Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate.

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.     

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.     

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.     

DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium.

DNA probes specific for Mycoplasma pneumoniae and Mycoplasma genitalium were selected from genomic libraries prepared in pUC13. The 32P-labeled probes could detect, by dot blot hybridization, down to about 0.1 ng of the specific mycoplasma DNA or 10(5) CFU. Biotinylation of probe decreased the sensitivity of detection and produced nonspecific background reactions with nonhomologous DNAs. Sulfonation of probe yielded a similar level of sensitivity with less background.     

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.     

配对母婴血清和羊水β2—微球蛋白测定

目的：建立本地区母婴血清和羊水β２－微球蛋白（β２－ＭＧ）正常参考值，探讨其变化规律及作为母婴产前与前后监护指标的科学性与可行性的依据。方法：采用放射免疫法测定４０名正常未孕妇女血清和５５对正常母婴及１２对重度妊高征母婴血清和羊水中β２－ＭＧ水平。结果：（１）正常孕妇组中母血β２－ＭＧ水平极显著低于脐动脉血与羊水（均Ｐ＜０．００１）；后二者间也有差异（Ｐ＜０．０５）；三者中仅母血与脐动脉血间存在相关性（ｒ＝０．５８６，Ｐ＜０．００１）。（２）妊高征组母血和脐血β２－ＭＧ水平均显著高于正常孕妇组（分别Ｐ＜０．００１，０．００５）。结论：（１）正常参考值范围的确定有待商讨。（２）高水平的β２－ＭＧ可能是妊高征形成的原因之一，而不仅仅是其后果；且测定β２－ＭＧ可作为评估妊高征病情严重程度的敏感指标     

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.     

Immunological cross-reactivity of a Mycoplasma pneumoniae membrane-associated protein antigen with Mycoplasma genitalium and Acholeplasma laidlawii.

A murine monoclonal antibody, OC2F5, reacts with a Mycoplasma pneumoniae antigen with an approximate Mr of 43,000. This antigen is trypsin and proteinase K sensitive and partitions in the detergent phase of a Triton X-114 solution. The monoclonal antibody cross-reacts with an antigen from both Mycoplasma genitalium and Acholeplasma laidlawii with a similar molecular weight. This cross-reactivity should be considered in the development of M. pneumoniae antigen detection systems based on the use of antibodies directed to this protein antigen.     

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.     

A novel polymerase chain reaction assay to detect Mycoplasma genitalium.

AIMS: To design and validate a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Mycoplasma genitalium. METHODS: Primers were designed that were complementary to the 16S rRNA gene sequence of M genitalium. After optimisation of the reaction conditions, the PCR was tested against nine M genitalium strains, a dilution series of M genitalium DNA, and a panel of common microorganisms. The PCR was also challenged in parallel with a published assay against 54 urine specimens from men with urethritis. RESULTS: The expected 341 bp product was produced on amplification of material from all M genitalium strains and from none of the other microorganisms tested. The lower limit of detection was 50 genome copies. The new assay detected M genitalium DNA in nine of 54 men with urethritis, in comparison with eight positive specimens detected with the alternative PCR. CONCLUSIONS: This novel PCR targeting the M genitalium 16S rRNA gene has been optimised and now provides a sensitive and specific alternative or addition to the available MgPa gene targeting assays.     

Amino Acid Changes in Elongation Factor Tu of Mycoplasma pneumoniae and Mycoplasma genitalium Influence Fibronectin Binding

Mycoplasma pneumoniae and Mycoplasma genitalium are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized M. pneumoniae elongation factor Tu (EF-Tu_Mp) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated M. genitalium EF-Tu (EF-Tu_Mg), in spite of sharing 96% identity with EF-Tu_Mp, does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu_Mp that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu_Mg. Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu_Mp. Synthetic peptides corresponding to this region of EF-Tu_Mp (EF-Tu_Mp 340-358) blocked both recombinant EF-Tu_Mp and radiolabeled M. pneumoniae cell binding to Fn. In contrast, EF-Tu_Mg 340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.Many pathogens express surface proteins that facilitate colonization and cellular invasion (12, 39, 44, 49, 55). The human mycoplasmas, Mycoplasma pneumoniae and Mycoplasma genitalium, have genome sizes of 816,394 bp (20) and 580,070 bp (12), respectively, with the latter considered the smallest self-replicating biological cell (14, 38). These bacterial pathogens possess terminal tip-like structures comprised of specific membrane adhesins and adherence-related accessory proteins that mediate surface parasitism of target cells (5) and are essential for virulence (4). While adherence of virulent M. pneumoniae is mediated primarily by tip organelle-associated adhesins (10, 24), the absence of these proteins in hemadsorption-negative mutants (HA⁻ class II mutants) (17) still permits detectable adherence (18), suggesting the involvement of alternative mechanisms by which mycoplasmas bind to host cells.Recently, we showed that M. pneumoniae surface-associated elongation factor Tu (EF-Tu_Mp; MPN665) and the pyruvate dehydrogenase E1 beta subunit (MPN392) interact with fibronectin (Fn) (11). In addition, we demonstrated that HA⁻ class II mutants also bind Fn through EF-Tu (11). Fn is an abundantly available pathogen target (22) that exists in soluble form in blood fluids and plasma and in fibrillar form in the extracellular matrix (56). M. pneumoniae could readily access the extracellular matrix through virulence-related determinants following epithelial cell damage (29) and could directly bind to subepithelial tissue targets through EF-Tu interactions with Fn. Furthermore, these distinct pathogenic pathways may also contribute to the ability of M. pneumoniae to invade and to establish intracellular and perinuclear residence (9, 57).Detailed analyses of EF-Tu_Mp-Fn interactions revealed the critical role of the carboxyl region of EF-Tu (amino acids 192 to 219 and 314 to 394) in Fn recognition (3). Other mycoplasmas with tip organelles, such as Mycoplasma penetrans and Mycoplasma gallisepticum, have been reported to bind Fn through a 65-kDa protein (13) and the PlpA and Hlp3 proteins (34).Following our initial findings of EF-Tu_Mp-Fn interactions, surface-associated EF-Tu proteins from other microorganisms, including Lactobacillus johnsonii, Listeria monocytogenes, and Pseudomonas aeruginosa, were reported to bind mucin (16), fibrinogen (43), plasminogen, and factor H (32). Since EF-Tu is one of the most highly conserved proteins in mycoplasmas, it has been used to create an EF-Tu sequence-based mycoplasma phylogeny tree. This allows the classification of the human pathogens, M. genitalium and M. pneumoniae, along with M. gallisepticum, a poultry pathogen, in the same group (28). M. pneumoniae is an established pathogen of the respiratory tract (54) but has also been isolated from the urogenital tract (15). M. genitalium, an emerging sexually transmitted disease pathogen (27, 51), has also been associated with respiratory (6) and joint (50) pathologies. It has been suggested that the tissue-specific tropisms and pathogenic mechanisms of these two mycoplasmas are determined by genetic distinctions between them (19). Most of the open reading frames proposed for M. genitalium are present in M. pneumoniae. Overall, M. pneumoniae and M. genitalium share 67.4% average identity at the amino acid level, while conserved housekeeping proteins exhibit 70 to 97% identity (19). Among the latter proteins, EF-Tu displays a high sequence identity (96%).In this study, we compared EF-Tu-Fn binding between M. pneumoniae and M. genitalium and discovered biological and biochemical differences that facilitated the identification of key amino acids responsible for these interactions. Such distinctions provide evidence of unique colonization capabilities of these bacteria.     

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.     

Mycoplasma pneumoniae in hamster tracheal organ culture studied by scanning electron microscopy.

Hamster tracheal rings in organ culture were inoculated with a virulent strain of Mycoplasma pneumoniae and examined by scanning electron microscopy. A progressive increase in epithelial cell injury was detected from 48 to 96 h post-inoculation and was characterized by apparent loss of the apical portion of ciliated cells. M. pneumoniae attaching to the epithelial cell surfaces could be identified by comparison with the surface morphology of mycoplasmas grown on glass cover slips.     

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.     

Immune electron microscopy of cross-reactions between Mycoplasma pneumoniae and human erythrocytes.

Respiratory infection with Mycoplasma pneumoniae evokes immunoglobulin M autoantibody which agglutinates human erythrocytes at 4 degrees C (cold agglutinin) and is specific for I antigen. Cross-reactions between surface antigens of M. pneumoniae and human erythrocytes, previously examined by serological analysis, were examined by transmission and scanning electron microscopy. Ferritin-labeled human antimycoplasmal and rabbit antisera to erythrocyte membrane components reacted with antigens on the surface of both M. pneumoniae and erythrocytes. Adsorption of human erythrocytes to M. pneumoniae was blocked by the same antisera without ferritin label. It is proposed that the cross-reactive specificity lies in peripheral areas of the mycoplasmal cell, probably in a surface carbohydrate which has antigenic identity with erythrocyte glycoprotein.     

Failure of Mycoplasma pneumoniae infection to confer protection against Mycoplasma genitalium: observations from a mouse model

Mycoplasma pneumoniae and M. genitalium are genomically distinct but share antigens that induce some serological cross-reactivity. Therefore, the possibility that M. pneumoniae infection of the human respiratory tract might provide immunity to M. genitalium infection of the genital tract was considered. Because of the difficulty of assessing this proposition in man, it was evaluated experimentally in a mouse model. Female BALB/c mice were susceptible to infection of the vagina with M. pneumoniae, whereas those infected previously in the oropharynx with M. pneumoniae were completely immune to infection of the vagina with this mycoplasma. However, all mice with such a respiratory tract infection were susceptible to infection of the vagina with M. genitalium. The findings suggest that an M. pneumoniae infection of the human respiratory tract is unlikely to influence infection of the genital tract by M. genitalium.     

Development of a quantitative real-time PCR assay for detection of Mycoplasma genitalium

Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/microl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.