Inhibition of mycoplasma cell division by cytochalasin B

Mycoplasma gallisepticum has subcellular organelles which may function as a primitive "mitotic-like" apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time "mitotic-like" structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle.     

Utilization of Neuraminic Acid Receptors by Mycoplasmas

Erythrocytes and H-HeLa cells were treated with neuraminidase and then compared with untreated cells for their ability to adsorb to mycoplasma colonies or be agglutinated by suspensions of the mycoplasmas. Of the 17 mycoplasma serotypes examined, only 4 were found to use neuraminic acid receptors; these were Mycoplasma pneumoniae, M. gallisepticum, M. synoviae, and mycoplasma WR1. Not all strains of a serotype behaved alike. Thus, removal of receptors on erythrocytes for one strain of M. gallisepticum required at least 100 times the concentration of neuraminidase needed to remove them for another strain. The mechanism of attachment of erythrocytes to mycoplasma colonies does not appear to be the same as that for attachment to mycoplasmas in suspension.     

Identification and molecular characterization of macrophage-associated antigens of the rat

Rabbit antiserum to rat peritoneal exudate (PE) macrophage (M phi) antigens was prepared and its reactivity with cell surface proteins of M phi, granulocytes, and lymphocytes was studied by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). A total of 14 membrane antigens were identified of which three were found to be expressed only by M phi and granulocytes. By one-dimensional analysis, a protein with an approximate m.w. of 105,000 was present on splenic and PE M phi and on splenic lymphocytes. Two-dimensional analysis revealed that this band was heterogeneous and contained at least three species, one of which was restricted to expression on M phi and granulocytes. A second protein of 150,000 daltons was resolved into two species by two-dimensional analysis. Both of these species were present on M phi and granulocytes but not on lymphocytes. Both the 105,000- and 150,000-dalton proteins were glycosylated. Because the 105,000- and 150-000-dalton proteins expressed by M phi were also expressed by granulocytes, is is likely that these are differentiation antigens whose expression is a characteristic property of cells within both monocytoid and myeloid lineages. All three 105,000-dalton species and one of the two 150,000-dalton species were detected on mouse M phi, indicating their expression is not unique to the rat.     

A 41-kDa variable surface protein of Mycoplasma gallisepticum has a counterpart in Mycoplasma imitans and Mycoplasma iowae

Abstract Mycoplasma gallisepticun, M. imitans and M. iowae are three morphologically similar avian Mycoplasma species, and M. gallisepticum and M. imitans have been shown to be antigenically related. Using a monoclonal antibody that binds to the previously described size- and phase-variant integral membrane surface protein PvpA of M. gallisepticum , we have identified in all three avian Mycoplasma species a 41-kDa surface antigen, which in M. gallisepticum and M. imitans was identified as peripheral membrane protein undergoing variation in expression among clonal isolates. Southern blot analysis using the pvpA gene as a probe demonstrated sequence homology with M. imitans and M. iowae genomic DNA and suggested that a pvpA -related gene that may encode the 41-kDa product exists in these two Mycoplasma species. These studies establish (i) that M. iowae is antigenically related to M. gallisepticum and M. imitans , (ii) that the three species share non-ribosomal gene sequences, and (iii) that peripheral membrane proteins contribute to Mycoplasma surface variation.     

Liposomes replace serum for cultivation of fermenting mycoplasmas.

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.     

Liposomes replace serum for cultivation of fermenting mycoplasmas

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.     

Detection and differentiation of avian mycoplasmas by surface-enhanced Raman spectroscopy based on a silver nanorod array

Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to contain M. gallisepticum infections and ensure mycoplasma-free avian stocks, but several factors make detection of M. gallisepticum and diagnosis of M. gallisepticum infection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array-surface-enhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection of M. gallisepticum in choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.     

Differential inhibition of mollicute growth: an approach to development of selective media for specific mollicutes

The energy-generating pathways of Mycoplasma spp. are diverse. Thus, it was predicted that the ability of inhibitors of these pathways to block growth would vary among species. This prediction was tested with 14 Mycoplasma species and potential inhibitors. The greatest differentiation among test species was obtained using fluoride, iodoacetate (IAA), beta-fluoropyruvate (FP), cibacron blue (CB), L-citrulline, and carbonyl cyanide m-chlorophenylhydrazone. A range of other potential inhibitors, including L-arginine analogues, had little inhibitory effect on growth, and D-arginine was shown to be a growth substrate for arginine-hydrolyzing species. Fluoride selectively inhibited the growth of mycoplasmas that were able only to ferment sugars to lactate and/or to hydrolyze arginine. In contrast, IAA was most effective against organic acid-oxidizing species, and L-citrulline inhibited arginine-hydrolyzing species. Mycoplasma verecundum, a species for which energy sources have not been identified, was relatively resistant to FP. Similarly, Acholeplasma laidlawii was distinguished by its CB resistance.     

Identification of an isozymic form of acetyl-CoA carboxylase

Acetyl-CoA carboxylase (ACC) is a major rate-limiting enzyme of fatty acid biosynthesis; its product, malonyl-CoA, also contributes to the regulation of fatty acid oxidation and elongation. Using monospecific antibodies directed against rat liver ACC and N- and C-terminal antipeptide antibodies raised against predicted sequences of the cloned ACC of Mr 265,000, we have identified a unique biotin-containing cytosolic protein of molecular mass 280,000 daltons that is distinct from this 265,000-dalton protein. This protein is uniquely expressed in rat cardiac and skeletal muscle but is co-expressed with the 265,000-dalton protein in rat liver, mammary gland, and brown adipose tissue. In the fed rat, white adipose tissue contains only the 265,000-dalton protein. Like the 265,000-dalton protein, the 280,000-dalton protein is present predominantly in the cytosolic fraction of liver. In the liver, the content of both proteins is diminished on fasting and increases on fasting/refeeding with a high carbohydrate diet. In contrast, the cardiac and skeletal muscle 280,000-dalton protein content is unaltered by nutritional manipulation. Avidin-Sepharose isolates of citrate-dependent ACC from the heart reveal only the 280,000-dalton protein, while white adipose tissue isolates show only the 265,000 form. These species differ in the sensitivity to citrate activation and in the Km for acetyl-CoA. Antibodies reactive with the 280,000-dalton protein on immunoblotting precipitate ACC activity in heart isolates, while white adipose ACC is precipitated only by antibodies specific for the 265,000-dalton species. However, in ACC isolates where both proteins are present, a heteroisozyme complex can be detected both by immunoprecipitation and by a sandwich enzyme-linked immunosorbent assay. We conclude that the 280,000-dalton protein is an isozyme of ACC, distinct from the previously cloned 265,000-dalton species. Its presence in cardiac and skeletal muscle, where fatty acid synthesis rates are low, suggest that it might play alternative roles in these tissues such as regulation of fatty acid oxidation or microsomal fatty acid elongation.     

Localization of an immunodominant 64kDa 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%.     

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.     

The beta-subunit of the F1F0-ATPase is conserved in mycoplasmas

Monospecific polyclonal antibodies that were generated against the beta-subunit of Escherichia coli ATPase (F1Fo) cross-reacted with a protein present in the cells of several Mycoplasma and Acholeplasma species. In Mycoplasma gallisepticum, the reactive protein was found almost exclusively in the cell membrane. This protein had an apparent molecular mass of approximately 52 kDa and could not be released from the membranes by repeated washings with either low or high salt solutions in the presence or absence of EDTA. The reactive protein was found to be catalytically active, exhibiting up to 44% of the total membrane-bound ATPase activity. We suggest that mycoplasmas possess a F1Fo-ATPase which undergoes structural modification(s) allowing its integration into the membrane.     

Cultivation of Mycoplasmas on Glass

Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.     

Cultivation of Mycoplasmas on Cellulose Ester Substrates

The ability of mycoplasmas to grow on cellulose ester substrates was evaluated. Mycoplasma pneumoniae, M. hominis, M. arthritidis, M. gallisepticum, and Acholeplasma laidlawii grew on Millipore (mixed cellulose ester) filters and Sepraphore III (cellulose polyacetate) membranes.     

Transposon mutagenesis of Mycoplasma gallisepticum by conjugation with enterococcus faecalis and determination of insertion site by direct genomic sequencing

Few genetic systems for studying mycoplasmas exist, but transposon Tn916 has been shown to transpose into the genomes of some species and can be used as an insertional mutagen. In the current study, the ability of Enterococcus faecalis to serve as a donor for the conjugative transfer of transposon Tn916 into the genome of the avian pathogen Mycoplasma gallisepticum strain PG31 was examined. Transconjugants were obtained at a frequency of > or =6 x 10(-8) per recipient CFU. To determine the transposon insertion site, an oligonucleotide primer corresponding to the 3' end of Tn916 was designed for the purpose of directly sequencing genomic DNA without PCR amplification. Using the direct sequencing approach, Tn916 was shown to insert into any of numerous sites in the M. gallisepticum genome. This is the first report of conjugal transposition of Tn916 into the M. gallisepticum genome. The ability to determine transposon insertion sites in mycoplasmas by genomic sequencing has not been previously described and allows rapid sequence analysis of transposon-generated mutants.     

Detection and identification of Mycoplasma infections by DNA hybridization

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.     

Incorporation and modification of exogenous phosphatidylcholines by mycoplasmas.

The uptake and modification of exogenous phosphatidylcholine (PC) by several Mycoplasma and Spiroplasma species was investigated. While in most Mycoplasma species and in all Spiroplasma species tested the PC appears to be incorporated unchanged from the growth medium, the PC of M. gallisepticum, M. pulmonis, and M. pneumoniae was disaturated PC, apparently formed by modification of 1-saturated-2-unsaturated PC from the growth medium. The modification of the exogenous PC by M. gallisepticum was inhibited by chloramphenicol under conditions that did not affect de novo synthesis of phosphatidylglycerol. A low activity of an endogenous phospholipase A was detected in native M. gallisepticum membranes. The activity was markedly stimulated by treating the membranes with low concentrations of the nonionic detergents. The PC modification was affected by the fatty acid composition of the exogenous PC species. Diunsaturated, 1-saturated-2-unsaturated, and 1-unsaturated-2-saturated PCs were modified to various extents, whereas the disaturated dipalmitoyl PC (DPPC) was not. Both modified and unmodified PCs were incorporated by the cells, but the unmodified DPPC was incorporated at a lower rate and to a lesser extent. The possibility that the incorporation of DPPC into M. gallisepticum cells is associated with the formation of intracytoplasmic membranes is discussed.     

Comparative proteomic characteristic of Mycoplasmas (Mollycutes)

Saturating proteome identification and the study of post-translational protein modifications of Acholeplasma laidlawii using combination of single- and two-dimension gel electrophoresis followed by mass-spectrometry analysis have been carried out. Results were compared to the earlier identified proteome of Mycoplasma gallisepticum. It was found that M. gallisepticum and A. laidlawii express 61 and 58% of the annotated ORFs respectively. All subunits of DNA-polymerase III were identified during our study which indicates that our methods can detect single copies of a given protein per cell. Metabolic pathways of the respective mycoplasmas were compared further in this work.     

Nucleosome mono, di, tri-, and tetramers from chicken embryo chromatin.

The fractionation of gram quantities of nuclease digested chromatin from chicken embryos into nucleosome mono-, di-, tri-, and tetramers is described in detail. Each of these nucleosomal species contains a fraction soluble in 0-1 M KC1 that decreases with increasing repeat number. Less histone H1 is associated with the nucleosome fractions soluble as compared to the respective fractions precipitated in 0.1 M KC1. Thermal denaturation profiles of the four nucleosomal species are monophasic. The same Tm of 78 degrees C has been determined for the KC1-soluble nucleosomes and for the KC1-insoluble monomer. The Tm of the KC1-insoluble oligomers is 79.8 degrees C. Multiphasic melting curves were recorded for nucleosomal material that was concentrated by lyophilisation or stored at 4 degrees C in 0.25 mM EDTA. Total nucleosome mono-, di-, tri-, and tetramers (consisting of both the fraction soluble and insoluble in 0.1 M KC1) have been analyzed concerning their sedimentation, diffusion, partial specific volume, and molecular weight and compared with the sedimentation and molecular weight data of KC1-soluble nucleosome mono- and tetramers.     

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.