Localization of an immunodominant 64 kDa lipoprotein (LP 64) in the membrane of Mycoplasma gallisepticum and its role in cytadherence.

A 64 kDa lipoprotein (LP 64) haemagglutinin (pI 4.9-5.0) was isolated from the membrane of Mycoplasma gallisepticum. Triton X-114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]-palmitate-labelled M. gallisepticum revealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels of M. gallisepticum showed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots of M. gallisepticum indicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post-infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains of M. gallisepticum, but its presence could not be detected in Mycoplasma synoviae. Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one- or two-dimensional immunoblots of M. gallisepticum. This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of 14C-labelled M. gallisepticum to chicken tracheal epithelium in vitro by 62%.

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function.

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.

Morphologic changes following intraarticular inoculation of Mycoplasma bovis in calves.

Intraarticular inoculation of Mycoplasma bovis into the joints of six-week-old calves caused severe arthritis in five inoculates and mild arthritis in a sixth. Intraarticular inoculation of killed M. bovis did not cause arthritis. Arthritic calves had fever, joint swelling, lameness, neutrophilia, and intercurrent pneumonia from which M. bovis could not be recovered. Gross lesions were massive fibrinosuppurative synovitis and tenosynovitis, erosion of cartilage, and its replacement by polypoid granulation tissue. Histologic lesions were extensive ulceration of synovial membranes, leukocytic infiltration of the subsynovium, congestion, hyperemia, and thrombosis of the subsynovial vessels. Cartilage erosion was accompanied by chronic osteomyelitis and formation of pannus tissue. The presence of thrombi and platelet aggregates suggests that the inflammatory process in the synovium may arise from the interaction of M. bovis with the vasculature and the coagulation process of the host.

Inflammatory toxin from Mycoplasma bovis: isolation and characterization.

An inflammatory toxin was extracted from Mycoplasma bovis with 75 percent aqueous ethanol. The toxin is a complex polysaccharide composed of glucose, glucosamine or galactosamine, and a heptose, is heat-stable, devoid of protein and lipid, and has a molecular weight of 73,000. The holotoxin in the cell membrane is a glycoprotein; however, it is the polysaccharide portion that is toxic. This inflammatory toxin increases vascular permeability and is capable of activating complement. Infusion of 0.9 milligram of toxin into the bovine udder resulted in the characteristic eosinophilic mastitis produced by Mycoplasma bovine.

Infertility of cattle caused by mycoplasmas.

Mycoplasmas have been well established as pathogens of the bovine urogenital tract, and produce pathologic lesions resulting in infertility. Serologic examination of cattle with infertility problems with Mycoplasma bovigenitalium and Mycoplasma agalactiae subsp. bovis show a high incidence of positive reactors suggesting that mycoplasmas play an important role in bovine infertility. The similarities of pathologic lesions in the urogenital tract of cattle and women with infertility problems and the frequency of isolation of mycoplasmas from human females suggest closer examination for mycoplasmas in human infertility. Studies with bulls suggest that mycoplasmas as a cause of human male infertility should not be ignored.

Freeze-fractured Acholeplasma laidlawii membranes: nature of particles observed.

Freeze-fracture of Acholeplasma laidlawii membranes from cells incubated in the presence of puromycin or omission of amino acids reveals a decrease in the number of particles between 50 and 100 angstroms in the hydrophobic fracture plane, which strongly suggests that these particles are protein. Additional evidence indicates that they may be involved in substrate transport.

Membrane structure: spin labeling and freeze etching of Mycoplasma laidawii.

A spin-labeled fatty acid was incorporated in vivo into the polar lipids of Mycoplasma laidlawii membranes. The electron paramagnetic resonance signal from either intact cells or their extracted lipids reflected the fatty acid composition of the Mycoplasma membranes. Comparison of signals from intact cells, gramicidin-treated cells, heat-treated cells, and extracted lipids indicates that a major portion of the membrane lipids is in a semiviscous hydrocarbon environment. The results also show that the spin label in the intact membrane is slightly but significantly less mobile than it is in protein-free lipid extracts made from these membranes. Correlated electron microscope examinations using the freeze-etch technique reveal particulate components in the hydrophobic region of the membrane. The mobility of the lipids in the intact cell membrane may be influenced by their association with these particles.

Characterization and comparative genital tract pathogenicity of bovine mycoplasmas.

Sixteen bovine genital mycoplasmal isolates obtained from semen and prepuce of bulls and from aborted fetuses were compared physiologically and serologically with the Donetta strain (tentatively Mycoplasma agalactiae var. bovis), a known pathogen. All isolates were distinct from the Donetta organism. Four appeared to be saprophytes, and the remainder were placed in one group which could not be further separated by the biochemical or serological methods used. Two of the organisms in the latter group have been subsequently identified as M. bovigenitalium. Uterine infusion of broth cultures of four isolates into virgin heifers failed to produce clinical evidence of disease, and significant lesions were not present at necropsy. The mycoplasmas were recovered from cervicovaginal mucus of only three heifers, and never for more than 3 days postinfusion. Since the organisms were not recovered from any organs at necropsy, it appears that the mycoplasmas were incapable of surviving in the clinically normal virgin female reproductive tract.

Cytopathic Effects and Ultrastructure of Mycoplasma agalactiae var. bovis (Donetta strain).

An electron microscopic examination of the pathogenic Donetta strain mycoplasma (tentatively Mycoplasma agalactiae var. bovis), in broth and cell cultures, revealed that this organism possessed considerable pleomorphism. A membranelined space occurred in a number of the organisms, and some of these were considered to represent deep, cuplike invaginations of the outer limiting membrane. In cell cultures, the mycoplasmas were located almost exclusively in extracellular locations but often in close association to the host-cell membrane. The cytopathic effects (CPE) were not considered to be caused primarily by cytoplasmic invasion. Serum was necessary for the development of the CPE and multiplication of the mycoplasmas in cell cultures. An effort to determine the basic cause of the CPE was unsuccessful but did eliminate causes previously reported for other strains of mycoplasma.

Metabolic turnover of the polar lipids of Mycoplasma laidlawii strain B.

The fatty acyl groups of the membrane polar lipids of Mycoplasma laidlawii B were radioactively labeled by growth in the presence of (14)C-palmitic, oleic, or linoleic acids. No loss of radioactivity from any of the polar lipids occurred during incubation of radiolabeled cells in growth medium containing various nonradioactive fatty acids, although growth and lipid biosynthesis continued throughout the incubation period. The metabolism of the glucose and phosphate moieties was also studied in a similar fashion by utilizing growth in (14)C-glucose or (32)P-inorganic phosphate to radioactively label these groups. As before, no loss of radioactivity from any of the polar lipids occurred during subsequent growth in medium containing unlabeled glucose or phosphate. The results establish that the glyco- and phospholipids of M. laidlawii B are metabolically stable in actively growing cells. The absence of metabolic turnover is discussed in terms of proposed relationships between lipid metabolism and function in this organism.

Calorimetric evidence for the liquid-crystalline state of lipids in a biomembrane.

Both membranes of Mycoplasma laidlawii and water dispersions of protein-free membrane lipids exhibit thermal phase transitions that can be detected by differential scanning calorimetry. The transition temperatures are lowered by increased unsaturation in the fatty acid residues, but in each case they are the same for membranes and lipids. The transitions resemble those observed for synthetic lipids in the lamellar phase in water, which arise from melting of the hydrocarbon chains within the phospholipid bilayers. Such melts are cooperative phenomena and would be greatly perturbed by apolar binding to protein. Thus the identity of membrane and lipid transition temperatures suggests that in the membranes, as in water, the lipids are in the bilayer conformation in which the hydrocarbon chains associate with each other rather than with proteins. Observations of morphological changes indicate that osmotic imbalance occurs when the membrane transition temperature exceeds the growth temperature, and that for transport processes to function properly the hydrocarbon chains must be in a liquid-like state.

Mycoplasma membrane lipids: variations in fatty acid composition.

The fatty acid composition of the membrane polar lipids of Mycoplasma laidlawii B can be dramatically altered. These variations reslut in characteristic morphological chlanges, and in most cases the cells remain viable. This organism should provide a useful system for clarifying the role of fatty acyl chains in biological membranes.

Genetic relationship among bacteria classified as vibrios.

Genetic relatedness among six bacterial strains currently classified as vibrios was determined by biochemical tests, deoxyribonucleic acid base composition analysis, and the detection of nucleic acid homology by the membrane filter method. Varying degrees of genetic homology existed among vibrios having similar base compositions. The bovine isolate which had a substantially different per cent guanine plus cytosine content was shown to be genetically different from the remaining vibrio strains. The use of nucleic acid homology studies in classifying vibrio strains is discussed.

Synthesis of saturated long chain fatty acids from sodium acetate-1-C14 by Mycoplasma.

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C(14). The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C(14) and methyl palmitate-1-C(14). This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.