermentans (Fig. (Fig.1A),1A), was the only fusion protein that showed a distinct increase in fluorescence above background levels. The striking pattern of selective binding provided the impetus to examine FP29 rigorously for HeLa cell binding activity.

To better characterize the interaction of fusion proteins with HeLa cells, a versatile assay to quantitate fusion protein binding to viable, intact HeLa cells was devised based on flow cytometric analysis. Initially, a known concentration of fusion protein was first allowed to bind to HeLa cells. Bound fusion proteins were detected with an excess of reporter MAb C15 (to MBP) followed by an FITC-conjugated Ab to mouse IgG. Binding of fusion protein was quantitated by flow cytometry as an increase in the geometric mean fluorescence in the population measured, over the levels obtained without fusion protein. By this assay system, FP29 was shown to bind to HeLa cells in a quantifiable manner (Fig. (Fig.2A2A ). This pattern coincided precisely with marked binding observed by fluorescence microscopy (Fig. (Fig.2B).2B). Assorted fusion proteins including FP78, FPVlpB, or MBP alone did not show significant binding, as no increase in the fluorescence signal was observed when HeLa cells were treated with these proteins. Fluorescence histograms also revealed a symmetrically distributed population, indicating uniformity of binding, consistent also with the pattern observed with fluorescence microscopy. By this assay system, an initial time course for FP29 binding to HeLa cells to reach a plateau level was determined. When 500 nM FP29 was added to HeLa cells, approximately 80% of the binding occurred within 30 min, with no significant difference in binding seen between 60 and 120 min (data not shown). A 60-min incubation (at 22°C) was used for all subsequent experiments.

FIG. 2.
Analysis of fusion protein binding to HeLa cells. (A) HeLa cells treated with various fusion proteins were subsequently analyzed by flow cytometry, with reporter MAb C15 and an FITC-conjugated secondary Ab to measure bound MBP fusion constructs, as described ...

The inability of MBP-containing constructs lacking the P29 sequence to bind to HeLa cells suggested that the P29 portion of the FP29 molecule is most likely responsible for HeLa cell binding activity. To assess this directly, FP29t, a subsequent construct (Fig. (Fig.4)4) simultaneously used to show that C-terminal sequences of P29 were dispensable for binding function, was partially digested with factor Xa, and the mixture of cleaved and uncleaved proteins was incubated with HeLa cells as described above. Washed cells were analyzed by SDS-PAGE and immunoblotting, with MAbs C15 and C19 to detect MBP and P29 epitopes, respectively. As depicted in Fig. Fig.2C,2C, both rP29t (the ~29-kDa product released by factor Xa) and residual FP29t remained associated with the cells after washing. In contrast, no released MBP was detected in association with cells, indicating that the HeLa cell binding activity resides exclusively in the P29 portion of the molecule.

FIG. 4.
Mapping of the P29 binding domain with FP29 truncation constructs. FP29 truncation constructs were tested for their ability to inhibit M. fermentans binding to HeLa cells, by assays described in the Fig. Fig.33 legend and in Materials and Methods. ...

FP29 binding to HeLa cells is saturable.

To further assess the characteristics of FP29 binding to HeLa cells, cells were treated with increasing concentrations of fusion proteins FP29, FP78, FPVlpB, and MBP. Bound fusion protein was detected as described previously with MAb C15, and binding results were quantitated by flow cytometry. HeLa cells treated with FP29 showed a strong correlation between fusion protein concentration and fluorescence, which appeared to reach saturation as it approached a concentration of 5 μM (Fig. (Fig.2D).2D). The half-maximal concentration of FP29 was calculated to be approximately 600 nM. FPVlpB and MBP both showed only background levels of fluorescence when tested at concentrations up to 5 μM. FP78 did not appear to bind to HeLa cells when tested at the lower protein concentrations, although a slight increase in fluorescence consistent with some FP78 binding was seen at much higher concentrations (Fig. (Fig.2D).2D). The binding activity of FP29 suggests that the HeLa cell binding activity of M. fermentans could be mediated via this single polypeptide. Consequently, to test whether P29 was responsible for M. fermentans binding to HeLa cells, the ability of FP29 to inhibit M. fermentans binding to HeLa cells was determined.

P29 mediates the interaction of M. fermentans with HeLa cells.

Prior to measuring the ability of FP29 to inhibit the binding of M. fermentans to HeLa cells, binding parameters for the M. fermentans-HeLa cell interaction were established. The versatile, lipophilic fluorochrome DiIC18 was used as described in Materials and Methods to label broth-grown organisms. Labeling conditions resulted in no detectable decrease in mycoplasmal viability, as measured by standard assay for CFU (24). After initial experiments to determine conditions for binding, incubation of M. fermentans with HeLa cells for 120 min was chosen because both qualitative and quantitative differences in binding were consistently observed during this period (data not shown). HeLa cells were treated with increasing concentrations of DiIC18-labeled M. fermentans to determine whether saturation could be achieved and to define an appropriate amount of mycoplasmas for use in further binding inhibition assays. Saturation of M. fermentans binding to HeLa cells was achieved when approximately 6 × 102 viable mycoplasmas were added per HeLa cell (Fig. (Fig.3A).3A). In subsequent inhibition assays, a normalized concentration of approximately 1.6 × 102 viable mycoplasmas per HeLa cell was used.

FIG. 3.
Inhibition of M. fermentans binding to HeLa cells by FP29. (A) Dose-dependent binding of M. fermentans to HeLa cells. Twofold-increasing concentrations of DiIC18-labeled mycoplasmas were added to HeLa cells and incubated for 120 min, after which the HeLa ...

With these standardized conditions established for M. fermentans binding to HeLa cells, experiments were performed to determine the effects of FP29 on the interaction of M. fermentans with HeLa cells: specifically, whether occupancy of the HeLa cell ligand recognized by FP29 would inhibit binding of the organism. In preliminary qualitative experiments, HeLa cells treated with FP29 showed a dramatic decrease in mycoplasma binding by fluorescence microscopy compared to those receiving no treatment or treatment with FP78 or MBP (data not shown). When the effects of FP29 on M. fermentans binding to HeLa cells were quantitated by flow cytometric analysis, treatment of HeLa cells with 500 nM FP29 resulted in a 90% decrease in mycoplasma binding (10% residual binding) compared to the no-FP control (Fig. (Fig.3B).3B). Treatment of HeLa cells with 500 nM FP78 had no effect on M. fermentans binding. Quantitative cytometric data were consistent with the fluorescence microscopic patterns observed under these same conditions (Fig. (Fig.3C).3C). The ability of FP29 to inhibit the interaction of M. fermentans with HeLa cells was also shown to result from direct interaction between FP29 and HeLa cells. Pretreatment of HeLa cells with FP29 followed by washing to remove unbound fusion protein before addition of M. fermentans led to inhibition of M. fermentans binding, whereas pretreatment of M. fermentans with FP29 prior to addition to HeLa cells did not affect M. fermentans binding (data not shown).

Since it was shown that FP29 was able to inhibit the binding of M. fermentans to HeLa cells, quantitative inhibition by FP29 was determined over a range of fusion protein concentrations, in parallel with control FP78 and other fusion proteins. By using fusion proteins at concentrations ranging from 8 nM to 2 μM, it was shown that FP29 was able to inhibit the binding of M. fermentans to HeLa cells completely and in a concentration-dependent manner (Fig. (Fig.3D).3D). Half-maximal inhibition of M. fermentans binding occurred at a fusion protein concentration of approximately 125 nM FP29 with near-maximal inhibition occurring at 1,000 nM. FP78 was unable to inhibit the binding of M. fermentans to HeLa cells (Fig. (Fig.3D),3D), even when tested at a concentration of 4 μM (data not shown). FPVlpB and MBP were also unable to inhibit M. fermentans binding to HeLa cells (Fig. (Fig.3D).3D). These data indicated that a ligand bound by soluble recombinant protein FP29 could be completely saturated, with the result that M. fermentans was unable to bind cells. This also directly implicated the P29 surface protein as the primary and perhaps exclusive adhesin mediating initial binding of the organism to HeLa cells.

The functional binding of P29 is localized to a central 78-amino-acid region.

Definition of an adhesin function by direct ligand binding involving a soluble protein and an intact cell has advantages over indirect methods such as Ab inhibition or binding interactions measured with acellular preparations. An immediate application, particularly pertinent in understanding the overall structure and surface display of the P29 protein, is the ability to test whether a localized portion of P29 is responsible for direct ligand binding. Initial identification and mapping of the putative binding region were therefore undertaken by creating MBP fusion constructs deleting regions of the P29 sequence and testing their ability to inhibit the binding of M. fermentans to HeLa cells, with the objective of defining deletion constructs retaining inhibition activity relative to FP29. Specific regions of the p29 gene were amplified by PCR with the primers listed in Table Table1.1. These were cloned into pMAL-c2 to create a nested set of FP29 constructs (Fig. (Fig.4)4) that were expressed and purified (Fig. (Fig.1B)1B) as described in Materials and Methods. By using this set of N- or C-terminal truncation constructs, it was possible to map the approximate (maximal) boundaries of the domain responsible for the inhibitory activity. The study was performed with a single concentration of fusion proteins in the range most sensitive to fluctuation, and only those constructs showing inhibition equivalent to that of FP29 were scored as positive. Results in Fig. Fig.44 show that function was retained when amino acids 2 to 87 of the P29 sequence were removed. Similarly, the C terminus of P29 was shown not to be necessary for inhibition of M. fermentans binding, since removal of amino acids 166 to 219 did not result in the loss of FP29 activity in this assay. This provisionally identified a portion of the P29 sequence, amino acids 88 to 165, as the putative region sufficient for ligand binding.

The proposed functional domain contains the two internal Cys residues which were previously shown to form an intrachain disulfide bond in the native P29 expressed in mycoplasmas (44). To determine if the disulfide bond was necessary for ligand binding function, an FP29 truncation construct (FP29-315) was made in which the Cys residue at position +91 was mutated to a Ser residue. With only one Cys residue per molecule, this construct was unable to form an intrachain disulfide bond and yet was able to inhibit M. fermentans binding to the same extent as FP29 and other active truncation constructs (Fig. (Fig.4).4). This suggests that formation of the disulfide bond is not required for function of the recombinant P29 adhesin. Parenthetically, while it is possible for FP29-315 and other constructs to form interchain disulfide bonds, the major species observed following expression and purification was found to be monomeric (24) (data not shown).

FP29 selectively inhibits the binding of some other mycoplasma species.

Since it was shown that FP29 is able to bind to HeLa cells and to inhibit the binding of M. fermentans, the possible utilization of the same HeLa cell ligand by other mycoplasma species was evaluated, by using the direct measurement of inhibition of mycoplasma binding. Two additional mycoplasma species were used in this initial comparison. M. hominis, a known human pathogen (22), had previously been shown in a similar flow cytometric assay to adhere to HeLa cells by the Vaa lipoprotein adhesin (54). M. capricolum, an unrelated mycoplasmal pathogen of sheep and goats (38), was used as a control that also bound HeLa cells, although little is known about its adherence mechanism or the host cell ligand(s) which it binds.

All three mycoplasma species were tested for the ability of FP29 to inhibit their binding in the assay already established for M. fermentans, with a normalized amount of labeled mycoplasmas and a fixed concentration of FP29 or control fusion proteins (Fig. (Fig.5).5). In direct comparison to the inhibition of M. fermentans (resulting in 10% residual binding), M. hominis binding was also significantly and reproducibly inhibited by FP29 (to approximately 40% residual binding). In contrast, FP29 had no effect on the binding of M. capricolum to HeLa cells. Neither FP78 nor MBP affected binding of any of these mycoplasmas. Together these preliminary results raise the possibility that M. hominis may share a common ligand with the M. fermentans adhesin P29, which is distinct from that used by M. capricolum.

FIG. 5.
Effect of FP29 treatment of HeLa cells on the binding of M. hominis and M. capricolum. FP29, FP78, and MBP were assayed at a concentration of 500 nM for the ability to inhibit the binding of M. fermentans, M. hominis, or M. capricolum to HeLa cells. The ...


This study provides direct evidence that the surface lipoprotein P29 of M. fermentans serves as the primary adhesin in the binding of the organism to cultured human HeLa cells and delineates a region of the P29 molecule that mediates binding to a ligand on those cells. Importantly, this was determined in an assay system developed explicitly for measuring direct mycoplasma-host interactions without perturbation of the host cell or organism. Because highly viable HeLa S3 cells can be grown and harvested without any treatment and freshly broth-grown mycoplasmas can be labeled with DiIC18 without measurable effect on viability, the resulting binding events reflect an authentic interplay. Additional technical and quantitative aspects of this assay are also noteworthy. First, the measurements of gated cell populations reflect only those interactions with cells. Moreover, the fluorescence measurements represent the integrated sum of individual cell binding events, providing assessment of the range of interactions within the population, in this case showing a very homogeneous characteristic. Finally, the use of the DiIC18 label provides a versatile method that can be readily applied to multiple mycoplasma species. It circumvents the need to generate and monitor binding by MAbs that (i) must be specific for a species, (ii) may differ in their affinity and efficiency of detection with secondary reporter Ab, and (iii) must bind to “irrelevant” surface structures that may in fact affect the surface architecture of the organism.

Importantly, we could demonstrate adhesin binding to HeLa cells as the interaction of a soluble recombinant fusion protein, FP29 (or the factor Xa-released P29 sequence), that represents the extracellular hydrophilic sequence of the surface lipoprotein. Consequently we could directly measure molecular interactions that defined a ligand on the HeLa cell surface. This ligand, while not identified in this study, is completely saturable and mediates rapid, strong binding of the P29 polypeptide sequence compared to unrelated fusion constructs. Neither the MBP fusion containing the VlpB protein sequence of M. hyorhinis nor that containing the full-length P78 lipoprotein sequence showed significant binding to HeLa cells. The latter control is noteworthy, since it contains Cys residues and also has a high pI, like P29. The basis for the very low binding of FP78 is not apparent. We reported earlier (45) that P78 is a putative substrate binding protein of an ABC transporter. Other substrate binding proteins have been reported elsewhere to serve as adhesins in some gram-positive organisms, including the ScaA protein of Streptococcus gordonii (40) and the Lmb protein of Streptococcus agalactiae (39). The relevance of FP78 interaction is unclear, but the binding characteristics do not suggest an adhesin function such as that demonstrated for P29.

Like the interaction of the soluble FP29 recombinant adhesin protein, the binding of M. fermentans cells to HeLa cells was also readily measured by this flow cytometric method and similarly demonstrated a dose-dependent increase and saturation. This again suggested binding to a finite ligand exposed on the surface of the intact host cell. Fluorescence microscopy revealed a pattern consistent with the distribution of mycoplasmas on the HeLa cell surface. This species has been shown elsewhere to interact with (and in some cases be internalized by) HeLa and other cells (3, 4, 42, 43). While internalized cells would be counted during flow cytometry, the relatively short time period of incubation and lower temperature of our assay would largely preclude this event, which in any case would occur after binding and without effect on measurements. Saturation was achieved with an input of approximately 600 mycoplasmas per HeLa cell, but the actual number bound at saturation has not yet been determined. A recent report (50) indicating that internalization of M. fermentans in HeLa cells per se was enhanced by the binding to mycoplasmas and activation of exogenous plasminogen probably reflects a mechanism distinct from, but possibly subsequent to, the initial events measured in this study.

We formally established the functional relation between the authentic mycoplasmal P29 surface protein and the soluble recombinant adhesin by demonstrating complete, quantitative inhibition of mycoplasma binding by FP29, whereas control constructs had no effect on this mycoplasma-host interaction. Moreover, inhibition was achieved in the submicromolar range, reflecting a limited amount of ligand and very specific interaction. Somewhat surprising was the finding that mycoplasma binding could be inhibited at about half saturation of ligand by FP29. The basis for this is not clear but could reflect a requirement for a minimal density of ligand to effect binding by the organism. At present, we do not know the biochemical nature of the ligand, its distribution or lateral mobility in the membrane, or its interaction, if any, with the HeLa cell cytoskeleton. In addition, while our present data clearly define the adhesin and the presence of a ligand, details of their molecular interactions will require additional understanding of the ligand and more detailed binding analysis. These aspects should be readily accessible with the present recombinant constructs and further refined derivatives, as tools to measure these interactions directly and manipulate the host cell ligand for this organism.

These results define the entire interaction of M. fermentans with a host cell in this system, which resembles the overall binding characteristics observed for some other mycoplasmas including Mycoplasma bovis (36) and M. arthritidis (47). However, better definition of the molecular details of mycoplasmal adhesins, their interaction with host ligands, and the presentation of their binding structures in the architectural context of the mycoplasma membrane surface will be required for a complete understanding of the host interaction of these organisms. Evolutionary solutions to binding (as manifest in protein structure and interactions) may be highly varied and will likely reflect the strong adaptive pressures that characterize the coevolution of a mycoplasma species and its host. Because P29 is fully active as a purified soluble protein, it apparently requires no complex structure or interacting partners to create the actual binding interface. Thus, the regions of the protein involved in binding could be assessed by mutagenesis. The very preliminary results obtained with truncation constructs identified a region of the molecule (amino acids 88 to 165) that is predicted to be sufficient for binding activity. Again, this contrasts with some more complex systems wherein the structure of the entire protein or specific interactions of partners are required for correct spatial presentation. No known binding motifs were apparent from inspection of the P29 sequence. Although we have not determined the minimal region required for activity, the active region delineated by these experiments was hydrophilic and included a disulfide loop of 36 amino acids. No strong motifs of secondary structure were indicated in this region, with the exception of a previously reported (44) alpha-helical repeat (Rep2) within the loop that was also reiterated near the N-terminal portion (residues 40 to 53) of the mature lipoprotein (Rep1). Further truncations and mutation of the binding region with constructs containing the putative adhesin binding domain should refine our understanding of the minimal domain and specific residues required for ligand binding on the natural host cell surface. This should be straightforward, since the actual intrachain disulfide bond is apparently not required for an active binding structure.

Other portions of the P29 sequence are of interest, both from structural predictions and from our knowledge of surface-accessible epitopes that reside on this molecule. The strongest predicted secondary structural element was a pronounced coiled-coil region spanning residues 18 to 53 and comprising five uninterrupted heptad repeat motifs of heterogeneous sequence. Thus, Rep1 corresponds approximately to the most distal of these heptad units. The role of this coiled-coil segment is not clear, but a construct (FP29-307) containing residues 2 to 70 (encompassing the entire motif) was inactive in binding to HeLa cells or inhibition of mycoplasma binding. It is perhaps more likely that this region plays a role in the overall surface topology or scaffolding of the protein on the mycoplasma membrane. Whether this membrane-proximal region may also mediate interactions between P29 lipoproteins, as predicted by one algorithm (http://nightingale.lcs.mit.edu/cgi-bin/multicoil), or promote an internally folded structure within the P29 monomer is not yet clear. Biophysical measurements of this domain in the recombinant construct may distinguish among these possibilities.

We were also able to refine the localization of two epitopes previously defined (44) by MAbs C19 and E12. The lack of E19 binding to construct FP29t, which replaced only the nine C-terminal residues of P29 (Fig. (Fig.1C),1C), confirmed the importance of this distal region of the protein in Ab binding. Our previous mapping of the C19 epitope indicated its presence on a construct containing the first 60 residues but not the first 40 residues of P29. The present study further localized the epitope by the observation that it was not demonstrable in a construct (FP29-308) eliminating only the first 37 residues of the protein (Fig. (Fig.1C).1C). Thus, the cognate epitope of C19 appears to reside in the vicinity of the coiled-coil region of the protein. Interest in these locations comes from an earlier study (44) showing that, within clonal lineages of M. fermentans, surface accessibility of these epitopes is phase variable, despite the constitutive expression of P29 and the integrity of the disulfide loop in these variants. Moreover, variation of accessibility occurred independently for these two structures. While it is not clear what other surface events might govern this variation, it was shown that the terminal regions of the P29 lipoprotein can be differentially displayed. With our new findings from the present report, we know that the ligand binding domain resides in a different, possibly unstructured region of P29. The M. fermentans (clone 39) population used in the cell binding studies described here displays both epitopes on the surface. A major, unanswered question raised by our present findings concerns whether the ligand binding domain of P29 per se is differentially presented or is subject to phase-variable presentation, leading to a phase-variable adherence phenotype. The nature of the events or structures mediating phase variation observed for P29 is not yet clear. One possible explanation could be the phase-variable expression of an interacting partner, as we have recently documented for another mycoplasma (55). Studies to address this possibility are in progress.

Finally, we were able to show even in our very preliminary experiments that P29 interacted with a HeLa cell surface ligand that apparently mediates binding of at least one other human mycoplasma, M. hominis. We also demonstrated that the ligand was not universally used, by showing a lack of inhibition of M. capricolum binding. This also raises an interesting possibility, that diverse adhesins of mycoplasmas may utilize very similar host cell ligands in some cases. Because the identity and properties of the HeLa cell ligand are unknown, speculation on the specificity of other mycoplasmal adhesins is premature. It is perhaps most interesting that we showed earlier (54) that binding of M. hominis to the same HeLa cell line, by essentially the same assay procedure, was mediated by the Vaa lipoprotein, as revealed by loss and reacquisition of adherence function in spontaneous phase variants differing in expression of the protein. If the ligands bound are similar, comparison of P29 and the entirely dissimilar Vaa protein (5, 20, 53) may be particularly instructive in understanding the convergent evolution of these binding structures.


We thank Robyn Watson-McKown for production and purification of MBP, Jeong Im for assistance in purification of proteins, and Mary Kim for preparation of MAbs.

This work was supported in part by DHHS grant AI32219 (to K.S.W.) from the National Institute of Allergy and Infectious Diseases. S.A.L. was supported by grant T32 GM08396 from the National Institute of General Medical Sciences as a trainee of the University of Missouri—Columbia Molecular Biology Program.


Editor: D. L. Burns


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