Shared epitopes between Mycoplasma pneumoniae major adhesin protein P1 and a 140-kilodalton protein of Mycoplasma genitalium

Previous serological data have demonstrated cross-reactive antigens between two pathogenic species of mycoplasmas, M. pneumoniae and M. genitalium. Preliminary analysis of sera and monoclonal antibodies (MAbs) to protein antigens of these species showed an immunodominance of adhesin P1 (165 kilodaltons [kDa]) of M. pneumoniae in mice and hamsters and a 140-kDa protein of M. genitalium in mice and experimentally infected chimpanzees. To further characterize these two proteins, we assayed multiple anti-P1 and anti-140-kDa protein MAbs by enzyme-linked immunosorbent assay, immunoblot, and radioimmunoprecipitation techniques. The 140-kDa M. genitalium protein was shown to be surface accessible and insensitive to levels of trypsin which readily degrade protein P1. Peptide mapping was used to identify a unique class of MAbs which bound a cross-reactive molecule common to both the major adhesin protein P1 of M. pneumoniae and the 140-kDa protein of M. genitalium. MAbs generated against both M. pneumoniae and M. genitalium which were reactive with this determinant blocked M. pneumoniae attachment to chicken erythrocytes.     

DNA and protein sequence homologies between the adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae.

Mycoplasma genitalium and Mycoplasma pneumoniae are morphologically and serologically related pathogens that colonize the human host. Their successful parasitism appears to be dependent on the product, an adhesin protein, of a gene that is carried by each of these mycoplasmas. Here we describe the cloning and determine the sequence of the structural gene for the putative adhesin of M. genitalium and compare its sequence to the counterpart P1 gene of M. pneumoniae. Regions of homology that were consistent with the observed serological cross-reactivity between these adhesins were detected at both DNA and protein levels. However, the degree of homology between these two genes and their products was much higher than anticipated. Interestingly, the A + T content of the M. genitalium adhesin gene was calculated as 60.1%, which is substantially higher tham that of the P1 gene (46.5%). Comparisons of codon usage between the two organisms revealed that M. genitalium preferentially used A- and T-rich codons. A total of 65% of positions 3 and 56% of positions 1 in M. genitalium codons were either A or T, whereas M. pneumoniae utilized A or T for positions 3 and 1 at a frequency of 40 and 47%, respectively. The biased choice of the A- and T-rich codons in M. genitalium could also account for the preferential use of A- and T-rich codons in conservative amino acid substitutions found in the M. genitalium adhesin. These facts suggest that M. genitalium might have evolved independently of other human mycoplasma species, including M. pneumoniae.     

Expression of Mycoplasma pneumoniae antigens in Escherichia coli.

A genomic library of Mycoplasma pneumoniae was generated by using bacteriophage lambda EMBL3 as the vector. Screening of the library for the expression of M. pneumoniae protein antigens with adsorbed anti-M. pneumoniae serum revealed strong reactivity from a third of those clones which contained mycoplasma DNA inserts. Three of the most highly reactive clones were analyzed in detail and found to synthesize discrete mycoplasma proteins. Two carried overlapping fragments of mycoplasma DNA which encoded a protein that was readily detected in Escherichia coli after infection with recombinant bacteriophage. The third clone contained a novel mycoplasma DNA fragment which directed the synthesis of two additional mycoplasma proteins. Further screening of the library with antiserum raised against the major M. pneumoniae adhesin protein P1 (165 kilodaltons [kDa]) yielded one clone which produced an immunologically reactive protein of 140 kDa. Adsorption of anti-P1 serum by this clone selected a population of antibodies that were reactive with M. pneumoniae adhesin P1 (165 kDa). These results demonstrate that immunologically active M. pneumoniae proteins are synthesized in E. coli.     

Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract.

Mycoplasma genitalium, an organism first isolated from the urethras of two men with nongonococcal urethritis, has been found in throat specimens from military recruits participating in an inactivated Mycoplasma pneumoniae vaccine field trial in 1974-1975. Four of 16 preserved throat isolates, previously identified as strains of M. pneumoniae, have now been shown to be mixtures of M. pneumoniae and M. genitalium. Purification of these mixed mycoplasmas by selection of single colonies confirmed the presence of M. genitalium. Identification of M. genitalium was based upon the occurrence of a species-specific 140-kilodalton protein adhesin in these isolates and their serologic reactivity to an M. genitalium antiserum. The frequent occurrence of both M. pneumoniae and M. genitalium in a number of these throat specimens, in combination with their shared antigenic cross-reactivities, suggests the likelihood that M. genitalium strains are easily missed in the usual laboratory identification procedures. What role M. genitalium may play in human respiratory disease remains to be determined.     

A Mycoplasma genitalium protein resembling the Mycoplasma pneumoniae attachment protein.

In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.     

Expression and functional identification of the hypothetical adhesin P32 from Mycoplasma genitalium

Mycoplasma genitalium is the main causative agent for non-gonococcal and non-chlamydial urethritis. P32 is the putative surface-exposed membrane protein of M. genitalium and it has substaintial identity in amino acid sequence with adhesin protein P30 from M. pnewnoniae. Since M. pneumoniae mutants lacking P30 protein is defective in cytadherence, P32 protein has been proposed to be an essential adhesin implicated in the adherence of M. genitalium to host cells. The prokaryotic expression vector pET-30 ( )/p32 was constructed in the present study, and the recombinant protein was expressed in E. coli and purified under denaturing condition. As demonstrated by the immunoblotting analysis, the recombinant protein could react with rabbit antisera against M. genitalium, and adherence inhibition assays were petformed with antisera against this recombinant protein. It was demonstrated that P32 protein apperared to be an adhesion protein of M. genitalium, thus providing the experimental basis for better understanding of the pathogenesis of M. genitalium infection and for the development of the related vaccines against the infection.     

Adhesin gene of Mycoplasma genitalium exists as multiple copies

The structural gene of the 140 kDa adhesin of Mycoplasma genitalium was used to probe M. genitalium genomic DNA for gene copy number. Since multiple banding patterns were observed, the 140 kDa structural gene was subdivided into 10 contiguous fragments in size from 165 to 657 base pairs in order to determine which parts of the adhesin gene existed as multiple copies. Each fragment was labeled by nick translation and used to probe the entire M. genitalium genomic DNA. Approximately half the gene was present in single copy while the remaining sequences were multiple copied. Both single and multiple copy regions were interspersed throughout the structural gene.     

Cross-hybridization between the cytadhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium and genomic DNA of Mycoplasma gallisepticum

Immunological cross-reactivity was observed between the cytadhesin proteins of Mycoplasma pneumoniae and Mycoplasma genitalium and a 155 kDa protein of Mycoplasma gallisepticum. Furthermore, the cytadhesin genes of M. pneumoniae and M. genitalium were used to demonstrate homology with M. gallisepticum genomic DNA under low stringency conditions suggesting that a family of adhesin-related genes exists among these pathogenic mycoplasmas.     

Absence of Mycoplasma pneumoniae cytadsorption protein P1 in Mycoplasma genitalium and Mycoplasma gallisepticum

Polyclonal and monoclonal antibodies to Mycoplasma pneumoniae protein P1 were nonreactive with whole-cell or soluble preparations of M. genitalium and M. gallisepticum. However, radioimmunoprecipitation performed with hyperimmune rabbit sera raised against each mycoplasma species indicated antigenic cross-reactivity between M. pneumoniae and M. genitalium.     

Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene

In order to develop a species-specific PCR for the detection of Mycoplasma genitalium, the sequence of 1,490 bases of the 16S rRNA gene was determined for M. genitalium G37 (type strain) and four Danish isolates of M. genitalium. The sequences of the four Danish strains, mutually different with respect to their MgPa gene, were 100% homologous, although they carried a single common base substitution compared to the type strain. Among members of the Mycoplasma pneumoniae phylogenetic cluster, M. genitalium showed the most-prominent homology to the 16S rRNA sequence of M. pneumoniae (98% homology). From regions showing the least homology to the M. pneumoniae 16S rRNA gene sequence, primers were chosen to amplify DNA from M. genitalium only. Two sets of primers were selected for their ability to detect <10 to 50 M. genitalium genome copies without cross-reactions with M. pneumoniae. The performance of these primers was compared to the performance of two pairs of primers amplifying parts of the MgPa adhesin gene; 1,030 randomly selected specimens submitted for Chlamydia trachomatis culture were screened with one of the 16S rRNA gene primer sets. A total of 41 specimens were found to be positive for this gene; 40 of these could be confirmed by one of the MgPa primer sets, whereas the other MgPa primer set detected only 21 positive specimens out of 40. These results indicate that estimates of the prevalence of M. genitalium in various populations using MgPa PCR primers could be incorrectly low if the PCR primers are located in variable regions of the MgPa gene.     

Comparison of host responses after intranasal infection of guinea-pigs with Mycoplasma genitalium or with Mycoplasma pneumoniae

Guinea-pigs were infected intranasally with Mycoplasma genitalium or Mycoplasma pneumoniae. The lung lesions produced by the two mycoplasmas were comparable in extent and histological pattern. Sera of both animal groups taken 2 weeks after infection reacted strongly in the complement fixation test with the M. pneumoniae glycolipid extract. In an ELISA using the respective adherence proteins (P1-protein of M. pneumoniae and MgPa of M. genitalium), strong specific activity, but also considerable cross-reactions were found. Epitope analysis by using overlapping octapeptides of a P1-region immunologically active in human M. pneumoniae infections and of the corresponding MgPa-region revealed six common epitopes but also one M. genitalium and two M. pneumoniae specific determinants. For analysis of a possible pathogenicity of M. genitalium in the human respiratory tract species-specific tests have to be developed.     

Antigenic determinants of the attachment protein of Mycoplasma pneumoniae shared by other pathogenic Mycoplasma species.

In previous studies with hyperimmune rabbit antisera, we found evidence of serologic cross-reactivity among Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. Because of certain biologic and morphologic similarities of these species, attempts were made to determine if this cross-reactivity related to the attachment protein (P1) of M. pneumoniae. Monoclonal and monospecific antibodies against P1 were used to probe proteins of the other species by immunoblotting. One of the P1 monoclonal antibodies was reactive with a smaller protein of M. genitalium; rabbit antiserum raised by immunization with P1 excised from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was found to react with a similar-sized protein of M. gallisepticum. These preliminary findings suggest antigenic sharing among the species examined; however, limitations of the methods used are discussed.     

Serological investigation of Mycoplasma genitalium in infertile women

BACKGROUND: The role of Mycoplasma genitalium in the pathogenesis of pelvic inflammatory disease has not been characterized. METHODS: Sera from 308 infertile women were investigated for antibodies to M. genitalium by immunoblotting. Women with tubal factor infertility (TFI) made up 132 of the patients, 67 of the women had an infertile male partner and 109 were infertile for unknown reasons. RESULTS: Of the TFI patients 29 (22.0%) were seropositive to the major adhesin, MgPa, of M. genitalium versus 11 (6.3%) in the group of women with normal tubes. No cross-reactions between MgPa and P1 of the related Mycoplasma pneumoniae were found. Besides, MgPa positive sera were confirmed by immunoblotting using a cloned fragment of the C-terminal part of MgPa specific to M. genitalium. Chlamydia trachomatis is known to be able to cause infertility as a result of salpingitis. Therefore, the sera were tested against C. trachomatis using a commercial ELISA test. Seventy-five (56.8%) of the TFI patients were seropositive to C. trachomatis. Eight (27.6%) TFI patients seropositive to MgPa were negative to C. trachomatis. CONCLUSIONS: This study indicates that M. genitalium may be an independent risk factor in the development of an inflammatory process leading to scarring of the uterine tubes in women and thereby causing infertility.     

Adhesion and inhibition assay of Mycoplasma genitalium and M. pneumoniae by immunofluorescence microscopy

Adhesion of Mycoplasma pneumoniae and the closely related M. genitalium to HEp-2 cells was investigated. The main surface proteins known to be involved in adhesion are P1 of M. pneumoniae and its homologue, MgPa, of M. genitalium. Both proteins are also immunodominant proteins. Protein P116 is another immunodominant protein of M. pneumoniae. These immunogenic proteins were investigated for their surface exposure and involvement in adhesion to host epithelial cells. Immunofluorescence microscopy (IFM) was used to detect M. pneumoniae and M. genitalium adhering to HEp-2 cells. Monospecific antibodies were produced against fragments of the surface proteins lacking tryptophan stop codons and were used for adhesion detection, surface exposure and adhesion inhibition IFM assays. Three monospecific antibodies were made against MgPa covering regions in the N-terminal, the middle and the C- terminal part; two monospecific antibodies were produced against P1 covering regions of the N- and the C-terminal part and one monospecific antibody was made against most of P116. Only the C-terminal parts of P1 and MgPa were surface exposed and blocking of these regions with the monospecific antibody resulted in inhibition of cytadsorption. Protein P116 was shown to be surface exposed and an essential protein involved in adhesion because the anti-P116 antibody prevented attachment of M. pneumoniae to the HEp-2 cells independently of P1. This study adds to the understanding of the molecular biology of M. genitalium and M. pneumoniae and presents a method to study the proteins involved in adhesion of these mycoplasmas.     

The mature MgPa-adhesin of Mycoplasma genitalium

A high molecular weight protein of Mycoplasma genitalium (MgPa-protein) was isolated by fractionated solubilization with 1% CHAPS, followed by subsequent extraction with 2% octylglucoside and size exclusion chromatography. The comparison of the N-terminal sequence reported here with published nucleotide sequence data revealed the existence of a signal sequence; the molecular weight of the mature MgPa-protein was calculated to be 153, 134 dalton. The protein shares antigenic determinants with the adhesin of Mycoplasma pneumoniae (P1-protein). Therefore the amino acid sequence of the MgPa-protein was matched to the P1-protein sequence. Five of seven computer predicted hydrophobic regions of both amino acid sequences were located in corresponding regions.     

Rapid-cycle PCR for detection and typing of Mycoplasma pneumoniae in clinical specimens

We designed several new primers and modified previously described species- and type-specific primers targeting the Mycoplasma pneumoniae P1 adhesin gene. Optimized thermal profiles allowed one-step or nested PCR to be completed in less than 1 h. In 10 patients with pneumonia, M. pneumoniae type 1 was identified in 3 and type 2 in 7.     

Host reactions to Mycoplasma pneumoniae infections in guinea-pigs preimmunized systemically with the adhesin of this pathogen

Guinea-pigs developed systemic and local humoral responses after intraperitoneal immunization with the isolated adhesin (168 kDa protein) of Mycoplasma pneumoniae cells. Hilar lymphocytes of these animals showed proliferation after in vitro stimulation with the 168 kDa protein or sonicated M. pneumoniae whole cell antigen. Animals preimmunized and subsequently infected with M. pneumoniae showed increased M. pneumoniae-specific IgG, IgA and adherence inhibiting antibody activities. Nevertheless these animals developed severe lung lesions of lympho-histiocyte infiltrations. Furthermore hilar lymph nodes were depleted of immunocompetent lymphocytes, suggesting a cell transfer of specific stimulable lymphocytes to the inflammation sites.     

The principal protein antigens of isolates of Mycoplasma pneumoniae as measured by levels of immunoglobulin G in human serum are stable in strains collected over a 10-year period.

To determine whether antigenic variation in protein antigens of Mycoplasma pneumoniae occurred over time, 12 isolates obtained from pneumonia patients over a 10-year period (1964 to 1974) were compared by immunoblotting (Western blotting) against acute and convalescent human serum samples obtained from the same patients. The strains selected were isolated from patients who had low anti-lipid complement-fixing antibody titers in their acute-phase serum samples and high titers in their convalescent-phase serum samples. The polypeptide composition of the strains was closely similar by protein staining even when compared with prototype FH-Liu. On immunoblotting, all strains showed five bands (170, 130, 90, 45, and 35 kilodaltons [kDa]) which were stained more intensely by convalescent-phase than by acute-phase specimens. A sixth band (62 kDa) was detected by the conjugate alone. In FH-Liu, one band (110 kDa) was prominently stained by convalescent-phase specimens; this band was much less apparent in all of the clinical isolates. Two isolates possessed an additional band (92 kDa) which was stained more prominently by some but not all convalescent-phase specimens. Because of its known antigenic relationships and culture similarities, Mycoplasma genitalium was used for comparison. More polypeptides of M. genitalium than of M. pneumoniae were recognized by acute-phase serum samples, and 4 of 12 convalescent-phase serum samples showed increases in antibodies to certain M. genitalium polypeptides. However, these reactive polypeptides did not correspond in molecular mass to polypeptides recognized in M. pneumoniae; thus the signature profile of human convalescent-phase specimens with M. pneumoniae was distinct. These five polypeptides, individually or in combination, are especially promising for use in detection of human serum antibodies by enzyme-linked immunosorbent assay because they were found in all M. pneumoniae isolates tested.     

Expression and immunological characterization of the carboxy-terminal region of the P1 adhesin protein of Mycoplasma pneumoniae

Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia in humans. Adherence of M. pneumoniae to host cells requires several adhesin proteins, such as P1, P30, and P116. A major limitation in developing a specific diagnostic test for M. pneumoniae is the inability to express adhesin proteins in heterologous expression systems due to unusual usage of the UGA stop codon, leading to premature termination of these proteins in Escherichia coli. In the present study, we successfully expressed the C-terminal (P1-C1) and N-terminal (P1-N1) regions of the P1 protein in E. coli. On screening these recombinant proteins with sera from M. pneumoniae-infected patients, only the P1-C1 protein was found to be immunogenic. This protein can be used as an antigen for immunodiagnosis of M. pneumoniae infection, as well as in adherence inhibition studies to understand the pathophysiology of the disease.