Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma,Spiroplasma mirum,strain SMCA

Summary A helical mycoplasma,Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T cells. The time of inoculation ofS. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation. Editor's statement This paper describes the possible role of a mycoplasma organism, which induces cataracts and brain pathology similar to Creutzfeld Jacob and other degenerative diseases, in malignant transformation of mammalian cells. In addition to this surprising and novel finding is the observation that the mycoplasma resides in an intracellular position. These findings may have important implications for understanding malignant transformation and the nature of the diseases produced by this organism.     

The use of ciprofloxacin for the elimination of mycoplasma from naturally infected cell lines

The fluoroquinolone antibiotic Ciprofloxacin, has been used to eliminate mycoplasma from 26 naturally infected cell lines with no evidence of remergence of infection and with no treatment failures.     

Identification of Phosphocholine-Containing Glycoglycerolipids Purified from Mycoplasma fermentans-Infected Human Helper T-Cell Culture as Components of M. fermentans

Previously, we have reported the occurrence of novel phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) in human helper T-cell culture (MT-4 cell line) (Matsuda et al, Glycoconjugate J. 10: 340). However, the GGPLs disappeared from the MT-4 cells after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent-untreated MT-4 cell culture produced mycoplasma-like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated MT-4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL-I and GGPL-III. Thin-layer chromatography-mass spectrometry and immunological analyses showed that these glycoglycerolipids which were derived from the isolated M. fermentans were identical with GGPL-I and GGPL-III previously obtained. This is the first report that shows mycoplasma has phosphocholine-containing glycoglycerolipids.     

Influence of five spiroplasma strains on growth rate and survival of Galleria mellonella (lepidoptera: Pyralidae) larvae

Infection of Galleria mellonella larvae with five spiroplasma strains caused increased mortality and decreased growth rates of larvae. Reductions in growth rate and survival were related to spiroplasma strain and multiplication rate of the spiroplasma in the larvae. Three strains, considered to be ephiphytic on flowers (23-6, SR-3, PPS1), proved highly pathogenic to G. mellonella larvae, whereas strains known to be pathogenic to plants (SC-27) or honeybees (G-1) had a lesser impact.     

Microbiological cultivation ofMycoplasma hyorhinis from cell cultures

The failure of many cell culture isolates ofMycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly prepared yeast extract in mycoplasmal media together with incubation in 5% CO₂/air successfully detectedM. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures. This study was supported by grant AI 15748 from NIH. We thank Ronnie Vanaman and Judi Sarama for their technical assistance.     

Inhibition of growth of mammalian cell cultures by extracts of arginine-utilizing mycoplasmas

Summary Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas. In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas. These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas. In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth. A highly positive correlation (r=0.96,P<0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas.     

Gliding mycoplasmas are inhibited by cytochalasin B and contain a polymerizable protein fraction

Studies are presented on the effect of cytochalasin B (CB) on the growth of five Mycoplasma species, three Acholeplasma species, and one Spiroplasma species. The three gliding mycoplasma species (M) gallisepticum, M pneumoniae and M pulmonis are the only mycoplasmas inhibited by CB. These are the only prolaryotes reported to be inhibited by CB. This suggested that these three mycoplasmas might have some sort of cytoskeletal structure. A protein fraction has been isolated from M gallisepticum which polymerizes in 0.6 M KC1 and depolymerizes when KC1 is removed. This fraction contains a major 58,000-dalton protein, a 46,000-dalton protein, and a minor 87,000-dalton protein.     

Lactoperoxidase-catalyzed iodinaton of cell cultures infected with mycoplasma

Summary Hamster BHK cells or secondary cultures of mouse embryo fibroblasts are not iodinated by lactoperoxidase in the absence of hydrogen peroxide. When such cell cultures are infected with a noncultivable strain ofM. hyorhinis, endogenous peroxide generation is sufficient to permit nearly maximal iodination. The SDS-polyacrylamide gel pattern of iodinated cell surface polypeptides is essentially the same regardless of the source of peroxide and whether or not the cultures are infected with mycoplasma.     

Virus capping on mycoplasma cells and its effect on membrane structure

The capping of mycoplasmavirus L3 on the surface of Acholeplasma laidlawii was investigated. In electron microscope studies we observed a reduced capping after treatment of the host cell with energy-blocking agents. Other drugs inhibiting ligand capping on eucariotic cells had no effect. Changes in membrane structure after virus adsorption were observed spectroscopically using the excimer fluorescence technique. The results are interpreted in terms of a lipid-protein phase separation in connection with virus capping.     

Endogenous HPRT activity in mycoplasmas isolated from cell cultures

Summary Five mycoplasma species most frequently isolated from cell cultures were tested for the presence of endogenous hypoxanthine phosphoribosyl-transferase (HPRT), activity. All of the five, cultured in cell-free medium, contained variable but significant levels of HPRT. Two strains ofM. hyorhinis exhibited a 13-fold difference in their specific HPRT activity. When infected with any of these mycoplasma species, HPRT-deficient mouse cell mutants rapidly acquired a cell-associated HPRT activity; however, the cells remained sensitive to HAT medium and resistant to 6-thioguanine. On the other hand, normal HPRT-positive cells deliberately infected with the mycoplasmas uniformly became sensitive to HAT medium. The apparent transfer of mycoplasma-specific HPRT activity to HPRT-deficient cells may be used as a sensitive measure of cell infection by these mycoplasma strains. The HPRT activities of mycoplasmas share several common properties so that they can be distinguished easily from the mammalian HPRT isozymes. Compared to the animal cell enzymes, the mycoplasmal HPRT activities are less heat stable, more strongly inhibited by 6-thioguanine, and in general migrate more slowly in electrophoresis at a neutral pH. This work was supported in part by PHS Research Grants 5 R01 GM21014 and 1 P03 GM19100 (Genetics Center Grant to Albert Einstein College of Medicine), and PHS Research Contracts N01 GM 6-2119 and N01-AG-4-2865 (to the Institute for Medical Research), from the National Institute of General Medical Sciences and National Institute on Aging. S. S. is a recipient of a Faculty Research Award from the American Cancer Society.