Contrasting effects of Mycoplasma fermentans and M. felis on the viability and chemiluminescence response of human polymorphonuclear leukocytes

Abstract Trypan blue exclusion was used to estimate the viability of human polymorphonuclear leukocytes (PMNL) in the presence of Mycoplasma felis and two strains of M. fermentans (PG18 and incognitus). The competence of PMNL to mount a respiratory burst when challenged with the mycoplasmas was also monitored by luminol-dependent chemiluminescence (CL). Both un-opsonised and non-immune human serum opsonised M. felis cells had little effect on PMNL viability. In contrast, PMNL viability was reduced markedly by un-opsonised cells of M. fermentans strain incognitus and, to a lesser extent, strain PG18, and opsonisation of these mycoplasmas further enhanced killing. Death of PMNL in the presence of M. fermentans was not associated with the autonomous production of active oxygen species during the respiratory burst as M. felis induced a high CL response from PMNL, whereas that induced by M. fermentans strain incognitus was significantly lower. M. fermentans may invade mammalian cells and it is suggested that the mechanism of PMNL death could be related to the ability of M. fermentans to penetrate host cell membranes.     

Relation between Mycoplasmas and Leukaemia and Related Diseases

Forty-five bone marrow specimens from leukaemia patients and 40 marrows from nonleukaemia patients were cultured for mycoplasmas. No mycoplasmas were isolated. Sera from patients with leukaemia or reticuloses and from non-leukaemic subjects were examined for antibodies to the Negroni and 880 strains of Mycoplasma pulmonis and also to the K7, K10, and prototype (PG18) strains of M. fermentans. No significant differences were observed between the two groups of patients with respect to antibodies to these mycoplasmas. These findings support those of the majority of other workers in failing to show any relationship between mycoplasmas and neoplastic disease in man.     

Elimination of mycoplasmas from cell cultures utilizing hyperimmune sera

Eighteen cell lines contaminated with various mycoplasmas have been treated with hyperimmune sera and mycoplasmas have been eradicated from all. After treatment the cell lines have been observed for a least one year and they are still free from mycoplasma contamination as ascertained by four independent mycoplasma detection assays. The hyperimmune sera used were of high titer, type-specific and growth-inhibiting. These sera were produced by immunization of rabbits with purified membranes from Mycoplasma orale, M. arginini, M. hominis, M. fermentans, M. hyorhinis and Acholeplasma laidlawii. In addition to elimination of mycoplasmas from cell cultures we have successfully used these sera for detection and typing of mycoplasma contamination in cell cultures.     

Mycoplasma hyorhinis and Mycoplasma fermentans induce cell apoptosis and changes in gene expression profiles of 32D cells

Infection of mycoplasmas has been linked to various human diseases including arthritis, pneumonia, infertility and cancer. While Mycoplasma hyorhinis and Mycoplasma fermentans have been detected in gastric adenocarcinomas, the mechanisms underlyine the pathogenesis are unknown. In this study, cell growth kinetics, Hoechst 33258 staining, DNA ladder assays, Western blotting analysis and cDNA microarray assays were performed to investigate the roles of M. hyorhinis and M. fermentans during infection of mammalian cells. Our data demonstrated that these mycoplasmas inhibid the growth of immortalised cell lines (32D and COS-7) ane tumor cell lines (HeLa and AGS). In addition, the infection of the 32D cell line with M. hyorhinis and M. fermentans induced compression of the nucleus, degradation of the cell genome and dysregulation of the expression of genes related to proliferation, apoptosis, tumorigenesis, signaling pathway and metabolism. Apoptosis related proteins Bcl-2, Bid and p53 were down-regulated, Fas was up-regulated and Bax was dysregulated in mycoplasma-infected 32D cells. Together, our data demonstrated that infection of mycoplasmas inhibitd cele growts through modification of gene expression profiles and post-translation modification of proliferation and apoptosis related proteins.     

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.     

Characterization of DNA topoisomerase activity in two strains of Mycoplasma fermentans and in Mycoplasma pirum.

DNA topoisomerases (topos) are essential enzymes that participate in many cellular processes involving DNA. The presence of the DNA-gyrase genes in various mycoplasmas has been reported elsewhere. However, the characterization of DNA topo activity in mycoplasmas has not been previously undertaken. In this study, we characterized the topo activity in extracts of Mycoplasma fermentans K7 and incognitus and in Mycoplasma pirum, as well as in partially purified extract of M. fermentans K7. The topo activity in these microorganisms had the following properties. (i) The relaxation of supercoiled DNA was ATP dependent. (ii) ATP independent relaxation activity was not detected. (iii) Supercoiling of relaxed topoisomers was not observed. (iv) The relaxation activity was inhibited by DNA gyrase and topo IV antagonists (novobiocin and oxolinic acid) and by eukaryotic topo II (m-AMSA [4'-(9-acridylamino)methanesulfon-m-anisidide]) and topo I antagonists (camptothecin). Other eukaryotic topo II antagonists (teniposide and etoposide) did not affect the topo relaxation activity. (v) Two polypeptides of 66 and 180 kDa were found to be associated with the mycoplasma topo activity. These results suggest that the properties of the topo enzyme in these mycoplasma species resemble those of the bacterial topo IV and the eukaryotic and the bacteriophage T4 topo II. The findings that mycoplasma topo is inhibited by both eukaryotic topo II and topo I antagonists and that m-AMSA and camptothecin inhibited the growth of M. fermentans K7 in culture support our conclusion that these mycoplasma species have topo with unique properties.     

Increased sister chromatid exchange in human lymphocyte cultures infected with mycoplasma

The effects of fermenting, poorly arginine-utilizing Mycoplasma fermentans and arginine-utilizing Mycoplasma salivarium on the frequency of sister chromatid exchange (SCE) in cultured human lymphocytes were examined. M. fermentans caused no apparent mitosis inhibition of lymphocytes and the increase in SCE frequency was dependent on the inoculum size of the mycoplasma. An evident increase in SCE frequency was observed in lymphocytes infected with smaller inoculum sizes of M. salivarium whereas there was mitosis inhibition of lymphocytes infected with larger inoculum sizes of the mycoplasma. In lymphocyte cultures infected with M. salivarium, the addition of arginine to the culture medium reduced mitosis inhibition but did not diminish the increase in SCE frequency, indicating that arginine depletion was not involved in causing the induction of SCEs in mycoplasma-infected lymphocytes. With regard to the genetic effectiveness of SCE, these results suggested that mycoplasmas are capable of inducing cytogenetic changes in infected host cells.     

The reducing antioxidant capacity of Mycoplasma fermentans

Mycoplasma fermentans is an extracellular microorganism capable of adhering to the surface of host cells. It has been recently shown that plasminogen binding to M. fermentans in the presence of the urokinase-type plasminogen activator promotes the invasion of host cells by this organism. In this report, we show that viable mycoplasmas persist within the infected HeLa cells for prolonged periods of time despite the expectation that within host cells the organism may be exposed to oxidative stress. Using cyclic voltammetry and luminol-enhanced chemiluminescence assays, we detected a potent reducing antioxidant activity in M. fermentans. The reducing antioxidant activity was heat stable, not affected by proteolysis and was almost totally lost upon dialysis suggesting that the activity is due to a nonproteinaceus low molecular weight antioxidant. This antioxidant was partially purified by Bio-Gel column chromatography followed by high-pressure liquid chromatographic analysis. We suggest that the high reducing antioxidant capacity in M. fermentans is a principal defense mechanism playing a major role in the battle of the organism against oxidative stress within the host cells.     

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell’s interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.     

Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes

A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a K(m) of 2.2x10(-5) M and a V(max) of 0.15 nmol 10 min(-1) mg(-1) cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium.     

Ether lipids in the cell membrane of Mycoplasma fermentans.

Two new ether lipids, 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine and its lyso form, 1-O-alkyl/alkenyl-glycero-3-phosphocholine, were identified in the cell membrane of Mycoplasma fermentans using chemical analyses, GLC-MS, MALDI-TOF MS, and 1D and 2D NMR spectroscopy. The lipids are heterogeneous with respect to both acyl and alkyl/alkenyl residues. The acyl residues at position 2 of glycerol are hexadecanoyl and octadecanoyl in a molar ratio of 3.6 : 1 with a trace amount of octadecenoyl. The alkyl/alkenyl residues at position 1 of glycerol are hexadecyl (78%), octadecyl (7%), octadecenyl (14%), and hexadecenyl (traces). In the octadecenyl residue, the double bond has a cis configuration and is located at either position 1' (plasmalogen-type lipid) or 9' in a ratio approximately 1 : 1. This is the first report of the presence of alkyl and vinyl (alk-1'-enyl) ether lipids in the cell membrane of aerobically grown mycoplasmas. Lipids of this type have been found in some Gram-positive bacteria, thus supporting the hypothesized close taxonomical relationship of these bacteria to mycoplasmas. The ether lipids of M. fermentans are structurally similar to platelet activating factor; it was demonstrated that the 2-O-acetylated lyso form lipid can mimic platelet-activating factor activity in isolated perfused and ventilated rat lungs.     

Occurrence and properties of lactic dehydrogenases of fermentative mycoplasmas

Eight fermentative mycoplasmas differing in genome size, deoxyribonucleic acid (DNA) base composition, or sterol dependence were examined for lactic dehydrogenase composition by spectrophotometric assay and polyacrylamide gel electrophoresis. Three completely different patterns of lactic dehydrogenase composition were found. (i) A nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactic dehydrogenase was found in Mycoplasma pneumoniae, M. gallisepticum, M. mycoides var. mycoides, mycoplasma UM 30847, M. neurolyticum, and Acholeplasma axanthum. Electrophoresis of cell-free extracts of each of these mycoplasmas produced, with the exception of M. mycoides var. mycoides and UM 30847, single, different enzyme bands. M. mycoides var. mycoides and UM 30847 were similar and formed multiple bands of enzyme activity. We were unable to establish whether these multiple bands were due to lactic dehydrogenase isoenzymes or artifacts. (ii) An NAD-dependent d(-)-lactic dehydrogenase which could not be reversed to oxidize lactate was found in M. fermentans. (iii) A. laidlawii A possessed an NAD-independent d(-)-lactic dehydrogenase capable of reducing dichlorophenol-indophenol, and an NAD-dependent l(+)-lactic dehydrogenase which is specifically activated by fructose-1,6-diphosphate. Heretofore, this enzyme regulatory mechanism was known to occur only among the Lactobacillaceae. No yeast-type lactic dehydrogenase activity was found in any of the mycoplasmas examined. The stereoisomer of lactic acid accumulated during growth correlated perfectly with the type of NAD-dependent lactic dehydrogenase found in each mycoplasma. The types of lactic dehydrogenase activity found in these mycoplasmas were not related to genome size, DNA base composition, or sterol dependence.     

The Mycoplasma fermentans prophage phiMFV1: genome organization, mobility and variable expression of an encoded surface protein

The approximately 16 kb genome of the Mycoplasma fermentans phiMFV1 prophage is described, and its mobility, replication and effect on the mycoplasma surface phenotype are demonstrated. In various M. fermentans strains, phiMFV1 was either absent or integrated at diverse (and sometimes multiple) chromosomal sites, each marked by a conserved TTTTTA target sequence that is duplicated upon integration. Precise excision, replication of an extrachromosomal form and loss of phiMFV1 from the mycoplasmal genome were documented in a series of clonal derivatives of M. fermentans propagated in culture. Of 18 open reading frames (ORFs) encoded by phiMFV1, most can be ascribed functions related to phage biology, whereas one encodes a unique coiled-coil membrane surface protein, Mem, that was confirmed to be expressed in propagating populations of M. fermentans. With the exception of Mem and other minor ORFs, the striking similarity between the deduced proteomes of phiMFV1 and the recently described phiMAV1 of arthritogenic strains of Mycoplasma arthritidis, along with the prominent gene synteny between these elements, provides the taxonomic basis for a new family of prophage. Their coding features are consistent with long-term residence in mycoplasma genomes and the divergence of species within a phylogenetic clade of mycoplasmas. The unique Mem protein expressed from phiMFV1 and the unique hypothetical surface lipoproteins encoded by phiMAV1 and phiMFV1 also suggest that prophage-associated genes may provide specific, selectable phenotypic traits during co-evolution of mycoplasma species with their respective mammalian hosts. Retention of these labile prophage elements in organisms with such drastically reduced genome sizes implies a significant role in adaptation and survival.     

Detection of mycoplasma contaminations by the polymerase chain reaction

The polymerase chain reaction (PCR) has been used for the general detection ofMollicutes. 25Mycoplasma andAcholeplasma species were detected including important contaminants of cell cultures such asM. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1–2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.     

Cell death in trichomonads: new insights

Tritrichomonas foetus is an amitochondriate parasite that possesses hydrogenosomes, unusual anerobic energy-producing organelles. In these organisms the “mitochondrial cell death machinery” is supposed to be absent, and the mechanisms that lead to cell demise remain to be elucidated. The presence of a cell death program in trichomonads has already been reported, suggesting the existence of a caspase-like execution pathway in such organisms. Here we demonstrate the alterations provoked by the fungicide griseofulvin and raise the possibility that other cell death pathways may exist in T. foetus. Dramatic changes in trichomonads morphology are presented after griseofulvin treatment, such as intense plasma membrane and nuclear envelope blebbing, nucleus fragmentation, and an abnormal number of oversized vacuoles. One important finding was the exposition of phosphatidylserine (PS) in the outer leaflet of the plasma membrane in cells after drug treatment, and also the presence of a high amount of misshapen flagella and tubulin precipitates as vacuolar contents, suggesting an autophagic process of abnormal cellular elements. Interestingly, immunoreactivity for activated caspase-3 was not detected during griseofulvin treatment, a finding distinct from the observed when this cell was treated with H₂O₂. The possibility of the existence of different pathways to cell death in trichomonads is discussed.     

Mycoplasmal infections prevent apoptosis and induce malignant transformation of interleukin-3-dependent 32D hematopoietic cells

32D cells, a murine myeloid cell line, rapidly undergo apoptosis upon withdrawal of interleukin-3 (IL-3) supplement in culture. We found that 32D cells, if infected by several species of human mycoplasmas that rapidly activated NF-kappaB, would live and continue to grow in IL-3-depleted culture. Mycoplasma-infected cells showed no evidence of autocrine production of IL-3. Pyrrolidine dithiocarbamate (PDTC) blocked activation of NF-kappaB and led to prominent cell death. Heat-killed mycoplasmas or mycoplasmal membrane preparations alone could support continued growth of 32D cells in culture without IL-3 supplement for a substantial period of time. However, upon removal of heat-inactivated mycoplasmas, 32D cells quickly became apoptotic. In comparison, live Mycoplasma fermentans or M. penetrans infection for 4 to 5 weeks induced malignant transformation of 32D cells. Transformed 32D cells grew autonomously and no longer required support of growth-stimulating factors including IL-3 and mycoplasmas. The transformed 32D cells quickly formed tumors when injected into nude mice. Karyotyping showed that development of chromosomal changes and trisomy 19 was often associated with malignant transformation and tumorigenicity of 32D cells. Mycoplasmal infections apparently affected the fidelity of genomic transmission in cell division as well as checkpoints coordinating the progression of cell cycle events.     

Membrane-associated hemolysin activities in mycoplasmas

Abstract Mycoplasmas are cell wall-less organisms that require membrane precursors for growth. Activities involved in the acquisition of these materials have been hypothesized as mycoplasmal virulence factors because of the effects these activities might have on host cells. Twenty-nine species or strains of mycoplasmas were examined for membrane-associated hemolysis activity similar to that previously identified in Mycoplasma pulmonis . Membrane-associated hemolytic activity was found in most mycoplasma species, but the amount of activity varied between and within the species. All of the arginine-utilizing mycoplasmal species, one M. pulmonis strain, one Acholeplasma species, and the intracellular human pathogens M. penetrans and M. fermentans ssp. incognitus were devoid of activity. The wide distribution of the membrane-associated hemolysis activity suggests that it may be important to the survival of the organism.     

Evidence that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen.

It was demonstrated that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen with a molecular weight ranging from 42,000 to 48,000. Western blotting (immunoblotting) with an antibody specific to a 43-kDa membrane protein of Mycoplasma fermentans showed the existence of this protein antigen in all Mycoplasma spp. tested (14 species), Acholeplasma laidlawii (1 strain), and gram-negative bacteria (8 species) but only in Staphylococcus aureus of four gram-positive species tested. Neither Ureaplasma urealyticum nor mammalian cell cultures showed any cross-reactions with this antibody. These proteins were found in both cytoplasmic and membrane fractions of mycoplasma cells but were not exposed on the surface of mycoplasmal or bacterial cells.