Adsorption of Mycoplasma pneumoniae to Neuraminic Acid Receptors of Various Cells and Possible Role in Virulence

Monkey, rat, and chicken tracheal epithelial cells, as well as monkey, rat, guinea pig, and chicken erythrocytes, adsorbed firmly to colonies of Mycoplasma pneumoniae and M. gallisepticum. Colonies of M. pulmonis also adsorbed erythrocytes but with less avidity than M. pneumoniae or M. gallisepticum; unlike the latter organisms, M. pulmonis did not adsorb tracheal epithelial cells. Colonies of M. orale type 1 and M. orale type 3 adsorbed only chicken red cells. Other mycoplasma species tested, including four of human origin and one of animal origin, did not adsorb red cells or epithelial cells. M. pneumoniae and M. gallisepticum appeared to attach to erythrocytes or tracheal epithelial cells by neuraminic acid receptors on these cells, whereas M. orale types 1 and 3 and M. pulmonis seemed to utilize another type or other types of receptors. Pretreatment of red cells or tracheal epithelial cells with receptor-destroying enzyme, neuraminidase, or influenza B virus removed the adsorption receptors for M. pneumoniae. Similarly, pretreatment of M. pneumoniae colonies with neuraminic acid-containing materials prevented adsorption of erythrocytes or respiratory tract cells. The adsorption sites on M. pneumoniae were specifically blocked by homologous but not heterologous antisera. This property made it possible to study the nature of the mycoplasma adsorption sites by testing the capacity of different fractions of the organism to block the action of adsorption-inhibiting antibodies. Such studies suggested that the mycoplasma binding sites were probably lipid or lipoprotein in nature. The glycerophospholipid hapten was implicated as one such site, since this serologically active hapten blocked the action of hemadsorption-inhibiting antibodies in M. pneumoniae rabbit antiserum. The affinity of M. pneumoniae for respiratory tract epithelium, unique among the mycoplasmas that infect man, may play a role in virulence, since this type of attachment provides an unusual opportunity for peroxide, secreted by the organism, to attack the tissue cell membrane without being rapidly destroyed by catalase or peroxidase present in extracellular body fluids.     

Demonstration of cross-reactive antibodies to mycoplasmas in human sera by ELISA and immunoblotting

Varying levels of cross-reactivity to some mycoplasma species were observed in the sera of patients infected with Mycoplasma pneumoniae and even in normal human sera by enzyme-linked immunosorbent assay (ELISA). The absorption of the patients' sera with M. pneumoniae lysate showed the decrease in ELISA titers not only to M. pneumoniae, but also to other mycoplasma species. These results suggested the existence of cross-reactive antibodies to mycoplasmas in human sera. Cross-reactive antibodies to M. pneumoniae and other mycoplasmas in the patients' sera were also demonstrated by Western blotting technique.     

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.     

Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice

Lutsky, Irving I. (Marquette University School of Medicine, Milwaukee, Wis.), and Avrum B. Organick. Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice. J. Bacteriol. 92:1154-1163. 1966.-Two species of mycoplasma of human origin, Mycoplasma pneumoniae and M. salivarium, were tested for their ability to produce respiratory disease in the Ha/ICR mouse when inoculated by the intranasal route. The mouse pathogen M. pulmonis was studied as a positive control. Conventional and gnotobiotic Ha/ICR mice were employed, the latter to provide a system free from indigenous mycoplasma and bacteria. Pneumonia from which mycoplasma were isolated was produced in all groups of the conventional Ha/ICR mice, including those inoculated with sterile broth. Only M. pulmonis produced disease when inoculated intranasally into the gnotobiotic mice, and the gross and microscopic lesions resembled those described in conventional mice. The gnotobiotic mouse provided a tool to study the pathogenicity of different mycoplasma species, and indicated marked differences in host specificity that could not be clearly seen when conventional mice were used.     

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.     

Comparative Studies of Antigens from Mycoplasma mycoides and Escherichia coli

Extracts of sonically disrupted Mycoplasma mycoides and Escherichia coli were fractionated by sucrose density gradient centrifugation. The presence of antigen in each of the fractions was determined by complement-fixation and agar-gel diffusion precipitin tests, in which cow, pig, and rabbit anti-M. mycoides sera and rabbit anti-E. coli serum were used. Fractions of M. mycoides, with a buoyant density of 1.225 or lower, fixed complement with cow and pig anti-M. mycoides sera. These fractions also formed precipitin lines with pig antiserum. Fractions in the buoyant density range of 1.10 to 1.20 fixed complement with rabbit anti-E. coli serum, but precipitin lines were not formed. All E. coli fractions fixed complement and gave precipitin lines with homologous serum. But fractions in the buoyant density range of 1.10 to 1.20 had minimal complement fixation with heterologous M. mycoides sera. The cross-reacting antigens in M. mycoides and E. coli had a buoyant density of 1.10 to 1.20; the specific antigens were isolated from M. mycoides at a buoyant density of 1.08 to 1.02.     

Cimulative hemagglutination by Mycoplasma pneumoniae and other agglutinins

John, T. Jacob (University of Colorado School of Medicine, Denver), Marlene Stahl, and Vincent A. Fulginiti. Cumulative hemagglutination by Mycoplasma pneumoniae and other agglutinins. J. Bacteriol. 92:1002-1004. 1966.-The phenomenon of cumulative hemagglutination, or hemagglutination by two agglutinins, each in subagglutinating concentration, was demonstrated by use of four different systems, namely, horse serum and Mycoplasma pneumoniae, horse serum and measles antigen, M. pneumoniae and measles antigen, and parainfluenza 2 virus and M. pneumoniae. Cumulative hemagglutination appears to be the mechanism by which a horse-serum diluent causes high hemagglutination titers of M. pneumoniae, since both contain hemagglutinins against vervet erythrocytes. It was also shown that antibodies against either one of the two antigens may cause inhibition of such hemagglutination.     

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.     

The skin-reactive antigens of Mycoplasma pneumoniae.

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the orgnaism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 mug. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform-methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

Characterization of a Newly Identified Mycoplasma (Mycoplasma orale Type 3) from the Human Oropharynx

Six mycoplasma strains, isolated under anaerobic conditions from the human oropharynx, were studied by biologic and serologic means. The strains produced nippled colonies with weak hemolytic activity for guinea pig erythrocytes on agar medium. In addition, the strains metabolized arginine with a concomitant alkaline shift in the pH of the medium but did not produce a pH shift when grown in the presence of glucose or urea. The strains failed to reduce 2-3-5 triphenyl tetrazolium and were inhibited by 0.001% methylene blue. In addition, they required fresh yeast extract for growth. When compared by several serologic methods, the strains were found to be related to each other but distinct from 23 serotypes of human, animal, and avian origin. However, one-way serologic relationships between one of the new strains and Mycoplasma orale type 1 and M. salivarium were observed when they were tested by complement fixation. Furthermore, partial relationship of one of the new strains to all of the arginine-utilizing mycoplasma species of human origin was demonstrated with the agar gel diffusion technique. Thus, the new strains appear to constitute a new mycoplasma species, for which the name M. orale type 3 is tentatively proposed. M. orale type 3 accounted for 1.4% of 437 mycoplasma isolates from the oropharynx of adults. The new species probably is a rare member of the normal mycoplasmal flora of man.     

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.     

Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas.

Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.     

Monoclonal antibodies specific for cell culture mycoplasmas

Mycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised in rabbits or donkeys. In addition, a monoclonal antibody to Mycoplasma pneumoniae was produced. M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful in the clinical diagnosis of M. pneumoniae infection in man.     

Immunological responses of the rat to Mycoplasma arthritidis

Arthritis was produced in rats by the intravenous injection of Mycoplasma arthritidis. Metabolic inhibiting antibody and indirect hemagglutinating antibody could not be detected in the sera of arthritic or convalescent animals. Nonmurine species of mycoplasma were capable of inducing metabolic inhibiting antibody in the rat. A hypothesis based upon the possible occurrence of heterogenetic antigens common to M. arthritidis and rat tissue was brought forward to explain these findings. Complement-fixing antibody to M. arthritidis was detected 3 to 4 days after injection and subsequently rose to high levels, depending upon the severity of arthritis and number of organisms injected. Animals that had recovered from intravenous or subcutaneous inoculation with M. arthritidis were resistant to subsequent infections by the organism. Immunity could be passively transferred by the intravenous injection of convalescent serum. Adsorption of the convalescent serum with antigen greatly reduced the complement fixation titer but did not significantly alter the protective properties of the serum. The presence of complement-fixing antibody could not be related to the development of immunity. An avirulent strain of M. arthritidis and a strain previously classified as M. hominis type 2 were capable of inducing resistance to subsequent injection by virulent M. arthritidis.     

Adherence of Mycoplasma pneumoniae to human alveolar macrophages

Abstract The human pathogen Mycoplasma pneumoniae causes primary atypical-cold agglutinin-positive pneumonia. Since alveolar macrophages internalize mycoplasma as part of their immune defense, we studied characteristics of the human macrophage receptor for opsonized and nonopsonized M. pneumoniae . The glass-adhering subpopulation of M. pneumoniae attached more than the non-adherent subpopulation. The attachment was dose-dependent and enhanced by opsonization in the presence of human serum. It is inhibited by sulfated compounds such as dextran-sulfate and polyanetholsulfonic acid, but not by dextran or several monosaccharides, suggesting that sulfated glycolipids on the macrophage surface may act as receptors for M. pneumoniae binding. In addition, sialylated compounds, such as fetuin and α 1-acid glycoprotein, were found to be potent inhibitors of the attachment, also indicating the role of sialic acid residue in recognition and attachment of M. pneumoniae to human alveolar macrophages.     

Detection of mycoplasma antigens and immune complexes in sera of the children with bronchial asthma

The study was targeted at revealing Mycoplasma pneumoniae (M.p.) and Mycoplasma hominis (M.h.) antigens in blood samples of children with bronchial asthma (BA),both in a free state and those included in circulating immune complexes (CIC). The mycoplasma antigens of one or both species have been detected in one third out of 62 patients with BA. In this group of patients the frequency of detection of specific antibodies twice exceeded that of mycoplasma antigens. Testing paired blood samples of children with BA (n=26) showed, that at receipt in a hospital and a month after primary examination the mycoplasma antigens were detected in 16 and 12 patients, respectively, the association of M.p. and M.h. antigens being more frequent. Data on distribution of antibodies according to immunoglobulin classes testify that basically two (M, G), or four (M, G, A, E) classes were registered in children, the M class antibodies in high percentage of cases (from 36.6 up to 50.0%)being detected in every term of examination. These data indirectly testify that the antigens can be partly included in the CIC structure. The level of the total CIC content in BA children's blood sera one month after hospitalization twice exceeded the value detected at primary examination. Three months later later this parameter decreased not reaching the control value. The differential analysis of the precipitated CIC within the whole period of examination showed that mycoplasma antigens were present in the CIC structure in 87.4 - 65.0% of cases. The data obtained precondition future studies on the role of mycoplasma and M.p. and M.h. antigens included in the CIC in the pathogenesis of BA.     

Disposition of exposed antigens on the faces of isolated Mycoplasma gallisepticum membranes.

The transverse disposition of exposed protein antigens on the two faces of isolated Mycoplasma gallisepticum membranes have been investigated by using indirect immunoferritin labeling to accomplish visualization of the antigens at the ultrastructural level. Comparison between the labeling patterns obtained with unabsorbed specific mycoplasma antiserum and antiserum from which antibodies directed against outer side determinants had been removed revealed that the majority of protein antigens were the same on the opposed membrance faces or at least displayed extensive interside cross-reactivity. The relatively scarce tagging of isolated Acholeplasma laidlawii membranes, contrary to membranes on intact organisms observed in this investigation, precluded conclusions regarding the disposition of membrane antigens of this species. The advantages and limitations of the employed method in disposition studies and the factors influencing the transverse distribution of membrane proteins in mycoplasmas are discussed.     

Elimination of Mycoplasma from various cell cultures

Elimination ofMycoplasma orale-I from chronically infected cell lines was achieved either by treatment with a mixture of antibiotics in a hypotonic solution, or with 10 vol % of anti -M.orale rabbit serum in tissue culture medium. The latter treatment was preferable in most cases, as it was practically harmless to the cells. Inactivation of this antiserum had no effect on its potency. The antibiotic-hypotonic treatment was rather destructive, but to a different degree for the various cell cultures. Both methods were equally useful for the treatment of a monkey kidney cell line contaminated with a mycoplasma strain related toM.hyorhinis. The available anti -M.hyorhinis rabbit serum was toxic for the monkey cells when not inactivated. The potency of the antiserum was rather low and even lower after inactivation. However, prolonged treatment successfully eliminated the mycoplasma. Pre-incubation of the inactivated anti -M.hyorhinis serum with tissue culture medium to which 10% non-inactivated calf serum had been added, favoured the elimination of the mycoplasma.During the treatment of contaminated cell cultures with single antibiotics a strain related toM.hyorhinis became resistant to chlortetracyclin.M.orale- I was found to be resistant to various single antibiotics.We are grateful to Professor Dr. A. Ch. Ruys (University of Amsterdam) and Dr. R. H. Leach (Mycoplasma Reference Laboratory, London) for helpful discussions and for identifying some of our mycoplasma strains; Dr. Leach also for kindly supplying us with his G. D. L.-strain. We thank Dr. H. Cohen and Dr. A. C. Hekker for their criticism and Mr. N. L. M. van Zwetselaar for his accurate technical assistance.     

Use of the Mycoplasma hominis 102-116 kD proteins as antigen in an enzyme-linked immunosorbent assay

Mycoplasma hominis surface structures involved in human immune response and in the pathogenesis of this bacterial infection are inadequately defined. Attempts have been made to identify M. hominis surface proteins, to determine the antigenicity of these polypeptides, and to examine antigens which could lead to the development of species-specific diagnostic tests. By means of Western blotting, using a pool of sera from patients with culturally proven vaginal infection, most antigens recognized were surface exposed. Among these proteins, antigens of molecular weights between 102 and 116 kD were most consistently revealed. These polypeptides were recovered by electroelution and assayed in an IgG-ELISA. The electroeluted antigen specificity was examined by ELISA and immunoblotting with different mycoplasma species. Electroeluted proteins may be effective and specific for establishing a reliable diagnosis test.     

Construction and analysis of a modified Tn4001 conferring chloramphenicol resistance in Mycoplasma pneumoniae

The Staphylococcus aureus transposon Tn4001 and derivatives thereof have been transformed successfully in several mycoplasma species. In order to expand the versatility of Tn4001 for other genetic manipulations and for use in mycoplasma species resistant to gentamicin (Gm), chloramphenicol acetyltransferase (Cat) from S. aureus was evaluated as a selectable marker. The cat gene was cloned in both orientations into a modified Tn4001 and transformed into Mycoplasma pneumoniae, conferring resistance to Cm and Gm. Replacement of the gene for GmR in Tn4001 with cat likewise conferred CmR when transformed into M. pneumoniae. The minimum inhibitory concentration to Cm in transformants with cat derivatives of Tn4001 was 300-500 microg/ml, and Cat enzyme activity was demonstrated by using a fluorescent substrate.