Transmission of a common intestinal neoplasm in zebrafish by cohabitation.

Intestinal neoplasms are common in zebrafish (Danio rerio) research facilities. These tumours are most often seen in older fish and are classified as small cell carcinomas or adenocarcinomas. Affected fish populations always contain subpopulations with preneoplastic lesions, characterized by epithelial hyperplasia or inflammation. Previous observations indicated that these tumours are unlikely caused by diet, water quality or genetic background, suggesting an infectious aetiology. We performed five transmission experiments by exposure of naïve fish to affected donor fish by cohabitation or exposure to tank effluent water. Intestinal lesions were observed in recipient fish in all exposure groups, including transmissions from previous recipient fish, and moribund fish exhibited a higher prevalence of neoplasms. We found a single 16S rRNA sequence, most similar to Mycoplasma penetrans, to be highly enriched in the donors and exposed recipients compared to unexposed control fish. We further tracked the presence of the Mycoplasma sp. using a targeted PCR test on individual dissected intestines or faeces or tank faeces. Original donor and exposed fish populations were positive for Mycoplasma, while corresponding unexposed control fish were negative. This study indicates an infectious aetiology for these transmissible tumours of zebrafish and suggests a possible candidate agent of a Mycoplasma species.

Potential malignant transformation in the gastric mucosa of immunodeficient mice with persistent Mycoplasma penetrans infection.

Mycoplasma infection has been reported in immunocompromised cancer patients; nevertheless, it is not clear if persistent Mycoplasma infection could facilitate the proliferation of cancer cells in immunocompromised organisms. The aim of this study was to examine the relationship between persistent Mycoplasma infection and malignant transformation in an immunodeficient host model. Immunodeficient mouse model was established using cyclophosphamide and mice gastric mucosal cells were infected with Mycoplasma penetrans (Mpe). After 18 weeks, mice were sacrificed and gastric mucosal Mpe infected cells were identified by fluorescence in situ hybridization (FISH). Moreover, pathological and ultrastructural changes in mice gastric mucosa were evaluated and the expression of multiple proto-oncogenes was examined by Western blot. Our data show that Mpe infection was detected in the blood of immunodeficient mice and Mpe persistent infection in mice gastric mucosa was confirmed by FISH. There were pathological and ultrastructural malignant transformation occurred in the gastric mucosa of infected mice compared to control mice. Mpe infected mice showed lower expression of p53 and p21 and higher H-ras expression compared to the control group. Moreover, expression of NF-κB p65 subunit increased in Mpe infected mice, similar to the TNF-α expression. Bax expression in gastric mucosa of Mpe infected mice was lower while Bcl-2 expression was higher than in the uninfected control group. Collectively these data demonstrate that persistent Mpe infection is associated with aberrant expression of multiple proto-oncogenes in gastric mucosa of immunodeficient mice which potentially facilitate the malignant transformation.

Analysis of energy sources for Mycoplasma penetrans gliding motility.

Mycoplasma penetrans, a potential human pathogen found mainly in HIV-infected individuals, uses a tip structure for both adherence and gliding motility. To improve our understanding of the molecular mechanism of M. penetrans gliding motility, we used chemical inhibitors of energy sources associated with motility of other organisms to determine which of these is used by M. penetrans and also tested whether gliding speed responded to temperature and pH. Mycoplasma penetrans gliding motility was not eliminated in the presence of a proton motive force inhibitor, a sodium motive force inhibitor, or an agent that depletes cellular ATP. At near-neutral pH, gliding speed increased as temperature increased. The absence of a clear chemical energy source for gliding motility and a positive correlation between speed and temperature suggest that energy derived from heat provides the major source of power for the gliding motor of M. penetrans.

Conserved terminal organelle morphology and function in Mycoplasma penetrans and Mycoplasma iowae.

Within the genus Mycoplasma are species whose cells have terminal organelles, polarized structures associated with cytadherence and gliding motility. Mycoplasma penetrans, found mostly in HIV-infected patients, and Mycoplasma iowae, an economically significant poultry pathogen, are members of the Mycoplasma muris phylogenetic cluster. Both species have terminal organelles that interact with host cells, yet the structures in these species, or any in the M. muris cluster, remain uncharacterized. Time-lapse microcinematography of two strains of M. penetrans, GTU-54-6A1 and HF-2, and two serovars of M. iowae, K and N, show that the terminal organelles of both species play a role in gliding motility, with differences in speed within and between the two species. The strains and serovars also differed in their hemadsorption abilities that positively correlated with differences in motility speeds. No morphological differences were observed between M. penetrans and M. iowae by scanning electron microscopy (SEM). SEM and light microscopy of M. penetrans and M. iowae showed the presence of membranous filaments connecting pairs of dividing cells. Breaking of this filament during cell division was observed for M. penetrans by microcinematography, and this suggests a role for motility during division. The Triton X-100-insoluble fractions of M. penetrans and M. iowae consisted of similar structures that were unique compared to those identified in other mycoplasma species. Like other polarized mycoplasmas, M. penetrans and M. iowae have terminal organelles with cytadherence and gliding functions. The difference in function and morphology of the terminal organelles suggests that mycoplasmas have evolved terminal organelles independently of one another.

Mycoplasma penetrans under nutritional stress: influence on lipid and lipoprotein profiles and on the binding to and invasion of HeLa cells.

By serial passages through media containing decreasing concentrations of horse serum, the sterol-requiring Mycoplasma penetrans strain HF-2 was adapted to grow in a serum-free medium supplemented with bovine serum albumin, cholesterol and free fatty acids. Chemical analysis of membrane preparations obtained from the native and adapted strains revealed two major differences. (1) The polar lipid fraction of the native strain contains, in addition to the de novo-synthesized phospholipids, exogenous lipids incorporated unchanged from the growth medium, whereas exogenous lipids were not detected in the adapted strain. (2) Protein analyses of the native and adapted strains showed that upon adaptation, the 42-kDa membrane lipoprotein, one of the two major lipoproteins of this organism, was missing. Studies on the adhesion to, and invasion of HeLa cells by the native and adapted strains revealed that whereas the adherence to HeLa cells of the adapted strain was almost the same as that of the native strain, the invasiveness of the adapted strain into HeLa cells was very low or nonexistent.

Mycoplasmal infections alter gene expression in cultured human prostatic and cervical epithelial cells.

To better understand how infections by mycoplasmas affect gene expression in human cells, we quantitatively measured the transcripts of 38 cytokine genes in HPV E6- and E7-immortalized cervical and prostatic epithelial cells before and after infection by four human urogenital mycoplasmas, M. fermentans, M. genitalium, M. hominis and M. penetrans. Using the multi-probe RNase protection assay (RPA), 22 and 23 cytokine gene transcripts were detected in the non-infected control prostatic and cervical epithelial cells, respectively. Although there were no discernible changes in cell morphology and growth kinetics following 72 h of mycoplasmal infection, 55-74% of the cytokine genes expressed in the two human epithelial cell lines were altered. Most changes reflected an increased expression of these cytokine genes, while expression of some cytokine genes significantly decreased. The effects varied with host cell type and species of infecting mycoplasmas. These alterations in gene expression were more profound in the cervical epithelial cells than in the prostatic cells. M. fermentans produced the most significant effects, followed by M. penetrans, M. genitalium and M. hominis. Some alterations in the gene expression were transient, but most persisted over the course of chronic (9 months) mycoplasmal infection. Prolonged gene expression changes induced by chronic mycoplasmal infection may gradually alter important biological properties in the infected mammalian cells and produce a unique form of disease process.

Protein kinase C activation and vacuolation in HeLa cells invaded by Mycoplasma penetrans.

The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. This study investigated the invasion of HeLa cells and its consequences by confocal laser scanning microscopy. Invasion was dependent on the duration of infection and temperature, diminished by inhibiting microfilament assembly with cytochalasin D and almost completely abolished by disorganising microtubules with vinblastine or taxol. After a short infection period (< 20 min), pronounced activation of protein kinase C was detected in host cells, whereas prolonged infection resulted in intensive vacuolation of the host cells and a pronounced increment in intracellular organic peroxide levels. A marked decrease in the extent of vacuolation was observed when peroxide accumulation was partially prevented by alpha-tocopherol. The possibility that M. penetrans entry into HeLa cells involves the activation of protein kinases and the recruitment of cytoskeleton components is discussed.

Absence of mycoplasmal gene in malignant mammalian cells transformed by chronic persistent infection of mycoplasmas.

Chronic persistent infections by mycoplasmas induced malignant transformation of C3H mouse embryo cells that normally had never been reported to undergo spontaneous transformation. This mycoplasma-mediated oncogenic process had a long latency (more than 7 weeks of continuous mycoplasmal infection) and showed a multistage progression characterized by reversibility (at least up to 11 weeks of mycoplasmal infection) and irreversibility of malignant properties upon removal of the mycoplasma from culture. Further prolonged infections (18 weeks) by Mycoplasma fermentans or M. penetrans resulted in permanent transformation of these C3H cells that no longer required the continued presence of the transformation-inducing mycoplasmas in cultures to retain their malignant properties. Previous studies of viral oncogenesis revealed that virus-transformed cells always had viral gene(s) present. Integration of viral gene(s) apparently played an important role in the process of oncogenesis. In this study, we examined if the continued presence of any mycoplasmal gene(s) in mammalian cells, in whatever form, was also crucial in causing malignant cell transformation. Representational difference analysis (RDA) was a recently developed powerful technique to compare differences between two complex genomes. In the RDA system, subtractive and kinetic enrichment was used to purify and isolate restriction endonuclease gene fragment(s) of mycoplasmal origin, presumably present only in mycoplasma-transformed C3H cells, but not in nonmycoplasma-exposed control C3H cells. After three rounds of subtractive hybridization following PCR enrichment for each of three different restriction enzymes DNA digests, no gene fragment of mycoplasmal origin was amplified or identified in the permanently transformed C3H cells. Differing from tumorigenesis in animal cells induced by most oncogenic viruses or in plant cells induced by Agrobacteria, mycoplasmas evidently did not cause malignant transformation by integrating their gene(s) into the mammalian cell genome.

Mycoplasma penetrans infection of Molt-3 lymphocytes induces changes in the lipid composition of host cells.

The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. The ability of M. penetrans to both adhere to and invade Molt-3 lymphocytes was markedly increased in the presence of polyethylene glycol 8000 (PEG). The effect of PEG was more pronounced in the more alkaline pH range, where the binding kinetics were much faster and almost unaffected by temperature (4-37 degrees C). Incubation of [14C]oleic-acid-labelled Molt-3 cells with viable M. penetrans resulted in a substantial release of radioactive fatty acids, whereas treating the host cells with heat-inactivated mycoplasmas, isolated M. penetrans membrane preparations, or M. penetrans growth medium, had no effect. Total lipid analysis of Molt-3 lymphocytes infected by M. penetrans revealed an augmented level of the neutral lipid fraction that was associated with a decrease in the relative amounts of polar lipids, mainly a decrease in the amount of phosphatidylserine and diphosphatidylglycerol. Analysis of the neutral lipid fraction in the infected Molt-3 cells revealed a fivefold increase in the relative amount of diacylglycerol and a marked increase in the free fatty acid (FFA) fraction. The profile of the FFAs released was dominated by a relatively high concentration of the polyunsaturated fatty acid docosahexaenoic acid. The release of lipid intermediates suggests that the degradation of Molt-3 cell phospholipids induced by M. penetrans may initiate a signal transmission cascade in the host cell.

Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans.

Mycoplasma penetrans adhered to cultured human cells, forming clusters that localized to specific areas of the host cell surface. Adherence and cluster formation were inhibited by anti-M. penetrans antibodies, suggesting the involvement of specific adhesin-receptor interactions. Ultrastructural studies showed that after 2 h of infection, mycoplasmas attach to and penetrate the host cell surface. M. penetrans bound selectively to immobilized fibronectin, an interaction which was not inhibited by a 70-kDa fragment containing a heparin-gelatin-binding domain of fibronectin, other matrix glycoproteins, or an RGD tripeptide, suggesting the recognition of other specific binding sites on the fibronectin molecule. A ca. 65-kDa fibronectin-binding protein of M. penetrans was eluted following Sepharose-fibronectin affinity chromatography. Confocal, light, and immunofluorescence microscopy demonstrated that the interaction of M. penetrans with target cells triggers a signal that causes recruitment of several cytoskeletal components, including tubulin and alpha-actinin, and aggregation of phosphorylated proteins. Detergent-soluble mycoplasma proteins with apparent molecular masses of 18, 28, 32, 36, 39, and 41 kDa selectively bound to glutaraldehyde-fixed HEp-2 cells. Our findings offer new insights into understanding the interaction of this human mycoplasma with host target cells.

[Induction of tumor necrosis factor alpha by Mycoplasma penetrans isolated from patients with AIDS].

The activity of induce tumor necrosis factor alpha (TNF alpha) production of several mycoplasmas, including AIDS associated mycoplasmas was investigated. M. penetrans which was detected and isolated from urine and tissue of Kaposi's sarcoma of patients with AIDS markedly exhibited the induction of TNF alpha production of both THP-1 cells and murine peritoneal macrophages to compare to other mycoplasmas. Each amount of M. penetrans, M. fermentans, M. incognitus, Acholeplasma ladilawii, M. orale, M. salivarium, M. hominis required for induction of 50% cytotoxicity to L cells in the supernatants of mouse peritoneal macrophages cultured with those microorganisms was 0.65 micrograms/ml, 11.3 micrograms/ml, 19.6 micrograms/ml, 6.6 micrograms/ml, 7.7 micrograms/ml, 6.3 micrograms/ml, 5.7 micrograms/ml respectively. Next, the components of M. penetrans were extracted by Bligh-Dyer method, in order to investigate chemical component to induce TNF alpha-production. The activity of TNF alpha induction was mainly found in the methanol-phase, but not in the chloroform-phase, where lipid and glycolipid of the microorganisms were generally thought to be accumulated. The binding of the active component to concanavalin A-Sepharose was blocked in the presence of Methyl alpha-D-mannopyranoside and Methyl alpha-D-glucopyranoside. These results suggest that the component possess mannoside and glucoside active site.

Induction of tumor necrosis factor alpha (TNF alpha) and enhancement of HIV-1 replication in the J22HL60 cell line by Mycoplasma penetrans.

Mycoplasma penetrans isolated from clinical specimens of AIDS patients showed potent activity in tumor necrosis factor alpha (TNF alpha) production in THP-1, U937 and J22HL60 cell lines, and in the enhancement of HIV-1 replication in a dormantly-infected J22HL60 cell line as compared with the activities of other mycoplasmas. Both activities were found in the methanol layer but not in the chloroform layer of the membrane extracted by the Bligh-Dyer method. TNF alpha production was observed in the peritoneal macrophages from both lipopolysaccharide-responsive and -unresponsive mouse strains, and was not inhibited by polymyxin B. The induction of TNF alpha production and enhancement of HIV-1 replication were strongly inhibited by Concanavalin A-Sepharose. The inhibitory effect of Concanavalin A-Sepharose was partially prevented by sugars in the order methyl-alpha-D-mannopyranoside and methyl-alpha-D-glucopyranoside but not methyl-alpha-D-galactopyranoside. Anti-human TNF alpha antibody, however, did not reduce the activity of the methanol layer to enhance HIV-1 replication, suggesting that the methanol layer could enhance HIV-1 replication directly. These results suggest that the carbohydrate derived from M. penetrans might be responsible for the progression of HIV-1 infection.

Interplay between mycoplasmas and host target cells.

The infectious pattern of mycoplasmas (Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma genitalium) in mammalian cells was examined using confocal microscopy and flow cytometry combined with cell fractionation and mycoplasma viability determinations. Within 2 h postinfection mycoplasmas parasitize cell surfaces, enter the intracellular spaces and locate throughout the cytoplasmic and perinuclear regions. These mycoplasmas can be cultivated from cytoplasmic and nuclear fractions 96 h later and continue to persist intracellularly for at least 7 days, suggesting a much more active intracellular role for mycoplasmas than had been considered previously.