Induction of interleukins by mycoplasma in culture of human mononuclear leukocytes

Mycoplasma shows a variety of effects on immune system, including the activation of macrophage, the increase in T cell cytotoxicity, and the enhancement of the proliferation and maturation of B cells, etc. As it is well known that many cytokines regulate the immune system, it is interesting to examine whether or not human peripheral blood mononuclear cells (PBMC) produce interleukin (IL) in response to mycoplasmas. In the present study, human PMBC were incubated with 7 species of mycoplasmas for 48 hours, and IL-1 beta, IL-2 and IL-6 activities in the supernatants were determined by ELISA. All the species of mycoplasmas were able to induce IL-1 beta and IL-6, although IL-2 was induced only by M. pneumoniae. These results suggest that the influence of mycoplasma infection on immune system may be partly due to the interleukins induced by mycoplasmas.     

Effects of mycoplasma infection on the host organism response via p53/NF-κB signaling

Mycoplasmas are bacteria lacking the cell wall, which is the major characteristic of this taxonomic class (Mollicutes). Among bacteria, mycoplasmas possess the smallest genome known for free-living organisms. This feature limits the autonomy of bacteria and makes them increasingly susceptible to changes in the host organism. Many mycoplasmas themselves cause pathological changes in the host organism, often complicated by immune disorders. Infection with certain strains of mycoplasma results in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells, which is the major mediator of the inflammatory response. Furthermore, mycoplasmas can inhibit p53-mediated checkpoint control of cell cycle and apoptosis. Collectively, these properties indicate that mycoplasmas might act as cancer-promoting factors. In this review, we summarize the information known to date on the role of mycoplasmas in the regulation of the host immune response and their functional interactions with p53.     

Interactions between mycoplasma lipoproteins and the host immune system

Mycoplasmas typically have a number of distinct lipoproteins anchored on the outer face of the plasma membrane. These surface antigens have a potent modulin activity and are preferential targets of the host immune response. However, the variation of some of these lipoproteins provides mycoplasmas with an effective means of evading the host immune defence system.     

Mechanisms of Genetic Instability in Mollicutes (Mycoplasmas)

Mollicutes are unique microorganisms characterized by a great extent for the reduction in genetic material, which retained the capability of independent division on acellular nutrient media. Phenotypically mycoplasmas differed from other bacteria by their small size and lack of a cell wall (mollis, soft; cutis, skin). High dependence on metabolism components utilized in the cultivation medium and high metabolic plasticity due to the absence of many genome regulatory elements make mycoplasmas perfect parasites for cells of the eukaryotic origin. The ability of these microorganisms to pass through host cells and their assumed participation in AIDS activation facilitate the study of mycoplasma pathogenesis. Another important feature of mycoplasmas, which is expressed during their interaction with a macroorganism, is their ability to escape from the immune response of a host due to surface antigen variation. These adaptation capacities of mycoplasmas ensuring their life in various biological niches, given a limited genome and the direct metabolic dependence on an environment, cannot be adequately explained at present. In this review, we attempted to collect and systematize data that contribute to our understanding of the important feature of mycoplasmas, genetic instability, which may underlie many of their adaptive responses.     

Adherence of Pathogenic Mycoplasmas to Host Cells

The significant genome compaction in mycoplasmas was made possible by adoption of a parasitic lifestyle. During their evolution and adaptation to a parasitic mode of life the mycoplasmas have developed various genetic systems enabling their attachment to host tissues as well as a highly plastic set of variable surface proteins. The generation of a versatile surface coat through high-frequency phase and size variation provides the organism with a useful tool for immune system avoidance, allowing the mycoplasmas to escape antibody attack, explaining why these minute organisms are such successful parasites.     

Interaction of Acholeplasma laidlawii and Mycoplasma arthritidis with the lymphocytes of mice of different strains

The interaction of mycoplasmas and mouse lymphocytes has been studied by the microbiological and electron-microscopic methods. The experiments have shown that A. laidlawii and M. arthritidis are adsorbed on lymphocytes and thymocytes of (C57BL6 X A/He)F1, BALB and C57BL mice after 15 minutes of their joint incubation at 37 degrees C, 1 hour later adsorption reaches its maximum intensity and after further prolongation of the time of incubation the number of adsorbed microbial cells remains unchanged. The first stage of the interaction of mycoplasmas with splenic and thymic lymphocytes (adsorption) is the same in (C57BL6 X X A/He)F1, BALB and C57BL mice, and differences in the persistence of mycoplasmas in mice of the above strains are probably due not to different capacity of the cells for adsorbing mycoplasmas, but to differences in the immune status of these animals.     

Mechanisms of genetic instability of mollicutes (mycoplasma)

Mollicutes are unique microorganisms characterized by a great extent for the reduction in genetic material, which retained the capability of independent division on acellular nutrient media. Phenotypically mycoplasmas differed from other bacteria by their small size and lack of a cell wall (mollis, soft; cutis, skin). High dependence on metabolism components utilized in the cultivation medium and high metabolic plasticity due to the absence of many genome regulatory elements make mycoplasmas perfect parasites for cells of the eukaryotic origin. The ability of these microorganisms to pass through host cells and their assumed participation in AIDS activation facilitate the study of mycoplasma pathogenesis. Another important feature of mycoplasmas, which is expressed during their interaction with a macroorganism, is their ability to escape from the immune response of a host due to surface antigen variation. These adaptation capacities of mycoplasmas ensuring their life in various biological niches, given a limited genome and the direct metabolic dependence on an environment, cannot be adequately explained at present. In this review, we attempted to collect and systematize data that contribute to our understanding of the important feature of mycoplasmas, genetic instability, which may underlie many of their adaptive responses.     

Peculiar properties of mycoplasmas: The smallest self-replicating prokaryotes

Mycoplasmas are the smallest and simplest prokaryotes capable of self-replication, with information provided by a genome which may be as small as 600 kb, estimated to carry less than 500 genes. Keeping the number of structural elements, metabolic pathways and components of the protein synthesizing machinery to an essential minimum places mycoplasmas closest to the concept of 'minimum cells'. Mycoplasmas are, therefore, most adequate candidates for the complete deciphering of the machinery of a self-replicating organism, and studies towards this goal are already underway. Living as 'minimum cells' was made possible by adopting a parasitic mode of life, securing from the host the many nutrients which cannot be synthesized by the mycoplasmas themselves. When pathogenic, infections by mycoplasmas usually follow a chronic course, with host immune reactions playing an important role in symptom production. Recent studies on the possible association of mycoplasmas with rheumatoid arthritis and AIDS are reviewed.     

Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells

Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.     

Role of complement in immune inactivation of Mycoplasma gallisepticum

Studies were undertaken to evaluate the role of complement in the interaction between mycoplasmas and antiserum. A suspension of the A-1 strain of Mycoplasma gallisepticum in PPLO broth was incubated at 37 C with rabbit immune serum which had been heated for 30 min at 56 C. Samples were removed from the mixture at timed intervals for 1 hr for titration of the mycoplasmas in broth. When normal guinea pig serum was included in the mixture at a final dilution of 1:40, the titer fell rapidly from 10(6) to 10(2) organisms per 0.2 ml. When the guinea pig serum was heated for 30 min at 56 C or was omitted from the mixture, the immune serum did not reduce the titer. The rate of inactivation was related to the final concentration of antiserum and to the incubation temperature. The effect of the guinea pig serum was eliminated by the addition of 0.01 m sodium ethylenediaminetetraacetate or by prior absorption with an unrelated antigen-antibody complex. It was concluded that complement-like substances play an important role in immune inactivation of M. gallisepticum.     

Effect of mycoplasma infection on pyruvate dehydrogenase complex activity of normal and pyruvate dehydrogenase complex-deficient fibroblasts

The fermentative mycoplasmas A. laidlawii JS, M. hyorhinis DBS-50, M. hyorhinis GDL and M. pneumoniae FH have very high apparent activities of pyruvate dehydrogenase (PDH) (EC 1.2.4.1) and pyruvate dehydrogenase complex (PDHC). Infection of normal and PDHC-deficient fibroblasts with these mycoplasma species resulted in a marked increase of the specific activity of these two enzymes, and under certain conditions could conceal the enzymatic defect. The non-fermentative mycoplasmas M. salivarium VV and M. arthritidis PG-6 have very low apparent activities of these two enzymes. Normal fibroblasts infected with non-fermentative mycoplasmas could appear as deficient in these two enzymes. The degree of interference depends on the number of mycoplasmas associated with the harvested cells. Besides the mycoplasma species, this depends (1) on the duration of infection which determines mycoplasmal titers and also can have a killing effect on both host cells and/or mycoplasmas; (2) harvest of the cells by scraping or trypsinization; (3) centrifugal force used in the collection of the cells; (4) washing and the inherent mechanical treatment; and (5) other possibilities.     

Induction of antitumor activity in macrophages by mycoplasmas in concert with interferon

Summary The in vitro growth of tumor cells infected with mycoplasmas was suppressed by macrophages pretreated with interferon (IFN), but the growth of mycoplasma-free tumor cells was not suppressed. Pretreatment of macrophages with IFN plus mycoplasmas or their soluble factors either simultaneously or sequentially, IFN first and mycoplasmas second, but not in the reverse order, was effective in activating macrophages to suppress the growth of mycoplasma-free tumor cells. Macrophages from C3H/HeJ mice (which respond only slightly to lipopolysaccharide) were activated by IFN plus mycoplasmas or their soluble factor, and their action was not influenced by the addition of a lipopolysaccharide-neutralizing agent, polymyxin B. These results suggest that the macrophage-activating agent in mycoplasmas does not mimic lipopolysaccharide. The administration of mycoplasmas plus IFN to mice with ascitic or solid tumors resulted in the reduction of tumor growth. The survival rate of tumor-bearing mice was improved by the administration of mycoplasmas, and this was synergistically enhanced by the addition of IFN. These results indicate (a) that mycoplasmas can be useful as a biological response modifier, and (b) that care should be taken to prevent contamination with mycoplasmas in experiments on macrophage activation.     

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.     

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.     

Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention

The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

The influence of photoperiod on embryonic diapause in the tammar wallaby,macropus eugenii

After inoculation of mycoplasmas via ductus deferens in male dogs with a vas deferens fistula the influence on gonads and general state of health was investigated using clinical, andrological, spermatological, microbiological and histological methods.On day 23 and 29 respectively after inoculation two animals showed local reactions within the fistulated gonads which were identified as orchitis and epididymitis. Clinical abnormalities correlated well with the decrease of sperm motility and significant increase (p < 0,05) of aberrant spermatozoa.The microbiological and histological findings are discussed in the light of the incidence of clinical symptoms and the influence of a mycoplasmal infection.     

Detection of anaerobic mycoplasmas in cell cultures

Summary A commercially available anaerobic generator and incubation system that develops a low oxidation-reduction potential was used for the assay of cell cultures for mycoplasmal contamination. Mycoplasma broth and agar media supplemented with dextrose, yeast extract, and horse serum were used. This system supported growth of some mycoplasmas that failed to grow in incubators with 5% CO₂ in nitrogen previously used in culture of mycoplasmas in this laboratory. This work was supported by a grant from the John A. Hartford Foundation General Research Support Grant No. 5 SO1 RR05582-4 from the National Institutes of Health, and by a Grant-in-Aid Contract from the State of New Jersey.