Filamentous structures in adherent Mycoplasma pneumoniae cells treated with nonionic detergents.

Mycoplasma pneumoniae cells adhering to glass or Parlodion-coated grids were extracted with Triton X-100. The extracted cells showed a cytoskeleton consisting of a rodlike tip structure and a filamentous network in the cytoplasm. The tip structure was up to 300 nm long and approximately 40 nm wide ending at the distal end in a bleb-like structure, and seemed to consist of filaments arranged in parallel, 4.8 +/- 0.5 nm wide. In the cytoplasm the filaments formed an irregular lattice. Similar filaments were found in platinum replicated critical-point dried extracted cells. An actinlike nature of the filaments is suggested by some of their properties, but the degree of homology with respect to eucaryotic actin is still unknown. The filaments were sensitive to protease treatment but stable in high molar KCl solutions. They were apparently destroyed by incubation in high molar KI solution, leaving only some parts of the tip structure. Formaldehyde-fixed M. pneumoniae cells treated with Triton X-100 bound rhodamine-labeled phalloidin specifically. Furthermore, they could be stained with antiactin antibodies. Binding of myosin subfragment 1 to the filaments was not observed.

Hemin-dependent growth and hemin binding of Bartonella henselae.

Bartonella henselae causes cat-scratch disease and bacillary angiomatosis peliosis. The bacteria reside in erythrocytes of asymptomatic cats, which represent the natural reservoir for this pathogen. B. henselae is usually grown on blood-enriched media. Growth experiments on Brucella medium without blood demonstrated that heme compounds are essential for the growth of B. henselae and can completely substitute the addition of blood components. The heme precursor protoporphyrin IX alone, or in combination with FeCl(2) or FeCl(3), as well as transferrin or lactoferrin did not support growth, indicating that B. henselae cannot synthesize heme itself. Hemin supported growth even when free iron was chelated, indicating that hemin is also used as an iron source. Binding assays showed that hemin starvation increased the binding capacity of B. henselae for hemin, providing evidence that the bacteria carry a specific hemin uptake system, which might be regulated by hemin.

[Infectious non-gonococcal urethritis in males (author's transl)]. / Die infektiöse nicht-gonorrhoische Urethritis des Mannes.

Infectious non-gonococcal urethritis (NGU) is a sexually transmitted disease of considerable importance. The etiologic agents are bacteria, to lesser extent yeasts, parasites (Trichomonas) or viruses. A considerable part of NGU is caused by bacterial groups with peculiar properties: Mycoplasmas, which lack a rigid cell wall, and chlamydiae, which are intracellular parasites. Because of the diversity of etiologic agents and qualified microbiological diagnosis is a prerequisite for a specific and successful therapy.

Antigenic components used in vaccines and their possible role in protection.

The success of active immunization depends on the production of antibodies or immunologically active cells directed against antigens of the parasite, which are involved in pathogenesis. These could be substances mediating attachment, antiphagocytic materials, structures involved in motility, or toxins and toxic enzymes. An additional target mechanism in mycoplasmas could be the exposed membrane and its metabolic mechanisms. The results of these immune reactions appear as inhibition of attachment, as opsonization, immobilization, antitotic effects or metabolic inhibition. Often only an infection with attenuated organisms will provide sufficient protection, suggesting the necessity of more intensive immunologic stimulation or an involvement of antigens that are present only in small quantities. To minimize side effects, the optimal vaccine should contain only antigens that are known to be relevant for protection.

Phagocytosis by macrophages of mycoplasma pneumoniae after opsonization by complement.

Mycoplasma pneumoniae cells are not phagocytized by peritoneal macrophages without opsonization. They are taken up after treatment with complement or antibody.

Pathogenicity factors of mycoplasmas.

The pathogenicity of mycoplasmas is caused by several factors, e.g. exotoxin, toxic properties of membrane components, exoenzymes, peroxide, and immunological factors. The absence of a rigid cell wall and the small genome tend to influence the interactions between mycoplasmas and host tissue. Mycoplasmas do not have a cell wass and are therefore resistant to the action of the host's lysozymes. They appear in some patients to be immunologically inconspicuous and in other patients they have been reported to have an immuno-suppressive effect. Recently there have been reports of central nervous system disorders due to mycoplasma. The pathogenic factors involved in these reactions have not been elucidated. Other aspects of Mycoplasma pneumoniae pathogenicity are also discussed.

Estimation of Mycoplasma pneumoniae inoculum size by rate of tetrazolium reduction.

The rate of appearance of red-colored formazan indicating reduction of 2,3,5,-triphenyltetrazolium chloride by Mycoplasma pneumoniae was dependent on the inoculum size. The test permits estimation of M. pneumoniae cells in preparations that cannot be counted by dilution methods.

Celular morphology of newly isolated Mycoplasma hominis strains.

Cells of Mycoplasma hominis growing in laboratory media for the first time were observed by phase contrast and identified by their reaction to homologous antiserum.

[Comparison of media for the isolation of Ureaplasma urealyticum (author's transl)]. / Vergleichende Untersuchungen zum Nachweis von Ureaplasma urealyticum

Two liquid media and two agar media were compared for their sensitivity in the isolation of Ureaplasma urealyticum. 22 of the 144 urine specimens examined were positive. The U9-medium with low serum content showed a higher isolation rate and earlier results than a medium with 20% serum (Table 1). However some cultures grew sometimes better in the serum rich medium, suggesting a growth enhancing effect of urine in the U9-cultures. On agar more positive cases were detected with the A6-differential agar which showed urease-activity by brown color (MnO2), and less specimens were positive on A5C-agar without manganese sulfate (19 vs. 16). The number of colonies was only slightly lower on A5C-agar (Fig. 3). The darkbrown colonies on A6 (Fig. 1) were easy to detect and to count. Filtration of the urine specimens through a polycarbonate filter (0.4mum) reduced the number of bacterial contaminations, but resulted also in a lower isolation rate of ureaplasmas (13 of 22). This is probably caused by the tendency of ureaplasmas to attach to other structures e.g. epithelial cells (Fig. 2). For isolation of U. urealyticum from clinical specimens a combination of a liquid medium and a differential agar-medium is recommended.

Interaction of Mycoplasma pneumoniae with alveolar macrophages: viability of adherent and ingested mycoplasmas.

Guinea pig peritoneal or alveolar macrophages were inoculated with Mycoplasma pneumoniae cells. Extracellular mycoplasms were killed by complement treatment, and the effect of macrophage action on the number of the remaining viable mycoplasmas was observed. The complement killing was to some extent inhibited by the presence of the macrophages, but the mechanism of this protection remains unknown. Opsonized mycoplasmas were ingested, and approximately 98% were killed within 4 h. The killing rate was somewhat lower than comparable data for bacteria, but lack of cell wall and high lipid content of the membrane apparently do not cause a significant delay in intracellular destruction.

Analysis of polypeptides of mutants of Mycoplasma pneumoniae that lack the ability to haemadsorb.

Haemadsorption-negative mutants of Mycoplasma pneumoniae were isolated which varied in their capacity to adsorb erythrocytes of various animal species suggesting adherence to erythrocytes is mediated by different binding mechanisms. Trypsin treatment of the wild-type strain resulted in loss of haemadsorbing activity; several polypeptides, some of which regenerated with haemadsorbing activity following further incubation, were also trypsin sensitive. The haemadsorption-negative mutants could be divided into two groups according to their polypeptide pattern. In the first group (11 mutants) the PAGE pattern was identical to that of the wild-type strain. The second group comprised 7 mutants which differed from the wild-type by lack of one or more polypeptides with molecular weights of 190 000, 90 000 or 40 000. During growth attachment to glass was weak or absent in the mutants. Surface hydrophobicity as measured by hydrophobic-interaction chromatography was nearly comparable in mutants and parent strain.

Gliding motility of Mycoplasma pulmonis.

The gliding movements of freshly isolated Mycoplasma pulmonis cells were observed and measured. The motile cells had a characteristic appearance, an average speed of 0.4 to 0.7 micron/s, and a maximum speed of 1 micron/s.

Motility of Mycoplasma pneumoniae.

Cell of Mycoplasma pneumoniae FH gliding on a glass surface in liquid medium were examined by microscopic observation and quantitatively by microcinematography (30 frames per min). Comparisons were made only within the individual experiments. The cells moved in an irregular pattern with numerous narrow bends and circles. They never changed their leading end. The average speed (without pauses) was relatively constant between o.2 and 0.5 mum/s. The maximum speed was about 1.5 to 2.0 mum/s. The movements were interrupted by resting periods of different lengths and frequency. Temperature, viscosity, pH, and the presence of yeast extract in the medium influenced the motility significantly; changes in glucose, calcium ions, and serum content were less effective. The movements were affected by iodoacetate, p-mercuribenzoate, and mitomycin C at inhibitory or subinhibitory concentrations. Sodium fluoride, sodium cyanide, dinitrophenol, chloramphenicol, puromycin, cholchicin, and cytochalasin B at minimal inhibitory concentrations did not affect motility. The movements were effectively inhibited by anti-M. pneumoniae antiserum. Studies with absorbed antiserum suggested that the surface components involved in motility are heat labile. The gliding of M. pneumoniae cells required an intact energy metabolism and the proteins involved seemed to have a low turnover.

Isolation of the adherence protein of Mycoplasma pneumoniae by fractionated solubilization and size exclusion chromatography.

The 168-kDa adherence protein of M. pneumoniae was solubilized and purified to homogeneity. Optimal yield was obtained by pretreatment of whole M. pneumoniae cells with buffer containing 1% Chaps and subsequent extraction with octylglucosid at a detergent to protein ratio of 5 and at octylglycoside concentrations between 1.5 and 2%. Contaminating membrane proteins with high molecular masses were removed by pretreatment with 1% Chaps and proteins of low molecular masses by size exclusion chromatography.

Antibodies in the sera of Mycoplasma pneumoniae-infected patients against proteins of Mycoplasma genitalium and other mycoplasmas of man.

Frequency and pattern of anti-Mycoplasma genitalium antibodies in sera of 50 patients with Mycoplasma pneumoniae-infection were examined by the Western immunoblot method. The sera reacted with several proteins of M. genitalium. However, during the course of infection there was only a moderate increase of antibodies mainly against the bands of 135 and 105 kd in contrast to the more intense increase of numerous bands of M. pneumoniae. If antibodies against the 168 kd-adhesin of M. pneumoniae were isolated by affinity chromatography, only the IgG-, but not the IgM-fraction reacted with the 135 kd protein of M. genitalium. Results with rabbit antisera supported these findings, additionally indicating a lack of substantial cross reactions between M. pneumoniae and other species except M. genitalium. The development of anti M. genitalium antibodies at early age and their relatively constant pattern suggest an early immunization by so far unknown cross reacting microbial antigens. Antigenic cross reactions between proteins of M. pneumoniae and M. genitalium apparently do not play a substantial role in the diagnostic serology of M. pneumoniae disease.

Effect of monoclonal antibodies to the attachment-tip on experimental Mycoplasma pneumoniae infection of hamsters. A preliminary report.

Mycoplasma pneumoniae, an important human pathogen, is an extra-cellular parasite, colonizing mucosal surfaces. Attachment to epithelial cells of the host is therefore an important mechanism of pathogenicity, and inhibition of adhesion might protect the host. M. pneumoniae predominantly adheres with a special organelle, the attachment-tip, to host cells. In vitro studies confirmed the observation that monoclonal antibodies (MOAB) to the tip inhibited adherence to erythrocytes. In animal experiments, high numbers of virulent M. pneumoniae exposed for 4 h at 37 C to MOAB and kept in suspension with MOAB 1:100 were inoculated intranasally into hamsters. A significant reduction in the lung lesion score, but not in the numbers of organisms in lung tissue or wash fluid of the upper respiratory tract, was seen in hamsters 14 days after inoculation of MOAB-treated organisms, as compared with controls. These observations, although preliminary, may have implications for the understanding of pathogenesis and for vaccine development.

Adherence of erythrocytes to Mycoplasma pneumoniae.

The human pathogen Mycoplasma pneumoniae adheres to a variety of cells, including erythrocytes. A hemadsorption technique was developed to quantitate adherence by photometric measurement of lysates of erythrocytes that attached to sheets of M. pneumoniae grown in cups of Linbro plates. Attachment of sheep erythrocytes (SE) increased with higher ionic strength, was unaffected by minor pH variations (6 to 9), and was blocked by anti-M. pneumoniae antiserum, but was not inhibited by a variety of sugars, amino acids, and bovine serum albumin. The reaction was time and temperature dependent. The temperature curve showed peaks at 14 and 28 degrees C with untreated SE but only one peak at about 38 degrees C with glutaraldehyde-treated SE. The temperature dependence indicated involvement of either metabolic or membrane activities in the binding process. Trypsin treatment of the M. pneumoniae sheet abolished adherence of SE but was only partially effective with human erythrocytes and noneffective with rabbit erythrocytes. The binding capacity of the mycoplasma cells for SE was restored by incubation in growth medium for 3 to 4 h; this restoration was inhibited by 10 mug of chloramphenicol per ml. Neuraminidase treatment of SE removed their attachment capacity but had no effect on attachment of rabbit erythrocytes and only a slight effect on attachment of human erythrocytes. Pretreatment of M. pneumoniae with neuraminic acid partially blocked the adherence of SE, whereas rabbit erythrocyte attachment was not affected. Attached SE could be detached by trypsin, but not by neuraminidase. For human and rabbit erythrocytes, the results suggest binding mechanisms other than the interaction between neuraminidase-sensitive receptors and protein-containing binding sites shown for SE.

Adherence of Mycoplasma pneumoniae to glass surfaces.

Attachment of M. pneumoniae to glass was quantitated in an experimental system enabling the settling down of [3H]palmitic acid-labeled cells onto glass cover slips. Attachment of mycoplasmas suspended in buffer increased with temperature, decreased with higher ionic strength, and showed a maximum at about pH 5.5. The findings suggest a participation of ionic bonds in the attachment process. Trypsin did not detach glass-bound mycoplasmas, and treatment of the cells with glutaraldehyde did not reduce their attachment to glass, suggesting that membrane components other than proteins may be involved in the attachment. Low concentrations (up to 20 mg/ml) of bovine serum albumin buffer. However, during the next few hours, attachment increased far above the bovine serum albumin control. This marked increase was reduced by more than half in the presence of chloramphenicol. Increased attachment was also observed when glucose (0.1 to 2 mg/ml) was added to the bovine serum albumin-containing buffer. The findings suggest different mechanisms for the attachment in protein-free buffer and in growth medium or glucose-containing bovine serum albumin buffer, respectively. The latter apparently requires metabolic activity of the mycoplasmas.

Role of energy metabolism in Mycoplasma pneumoniae attachment to glass surfaces.

Attachment values of Mycoplasma pneumoniae to glass are normally very low when tested in buffer containing bovine serum albumin (10 mg/ml). However, the addition of one of the metabolizable sugars glucose, fructose, or mannose increased attachment more than 10-fold. The effect was dose dependent with a distinct optimum at about 0.25 mg/ml. Higher concentrations reduced this effect. Not only the sugars themselves but also the products of their catabolism, pyruvate and phosphoenolpyruvate, enhanced attachment. Pyruvate was effective in the same range of concentrations as the sugars, whereas phosphoenolpyruvate enhanced attachment at a significantly lower concentration (0.001 mg/ml). Higher levels of these substances also resulted in a decrease of attachment. The glucose-induced increase could be partially inhibited by glucose analogs, especially by 3-O-methyl-glucopyranoside, and by various inhibitors or glycolysis. Furthermore, attachment was strongly reduced by the uncoupling agents carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, as well as by dicyclohexylcarbodiimide, an inhibitor of the membrane-bound Mg2+-adenosine triphosphatase, whereas the ionophore valinomycin increased attachment by about 30%. These findings provide strong evidence for coupling between the attachment process of M. pneumoniae to glass and the utilization of metabolic energy.