Mycoplasma pneumoniae attachment to glutaraldehyde-treated human WiDr cell cultures

Attachment of Mycoplasma pneumoniae to host cells initiates disease, and the attachment components may represent important protective immunogens for preventing disease. We have studied the mechanisms of attachment using in vitro cell culture systems and selected pathogenic and nonpathogenic strains of M. pneumoniae. Attachment of the pathogenic strains M129 and PI-1428 was several fold greater than attachment of the nonpathogenic strain, and attachment of strains M129 and PI-1428 was reduced by 21 to 63% when human WiDr cell monolayers were exposed to neuraminidase, supporting the concept that M. pneumoniae attaches to mammalian cells by a neuraminidase-sensitive glycoconjugate. While attachment of the two pathogenic strains was markedly reduced by treating the WiDr cells with glutaraldehyde, glutaraldehyde treatment produced minimal effects on the attachment of the nonpathogenic strain B176. Glutaraldehyde treatment also altered the temperature dependence of attachment by the pathogenic strains. Because glutaraldehyde-treated WiDr cell monolayers showed little difference in attachment between pathogenic and nonpathogenic strains, glutaraldehyde-treated cells are not appropriate cell substrates for studying M. pneumoniae attachment mechanisms or identifying immunogens for vaccine development.     

Presence of anaplerotic reactions and transamination, and the absence of the tricarboxylic acid cycle in mollicutes

Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Clinical complications of Mycoplasma pneumoniae disease--other organs

Although self-limited respiratory tract infections caused by Mycoplasma pneumoniae are well recognized in children and young adults, respiratory involvements and hepatic dysfunction may occur. The frequency and clinical features of these complications were investigated. Experimental studies with regard to bacterial superinfection were also carried out. The test animals which were first infected with Mycoplasma pneumoniae and then with Staphylococcus aureus showed more extensive bacteriological and pathological changes than those infected with Staphylococcus aureus only. Liver biopsies performed on three human patients showed hepatic dysfunction and the histological findings were diagnosed as non-specific reactive hepatitis in each case.     

Identification of gene products of the P1 operon of Mycoplasma pneumoniae

Gene P1 of Mycoplasma pneumoniae, which codes for a major adhesin, is flanked by two sequences with open reading frames designated ORF4 and ORF6 (Inamine et al., 1988b). In order to identify proteins translated from those ORFs, gene fusions between the N-terminus of the RNA replicase of the Escherichia coli bacteriophage MS2 and selected regions of ORF4 and ORF6 were constructed. The corresponding fusion proteins synthesized in Escherichia coli were used to immunize mice. Antisera directed against ORF4-related sequences did not recognize M. pneumoniae antigens in Western blot analysis, but antisera directed against ORF-6-derived fusion proteins reacted with two M. pneumoniae proteins of 40 kDa and 90 kDa. In addition, some of the antisera also recognized proteins that formed in a sodium dodecyl sulphate/polyacrylamide gel a protein ladder between 115 and 145 kDa.     

Mycoplasma genitalium and Mycoplasma pneumoniae share a 67 kilodalton protein as a main cross-reactive antigen

It was demonstrated that a 67 kilodalton (kDa) protein of Mycoplasma pneumoniae is a main cross-reactive antigen with similar molecular weight protein of Mycoplasma genitalium by Western blot analysis using monoclonal antibody to 67 kDa protein of M. pneumoniae and hyperimmune rabbit sera directed against each mycoplasma strain.     

Distribution of ecto 5'-nucleotidase on Mycoplasma species associated with arthritis

The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.     

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.     

Immune responses to Mycoplasma conjunctivae in alpine ibex, alpine chamois, and domestic sheep in Switzerland

The humoral immune response of three alpine chamois (Rupicapra rupicapra rupicapra), two alpine ibex (Capra ibex ibex) and three domestic sheep naturally affected with infectious keratoconjunctivitis (IKC), and four ibex and two sheep experimentally infected with Mycoplasma conjunctivae was analysed. In addition, the local immune response to M. conjunctivae was analysed using conjunctival washes from chamois and sheep. Immunoblot analysis of sera using whole cell antigens of M. conjunctivae revealed the major immunogenic proteins which had molecular masses of 175, 83, 68, 60, 50, 42, 36, and 33 kDa. Major antigens were found at 83, 68, 60, and 42 kDa in both sera and conjunctival washes from naturally infected animals of all three Caprinae species. In experimentally infected animals, antibodies to the 68 and 60 kDa antigens were dominant. Naturally infected animals showed much stronger immune reactions than those experimentally infected, and specific antibodies appeared 2 to 4 wk after experimental infection. To evaluate possible cross-reactions, whole cell antigen of M. conjunctivae was analysed by immunoblot against hyperimmune sera of closely related Mycoplasma spp. Antibodies to the 175, 73, 68, 60, and 33 kDa antigens appeared to be specific to M. conjunctivae. Cross-reactions mainly with 83, 50, and 42 kDa antigens were detected, in particular with M. ovipneumoniae and M. bovoculi hyperimmune sera, but also with antisera against M. capricolum capricolum and M. putrefaciens.     

Susceptibility of newly established mouse strain MPS to Mycoplasma pneumoniae infection

A newly established mouse strain, MPS, which is more sensitive to Mycoplasma pulmonis than ICR, ddY and other mouse strains was examined for its susceptibility to Mycoplasma pneumoniae. In experimental infections with M. pneumoniae, it was observed that M. pneumoniae attached to tracheas of MPS mice, and M. pneumoniae cells were isolated from tracheas and lungs of MPS mice even after four weeks of infection, while no mycoplasmas were isolated from ICR and ddY mice after one week of infection. Specific antibodies against M. pneumoniae were also observed by the Western blotting in the sera of MPS mice infected with M. pneumoniae. Although any lung lesion could not be observed in this work, this newly established mouse strain MPS may be useful for experiments of M. pneumoniae infection, especially for the analysis of strain differences in susceptibility to M. pneumoniae infection.     

Identification of an in vivo CD4+ T cell-mediated response to polymorphic membrane proteins of Chlamydia pneumoniae during experimental infection

Chlamydia pneumoniae is an obligate intracellular bacterium that causes upper and lower respiratory tract infection in humans. C. pneumoniae harbors the polymorphic membrane protein (Pmp) family with 21 different proteins with a molecular mass around 100 kDa. The Pmps are species-specific, abundant and, together with major outer membrane protein and outer membrane protein 2, the dominant proteins in the C. pneumoniae outer membrane complex. Nevertheless, it is unknown whether Pmps are recognized by the cell-mediated immune response. To address this issue, C57BL/6J mice were infected intranasally with C. pneumoniae and the immune response to primary infection was investigated. We demonstrate, as expected, that the primary response is of the Th1 type by IgG2a- and IgG1-specific sELISA (Medac) on serum. In vivo-primed spleen lymphocytes were found to be reactive to Pmp8, Pmp20 and Pmp21 in an interferon-gamma ELISpot assay. The responses were shown to be mediated by CD4(+) T cells. To our knowledge, this is the first identification of antigens recognized by CD4(+) T cells during murine C. pneumoniae infection.     

Immunological reaction of guinea-pigs following intranasal Mycoplasma pneumoniae infection and immunization with the 168 kDa adherence protein

Humoral responses to Mycoplasma pneumoniae proteins, especially the 168 kDa protein, were demonstrated by Western blotting in sera and bronchial washings of all groups of infected or immunized guinea-pigs. However, infection was not prevented by these local and systemic antibodies. Hilar lymphocytes of infected and immunized guinea-pigs were stimulated in vitro by sonicated M. pneumoniae antigen and by the 168 kDa protein. Stimulation was significantly lower in animals which had been infected twice or had been preimmunized and challenged by infection. Histologically the most severe lesions were seen in the twice-infected group followed by the preimmunized group which was subsequently infected.     

The interaction of alkylating derivatives of oligodeoxyribonucleotides and their methylphosphonate analogs with Mycoplasma cells]

Alkylating derivatives of decathymidylates and methylphosphonate analogs of oligodeoxyribonucleotides (MPAO) were studied for their interaction with cells of Acholeplasma laidlawii PG-8, Mycoplasma capricolum California Kid, M. pneumoniae FH and phytopathogenic strain (St. 118). It is shown that MPAO of octa- and hexadecathymidylates as well as decathymidylates 3'-terminal modified by phenazine and cholesterol groupings are sorbed by mycoplasma cells and can penetrate inside the cells. Efficiency of binding of alkylating derivatives and MPAO with mycoplasma cells depends on interaction time of reagents, their concentration in the reaction mixture and temperature.     

Protective effect of colostrum in Mycoplasma pneumoniae infection induced in infant mice

The immunological responses and mechanism of maternal immunity in Mycoplasma pneumoniae infection of mice were investigated. ICR female mice, 4 weeks old, and infant mice, 2 to 4 days old, were infected with M. pneumoniae. Anti-M. pneumoniae antibodies in serum and colostrum were determined by enzyme-linked immunosorbent assay. The specific IgG antibody production persisted for 9 months or longer in both the young and infant mice. These infected mice were protected from rechallenge with M. pneumoniae. In addition, the infected dams conferred passive immunity on their offspring. The infant mice born to uninfected normal dams were protected from the challenge with M. pneumoniae when fed by infected foster dams. Conversely, the infant mice born to infected dams were not protected from the challenge with M. pneumoniae when the infants were fed by uninfected dams. The specific IgG antibody appeared in serum of infant mice inoculated orally with M. pneumoniae-infected mouse serum and the infants were protected from challenge with M. pneumoniae, while the infants given protein A-absorbed serum were not protected from the challenge. These results suggest that one of the factors involved in the resistance of infant mice to M. pneumoniae infection is the specific IgG antibody present in the colostrum rather than the result of transplacental transfer.     

Demonstration of membrane association and surface location of Mycoplasma pneumoniae antigens using monoclonal antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.     

Development of mutants of the mosquitocidal bacterium Bacillus thuringiensis subspecies morrisoni (PG-14) toxic to lepidopterous or dipterous insects

The parasporal body of the mosquitocidal isolate (PG-14) of Bacillus thuringiensis subsp. morrisoni (BTM) contains five major proteins with molecular masses of, respectively, 27.3, 65, 128, 135, and 144 kDa. Proteins corresponding in mass to the first four of these also occur in the mosquitocidal strain, B. thuringiensis subsp. israelensis (BTI), and it is thought therefore that the mosquitocidal activity of both strains is due to these four proteins. In other studies it has been shown that each of these proteins exhibits from moderate to high toxicity to mosquitoes, though the specific toxicity of the 144 kDa protein in PG-14 to mosquitoes remains unknown. In the present study, two parasporal body mutants (M146 and M242) of PG-14 were developed growing the wild-type strain at 42 degrees C. The parasporal body of M146 contained less of the 65-kDa protein and was less toxic (LC50 = 108 ng/ml) to mosquitoes than the wild-type strain (LC50 = 8.3 ng/ml). The parasporal body of M242 consisted of a bipyramidal crystal composed of a 144-kDa protein that was not toxic to the mosquito, Aedes aegypti, but exhibited substantial toxicity (LC50 = 2.5 micrograms/ml) to the lepidopteran. Trichoplusia ni. Because the parasporal bodies of BTI and BTM PG-14 are similar in mosquitocidal toxicity on a weight basis, the latter results suggest the 144-kDa protein, though not mosquitocidal alone, can contribute to mosquitocidal, activity when in the presence of other mosquitocidal proteins.     

Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion

Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct.     

新型p1 基因型肺炎支原体分型方法的建立与应用

【目的】针对肺炎支原体新型p1基因型(V2c型)菌株检测工作的需要,建立相应PCR检测方法并进行评价。【方法】针对新型V2c型肺炎支原体菌株p1基因变异区域序列设计特异性扩增引物,建立对V2c型肺炎支原体菌株进行PCR检测的检测方法并用相关基因测序进行验证。使用所建立的巢式多重PCR对北京地区2008-2011年分离到的214株临床肺炎支原体进行分型分析。【结果】特异引物可有效检测出V2c菌株,在其它型别菌株均无阳性扩增。214株肺炎支原体临床分离株中1型菌株占90.2%(193/214),V2a型菌株占0.9%(2/214),V2c型菌株占8.9%(19/214);未检出2型菌株。【结论】针对V2c型肺炎支原体所建立的基于p1基因的PCR检测方法,能有效区分以往方法无法检测出的新型V2c型肺炎支原体菌株,对开展肺炎支原体流行病学调查和病原分析有重要意义。     

Antibody response to GroEL varies in patients with acute Mycoplasma pneumoniae infection

Although heat-shock proteins represent major antigens in a wide spectrum of bacterial infections, their immunogenicity is not known for Mycoplasma pneumoniae. M. pneumoniae is a major human respiratory pathogen and it has been suggested that its groEL gene might be dispensable in vitro. Using the specific monoclonal antibody 2C2/C3 we found an abundant synthesis of about 58 kDa GroEL in M. pneumoniae reference strains and in 15 clinical isolates examined at low and higher passages. In patients with acute respiratory disease caused by M. pneumoniae immunoblot analyses showed relatively low prevalence of systemic antibodies against its GroEL protein. Whereas all patients had strong antibody response to the P1 adhesin, only 5 of 29 patients (17.2%) had antibodies to GroEL. Among them, patient RI raised an early and very strong antibody response to GroEL. During the convalescent phase, levels of his serum IgG (mainly IgG2) to GroEL increased and were higher than levels of IgG to P1.     

The reactivity of antigens of Mycoplasma pulmonis derived from mice and rats to naturally infected rat sera

The reactivity of antigens of 4 mouse and 3 rat derived Mycoplasma pulmonis strains to 20 naturally infected rat sera was studied. The optical density values of the same serum by enzyme-linked immunosorbent assay using the 7 strains as the antigen revealed no marked difference among the strains. M. pulmonis antigens recognized by the antibodies were analyzed by the Western immunoblot method. The antigens with molecular weights of 92 K, 66 K, and 58 K were recognized in the 7 strains at a high frequency.