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1 Institute of Medical Microbiology and Immunology, Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark
2 Department of Molecular Biology, Gustav Wieds Vej 10C, University of Aarhus, DK-8000 Aarhus C, Denmark
3 Loke Diagnostics ApS, Sindalsvej 17, DK-8240 Risskov, Denmark
Correspondence
M. Drasbek
drasbek{at}medmicro.au.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A key characteristic of M. pneumoniae is the lack of a cell wall, which allows exchange of different components between the host membrane and the mycoplasma membrane after adhesion (Razin et al., 1981). Apart from respiratory-tract cells, M. pneumoniae adheres to many different types of eukaryotic cells in vitro, e.g. HEp-2, HeLa, macrophages and erythrocytes (Razin et al., 1981).
M. pneumoniae is elongated and consists of a longer tail-like rear end, a thicker body part and a frontal attachment organelle. This organelle is composed of a network of proteins, of which the adherence accessory proteins are thought to confer the tip structure, and to underlie mycoplasma mobility as well as the clustering of a network of adhesins at the tip (Bredt, 1979; Waites & Talkington, 2004). The P1 protein, which is mainly concentrated at the tip, is one of the major adhesins in M. pneumoniae, and the loss of P1 through mutation results in reduced adherence to host cells (Krause, 1996). Furthermore, a fragment of the C-terminal part of the P1 protein has been shown previously to be immunogenic, as sera from M. pneumoniae patients show a reaction with this fragment (Drasbek et al., 2004). Recently, it has been seen that anti-P1 antibodies reduce the gliding speed of M. pneumoniae, thus hampering the mobility of the bacterium and possibly its ability to find suitable host adhesion receptors (Seto et al., 2005).
Attachment of bacteria to host cells is one of the key steps of infection and often relies on an interaction between bacterial adhesins and oligosaccharides on the host cells. These interactions are usually mediated by binding of lectins present on the bacterial surface to oligosaccharide chains bound to glycoproteins and glycolipids on eukaryotic cells (Thomas & Brooks, 2004). This is probably also the case for M. pneumoniae attachment, since reduced adhesion of M. pneumoniae has been demonstrated when human alveolar epithelial cells (A549) are pre-treated with tunicamycin, which degrades oligosaccharides (Thomas & Brooks, 2004). In addition, other studies have shown that the adhesion of M. pneumoniae to tracheal epithelial cells decreases by 50–65 % when they are treated with neuraminidase, which cleaves terminal acylneuraminic residues from oligosaccharides, glycoproteins and glycolipids. Likewise, heat, methiolate, glutaraldehyde and formalin also inhibit the adhesion of M. pneumoniae to eukaryotic cells (Gabridge & Taylor-Robinson, 1979; Razin et al., 1981).
Adhering mycoplasmas contain several adhesins, and M. pneumoniae expresses at least three different adhesins, termed P1, P30 and P116, suggesting that there are multiple receptors for M. pneumoniae on the host cells (Razin & Jacobs, 1992; Svenstrup et al., 2002). Monospecific antibodies raised against P116 block the adhesion of M. pneumoniae to HEp-2 cells, as do antibodies against the P30 protein (Baseman et al., 1987). It is, however, unclear whether this inhibition is direct or results from steric hindrance of neighbouring structures. Recently, elongation factor Tu and the pyruvate dehydrogenase E1 
subunit have been shown to bind fibronectin, which is usually found in the extracellular matrix of eukaryotic cells (Dallo et al., 2002). In addition, the surface protein P65 contains an Arg-Gly-Asp (RGD) domain, raising the possibility that M. pneumoniae attaches to binding sites for fibronectin on the host cell (Krause, 1996).
Recently, it has been shown that antibodies against the C-terminal part of the M. pneumoniae P1 protein and the homologous Mycoplasma genitalium MgPa protein inhibit host-cell adhesion of M. pneumoniae and M. genitalium, respectively (Svenstrup et al., 2002). mAbs that recognize epitopes in this region (Dallo et al., 1988; Jacobs et al., 1990) also inhibit the binding of M. pneumoniae to host cells. However, no inhibition is observed using antibodies that target the N-terminal parts of the proteins. In mycoplasma-free extracts, P1 binds to host cells, suggesting a direct role for P1 in host receptor binding (Krause, 1998; Razin & Jacobs, 1992). Thus, the anti-P1 antibody inhibition of adhesion could originate from blocking of the adhesion region of P1. However, simple steric hindrance by the antibodies could also explain the observed inhibition. Therefore, in the present study, we applied a competitive-binding assay to determine the attachment-mediating region of the C-terminal part of P1. A recombinant protein covering this part of P1 (rP1-II; Fig. 1), as well as several synthetic overlapping peptides covering the same region, was utilized, and both rP1-II and some of the peptides inhibited M. pneumoniae adherence to receptors on HEp-2 cells. Direct binding of one peptide to the host cells was detected using biotinylated peptides, suggesting a direct interaction between the P1 protein and a host cell receptor. Furthermore, this receptor was shared between the peptide and rP1-II, as the binding of the biotinylated peptide was inhibited in a concentration-dependent manner by unmarked peptides and the rP1-II protein.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of M. pneumoniae and M. genitalium for infection of HEp-2 cells.
M. pneumoniae strain FH and M. genitalium G37 (ATCC) were cultured in 10 ml SP4 medium (Tully et al., 1979) in TPP tissue-culture flasks and incubated at 37 °C. After growth for 48 h, the medium changed colour from red to orange, indicating the exponential growth phase (Clausen et al., 2001). The mycoplasmas were scraped into 10 ml growth medium containing penicillin (100 U ml–1). To reduce the number of self-aggregating features of M. pneumoniae, the suspension was sheared through a 27 gauge needle five times and adjusted to a concentration of approximately 9x107 colour changing units ml–1 before infection of the HEp-2 cells.
Generation of the recombinant protein rP1-II.
The recombinant protein M. pneumoniae rP1-II aa 1107–1518 and the recombinant M. genitalium protein rMgPa aa 628–1350 were produced as described previously (Svenstrup et al., 2002). To reduce the concentration of urea in the purification buffer, the proteins were first diluted to 0.2 mg ml–1 in growth medium and then dialysed against the same medium with 10 µg gentamicin ml–1 for 2 h at 4 °C.
Synthesis of peptides.
Two epitopes, pepJ (Jacobs et al., 1990) and pepD (Dallo et al., 1988), of 8 and 13 aa, respectively, were synthesized (Fig. 1
). Additional peptides of 44–52 aa covering rP1-II were designed with an overlap of 6–10 aa (Fig. 1), together with a random peptide with the sequence RIYKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNSALGHVNCTIKELRRLFLVDDLVDSLK. Peptides were synthesized stepwise on a fully automated peptide synthesizer ABI 433 (Applied Biosystems) using the Fmoc strategy. Peptides were synthesized on a TentaGel S RAM resin [Fluka; s (substitution) 0.2 mM g–1] to generate peptide amides. Fmoc-protected amino acids in suitable side-chain-protected forms as well as the coupling reagent o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) were obtained from Fluka. After completion of assembly, peptides were cleaved from the resin, with simultaneous side-chain deprotection, using trifluoroacetic acid and triisopropylsilane/water as scavenger. Filtrates were concentrated in vacuo and peptides precipitated with ether. Precipitates were lyophilized from acetic acid/water. Molecular mass verification was made by MS using MALDI-TOF for all peptides except peptide 1, the size of which was instead verified by SDS-PAGE, since it fragmented during MS.
Biotinylation of peptides.
Biotinylation was carried out after completion of peptide assembly while the peptide, with protected side groups, was still attached to the resin, using biotin (Sigma-Aldrich) and TBTU as coupling reagents. The biotinylated peptide was cleaved from the resin and treated as described above.
Competitive-binding assay with rP1-II.
The dialysed rP1-II recombinant protein (10, 20, 40 or 80 µl) was incubated with HEp-2 cells grown in chamber slides for 30 min at 37 °C. The M. pneumoniae RPMI suspension (50 µl per well) was added and incubated overnight at 37 °C. The HEp-2 cells were washed twice in PBS and fixed in 100 % methanol at 4 °C for 1 min. To detect the adhering mycoplasmas, the infected HEp-2 cells were incubated with primary antibodies (PabFH or PabG37) diluted 1 : 500 for 30 min at 37 °C (Svenstrup et al., 2002), washed with PBS and incubated with 100 µl per well of secondary FITC-conjugated ‘Affinipure’ goat anti-rabbit (GaR) IgG (H+L) (Jackson Immuno Research Laboratories) diluted 1 : 100 in PBS with 0.002 % Evans blue, for 30 min at 37 °C. The wells were washed twice with PBS before attached M. pneumoniae were visualized using a fluorescence microscope.
Competitive-binding assay with synthetic peptides.
The M. pneumoniae suspension was incubated with each of the synthetic peptides (20 µg ml–1; 
1015 molecules) for 30 min at 37 °C. HEp-2 cells were incubated with the mycoplasma peptide suspension (50 µl per well) overnight. The samples were prepared for indirect immunofluorescence microscopy (IMF) as described in the competitive-binding assay with rP1-II.
IMF.
The samples prepared for the adhesion detection and the competitive-binding assays were investigated by IMF. A drop of antifade solution [p-phenyldiamine dihydrochloride (1 µg ml–1) in 10 % PBS and 90 %, v/v, glycerol, pH 9.0] was placed between the slide and the coverslip. Fluorescence microscopy was performed with a Leitz DMR fluorescence microscope (Leica).
Indirect immunofluorescence for fluorometric measurement by POLARstar.
The M. pneumoniae suspension was mixed with each of the synthetic peptides (20 µg ml–1) or a dilution series was made with M. pneumoniae to obtain a standard curve. HEp-2 cells were plated in black 96-well Nunclon cell-culture plates with optical bottoms, as described above, and incubated either with the mycoplasma peptide suspension or with diluted mycoplasmas (50 µl per well) overnight. The samples were prepared as described above for the rP1-II competitive-binding assay for IMF, with the exception that PBS with 6-diamino-2-phenylindole (DAPI; 1/1000) was added instead of antifade to each well before the fluorescence measurement was carried out using a POLARstar OPTIMA reader (BMC Labtech). Emission and excitation filters were 488 and 520 nm for FITC and 526 and 532 nm for DAPI, respectively.
Immunofluorescence with fluorescent beads.
HEp-2 cells were prepared as described above and incubated with biotinylated peptide 1 or peptide 7 (80 µg ml–1) for 30 min at 37 °C. In the peptide competition experiment, HEp-2 cells were preincubated with unmarked peptides or proteins for 30 min before the 30 min incubation with biotinylated peptides. The cells were fixed as described for the competition assay with rP1-II. To detect receptor-bound peptides, the cells were incubated with 10 µl NeutrAvidin-coated yellow/green fluospheres (Invitrogen), diluted 1 : 10 in PBS for 10 min at 4 °C, washed and investigated using IMF.
Statistical analysis.
In each experimental round, all samples were tested in duplicate or triplicate, as well as being repeated on different experimental days. Results are expressed as mean values with SDs. P values were calculated using Student's t test for normally distributed data (fluorescent intensity measurements), while the non-parametric Mann–Whitney test was used for the remaining data (microcolony counting) with a significance level of P<0.05 (two-tailed).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The recombinant rP1-II protein contained a 6xHis tag, which was used for nickel-affinity purification under denaturing conditions in a urea-containing buffer (Svenstrup et al., 2002). However, urea is toxic to HEp-2 cells, so rP1-II was diluted to 0.2 mg ml–1 and subjected to dialysis against growth medium for 2 h prior to use. Four different concentrations of rP1-II were incubated with HEp-2 cells for 30 min at 37 °C before live M. pneumoniae were added. The infected cells were incubated overnight at 37 °C, washed in PBS and fixed with methanol. Adherent M. pneumoniae were detected with pabFH, a polyclonal antibody raised against M. pneumoniae whole-cell proteins, and visualized using FITC-conjugated IgG antibody. To fluorescently visualize HEp-2 cells, the fixed cells were counterstained with Evans blue (red fluorescence, Fig. 2a) prior to immunofluorescent detection of attached M. pneumoniae. To estimate the inhibition of adhesion, the bound M. pneumoniae microcolonies were counted on individual HEp-2 cells that were not in contact with other cells. Each microcolony was counted as one, regardless of the individual size of the microcolonies. In the positive control without recombinant proteins, an average of 25 microcolonies per HEp-2 cell (range 16–33 microcolonies per HEp-2 cell) was counted (Fig. 2b). Preincubation with rP1-II prior to addition of M. pneumoniae to the HEp-2 cells clearly reduced the number of M. pneumoniae microcolonies associated with the HEp-2 cells (Fig. 2c) in a dose-dependent manner, as 10, 20, 40 and 80 µl rP1-II (2 µg ml–1) resulted in 17.3, 16, 8.5 and 6 M. pneumoniae microcolonies per HEp-2 cell, respectively. Furthermore, shortening the M. pneumoniae incubation time to 1 h showed a similar reduction in the number of microcolonies, suggesting that host cell receptors are persistently occupied by the recombinant proteins (data not shown). In contrast, no inhibition of M. pneumoniae adhesion was observed using a recombinant protein (rMgPa) from the closely related M. genitalium (data not shown). Thus, rP1-II seems to compete with M. pneumoniae for binding to receptors on HEp-2 cells.
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) which were recognized by inhibiting antibodies were tested in the competitive-binding assay. The number of microcolonies was counted and compared to the positive control without peptides. Both peptides showed a slight reduction in the number of M. pneumoniae microcolonies, 21.8 % for pepJ and 16.9 % for pepD, compared to the positive control without peptides. However, neither peptide reduced the binding of M. pneumoniae to HEp-2 cells significantly (P=0.27 and 0.41, respectively), as was also the case for a synthetic peptide spanning a region from pepJ to pepD (data not shown). To ascertain that the counting of microcolonies on the individual cells was not biased by the researcher, the fluorescence intensity of bound M. pneumoniae was measured using the POLARstar OPTIMA in black 96-well plates. The nuclear fluorescent stain DAPI was included as a measure of the number of host cells. The fluorescence measurements were fairly stable between wells and plates, showing a uniform distribution of HEp-2 cells in the plates. The amount of bound mycoplasma was calculated using a standard curve made from a dilution series of M. pneumoniae. Only slight variations in calculated M. pneumoniae concentration were seen when pepJ and pepD were tested for inhibition, confirming the manually counted results (data not shown).
Competitive-binding assay with 10 synthetic peptides
Because no significant competition was seen between M. pneumoniae and the two short peptides (pepJ and pepD), another approach to narrow down the adhesion region was utilized. Ten peptides (44–52 aa) covering the same region of P1 as rP1-II were designed with 6–10 aa overlaps (Fig. 1). These peptides were then tested in the competitive-binding assay to estimate the inhibitory effect of each peptide on M. pneumoniae adherence. As a negative control, the closely related M. genitalium was included. After incubation with the peptides, adherent M. pneumoniae on HEp-2 cells were detected as described for rP1-II, while M. genitalium were detected using an antibody raised against M. genitalium whole-cell proteins.
The attachment of mycoplasmas to host cells was estimated by counting microcolonies on solitary HEp-2 cells and by measuring fluorescence intensity using the POLARstar as before. Between 49 and 94 HEp-2 cells were selected for each peptide in order to estimate the number of M. pneumoniae microcolonies per HEp-2 cell (Fig. 3). Likewise, M. genitalium microcolonies on 17–26 HEp-2 cells were counted for each peptide. In the negative control (Fig. 3a), no M. pneumoniae microcolonies were detected, while a few mycoplasma microcolonies were detected with peptides 3–8 (Fig. 3e–j) compared to the positive control (Fig. 3b). In contrast, the numbers of microcolonies after competition with peptides 1–2 and 9–10 (Fig. 3c–d and k–l) were closer to the number in the positive control. These results were obtained after preincubating the peptides with M. pneumoniae before HEp-2 cell infection. However, preincubating the peptides with HEp-2 cells and/or reducing the M. pneumoniae incubation time to 1 h did not alter the results noticeably.
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, Table 1) with peptides 1–3 and 9–10 exhibiting the smallest reduction in adherence compared to peptides 4–8, with peptide 7 (amino acids 1347–1396) showing the greatest inhibition, 78.9 % (P<0.0001). The adherence of M. genitalium to HEp-2 cells was not inhibited by any of the 10 peptides (data not shown). A slightly higher number of M. genitalium microcolonies was detected when the peptides were added.
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). Five of these (peptides 4–7 and peptide 9) reduced binding significantly (P<0.05, Student's t test), and this corresponded to the results of microcolony counting. No inhibition of M. genitalium was observed (data not shown).
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). No fluorescence was seen from the biotinylated peptides without beads (Fig. 5a), while the beads showed a sporadic binding pattern without biotinylated peptide (Fig. 5b). The clustering of the beads on the HEp-2 cells after preincubation with peptide 1 suggested an interaction between the peptide and the HEp-2 cells (Fig. 5c). A more striking interaction was observed with peptide 7 (Fig. 5d), which correlates with the ability of the peptide to inhibit M. pneumoniae adhesion.
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). Likewise, increasing amounts of rP1-II decreased the number of biotinylated peptide 7 on the host cells. Neither a non-specific control peptide nor rMgPa from M. genitalium inhibited the binding of biotinylated peptide 7 (data not shown). These results show a pronounced direct interaction between the synthetic peptide 7 and the HEp-2 cells, an interaction that could be blocked by rP1-II, suggesting that the peptide inhibits M. pneumoniae adhesion by occupying receptors otherwise recognized by the P1 protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition, P1 is the most immunogenic M. pneumoniae protein, and antibodies recognizing this protein are always found in human serum samples from M. pneumoniae-infected patients (Hu et al., 1983; Jacobs et al., 1985, 1989). The C-terminal part of the protein has been found to be the immunogenic region, as determined using human sera, and monospecific and monoclonal antibodies (Chaudhry et al., 2005; Drasbek et al., 2004). Recently, using monospecific antibodies, it has been found that the C-terminal part of P1 is surface-exposed (Svenstrup et al., 2002). Furthermore, in the present study, a direct interaction between the P1 protein and host cell receptors was found, since the recombinant protein rP1-II clearly reduced adhesion of M. pneumoniae.
The model of P1 contains several surface-exposed loops, which have been suggested to form three domains, thereby building a tertiary adherence complex (Jacobs et al., 1989, 1990, 1995). Epitope mapping of the P1 protein has led to the suggestion that the N-terminal region and two additional domains, D1 and D2, are extracellular and arranged in close proximity (Fig. 6). This would enable the three different loops to form a triangular structure, which might exhibit a stronger adherence capacity due to co-operative binding to the receptor molecule (Gerstenecker & Jacobs, 1990; Razin & Jacobs, 1992). However, the adhesion-mediating protein sequences might not be identical to the sequences described as immunogenic epitopes. Indeed, adhesion-inhibiting mAbs targeting sequences not recognized by human serum samples have been described (Jacobs et al., 1990). In the present study, the region of the P1 protein which mediates adhesion to the host cells was mapped to the D2 domain. Sequences in this region (Fig. 6) have previously been linked to cytadherence, since mAbs targeting these sequences reduce M. pneumoniae adhesion to the host cells (Dallo et al., 1988; Jacobs et al., 1990). This inhibition is probably caused by steric hindrance of the large antibodies, because no significant reduction in the adhesion of M. pneumoniae to the HEp-2 cells was seen with pepJ and pepD, which cover these epitopes. In addition, peptide 7, which showed the greatest inhibitory effect, included pepJ and part of pepD, suggesting that the host receptor binding sequence of the P1 protein is to be found in the C-terminal half of rP1-II.
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). Five peptides inhibited the adhesion of M. pneumoniae to HEp-2 cells significantly, as measured by both manual counting and automatic measurements. However, the amount of reduction determined by the two methods differed. Manual counting of M. pneumoniae only included microcolonies associated with solitary host cells, giving a precise measure of attached M. pneumoniae microcolonies. However, these counts could be biased by the researcher, and therefore the unbiased fluorometric POLARstar measurements of the entire well of a black 96-well plate were included. Some basal background fluorescence was anticipated as M. pneumoniae attaches directly to plastic. These microcolonies would be unaffected by the host-receptor-blocking peptides, leading to lower relative reductions in M. pneumoniae-generated fluorescence. The synthetic peptides inhibited the adhesion of M. pneumoniae to host cells to varying degrees. A similar graduation of adhesion inhibition has been observed elsewhere in the investigation of the outer-membrane protein P5-homologous fimbrin adhesin protein from Haemophilus influenzae, in which both short and long peptides covering the protein show differing degrees of inhibition of bacterial adhesion (Novotny et al., 2000). The inhibition region, peptides 4–9, is flanked by the two membrane-associated regions, M4 and M5, in the extracellular D2 region (Fig. 6), suggesting that the D2 region binds directly to the host cell. Indeed, peptide 7 of P1 was found to bind a specific host receptor that was shared with the P1 protein, suggesting that the inhibiting peptides function through the blockage of specific host cell receptors. Similarly, a fragment of the P29 adhesin from Mycoplasma fermentans inhibits the adhesion of the bacteria to HeLa cells through the blockage of host cell receptors (Leigh & Wise, 2002).
In the present study, only a partial inhibition was seen, although an excess of peptides was added. This might be due to the difference in size and mobility of M. pneumoniae, which is larger and more mobile than the small and immobile peptides. Additionally, the observed partial inhibition could reflect the interaction of other M. pneumoniae proteins with receptors on the host cell, as a stronger inhibition of adhesion was seen previously when antibodies raised against P116 and the C-terminal part of P1 were combined (Svenstrup et al., 2002). This P116 protein has been identified elsewhere as a host-receptor-binding protein under the name of P2 together with the P1 protein and HMW3 (Krause et al., 1982), and it has been reported that the extracellular protein fibronectin is bound by elongation factor Tu and the pyruvate dehydrogenase E1 subunit from M. pneumoniae (Dallo et al., 2002). It is therefore likely that adhesion of M. pneumoniae to host cells is a more complex process than the receptor-mediated binding of peptide 7 of P1 to host cells, as described in the present study. To obtain a more complete understanding of the host–M. pneumoniae interaction, a model system of human ciliated respiratory cells should be used.
Such an understanding could be used to block host cell receptors to which pathogenic microorganisms adhere. This may be considered as an alternative strategy to prevent colonization by the pathogen. Indeed, a synthetic peptide mimicking an adhesion epitope of the oral bacterium Streptococcus mutans inhibits the colonization of tooth surfaces by this micro-organism (Younson & Kelly, 2004). Thus, the introduction of M. pneumoniae-derived peptides that block human adhesion receptors might in the future reduce or even prevent M. pneumoniae infections.
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ACKNOWLEDGEMENTS |
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Edited by: C. Citti
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Svenstrup, H. F., Nielsen, P. K., Drasbek, M., Birkelund, S. & Christiansen, G. (2002). Adhesion and inhibition assay of Mycoplasma genitalium and M. pneumoniae by immunofluorescence microscopy. J Med Microbiol 51, 361–373.
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Received 24 June 2007;
revised 17 July 2007;
accepted 20 July 2007.
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