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.     

Mycoplasma pneumoniae protects infected epithelial cells from hydrogen peroxide‐induced cell detachment

Epithelial cell shedding is a defence mechanism against infectious microbes that use these cells as an infection foothold and that eliminate microbes from infection foci by removing infected cells. Mycoplasma pneumoniae, a causative agent of respiratory infections, is known to adhere to and colonise the surface of ciliated airway epithelial cells; it produces a large amount of hydrogen peroxide, indicating its capability of regulating hydrogen peroxide‐induced infected cell detachment. In this study, we found that M. pneumoniae reduces exogenous hydrogen peroxide‐induced detachment of the infected cells from culture plates. This cell detachment occurred dependently of DNA damage‐initiated, poly (ADP‐ribose) polymerase 1 (PARP1)‐mediated cell death, or parthanatos. In cells infected with M. pneumoniae, exogenous hydrogen peroxide failed to induce DNA damage‐initiated poly (ADP‐ribose) (PAR) synthesis and concomitant increased cytoplasmic membrane rupture, both of which are biochemical hallmarks of parthanatos. The impairment of PAR synthesis was attributed to a reduction in the amount of cytosolic nicotinamide adenine dinucleotide (NAD), a substrate of PARP1, caused by M. pneumoniae. On the other hand, nonadherent mutant strains of M. pneumoniae showed a lower ability to reduce cell detachment than wild‐type strains, but the extent to which NAD was decreased in infected cells was comparable to that seen in the wild‐type strain. We found that NAD depletion could induce PARP1‐independent cell detachment pathways following stimulation with hydrogen peroxide and that M. pneumoniae could also regulate PARP1‐independent cell detachment in a cytoadhesion‐dependent manner. These results suggest that M. pneumoniae might regulate infected cell detachment induced by hydrogen peroxide that it produces itself, and such a mechanism may contribute to sustaining the bacterial infection.     

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas

Summary Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were:Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection withSpiroplasma mirum when this organisms was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98. 8, and 97.1%, respectively. The work was supported by grant AI-15748 from the National Institutes of Health, Bethesda, MD     

Factors in the Fermentation-Inhibition Test for Measurement of Growth-Inhibiting Antibody to Mycoplasma pneumoniae

Factors in the fermentation-inhibition test for the measurement of growth-inhibiting antibody in serum to Mycoplasma pneumoniae were studied. The fermentation-inhibiting antibody titer, as read on the day when the color of the control cup without serum had just changed to yellow, was constant among inoculum dilutions of 10^–2 to 10^–5 (10⁶ to 10³ CFU/ml) of a M. pneumoniae broth culture. The use of 10^–3 to 10^–5 dilutions (10⁵ to 10³ CFU/ml) was adequate for inoculation, inasmuch as one day delay in reading did not result in a significant decrease in the test. Heat inactivation of the serum gave no significant effect on the titer. The test was simple and reliable. The growth-inhibiting antibody was shown to be detectable in the test, when the growth of M. pneumoniae was suppressed at least to 1/100 of the growth of the control without serum. The growth-inhibiting antibody titer rose later than the complement-fixing antibody titer in some cases after M. pneumoniae infection. It is suggested that, when an erythromycin-sensitive strain of M. pneumoniae is used, the titer transiently rises and does not show a real growth-inhibiting antibody titer in sera from patients under erythromycin administration.     

Modelling persistent Mycoplasma pneumoniae infection of human airway epithelium

Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long‐term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways, however, remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air–liquid interface culture, showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread, including localisation to epithelial cell junctions. We extended those observations here, characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation, barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally, M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology, consistent with contrasting virulence in experimentally infected mice. In summary, this study reports pericellular invasion, NHBE cytoskeletal reorganisation, and tissue remodelling with persistent infection in a human airway epithelium model, providing clear insight into the likely route for extrapulmonary spread.     

Chlamydia and mycoplasma infections during pregnancy and their relationships to orofacial cleft

Serum antibodies to Mycoplasma pneumoniae and Chlamydia trachomatis have been studied in a group of newborns with orofacial cleft (OC) and their mothers (n = 59) as compared to a control group of healthy newborns and their mothers (n = 40) assayed by ELISA and Western blot analysis. In the first group, IgG antibodies to M. pneumoniae were found by ELISA in 12 newborns with OC and 22 mothers, while IgA antibodies were detected only in 5 and 11 cases, respectively. IgM antibodies indicating an acute infection were found in 2 mothers only. IgG antibodies to C. trachomatis were found in 2 newborns with OC and 4 mothers. In the control group, IgG antibodies to M. pneumoniae were found in 3 newborns and 7 mothers. IgG antibodies to C trachomatis were observed in 1 newborn and 1 mother, while IgM antibodies to C trachomatis were present in 1 mother only. Immunoblot analysis revealed in newborns with OC and their mothers C. trachomatis-specific bands associated with MOMP 1, 29 kDa, 45 kDa, and heat shock proteins (HSP) 60 and 70. Based on these results we suggest that the risk associated with the exposure to M. pneumoniae and/or C. trachomatis is so far unknown and further study is needed for its elucidation.     

Antimicrobial Activity of Inducible Human β Defensin-2 Against Mycoplasma pneumoniae

Defensins in innate immunity are known to play critical roles to protect the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between defensins and mycoplasmas. Human β defensin (hBD)-2 and hBD-3, but not hBD-1, were found to exert strikingly antimicrobial activity against Mycoplasma pneumoniae. To elucidate the role of defensins in M. pneumoniae infection, a human pulmonary squamous cell line EBC-1 was stimulated with M. pneumoniae and interleukin (IL)-1β. hBD-2 was markedly upregulated by IL-1β as well as M. pneumoniae, but neither hBD-1 nor hBD-3 was apparently upregulated. Thus, the results suggest that inducible hBD-2 would play a critical role in the protection of M. pneumoniae infection.     

Role of Humoral Antibodies in Resistance to Mycoplasma pneumoniae Pneumonia in Hamsters

Golden Syrian hamsters adoptively immunized with hyperimmune Mycoplasma pneumoniae rabbit antiserum, immunoglobulin (Ig) M-rich (IgM) fraction, IgG-rich (IgG) fraction, antiserum absorbed with either killed M. pneumoniae or killed Staphylococcus aureus organisms, or antiserum treated with 2-mercaptoethanol (2-ME) were examined for resistance against aerosol infection with virulent M. pneumoniae. Significant resistance to the establishment of infection in the respiratory tract was shown in hamsters pretreated with the untreated antiserum, IgG fraction or 2-ME-treated antiserum, whereas animals pretreated with the IgM fraction and the antisera absorbed with M. pneumoniae or S. aureus organisms were not significantly resistant. Histopathologically, lung lesions were markedly suppressed in animals with high resistance, but were typically pneumonic in animals with low or no resistance. The efficacy of adoptively administered serum preparations was closely related to their antibody titers. The results indicate that humoral antibody plays an important role in protection against experimental M. pneumoniae pneumonia in hamsters, although the participation of the cell-mediated immune response was not determined.     

Differential cytopathogenicity accompanyingMycoplasma pneumoniae infection of human lung fibroblasts maintained in newborn bovine serum or fetal bovine serum

Summary MRC-5 human lung fibroblasts maintained in Eagle's basal medium (BME) with either 10% fetal bovine serum (FBS) or 10% newborn bovine serum (NBS) did not respond identically to infection byMycoplasma pneumoniae. Fibroblasts grown in NBS did not develop any cytopathic effect (CPE) when infected withM. pneumoniae, whereas those maintained in FBS developed a pronounced CPE. There was also a difference in sensitivity to infection for fibroblasts maintained in the two sera before the infection. Fibroblasts maintained in NBS, then transferred to FBS 48 h before infection, were still less sensitive toM. pneumoniae infection than cells maintained constantly in FBS.Mycoplasma pneumoniae attached comparably to the fibroblasts grown in the two sera, so the differences in CPE development could not be attributed to differences in mycoplasma attachment. Measurements of DNA, RNA, and protein syntheses of the fibroblasts grown in NBS and FBS indicate that the cells in NBS were growing more rapidly than those in FBS. A determination of the doubling times shows that the doubling time of cells in NBS was 44 h, whereas that of cells in FBS was 51 h. Polyacrylamide gel electrophoresis of samples of NBS and FBS showed significant differences in serum protein composition. The NBS had several protein bands that were lacking in the FBS. This study demonstrates the importance of serum effects in the study ofM. pneumoniae infection. This work was supported in part by Public Health Service Grant AI 17795 from the National Institute of Allergy and Infectious Diseases, Bethesda, MD.     

Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins

Eleven hitherto unknown Mycoplasma pneumoniae proteins were identified and characterized with respect to their size and subcellular location. This was carried out through the construction of in vitro gene fusions between a modified mouse dehydrofolate reductase (dhfr) gene and selected regions (cosmid clones) of the M. pneumoniae genome and expressing them in Escherichia coli. Positive clones were identified using antibodies against specific fractions of M. pneumoniae. The deduced protein sequences of 11 out of 30 clones did not show significant homologies to known proteins in protein databank searches. Monospecific antibodies against these 11 fusion proteins were used to determine the size and cellular location of the corresponding M. pneumoniae proteins by immunoscreening Western blots of SDS-acrylamide gels from M. pneumoniae cell extracts.     

Metabolism inhibition as a result of interaction of antibody with a membrane protein ofMycoplasma bovoculi

The metabolism inhibition reaction is a test commonly used for the speciation of mycoplasmas. Cattle infected withMycoplasma bovoculi produce serum antibodies that are active in metabolism inhibition. For characterization of this reaction, affinity matrixes were prepared from lysates ofM. bovoculi bound to a monoclonal antibody that recognizes a 94,000 kDa membrane protein (p94) of the mycoplasma. These matrixes containing purified p94 were used to specifically remove metabolism-inhibiting activity from serum samples of cattle infected withM. bovoculi. The results provide evidence that p94 is the major and perhaps only target for the action of complement-independent, metabolism-inhibiting antibodies. This antibody reactivity may inhibit a vital membrane function associated with the p94 protein.     

Monoclonal antibodies against Mycoplasma hyorhinis: A secondary effect of immunization with cultured cells

Monoclonal antibodies were prepared against two different human tumour cell lines, the melanoma cell line SK-Mel-25 and the acute lymphoblastic leukemia T cell line CCRF-CEM. Presence of antibodies against human tumour cells in the supernatants of hybridoma cultures was tested by binding of ¹²⁵I-F(ab′)₂ anti-mouse IgG. On two occasions a hybridoma culture, initially selected for subsequent cloning as it seemingly produced antibodies against tumour cells, was later found to produce monoclonal antibodies specific for Mycoplasma hyorhinis. In immunofluorescent staining patchy structures were visible which seemed to be attached to the cell surface. By combined staining with FITC-conjugated anti-mouse immunoglobulin for monoclonal antibody, Evans blue for cytoplasm and Hoechst compound no. 33258 for DNA, the reaction against mycoplasma could be recognized. These results demonstrate that if cultured cells are used for preparation of monoclonal antibodies, there is a good chance that the selected hybridomas may produce antibodies against ‘culture artifacts’ such as mycoplasmas, in addition to the target antigens. Thus mycoplasma contamination of cell cultures poses a serious problem in the hybridoma research and the testing system for antibody specificity should be carefully monitored.     

Monoclonal antibodies to Mycoplasma hyorhinis surface antigens: tools for analyzing mycoplasma-lymphoid cell interactions

A library has been constructed of approximately 50 monoclonal antibodies that recognize antigens of Mycoplasma hyorhinis. Characteristics of six antibodies are discussed. Each reacts with a discrete determinant borne on a protein-containing molecule of distinct molecular size. Three of these respective antigens are expressed at the surface of mycoplasmas colonizing lymphoblastoid cells in culture. Of these three surface antigens, two are selectively expressed on strain GDL but not on strain BTS-7 of M. hyorhinis, thus defining strain-restricted immunological specificities within these species. One monoclonal antibody of the IgM class (mu, kappa) mediated marked complement-dependent growth inhibition of M. hyorhinis in broth culture. These monoclonal reagents should facilitate analysis of mycoplasma surface architecture, and the molecular interactions of these organisms with the host cell surface.     

Mycoplasma pneumoniae infection of intact guinea pig tracheas cultured in a unique matrix-embed/perfusion system

Summary A new method for the in vitro culture of entire, intact tracheas from adult guinea pigs is described. Matrix-embed/perfusion (MEP) culture is based on an immobilization of the tissue in nutrient agar. The tubular piece of agar-embedded organ was contained in a special perfusion block with two wells for liquid medium at either end. When incubated on a rocker platform, liquid medium flows through the trachea and supplies oxygen and nutrients. In this configuration, tracheas maintain near-normal metabolism (ATP content and dehydrogenase activity), structure (as determined by light and electron microscopy), and function (ciliary motion). Tissues could be maintained in vitro in a normal state for at least 4 wk, with reduced ciliary motion and cell metabolism detectable for at least 6 wk. Agarembedded tissues from the MEP cultures were nearly identical to those cultivated with standard tracheal ring explant techniques. Tracheas in the MEP cultures were infected withMycoplasma pneumoniae. Attachment was neuraminidase-sensitive. Mycoplasma attachment was lowest on the epithelium along the dorsal ridge, but was uniform along the length of the trachea. Ciliostasis and cytonecrosis induced byM. pneumoniae was dose dependent. The matrix-embed/perfuse technique appears to have considerable potential for several types of in vitro studies on trachea or other tubular organs. This study was supported in part by USPHS Grants AI 12559 and AI 17795 from the National Institutes of Allergy and Infectious Diseases, and Grants HL 23806 and HL 26880 from the National Heart, Lung, and Blood Institute.     

Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA

Background  

Serology is often used for the diagnosis of Mycoplasma pneumoniae. It is important to identify specific antigens that can distinguish between the presence or absence of antibodies against M. pneumoniae. The two proteins, P116 and P1, are found to be immunogenic. By using these in ELISA it is possible to identify an immune response against M. pneumoniae in serum samples.     

Cryptic I antigen activity andMycoplasma pneumoniae-receptor activity associated with sialoglycoprotein GP-2 of bovine erythrocyte membranes

The 250-kDa sialoglycoprotein of bovine erythrocyte membranes, GP-2, has been found to be an exception-ally rich source of branched sialo-oligosaccharides of poly-N-acetyllactosamine (I antigen) type with receptor activity for the human pathogenMycoplasma pneumoniae.Desialylated GP-2 is the most potent I-active substance thus far tested. Since this gtyco-protein is hydrophobic and can be readily re-incorporated into cell membranes, it should be useful in future studies of the mechanism of production of auto-antibodies to the I antigen which commonly arise following human infection withM. pneumoniae.     

Studies on Delayed Hypersensitivity of Antigens Isolated from Mycoplasma pneumoniae Cells

Cells of Mycoplasma pneumoniae Mac strain were fractionated into acetone-soluble and insoluble fractions. Acetone-insoluble fractions were digested with pronase and further purified by chromatography on Sephadex G-75, yielding three water-soluble fractions which were free from lipid and consisted mainly of polysaccharide-protein complex. All these water-soluble fractions possessed eliciting antigenicity to delayed hypersensitivity for M. pneumoniae as measured by skin reactions and macrophage migration inhibition tests, but not to complement-fixing antibodies. In contrast, the acetone-soluble fraction was reactive with the complement-fixing antibodies but not for the delayed hypersensitivity.     

Mpn491, a secreted nuclease of Mycoplasma pneumoniae,plays a critical role in evading killing by neutrophil extracellular traps

Neutrophils play an important role in antimicrobial defense as the first line of innate immune system. Recently, the release of neutrophil extracellular traps (NETs) has been identified as a killing mechanism of neutrophils against invading microbes. Mycoplasma pneumoniae, a causative agent of respiratory infection, has been shown to be resistant to in vitro killing by neutrophils, suggesting that the bacterium might circumvent bactericidal activity of NETs. In this study, we investigated whether M. pneumoniae possesses resistance mechanisms against the NETs‐mediated killing of neutrophils and found that the bacterium degrades the NETs induced upon M. pneumoniae infection. The NETs‐degrading ability of M. pneumoniae required the production of a secreted nuclease, Mpn491, capable of using Mg²⁺ as a cofactor for its hydrolytic activity. Moreover, the inactivation of the nuclease resulted in increased susceptibility of M. pneumoniae to the NETs‐mediated killing of neutrophils. The results suggest that M. pneumoniae employs Mpn491 as a means for evading the killing mechanism of neutrophils.     

Galactocerebroside biosynthesis pathways of Mycoplasma species: an antigen triggering Guillain–Barré–Stohl syndrome

Infection by Mycoplasma pneumoniae has been identified as a preceding factor of Guillain–Barré–Stohl syndrome. The Guillain–Barré–Stohl syndrome is triggered by an immune reaction against the major glycolipids and it has been postulated that M. pneumoniae infection triggers this syndrome due to bacterial production of galactocerebroside. Here, we present an extensive comparison of 224 genome sequences from 104 Mycoplasma species to characterize the genetic determinants of galactocerebroside biosynthesis. Hidden Markov models were used to analyse glycosil transferases, leading to identification of a functional protein domain, termed M2000535 that appears in about a third of the studied genomes. This domain appears to be associated with a potential UDP-glucose epimerase, which converts UDP-glucose into UDP-galactose, a main substrate for the biosynthesis of galactocerebroside. These findings clarify the pathogenic mechanisms underlining the triggering of Guillain–Barré–Stohl syndrome by M. pneumoniae infections.