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.     

Homologous regions shared by adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium

A lambda gt11 library of Mycoplasma genitalium genomic DNA was generated, and clones were identified using a pool of monoclonal antibodies directed against different epitopes of the 140 kDa adhesin protein. Because the 140 kDa protein of M. genitalium and the 170 kDa P1 adhesin of M. pneumoniae share biological properties such as a tip-associated location, cytadherence function and immunologic crossreactivity, we performed Southern blot analysis using these cloned partial 140 kDa gene fragments and 14 subclones that span the P1 structural gene of M. pneumoniae. Homologous regions of the two genes were identified.     

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.     

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.     

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.     

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.     

Characterization of P40, a cytadhesin of Mycoplasma agalactiae

An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGA(Trp) codon to the universal TGG(Trp) codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.     

Identification and characterization of a Mycoplasma hyopneumoniae adhesin.

An adhesin of Mycoplasma hyopneumoniae was identified and characterized in this study. A monoclonal antibody (MAb), F2G5, and its F(ab')2 fragments inhibited the adherence of M. hyopneumoniae to porcine tracheal cilia, the natural targets to which the mycoplasma binds during infection. MAb F2G5 detected multiple bands, but predominantly recognized a 97-kDa (P97) protein of M. hyopneumoniae on immunoblots. Affinity chromatography, conducted with immobilized MAb F2G5, mainly purified P97. The purified proteins were able to bind to cilia and blocked the adherence of intact M. hyopneumoniae cells to cilia. Immunolabeling of mycoplasmas with MAb F2G5 under electron microscopy demonstrated that the proteins recognized by MAb F2G5 were located at the surface of the mycoplasma, predominantly on a surface fuzzy layer. These results indicate that P97 functions as an adhesin of M. hyopneumoniae. The N-terminal amino acid sequence of P97 did not have significant homology with any known bacterial or mycoplasmal adhesins, suggesting that P97 is a novel protein. The predominant proteins detected by MAb F2G5 in different strains varied in size, indicating that the antigen bearing the epitope for MAb F2G5 undergo intraspecies size variation. Antigenic variation of adhesins may be a pathogenic mechanism utilized by M. hyopneumoniae to evade the porcine immune system.     

Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (O126:H27).

The adherence of diarrhea-associated Escherichia coli to the small-bowel mucosa is an important step in the pathogenesis of diarrheal diseases. In tissue culture systems, diarrhea-associated strains show three distinct patterns of adherence: localized adherence, diffuse adherence (DA), and the recently described aggregative adherence. To study the molecular basis of the DA phenotype, we investigated the diarrhea-associated DA strain 2787 (O126:H27), isolated from a case of infantile diarrhea. The DA phenotype is mediated by a 6.0-kb DNA fragment derived from a 100-kb plasmid harbored by the wild-type strain. This fragment codes for a 100-kDa protein which can be released from the bacterial cell into the supernatant by mild heat treatment. Recombinant DA+ strains as well as the isolated 100-kDa protein were used to engender specific antisera in rabbits. As demonstrated by Western blotting (immunoblotting), the antibodies engendered by the recombinant DA+ strain recognized a 100-kDa protein in the wild-type strain 2787 and in all recombinant strains showing DA. Immunogold electron microscopy localized the 100-kDa protein to the bacterial cell surface. Serologically related proteins of similar size were detected by Western blotting in other DA+ diarrhea-associated strains belonging to enteropathogenic E. coli serotypes. The 100-kDa protein denoted AIDA-I (adhesin involved in diffuse adherence) binds in a saturable fashion to HeLa cells. AIDA-I-specific immunoglobulin G antibodies--and, to an even greater extent, Fab fragments derived thereof--inhibited bacterial attachment to HeLa cells. This is direct evidence that the 100-kDa protein is the adhesin mediating the DA phenotype of these diarrhea-associated strains and is representative of a group of serologically related proteins in other DA+ strains.     

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.     

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.     

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.     

Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin

Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory ciliated epithelial cells. Cilium adherence is mediated by P97, a surface protein containing a repeating element (R1) that is responsible for binding. Here, we show that the cilium adhesin is proteolytically processed on the surface. Proteomic analysis of strain J proteins identified cleavage products of 22, 28, 66, and 94 kDa. N-terminal sequencing showed that the 66- and 94-kDa proteins possessed identical N termini and that the 66-kDa variant was generated by cleavage of the 28-kDa product from the C terminus. The 22-kDa product represented the N-terminal 195 amino acids of the cilium adhesin preprotein, confirming that the hydrophobic leader signal sequence is not cleaved during translocation across the membrane. Comparative studies of M. hyopneumoniae strain 232 showed that the major cleavage products of the cilium adhesin are similar, although P22 and P28 appear to be processed further in strain 232. Immunoblotting studies using antisera raised against peptide sequences within P22 and P66/P94 indicate that processing is complex, with cleavage occurring at different frequencies within multiple sites, and is strain specific. Immunogold electron microscopy showed that fragments containing the cilium-binding site remained associated with the cell surface whereas cleavage products not containing the R1 element were located elsewhere. Not all secreted proteins undergo multiple cleavage, however, as evidenced by the analysis of the P102 gene product. The ability of M. hyopneumoniae to selectively cleave its secreted proteins provides this pathogen with a remarkable capacity to alter its surface architecture.     

Passive transfer of antiserum specific for immunogens derived from a nontypeable Haemophilus influenzae adhesin and lipoprotein D prevents otitis media after heterologous challenge

We recently determined that passive transfer of serum directed against a synthetic peptide called LB1 or a recombinant fusion protein immunogen [LPD-LB1(f)(2,1,3)] could prevent otitis media after challenge with a homologous nontypeable Haemophilus influenzae (NTHI) isolate. NTHI residing in the nasopharynx was rapidly cleared from this site, thus preventing it from ascending the eustachian tube and inducing otitis media in chinchillas compromised by an ongoing viral upper respiratory tract infection. While LB1 is based solely on one NTHI adhesin, the latter immunogen, LPD-LB1(f)(2,1,3), was designed to incorporate two NTHI antigens shown to play a role in the pathogenesis of otitis media; lipoprotein D (LPD) and the P5-homologous fimbrin adhesin. The design of LPD-LB1(f)(2,1,3) also accommodated for the recently demonstrated existence of three major groupings, based on amino acid sequence diversity, in the third surface-exposed region of P5-fimbrin. LPD-LB1(f)(2,1,3) was thus designed to potentially confer broader protection against challenge by diverse strains of NTHI. Chinchillas were passively immunized here with serum specific for either LB1 or for LPD-LB1(f)(2,1,3) prior to challenge with a member of all three groups of NTHI relative to diversity in region 3. The transferred serum pools were also analyzed for titer, specificity, and several functional activities. We found that both serum pools had equivalent ability to mediate C'-dependent killing and to inhibit adherence of NTHI strains to human oropharyngeal cells. When passively transferred, both serum pools significantly inhibited the signs and incidence of otitis media (P 相似文献   

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.     

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

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.     

Intrastrain heterogeneity of the mgpB gene in Mycoplasma genitalium is extensive in vitro and in vivo and suggests that variation is generated via recombination with repetitive chromosomal sequences

Mycoplasma genitalium is associated with reproductive tract disease in women and may persist in the lower genital tract for months, potentially increasing the risk of upper tract infection and transmission to uninfected partners. Despite its exceptionally small genome (580 kb), approximately 4% is composed of repeated elements known as MgPar sequences (MgPa repeats) based on their homology to the mgpB gene that encodes the immunodominant MgPa adhesin protein. The presence of these MgPar sequences, as well as mgpB variability between M. genitalium strains, suggests that mgpB and MgPar sequences recombine to produce variant MgPa proteins. To examine the extent and generation of diversity within single strains of the organism, we examined mgpB variation within M. genitalium strain G-37 and observed sequence heterogeneity that could be explained by recombination between the mgpB expression site and putative donor MgPar sequences. Similarly, we analyzed mgpB sequences from cervical specimens from a persistently infected woman (21 months) and identified 17 different mgpB variants within a single infecting M. genitalium strain, confirming that mgpB heterogeneity occurs over the course of a natural infection. These observations support the hypothesis that recombination occurs between the mgpB gene and MgPar sequences and that the resulting antigenically distinct MgPa variants may contribute to immune evasion and persistence of infection.     

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.     

Epitope mapping of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae

To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae (NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8- to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8- to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.     

Inhibition of Mycoplasma pneumoniae Hemadsorption and Adherence to Respiratory Epithelium by Antibodies to a Membrane Protein

Antiserum and purified immunoglobulin directed against Mycoplasma pneumoniae membrane protein P1 were examined for their influence on mycoplasma viability, metabolism, and cytadsorption. Anti-P1 immunoglobulin inhibited adherence of M. pneumoniae to hamster tracheal rings in vitro by up to 80% and inhibited hemadsorption by greater than 90%. Cytadsorption was also inhibited by anti-P1 Fab fragments. Anti-P1 antibodies had no effect on M. pneumoniae viability or metabolism. The data indicate that anti-P1 antibody obstructs the interaction of M. pneumoniae adhesin P1 with its host receptors.