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M F Barile

Publications and source records attributed to M F Barile.

At least 37 records · Page 2Linked to original sources
PubMed1989

Nucleotide sequence of the MgPa (mgp) operon of Mycoplasma genitalium and comparison to the P1 (mpp) operon of Mycoplasma pneumoniae.

The attachment of Mycoplasma genitalium and Mycoplasma pneumoniae to ciliated epithelium involves two surface proteins designated MgPa and P1, respectively. We have previously cloned and sequenced the P1 (mpp) operon of M. pneumoniae, and report here the use of P1-derived probes to clone and sequence a 10.4-kb region of M. genitalium DNA that, by analogy to the P1 operon, contains the MgPa (mgp) operon. The deduced amino acid sequences of the 29-kDa (ORF-1), MgPa (160-kDa) and 114-kDa (ORF-3) proteins of the MgPa operon show extensive homologies with those of the 28-kDa, P1 (170-kDa) and 130-kDa proteins, respectively, encoded by the P1 operon. The common features and homology of these operons are consistent with previous observations that the MgPa and P1 proteins share cross-reactive epitopes, as well as similar biological function. The gene order of the MgPa operon is ORF-1, MgPa, ORF-3, with intervening regions of 6 and 1 nt, respectively. A consensus ribosome-binding site (RBS) sequence is found before ORF-1 and a sequence indicative of a transcription terminator is located beyond ORF-3; the absence of such sequences adjacent to the MgPa gene suggests that the operon is transcribed as a polycistronic message. The RBS sequence is followed by sequences of dyad symmetry that have the potential to form two alternative stem-and-loop structures, which could be involved in controlling initiation of translation.

Base Sequence↗
PubMed1989

Adhesion of Mycoplasma pneumoniae to sulfated glycolipids and inhibition by dextran sulfate.

A virulent strain of Mycoplasma pneumoniae was metabolically labeled with [3H]palmitate and studied for binding to glycolipids and to WiDr human colon adenocarcinoma cells. The organism binds strongly to sulfatide and other sulfated glycolipids, such as seminolipid and lactosylsulfatide which all contain terminal Gal(3SO4) beta 1-residues and weakly to some neolactoseries neutral glycolipids. M. pneumoniae do not bind gangliosides including the sialylneolacto-series and other neutral glycolipids that were tested. Only metabolically active M. pneumoniae cells bind to sulfatide, as binding is maximal in RPMI medium at 37 degrees C and almost completely abolished in nutrient-deficient medium or by keeping the cells at 4 degrees C. Dextran sulfate but not other sulfated or anionic polysaccharides at 10 micrograms/ml completely inhibits binding of M. pneumoniae to purified sulfatide. Dextran sulfate does not inhibit binding to the neolacto-series neutral glycolipids. Dextran sulfate partially inhibits adhesion of M. pneumoniae to cultured human colon adenocarcinoma cells (WiDr). The biological relevance of these data is suggested by our finding that sulfatide occurs in large amounts in human trachea, lung, and WiDr cells. Thus, there are at least two distinct receptors that mediate binding of M. pneumoniae to cells: glycolipids containing terminal Gal(3SO4) beta 1-residues as reported here, and glycoproteins containing terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc sequences (Roberts, D. D., Olson, L. D., Barile, M. F., Ginsburg, V., and Krivan, H. C. (1989) J. Biol. Chem. 264, 9289-9293).

Animals↗
PubMed1989

Sialic acid-dependent adhesion of Mycoplasma pneumoniae to purified glycoproteins.

Several purified glycoproteins including laminin, fetuin, and human chorionic gonadotropin promote dose-dependent and saturable adhesion of Mycoplasma pneumoniae when adsorbed on plastic. Adhesion to the proteins is energy dependent as no attachment occurs in media without glucose. Adhesion to all of the proteins requires sialic acid, and only those proteins with alpha 2-3-linked sialic acid are active. The alpha-subunit of human chorionic gonadotropin also promotes attachment, suggesting that a simple biantennary asparagine-linked oligosaccharide is sufficient for binding. Soluble laminin, asparagine-linked sialyloligosaccharides from fetuin, and 3'-sialyllactose but not 6'-sialyllactose inhibit attachment of M. pneumoniae to laminin. M. pneumoniae also bind to sulfatide adsorbed on plastic. Dextran sulfate, which inhibits M. pneumoniae binding to sulfatide, does not inhibit attachment on laminin, and 3'-sialyllactose does not inhibit binding to sulfatide, suggesting that two distinct receptor specificities mediate binding to these two carbohydrate receptors. Both 3'-sialyllactose and dextran sulfate partially inhibit M. pneumoniae adhesion to a human colon adenocarcinoma cell line (WiDr) at concentrations that completely inhibit binding to laminin or sulfatide, respectively, and in combination they inhibit binding of M. pneumoniae to these cells by 90%. Thus, both receptor specificities contribute to M. pneumoniae adhesion to cultured human cells.

Animals↗
PubMed1989

Mechanisms of macrolide resistance in Ureaplasma urealyticum: a study on collection and clinical strains.

Ureaplasma urealyticum is considered as a species which is intrinsically sensitive to macrolides (MIC less than 1 microgram/ml). Nevertheless, some of the strains recently isolated in our laboratories showed moderate to high levels of resistance (MICs ranging from 2 micrograms/ml to 100 micrograms/ml). In particular, a strain (CT28) isolated from a patient with nongonococcal urethritis long treated with erythromycin revealed a MIC greater than 100 micrograms/ml for this antibiotic. In order to investigate the mechanisms of resistance, strain CT28 and ten clinical and laboratory U. urealyticum strains were compared for the sensitivity to six antibiotics including three macrolides. Moreover the amount of macrolide uptake and the specific antibiotic binding to ribosomes were studied. Strain CT28 was resistant to josamycin, erythromycin, roxithromycin, lincomycin and clindamycin but sensitive to minocycline. When compared to a sensitive strain, strain CT28 showed a six-fold reduction in intracellular macrolide influx and accumulation and a reduction in antibiotic binding to ribosomes. The mechanisms implicated in these differences may be important for macrolide resistance in U. urealyticum.

Biological Transport↗
PubMed1989

Binding of thyrotropin to selected Mycoplasma species: detection of serum antibodies against a specific Mycoplasma membrane antigen in patients with autoimmune thyroid disease.

Radiolabeled human (hTSH) and bovine (bTSH) thyroid stimulating hormone was shown to bind to five species of Mycoplasma, the wall-less prokaryotes. The maximum binding capacity of 125I-bTSH to these five species was about 7.9 x 10(-13) moles-1.4 x 10(-12) moles for 50-100 micrograms protein with dissociation constants of approximately 1.7 to 2.2 x 10(-7)M. Approximately 50% of the 125I-bTSH binding was displaced by excess, unlabeled bTSH or hTSH, but labeled bTSH was not effectively displaced by growth hormone, LH, FSH, prolactin, or the beta subunit of hTSH, FSH and LH. Antisera prepared against Mycoplasma gallisepticum and Mycoplasma pneumoniae bound to human thyroid membranes and guinea pig fat cells, suggesting that receptors on human thyroid tissues and on Mycoplasma cells may have similarities in antigenicity. These findings were substantiated by the occurrence of TSH binding to Mycoplasma antisera. Further, sera from three of six patients with Graves' disease containing antibodies to thyroid tissues also reacted to a 108 Kd polypeptide of Mycoplasma gallisepticum.

Animals↗
PubMed1989

Biological activities of monoclonal antibodies to Mycoplasma pneumoniae membrane glycolipids.

A purified preparation of membranes was obtained by using a unique method of treating Mycoplasma pneumoniae with the ATPase inhibitor, diethylstilbestrol. This method was shown to yield highly purified membranes with little or no cytoplasmic contamination. These membranes were used to immunize mice for subsequent productions of monoclonal antibodies (MAbs). Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay with whole-cell M. pneumoniae and lipid extract antigens. Four stable MAbs were obtained and characterized. MAb CP3-46F5 reacted with a protein of a molecular weight of approximately 52,000 as determined by Western blot (immunoblot). MAbs CP3-50C2, CP3-53C5, and CP3-53C8 did not react with any antigens on Western blots but did bind to at least 10 distinct glycolipid bands as determined by orcinol staining on thin-layer chromatograms of M. pneumoniae lipid extracts. The MAbs did not react with similarly prepared lipid extracts from Mycoplasma genitalium, Mycoplasma neurolyticum, and Mycoplasma gallisepticum. These MAbs did not inhibit M. pneumoniae metabolism or attachment to WiDr cell cultures. The anti-glycolipid MAbs recognize determinants specific to M. pneumoniae, unlike polyclonal hyperimmune sera against M. pneumoniae, which cross-react with lipid extracts of M. genitalium.

Animals↗
PubMed1988

Protein phosphorylation in Mycoplasma gallisepticum.

Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.

Adenosine Triphosphate↗
PubMed1988

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.

Acholeplasma↗
PubMed1988

Parameters of Mycoplasma pneumoniae infection in Syrian hamsters.

An animal model for evaluating the potency of Mycoplasma pneumoniae vaccines was developed with hamsters. Factors that influence hamster infection by M. pneumoniae were defined, and parameters for assessment of intensity of pulmonary disease were established. Colonization of hamster lungs was determined by culture, and intensity of lung disease was assessed histopathologically and expressed numerically as a lung pathological score. Intratracheal inoculation of the challenge was superior to the intranasal or aerosol route for inducing a consistent degree of lung disease. A challenge dose of 10(6) CFU inoculated intratracheally produced lung colonization and significant reproducible lung pathological scores in essentially all unvaccinated animals. The peak of infection, as determined by these criteria, was at about 2 weeks after challenge. Animals over 6 weeks of age were preferable for the test, since younger animals exhibited a lower lung pathological score even though they showed the same degree of lung colonization. The hamster assay developed provides a dependable experimental system for testing the protective potency of M. pneumoniae vaccines.

Administration, Inhalation↗
PubMed1988

Hamster challenge potency assay for evaluation of Mycoplasma pneumoniae vaccines.

A hamster immunization challenge assay described in the accompanying paper (M. F. Barile, D. K. F. Chandler, H. Yoshida, M. W. Grabowski, R. Harasawa, and S. Razin, Infect. Immun. 56:2443-2449, 1988) was used to examine protection against Mycoplasma pneumoniae disease by passive immunization and to evaluate the protective potency of a Formalin-inactivated whole-cell and a cell extract M. pneumoniae vaccine. Passive immunization with a globulin fraction of hyperimmune mule antiserum to M. pneumoniae provided hamsters some protection against the challenge. When hamsters were actively immunized, a single dose of Formalin-inactivated vaccine provided only minimal protection, whereas multiple doses of this vaccine, particularly when combined with adjuvant, provided good protection. A single dose of the cell extract vaccine did not protect animals, but two doses caused a marked reduction of disease when a priming dose was given intraperitoneally, followed by a booster dose intratracheally. The correlation between the level of metabolism inhibition antibodies to M. pneumoniae in the sera of vaccinated hamsters and the degree of protection as measured by reduction of lung pathological scores and colonization was poor, indicating that seroconversion rates for metabolism inhibition antibodies are not by themselves adequate to measure the potency of M. pneumoniae vaccines.

Animals↗
PubMed1988

Characteristics of Ureaplasma urealyticum urease.

Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins. The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease. The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate. Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid. Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides.

Citrates↗
PubMed1987

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.

Antibodies, Monoclonal↗
PubMed1987

Ureaplasmas and mycoplasmas in chimpanzees of various breeding capacities.

Adult chimpanzees (24 male, 76 female) with low and high rates of conception were examined for ureaplasmas, arginine-metabolizing mycoplasmas and chlamydiae. Ureaplasmas were isolated from the throat of only 1 male and 1 female animal, but from the urethra of 29% of the males and from the vagina of 95% of the females. Mycoplasmas were isolated from the throat more often than were ureaplasmas, but from the genital tract with about the same frequency as ureaplasmas. The numbers of organisms, of either type, isolated from the vagina were larger than the numbers isolated from the male urethra. Chlamydiae were not isolated from any animal. The occurrence of ureaplasmas and mycoplasmas and the numbers of these organisms isolated were similar in animals with low or high rates of conception. Furthermore, no association was noted between the organisms in the lower genital tract and the occurrence of abortion and/or stillbirth.

Abortion, Veterinary↗
PubMed1987

Immunochemical evidence for an active (F1-F0)-ATPase in mycoplasmas.

In all Acholeplasma, Mycoplasma and Spiroplasma species tested, a protein capable of reacting with antibodies prepared against the beta subunit of the proton-ATPase complex from yeast, chloroplasts and Escherichia coli was detected. The reactive protein of M. gallisepticum was found to be catalytically active, suggesting that mycoplasmas, as other bacteria, possess a proton-translocating ATPase. Characterization of the ATPase activity of M. gallisepticum indicates that this organism also possesses a Na+-stimulated ATPase activity that differs from the proton-ATPase in its pH profile and its resistance to dicyclohexylcarbodiimide (DCCD).

Antibodies, Bacterial↗
PubMed1987

Superiority of the chimpanzee animal model to study the pathogenicity of known Mycoplasma pneumoniae and reputed mycoplasma pathogens.

As far as we know, humans are the only known natural hosts for M. pneumoniae disease. Whereas volunteer studies have provided useful data on the pathogenesis of disease and efficacy of vaccines, experimentally inducing disease in humans raises serious ethical questions and has become increasingly difficult to defend. Thus, there is a genuine need for a satisfactory animal model to study M. pneumoniae disease. Using the cotton rat and developing chick embryo models, Eaton and co-workers (9-13) have clearly shown that the infectious "Eaton agent" was the cause of primary atypical pneumonia. After the causative agent was identified as M. pneumoniae (14), more definite and quantitative studies were possible. The hamster animal model has provided most of our information on the pathogenicity of strains, the pathogenesis of disease and the potency of inactivated vaccines. However, protective data obtained in hamsters immunized with the TS mutant vaccines did not correlate with data obtained in humans, raising concern regarding the use of the hamster animal model to evaluate the potency of live TS vaccines. The chimpanzee animal model has a number of advantages. Chimpanzees become clinically ill, show positive X-ray findings, and develop cold agglutinin titers. In fact, the experimentally induced disease in chimpanzees is remarkably similar to naturally occurring primary atypical pneumonia in patients. Because of the close genomic relationship, immunologic reagents prepared and used for human studies can also be used successfully in chimpanzee studies. The chimpanzee model also has some serious disadvantages. They are expensive to house and maintain and are generally not available to the scientific community. Nonetheless, chimpanzees are probably the best, most meaningful animal models established thus far to examine the infectious process, the immune response and the pathogenesis of this disease and to determine approaches to effective therapy and immunization of diseases produced by known pathogens, like M. pneumoniae, as well as reputed mycoplasma pathogens, such as M. genitalium and M. hominis.

Animals↗
PubMed1987

Further studies on the Mycoplasma pneumoniae extract: ciliostatic and cell recruitment activities.

Since inactivated vaccines have proved disappointing in preventing Mycoplasma pneumoniae disease, we have initiated studies to identify protective immunogens as candidates for an acellular, purified component vaccine. A cell-free extract was prepared, and was shown to have hemagglutination, attachment inhibition, and ciliostasis activities. Ciliostatic activity in hamster tracheal organ cultures was dependent on the time of incubation and on the amount of added extract. The ciliostatic factor was resistant to boiling and to trypsin and chymotrypsin digestion. When administered intratracheally to Golden Syrian hamsters, the extract induced lung lesions with histopathologic characteristics similar to those caused by virulent M. pneumoniae infection. Thus, in addition to attachment and ciliostatic factors, the extract contains factors that induce inflammatory responses in hamster lungs and cause cytotoxic damage to lung tissues.

Animals↗
PubMed1987

Attachment of Mycoplasma hominis to human cell cultures.

Clinical isolates, cell-culture contaminants, and the type strain PG21 of Mycoplasma hominis were examined for attachment to erythrocytes and human cell cultures. Strain 13428 (from blood, postpartum fever) and strain 1184 (cell culture) attached to human and guinea pig erythrocytes, but there were no differences in attachment activities between these strains. However, five M. hominis strains isolated from different tissue sites showed quantitative differences in attachment to human WiDr (intestinal carcinoma cell cultures), MRC-5 (human embryonic lung fibroblasts) and HeLa (carcinoma of cervix) cell cultures. The relative attachment activities were, in descending order: strain 1184 (cell culture), strain 11932 (cervix), strain 13428 (blood, postpartum fever), 13408 (nongonococcal urethritis), and type strain PG21 (multiple passage, originally from human rectum). Trypsin and pronase treatment of M. hominis strain 1184 markedly reduced attachment, suggesting that surface proteins play a role in M. hominis attachment to mammalian cells. In subsequent studies, strain 1620 (septic arthritis) showed the highest attachment activity, whereas strain 1652 (surgical skin flap) and L01888 (cell culture) showed attachment activity similar to cell culture strain 1184. The differing attachment activities of these M. hominis strains isolated from different infected sites of patients with a variety of diseases may be relevant to the virulence of these strains.

Animals↗