Strategy To Create Chimeric Proteins Derived from Functional Adhesin Regions of Mycoplasma pneumoniae for Vaccine Development

Abstract

The cell wall-less bacterium Mycoplasma pneumoniae is one of the most common agents of respiratory tract diseases in humans. Adhesin-mediated binding of the bacteria to host cells is a crucial step in colonization and subsequent pathogenesis. For the first time, we expressed 16 recombinant proteins covering almost the whole major adhesin P1 and the adherence-associated protein P30 to characterize these proteins immunologically and functionally. We describe a new in vitro assay using several human cell lines in combination with fluorescence-activated cell sorting analysis to screen antisera raised against the recombinant proteins quantitatively for adherence inhibition activity. The protein derived from the nearly C-terminal part of the P1 adhesin (amino acids [aa] 1288 to 1518) and the protein P30 (aa 17 to 274) especially showed prominent immunoreactivity with sera from M. pneumoniae-immunized guinea pigs as well as with M. pneumoniae-positive patient sera. We demonstrate that the same protein regions are involved in mediating cytadherence since antibodies against these adhesin regions decrease mycoplasma adhesion to human cells significantly. For further vaccine studies, we optimized the immunogenic and adherence-mediating properties of the antigen by combining both the P1 and the P30 regions in a novel chimeric protein. Antibodies against this protein show an increased reduction of M. pneumoniae adherence to human bronchial epithelial cells by 95%, which is comparable to results with polyspecific anti-M. pneumoniae animal serum. Our strategy results in a promising defined antigen candidate for reducing or even preventing M. pneumoniae colonization of the respiratory tract in future vaccination studies.

Epidemiological studies confirm that between 5 and 10% of all community-acquired pneumonia cases, especially in children and young adults but also in elderly patients, are attributed to the cell wall-less bacterium Mycoplasma pneumoniae (49). Epidemic outbreaks in geographically close populations and the occurrence of extrapulmonary complications of the primary respiratory infections emphasize the significant impact of the agent on public health.

Adhesion of M. pneumoniae to the host respiratory epithelium (cytadherence) is an essential first stage of infection and a precondition for successful colonization (19). Even though M. pneumoniae is one of the smallest and simplest microorganisms, M. pneumoniae cells exhibit a complex differentiated cell extension, the attachment organelle, that functions in different processes including cytadherence, cell division, and gliding (1, 32, 39). The analysis of mutants has resulted in the identification of an increasing number of proteins associated with cytadherence of M. pneumoniae (reviewed in reference 33), such as the transmembrane proteins P1 and P30, both densely clustered at the attachment organelle (31). The results of binding experiments and of the characterization of P1-deficient (avirulent) mutants established the role of the protein P1 as the main adhesin of the bacterium (18, 20, 39, 41). In addition, the P1 adhesin gene serves as an important marker for subtyping M. pneumoniae clinical isolates. Three subtypes (1 to 3) and three variants are discriminated on the basis of sequence differences in one or both of the repetitive elements, RepMP4 and RepMP2/RepMP3 (RepMP2/3), within this gene (7, 11, 30, 40, 45). The circulation of different subtypes and variants of M. pneumoniae in the human population is discussed as a possible reason for the epidemic outbreaks which occur every 3 to 7 years (36, 43).

The protein P30 was first characterized by Dallo et al. (4). The C-terminal region of P30 is dominated by proline-rich repeating motifs similar to those found near the C terminus of P1 (4). In-frame deletions in these repeat regions resulted in a loss of cytadherence (34). Analysis of P30 mutants suggested an association of the protein with proper cell development, appropriate conformation of the adhesin P1 in the mycoplasma membrane, and receptor binding (16, 42).

Besides their role in the attachment process, the adhesion proteins P1 and P30 act as potent immunogens that result in high titers of specific antibodies in serum of patients with acute M. pneumoniae infections (21, 24, 48). However, immunocompetent hosts with significant titers of anti-P1 antibodies are not protected from reinfection (28). Results of screening patient sera for adherence-inhibiting activity suggested that the P1 protein induces antibodies that are mostly not directed to cytadherence-mediating sites of the P1 protein (29).

Considering the prominent role of adhesins in the interaction of M. pneumoniae and its host, the characterization of P1 and other adhesion-associated proteins of M. pneumoniae should identify regions that might serve as effective vaccine candidates. In the past, the investigation of mycoplasma adhesins was hampered by technical difficulties in protein expression. Unlike the universal genetic code, M. pneumoniae uses the TGA codon to incorporate tryptophan rather than as a stop codon (22, 44). To circumvent the problem, different strategies have been applied (14). The use of recombinant proteins covering defined adhesin regions turned out to be a suitable tool to identify protein regions with antigenic and adherence-mediating properties (3, 8, 46). However, these previous investigations were applied to only a few protein regions of P1.

In this study, we present for the first time 15 recombinant proteins that provide almost complete coverage of the P1 adhesin of M. pneumoniae. Together with a recombinant protein derived from the adherence-associated protein P30, we characterized these adhesin regions by their serological reactions with sera both of immunized guinea pigs as model animals and of patients suffering from M. pneumoniae infections. The development of a novel adhesion inhibition assay enabled us to identify the functional attachment sites of M. pneumoniae adhesins. The consecutive determination of antigenic properties of distinct protein regions in humans and animals and their role in the attachment process provided the basis for a promising vaccine candidate characterized by a combination of immunogenic and adherence-mediating protein regions in a chimeric protein.

MATERIALS AND METHODS

Bacteria and growth conditions.

The M. pneumoniae wild-type strain M129 (ATCC 29342) was grown in 50 ml of PPLO medium (Difco, Becton Dickinson, Sparks, MD) in 150-cm² tissue culture flasks (Greiner, Frickenhausen, Germany) at 37°C (10). After a slight color change of the medium from red to orange, the mycoplasmas attached to the bottom were washed twice in room-temperature phosphate-buffered saline (PBS) and finally scraped off the flasks into the same buffer. Bacteria were pelleted by centrifugation at 3,345 × g for 15 min and resuspended in 1 ml of PBS.

Expression and purification of recombinant proteins.

DNA isolation from M. pneumoniae was carried out with a QIAamp DNA minikit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Defined regions of the genes coding for proteins P1 (MPN141) and P30 (MPN453) between the TGA codons were expressed as recombinant proteins (Fig. 1). Fifteen fragments of P1 were amplified by PCR (for primer sequences, see Table S1 in the supplemental material). A gene region containing four TGA codons (nucleotide [nt] position 1105 to 1260 from the start of P1 [17], referred to as RP4) was amplified using a multiple-mutation reaction (15). A major part of the protein P30 covering nt 49 to 824 from the start of the gene was amplified. In addition, a recombinant protein derived from the N terminus (nt 69 to 234) of the protein adenylate kinase (ADK; MPN185) was produced (designated RPADK). PCR products were cloned into the pET-30/LIC vector with a Ligation-Independent Cloning kit (Novagen, Madison, WI) as prescribed by the manufacturer. Transformation of Escherichia coli NovaBlue and BL21(DE3), sequencing of inserts, and expression and purification of the N-terminal six-His-tagged recombinant proteins were performed according to a recent study (12).

FIG. 1.

Schematic illustration of proteins P1 (MPN141; according to reference 10) and P30 (MPN453) of M. pneumoniae strain M129 and location of recombinant proteins RP1 to RP15 and RP30.

SDS-PAGE and Western Blotting.

To confirm and compare the quality of recombinant protein expression, the protein eluate fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% NuPage Bis-Tris gels (Invitrogen, Carlsbad, CA), followed by Coomassie staining (Merck, Darmstadt, Germany). For immunoblotting, the separated protein fractions were transferred to nitrocellulose membranes (Invitrogen) by standard procedures. The recombinant proteins were detected by anti-His tag monoclonal mouse antibodies (1:500; Sigma, St. Louis, MO) combined with a peroxidase-conjugated anti-mouse immunoglobulin G ([IgG] 1:1,000; Pierce, Rockford, IL). Finally, the membrane was developed with the substrate 4-chloro-1-naphthol (Serva, Heidelberg, Germany) in the presence of hydrogen peroxide.

Immunoreactivity of recombinant proteins.

Antibody responses to the recombinant proteins were evaluated by immunoblotting of whole-cell preparations of M. pneumoniae M129. Human acute-phase serum that tested positive for IgA and IgM antibodies to M. pneumoniae by commercial enzyme-linked immunosorbent assay (ELISA) (Genzyme Virotech, Rüsselsheim, Germany) was used (1:500) in combination with a 1:5,000 dilution of anti-human Ig (Pierce). Furthermore, ELISAs of recombinant proteins with hyperimmune serum from guinea pigs immunized subcutaneously or infected intranasally with M. pneumoniae were performed as described previously (12). Briefly, Mikrolon plates (Greiner) were coated with 0.25 μg of recombinant protein (in 0.05 M carbonate buffer, pH 9.6) per well. After a blocking step, antigen-coated wells were incubated sequentially for 1 h with a 1:500 dilution of animal serum and a 1:5,000 dilution of peroxidase-conjugated anti-guinea pig IgG (Sigma). After the addition of substrate (TMB Super Slow; Sigma) the reaction was stopped, and the optical density (OD) was measured at 450 nm and a reference wavelength of 620 nm (OD_450/620). Carbonate buffer without antigen was used as a negative control. Test results with OD_450/620 values exceeding 0.3 were considered positive.

Production of guinea pig polyclonal antibodies.

The recombinant protein fractions were diluted to a concentration of 100 μg in 200 μl of PBS and mixed with 100 μl of complete Freund's adjuvant (Sigma). Male guinea pigs of the same age (Charles River, Sulzfeld, Germany) were given a subcutaneous injection of antigen (one guinea pig per recombinant protein). Booster immunizations and the collection of serum were performed as described previously (12). Serum was stored at −20°C until use.

The successful immunization was confirmed by immunoblotting of whole-cell preparations of M. pneumoniae M129 with the obtained antiserum (1:100) in combination with a 1:5,000 dilution of peroxidase-conjugated anti-guinea pig IgG.

The reaction of antibodies induced against the recombinant proteins to the whole-cell antigen of M. pneumoniae M129 was quantified by ELISA. A freshly grown M. pneumoniae culture was harvested as described and lysed by sonication for three cycles of 15 min each. The protein concentration was determined using a bicinchoninic acid protein assay (Pierce), 96-well plates were coated (0.75 μg of lysate per well), and an ELISA was performed with a 1:100 dilution of antiserum and a 1:5,000 dilution of peroxidase-conjugated anti-guinea pig IgG.

Preparation of human cells for infection with mycoplasma.

The cell lines HeLa (human cervical carcinoma cell line; ATCC CCL-2) and MRC-5 (human fetal lung fibroblasts; ATCC CCL-171) were cultured in tissue culture flasks containing Dulbecco's modified Eagle's medium or minimal essential medium (Gibco-Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) at 37°C in a 5% CO₂-95% air atmosphere. Primary human bronchial epithelial cells (HBEC; PromoCell, Heidelberg, Germany) were grown in Airway Epithelium Medium (PromoCell) according to the manufacturer's recommendations. Cells were detached using accutase (PAA Laboratories, Pasching, Austria) immediately prior to an adhesion inhibition assay. After centrifugation at 200 × g for 10 min, cells were resuspended carefully in HEPES buffer (PromoCell). Due to the different cell sizes of the cell types used, the optimal amount of cells per sample was determined by testing several cell concentrations (see Results).

In vitro adhesion inhibition assay using antiserum against recombinant proteins.

A freshly grown M. pneumoniae culture was harvested as described above and resuspended in 1 ml of PBS (pH 7.2). To avoid the noticeable self-aggregating feature of M. pneumoniae cells, the suspension was sheared through a 27-gauge needle. Fluorescent labeling of bacteria with fluorescein isothiocyanate ([FITC] 0.015 mg/ml; Fluka, Buchs, Switzerland) was performed by head-over-head rotation for 50 min at 37°C. Subsequently, labeled microorganisms were washed three times with PBS and resuspended to an OD₆₆₀ of 0.44. For opsonization, the mycoplasma suspension was diluted 1:40 (incubation with HeLa or MRC-5 cells) or 1:5 (incubation with HBEC) in 200 μl of PBS containing 25% PPLO medium and 10% heat-inactivated antiserum against a particular recombinant protein. The mycoplasma-antiserum mixtures were shaken for 1 h at 37°C. Following opsonization, human cells were added, and the suspensions were incubated at 37°C with head-over-head rotation. Adherence was reduced immediately after the addition of human cells (0 h) or after an incubation time of 1 h by diluting the mixtures 1:2 with ice-cold PBS (35). The samples were kept on ice and immediately subjected to flow cytometry.

The reproducibility of the assay was checked by including negative controls such as preimmune guinea pig serum and antiserum against a part of the cytosolic M. pneumoniae protein ADK. The results after incubation of the bacteria with this antiserum were considered to correspond to the mycoplasma adherence to human cells in the presence of antibodies against a His-tagged recombinant protein derived from a protein with no connection to the adhesion process. The hyperimmune serum from a guinea pig, stimulated subcutaneously three times with M. pneumoniae M129 whole cells, served as a positive control in each run.

To determine the detection threshold of added mycoplasmas to the human cell preparation using flow cytometric analysis, serial twofold dilutions of FITC-labeled mycoplasmas, adjusted to an OD of 0.44, were preincubated with preimmune guinea pig serum. After opsonization, each sample was divided into two aliquots. One aliquot was incubated with 8 × 10⁴ HBEC for 1 h and analyzed by flow cytometry as described above. The other aliquot was used to determine the number of CFU of M. pneumoniae. Therefore, appropriate dilutions in PBS were plated on PPLO agar plates (Becton Dickinson) and incubated at 37°C, and after at least 10 days the CFU were quantitated.

Analysis was performed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and monitored using CellQuest software, version 3.3 (Becton Dickinson). The cell population was gated in a forward-angle light scatter versus side-angle light scatter cytogram. Routinely, a total of 5,000 cells per sample were counted and analyzed for FL1 fluorescence. The resulting mean fluorescence intensity obtained after incubation of the mycoplasmas with the anti-RPADK serum was set to 100%.

Generation of chimeric protein.

To combine the gene regions coding for recombinant proteins RP14 (nt 3478 to 3630 from the start of the P1 gene) and RP30 (nt 49 to 824 from the start of the P30 gene), DNA of M. pneumoniae M129 was amplified using the primers listed in Table S1 in the supplemental material. PCR products were purified using a QIAquick PCR purification kit (Qiagen), and the restriction sites introduced were digested with PstI restriction enzyme (NEB, Ipswich, MA) as recommended by the manufacturer. The purified DNA fragments were ligated overnight at room temperature with T4 DNA ligase (Boehringer, Mannheim, Germany). The ligation product was integrated into the pET-30 Ek/LIC vector. Transformation of E. coli NovaBlue and BL21(DE3) and the expression and purification of the chimeric protein (denoted as HP14/30) as well as the generation of monospecific antiserum against the chimeric protein were performed as described above.

Comparison of the adherence-inhibiting activity of anti-RP14, anti-RP30, and anti-HP14/30 antisera.

Fluorescence-labeled mycoplasmas were preincubated with serial twofold dilutions (1:10 to 1:320) of antiserum against the single recombinant proteins RP14 and RP30 and against the chimeric protein HP14/30 before incubation with HBEC. Otherwise, the procedure was the same as described for the adhesion inhibition assay. Preimmune guinea pig serum and serum from an animal immunized subcutaneously with M. pneumoniae M129 again served as negative and positive controls, respectively.

Statistical methods.

In each experimental round, all samples of the adhesion inhibition assay were tested as duplicates or triplicates, as well as being repeated on at least three different experimental days. Results are expressed as mean values with standard deviations. Multiple group comparisons were performed with one-way analysis of variance, followed by a Dunnett T3 test (SPSS 13.0, Chicago, IL) to compare multiple experimental groups to a single control (anti-RPADK) or for pairwise comparisons. Significant levels were set at P values of <0.05 or <0.005.

RESULTS

Expression of recombinant proteins.

Circumventing the 21 TGA codons, 90% of the whole P1 gene was amplified and expressed successfully in E. coli as 15 recombinant proteins designated RP1 to RP15. Accordingly, the recombinant protein RP30 comprising the major part of the adhesin P30 was produced (Fig. 1). Analysis of all recombinant proteins by SDS-PAGE and subsequent Coomassie staining showed the migration of the recombinant proteins at their predicted molecular sizes. In Western blot analysis all expressed proteins reacted strongly with monoclonal His tag antibody (data not shown).

Immunological characterization of recombinant proteins.

To determine the antigenicity of the expressed protein regions derived from the adhesins P1 and P30 in humans, immunoblotting of each of the recombinant proteins with 14 serologically confirmed M. pneumoniae-positive patient sera was performed (Fig. 2A). The recombinant protein RP2 as well as the proteins RP8 and RP9, representing the middle region of P1, showed undetectable immunoreactions. Moreover, the recombinant protein RP13 covering a subtype-specific part of P1 within the repetitive element RepMP2/3 did not react with any tested patient serum. By contrast, the recombinant proteins RP1, RP5, RP6, and RP12 showed a specific reaction with at least 11 of 14 sera examined. The recombinant protein from P30 reacted with all patient sera, as did the recombinant proteins derived from the C terminus of P1 (RP14 and RP15).

FIG. 2.

(A) Results of immunoblot analysis of recombinant proteins from the P1 adhesin (RP1 to RP15) and the protein P30 (RP30) of M. pneumoniae with 14 human sera that tested positive for IgA and IgM antibodies to M. pneumoniae by commercial ELISA. (B) ELISAs of recombinant proteins RP1 to RP15 and RP30 with hyperimmune serum from guinea pigs immunized subcutaneously (s.c.) or stimulated intranasally (i.n.) with M. pneumoniae M129 (arithmetic mean of OD_450/620 values of four replicates with standard deviations). (C) Adhesion inhibition assay. Cytadherence of M. pneumoniae M129 to HBEC (HBEpC), MRC-5 cells, and HeLa cells after preincubation of mycoplasmas with monospecific antiserum against recombinant proteins RP1 to RP15 and RP30. Preimmune serum (NS; tested after 0 and 1 h of incubation of opsonized mycoplasmas with human cells) and anti-RPADK serum served as negative controls. Hyperimmune serum (PS) from an animal immunized subcutaneously with M. pneumoniae M129 served as a positive control. In each experimental run, all samples were tested as duplicates or triplicates, and tests were repeated on at least three different experimental days. Values represent the arithmetic means with standard deviations and are expressed as a percentage of the control (anti-RPADK). Significant levels for reduction of cytadherence in comparison to the effect of anti-RPADK were set at a P value of <0.05 (*) or <0.005 (**).

To confirm the detected immunological properties of the recombinant proteins, the ELISA reactivities with sera from guinea pigs obtained after subcutaneous immunization or intranasal infection with M. pneumoniae M129 were analyzed (Fig. 2B). The hyperimmune serum obtained after subcutaneous immunization showed no or only slight reactions with all recombinant proteins except with RP14 and RP30, where high OD_450/620 values were detected. In contrast, the animal serum obtained after intranasal stimulation with M. pneumoniae reacted with 6 of 15 P1-derived recombinant proteins including RP3 (comprising the subtype-specific region within RepMP4 of the P1 protein), RP5, RP7, RP10, and RP13 (genotype-specific region within RepMP2/3). The highest immunoreactions were again obtained with RP14 and RP30.

Generation of monospecific antiserum against recombinant proteins.

In order to produce monospecific antiserum for in vitro adhesion inhibition testing, guinea pigs were immunized with the purified recombinant proteins. In immunoblotting with whole-cell preparations of M. pneumoniae, all antisera obtained reacted exclusively (data not shown) with proteins P1 (169 kDa; anti-RP1 to -RP15) and P30 (30 kDa; anti-RP30).

To compare the reaction of antibodies induced against each of the recombinant proteins, an ELISA with M. pneumoniae cell lysates was performed (data not shown). All 15 antisera against P1 regions showed weak but detectable ELISA reactivities with comparable OD_450/620 values of about 0.3. In contrast, the reaction of mycoplasma lysate with anti-RP30 serum resulted in remarkably higher OD values of 1.7.

Screening of monospecific antiserum against the recombinant proteins for cytadherence-inhibiting activity using an in vitro adhesion inhibition assay.

The adhesion inhibition assay using fluorescence-activated cell sorting analysis was established to examine quantitatively whether monospecific antisera against defined regions of the adherence-related proteins P1 or P30 are able to decrease binding of M. pneumoniae M129 to several human cell lines. The highest level of cytadherence of M. pneumoniae in the presence of preimmune serum (negative control) and the highest reduction of cytadherence with anti-M. pneumoniae serum (positive control) were detected when cell suspensions were adjusted to 2.0 × 10⁵ HeLa cells, 8.0 × 10⁴ HBEC, and 7.5 × 10⁴ MRC-5 cells per sample (data not shown). Furthermore, preliminary tests were carried out to determine the minimal number of mycoplasmas that could be detected by fluorescence-activated cell sorting analysis (Fig. 3). The mean fluorescence of cells, equivalent to the number of adherent mycoplasmas per cell, seemed to be proportional to the mycoplasma-to-cell ratio used. Screening of recombinant protein-specific antiserum for adherence-inhibiting antibodies was performed with a mycoplasma dilution of 1:5. In this case the fluorescence intensity of cells (set to 100%) corresponded to a number of 171 (± 31) bacteria in the mycoplasma-cell preparation, whereas 86 (± 20) bacteria/assay (1:10 dilution of mycoplasma suspension) resulted in a fluorescence intensity of around 40%. When the mycoplasma suspension was diluted at 1:40 or higher, only a slight fluorescence of cells was detectable. This indicates that an interaction of M. pneumoniae and HBEC with a ratio of 7 or fewer bacteria/cell can hardly be detected by the flow cytometric approach used.

FIG. 3.

Determination of the detection threshold of the in vitro adhesion inhibition assay by flow cytometric analysis of mean fluorescence intensity of human cells and quantification of CFU of mycoplasmas. Serial twofold dilutions of mycoplasma suspensions were pretreated with preimmune guinea pig serum and incubated with HBEC (HBEp). The mean fluorescence intensity of HBEC was determined by flow cytometry. The value obtained under standard conditions (1:5) was set to 100%. The number of CFU of M. pneumoniae in each sample was determined by the plate count method, and the statistical number of mycoplasmas per cell was calculated. Values represent the arithmetic means ± standard deviations of three biological replicates.

Results of adherence experiments using mixtures of M. pneumoniae M129 and serum against the different recombinant proteins are summarized in Fig. 2C. FITC-labeled mycoplasma cells pretreated with preimmune serum showed a high binding capacity to all three human cell lines tested. Incubation of mycoplasmas with anti-RPADK antibodies resulted in 10% reduction of attachment to HeLa cells and HBEC and 30% reduction of attachment to MRC-5 cells compared to the cytadherence level obtained with preimmune serum. As expected, the incubation with the antiserum against M. pneumoniae whole cells obtained after subcutaneous immunization of the animal significantly reduced adherence of the mycoplasmas to all cell lines tested. The relative inhibition of adhesion ranged from 96% (HBEC) to 87% (HeLa cells) compared to anti-RPADK serum (P < 0.001).

Analysis of monospecific antiserum against the P1-derived recombinant proteins RP1 to RP13 and RP15 resulted in no significant reduction of mycoplasma attachment to bronchial epithelial cells (HBEC). The relative cytadherence in the presence of these sera varied only slightly from 94% (anti-RP11) to 110% (anti-RP12) compared to the negative control anti-RPADK. In contrast, antibodies against RP14, which covers nearly the entire C-terminal part of adhesin P1, were significantly able to inhibit M. pneumoniae from binding to HBEC by 95% (P = 0.0001), which is comparable to the effect of the M. pneumoniae whole-cell antiserum. Furthermore, a significant reduction of mycoplasma adhesion to HBEC by 28% was observed after treatment with anti-RP30 immunoglobulins (P = 0.017).

Screening of recombinant protein-specific antiserum on adherence-inhibiting activity using MRC-5 lung fibroblasts resulted in similar observations. Most antisera against P1 showed no reduction or only a slight reduction in mycoplasma adhesion in comparison with the nonspecific effect of anti-ADK serum (up to 5% in the case of anti-RP5). In agreement with the results using HBEC, preincubation with antibodies against RP14 reduced the adhesion of bacteria significantly (P = 0.015) but only by 58%. When mycoplasmas were pretreated with anti-RP30 serum, a reduction of adherence of up to 39% was measured (P = 0.143). In contrast to the observations with HBEC, antibodies against another P1 region (RP10) showed a detectable but not significant inhibition of M. pneumoniae binding to MRC-5 cells (24% inhibition; P = 0.183).

The performance of the assay using HeLa cells largely confirmed the results obtained with the other two cell lines. However, the level of adherence-inhibitory activity of anti-RP14 and anti-RP30 sera was much lower. Both antisera reduced mycoplasma adhesion to HeLa cells by nearly the same extent to a relative cytadherence of 75% (P = 0.112) and 68% (P = 0.045) only. All other recombinant protein-specific antisera showed no effect or only slight effects on mycoplasma binding.

The level of adherence inhibition detected in the presence of anti-RP14 antiserum differed significantly between the several cell types (HBEC versus MRC-5, P = 0.021; HBEC versus HeLa, P = 0.004; MRC-5 versus HeLa, P = 0.015). Comparing all data of the different experiments (n = 7 to 9) using mycoplasmas incubated with the M. pneumoniae whole-cell antiserum, an influence of the mycoplasma preparation on the relative adherence to the human cell lines could not be observed (mean relative adherence using HBEC, 5.8 ± 2.6%; MRC-5 cells, 8.7 ± 2.0%; and HeLa cells, 13.2 ± 5.1%).

Taking into account the differences in the adherence pattern between the cell lines tested, we performed further inhibition experiments using HBEC exclusively since the in vitro interaction between M. pneumoniae and this cell type seems to be closest to the natural host-pathogen interaction.

Despite determination of M. pneumoniae-specific antibody responses in each of the recombinant protein-specific antisera, one might suppose that a missing inhibitory effect on mycoplasma adhesion was due to a lower concentration of adherence-inhibiting antibodies. To exclude possible false-negative results, three randomly selected antisera against recombinant proteins derived from the P1 adhesin were tested together with the anti-RP30 serum in dilutions of 1:5, 1:10, and 1:20 (data not shown). Mycoplasma adherence to HBEC in the presence of serially diluted anti-RP2, anti-RP4, and anti-RP10 serum was equivalent and without effect. In contrast, pretreatment with anti-RP30 serum resulted in a concentration-dependent reduction of adherence of M. pneumoniae cells ranging from 25% relative adherence with antiserum diluted 1:5 to 110% relative adherence with a 1:20 dilution of serum.

Generation of chimeric protein.

The highly immunogenic and potentially functional domains within the recombinant proteins RP14 and RP30 were combined in one chimeric protein, HP14/30, in order to enhance the immunogenicity of the antigen and the adherence-inhibiting properties of the resulting antiserum.

The predicted molecular size of HP14/30 (57.3 kDa) was confirmed by SDS-PAGE, followed by Coomassie staining and Western blotting with monoclonal anti-His tag antibodies and polyclonal anti-M. pneumoniae M129, anti-RP14, and anti-P30 guinea pig serum (Fig. 4A and B). The antiserum raised against HP14/30 reacted specifically with the P1 and P30 proteins of M. pneumoniae M129 (Fig. 4C). ELISA studies using the single recombinant proteins RP14 and RP30, the chimeric protein HP14/P30, and the whole-cell antigen of M. pneumoniae M129 as antigens in addition to sera from guinea pigs immunized subcutaneously or infected intranasally with M. pneumoniae M129 confirmed the increased antigenicity of the chimeric protein. Both antisera showed higher reactivities with HP14/30 than with the single recombinant proteins RP14 and RP30 (data not shown).

FIG. 4.

Analysis of chimeric protein HP14/30 and the monospecific antiserum derived. (A) SDS-PAGE of purified protein HP14/30 (lane 1) stained with Coomassie blue. (B) Reaction of chimeric protein HP14/30 with anti-His monoclonal antibody (lane 1), polyclonal anti-M. pneumoniae M129 (lane 2), and anti-RP14 (lane 3) and anti-RP30 (lane 4) guinea pig serum. (C) Reaction of M. pneumoniae M129 whole-cell antigen with polyclonal guinea pig antiserum against the recombinant proteins HP14/30 (lane 1), RP14 (lane 2), and RP30 (lane 3). Lanes M, molecular mass markers.

Comparison of the adherence-inhibiting activity of anti-RP14, anti-RP30, and anti-HP14/30 antisera.

In order to compare the adherence-inhibitory effects of the antisera against the single recombinant proteins RP14 and RP30 to the blocking activity of the antiserum against the chimeric protein, serial twofold dilutions of antiserum were tested (Fig. 5). The anti-RP30 serum showed adherence inhibition up to a dilution of 1:10. The antiserum against RP14 was able to block cytadherence by around 60% up to a dilution of 1:80. In comparison to the serum against both of the single recombinant proteins, the adherence-inhibitory effect increased more than additively when the serum obtained after immunization with the chimeric protein HP14/30 was used. In this case a reduction of mycoplasma adhesion could be observed even with a 1:320 dilution of antiserum, which is comparable to the results from anti-M. pneumoniae M129 whole-cell serum.

FIG. 5.

Adherence of M. pneumoniae cells to HBEC after pretreatment of mycoplasmas with preimmune serum, anti-RP14 serum, anti-RP30 serum, anti-HP14/30 serum, and serum from an animal immunized subcutaneously with M. pneumoniae M129. In each experimental run, all samples were tested as duplicates or triplicates. Values represent the arithmetic means ± standard deviations of at least two independent experiments and are expressed as a percentage of the control (preimmune serum). The broken line represents the maximal level of attachment of M. pneumoniae cells when cells were preincubated with preimmune serum.

DISCUSSION

For initiating an infection, the mucosal pathogen M. pneumoniae uses a number of adherence-related proteins mainly clustered in the tip structure of the bacteria. However, only limited data are available describing antigenic adhesin regions of M. pneumoniae that play a functional role in the adhesion process targeting host cells. To study the P1 and P30 adhesins in more detail, 16 His-tagged recombinant proteins were expressed. Analysis of the immunoreactivity of these proteins with M. pneumoniae-positive patient serum and serum from guinea pigs stimulated or infected with M. pneumoniae confirmed the prominent antigenicity of the expressed part of protein P30 and of the nearly C-terminal region of protein P1 (2, 3, 23, 46, 47). In addition, at least five P1 regions (RP1, RP5/RP6, RP12, and RP15), reacted strongly with most or all patient sera examined. To illustrate the current knowledge about immunoreactive and adherence-related regions in the P1 adhesin, the published results are summarized in Fig. 6. In agreement with the data of the present report, epitope mapping studies (23, 26) identified immunoreactive epitopes within protein regions covered by the recombinant proteins RP1, RP6, and RP12. In contrast, the P1 regions expressed as RP2, RP8/RP9/RP10, and RP11 showed no or only slight ELISA reactivities with patient sera, which is in accordance with previous findings (3, 23). Differences between studies might be attributed to the length of the antigen used, i.e., short peptides previously (23) in contrast to larger recombinant proteins in the present study.

FIG. 6.

Schematic illustration of localization of immunoreactive and adherence-mediating sites of the P1 protein of M. pneumoniae based on the results of the present study (A) or previous studies (B). In panel B, the following are indicated: immunoreactive epitopes (A, B, and H [23]; C to F [26]; G [2]; and J [3]), nonimmunoreactive epitopes (a [3] and b to g [23]), adherence-mediating sites (I, III, V to VII, IX, and X [13]; II and IV [25]; VIII [6]; XI [46]; and XII [8]), and a noncytadherent region (i [46]).

Comparison of the reactivity of recombinant proteins with human serum and serum from intranasally stimulated guinea pigs revealed differences between the induced immune responses in both hosts after M. pneumoniae infection. The investigation of a recombinant protein derived from part (amino acids 9 to 315) of the adherence-associated protein P65 (MPN309) also resulted in no reaction with anti-M. pneumoniae M129 whole-cell serum, but the protein reacted strongly with sera of M. pneumoniae-infected patients (data not shown). These observations reinforce the hypothesis of a selective immunity (29), which is of importance in further vaccine and infection studies. The striking differences between the reactivity of serum obtained after intranasal infection or subcutaneous immunization of guinea pigs with M. pneumoniae were already described for genotype-specific P1 regions (12) and have an obvious influence on the immune response to particular regions of the P1 adhesin.

To investigate the role of certain protein regions in the adherence process, a new approach was developed to identify functional attachment sites. The use of flow cytometry to quantify the mycoplasmas attached to eukaryotic cells turned out to be a rapid method for obtaining quantitative, reproducible, and objective results. Thus, this approach could be an excellent alternative to replace time-consuming, more subjective or laborious techniques that are currently used to enumerate attached mycoplasmas (8, 16, 46). To summarize, only antibodies against recombinant protein RP14 of the P1 adhesin and against the recombinant protein derived from protein P30 decreased M. pneumoniae adherence to all three human cell lines tested. No significant adherence inhibition was detected with antiserum against other P1 regions, indicating that the conserved, nearly C-terminal P1 region plays a major role in receptor binding. Adherence-mediating epitopes within the nearly C-terminal region of P1 were recognized in previous studies (6, 13). Moreover, Svenstrup et al. (46) demonstrated a specific interaction of this domain with Hep-2 cells. Epitope mapping studies using monoclonal antibodies and sheep erythrocytes as target cells (13, 25) showed further adherence- mediating epitopes in several positions of P1 such as the regions covered by the recombinant proteins RP3, RP6, and RP9/RP10 (Fig. 6). In fact, antibodies against RP10 were able to decrease cytadherence of M. pneumoniae cells to MRC-5 lung fibroblasts by 42% but had no measurable effect on the interaction of mycoplasmas with HeLa or HBEC. The divergent findings of the studies mentioned in comparison with results of the present report might be explained by the use of different methods and/or different host cells such as Hep-2 cells or erythrocytes (8, 27, 29, 46), which might differ in the receptor configuration (37, 38, 50). Despite the fact that M. pneumoniae has a high binding capacity to HeLa cells, MRC-5 lung fibroblasts, and primary bronchial epithelial cells, it is still unclear if attachment of M. pneumoniae to different cell types is mediated by the same adherence mechanisms. Although differences in the level of adherence inhibition were observed, antisera against the recombinant proteins RP14 and RP30 were able to reduce mycoplasma adherence to all cell lines tested. This observation confirms that both the region near the C terminus of P1 and the part of P30 tested are crucial for adhesion to a variety of cell types. The results were additionally supported by previous studies (5, 34, 46) that provide convincing evidence of surface exposure of the protein regions expressed as RP14 and RP30.

In addition to the main P1 and the P30 adhesins, other surface proteins of M. pneumoniae have been characterized as adherence associated and are of interest for a selective approach to influence the adhesion of mycoplasma cells in vitro and in vivo. A portion of the protein P65 showed antigenicity to M. pneumoniae-positive human serum, but the antiserum against this protein region was unable to decrease the adherence of mycoplasmas to all three human cell lines investigated (data not shown). Svenstrup et al. (46) reported that antibodies against the surface protein P116 inhibited cytadherence to Hep-2 cells to the same extent as antiserum against the nearly C-terminal part of P1. Despite the immunogenicity of P116 (9), it was suggested that this protein might serve as an additional adhesin for attachment to Hep-2 cells. Further investigations are necessary to identify other protein regions that are useful for vaccination experiments. In the search for such candidate antigens, the results of the present study led to a chimeric protein combining the immunogenic and adherence-mediating properties of both the RP14 and RP30 regions. Results of the adhesion inhibition assay predict a protective immunity in vivo since antibodies against the chimeric protein show increased influence on the adhesion of M. pneumoniae cells, which is comparable to the effect of the polyspecific anti-M. pneumoniae serum. It is reasonable to expect antibodies to these conserved parts of proteins P1 and P30 to inhibit adherence of all subtypes and variants of M. pneumoniae to the epithelial cells of the host respiratory tract. To support this, the investigation of the subtype 2 strain FH (ATCC 15531) resulted in adherence inhibition rates to HBEC which were comparable with the data from subtype 1 strain M129 (data not shown).

In summary, the findings of the present study offer new insights into the functionality of P1 and P30 adhesins of M. pneumoniae. Further analysis of adhesin domains should lead to a better understanding of attachment mechanisms and the subsequent steps associated with pathogenesis of M. pneumoniae infections. We demonstrated not only that distinct protein regions of M. pneumoniae-specific adhesins show strong antigenic cross-reactivity with human and animal sera but also that antibodies against these regions reduce adherence of mycoplasmas to human cells. This strategy seems to be promising for further targeted animal studies testing optimized antigens as vaccines.