Apoptosis induced by lipid-associated membrane proteins from Mycoplasma penetrans is mediated by nuclear factor kappaB activation in mouse macrophage.

Mycoplasma penetrans was shown to be involved in alteration of several eukaryotical cells functions and a causative agent in urogenital infectious diseases. Lipid-associated membrane proteins (LAMPs) may be responsible for the pathogenicity of some mycoplamas. In this study, we investigated whether M. penetrans LAMPs have pathogenic potential by inducing apoptosis in mouse macrophages. As analyzed by annexin-V - fluorescein isothiocyanate staining, significant early- and late-stage apoptosis was induced in M. penetrans LAMPs-challenged mouse macrophages. And agarose gel electrophoresis of the DNA of M. penetrans LAMPs-challenged cells revealed a ladder-like pattern of migration of DNA indicative of apoptosis. The possible molecular mechanisms responsible for the induction of apoptosis were also investigated by characterizing the activation of nuclear transcription factor kappaB (NFkappaB). NFkappaB was activated and translocated into the nucleus in mouse macrophages stimulated by M. penetrans LAMPs. The activation of NFkappaB and M. penetrans LAMPs-induced apoptosis in mouse macrophages was partially inhibited by the NFkappaB-specific inhibitor pyrrolidine dithiocarbamate. Thus, this study demonstrates that M. penetrans LAMPs may be an important etiological factor owing to their ability to induce apoptosis in mouse macrophages, which is probably mediated through the activation of NFkappaB.

Lipid-associated membrane proteins of Mycoplasma fermentans and M. penetrans activate human immunodeficiency virus long-terminal repeats through Toll-like receptors.

Mycoplasmas are known to enhance human immunodeficiency virus (HIV) replication, and mycoplasma-derived lipid extracts have been reported to activate nuclear factor-kappaB (NF-kappaB) through Toll-like receptors (TLRs). In this study, we examined the involvement of TLRs in the activation of HIV long-terminal repeats (LTR) by mycoplasma and their active components responsible for the TLR activation. Lipid-associated membrane proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans and M. penetrans) that are associated with acquired immune-deficiency syndrome (AIDS), were found to activate HIV LTRs in a human monocytic cell line, THP-1. NF-kappaB deletion from the LTR resulted in inhibition of the activation. The LTR activation by M. fermentans LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and TLR6, whereas HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but not by DN TLR6. These results indicate that the activation of HIV LTRs by M. fermentans and M. penetrans LAMPs is dependent on NF-kappaB, and that the activation of HIV LTR by M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In contrast, the LTR activation by M. penetrans LAMPs is carried out through TLR1 and TLR2, but not TLR6. Subsequently, the active component of M. penetrans and M. fermentans LAMPs was purified by reverse-phase high-performance liquid chromatography (HPLC). Interestingly, the purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate NF-kappaB through TLR1 and TLR2. On the other hand, the activation of NF-kappaB by purified lipoprotein of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but not TLR1.

Intracellular location and survival of Mycoplasma penetrans within HeLa cells.

Mycoplasma penetrans invades HeLa cells and survives within them for prolonged periods of time. The intracellular distribution of M. penetrans within HeLa cells was studied utilizing the acidotropic dye LysoTracker (green), which permeates cell membranes and upon protonation remains trapped in acidic compartments. The excitation and emission spectra of the green LysoTracker are suitable for colocalization studies with rabbit anti- M. penetrans antibodies and red Cy5 goat anti-rabbit IgG. The images collected by confocal laser scanning microscopy revealed that in the infected HeLa cells almost all Cy5 fluorescent foci (red) were located within the LysoTrack-labelled intracellular compartments, apparently endosomes. Viable mycoplasmas were detected within endosomes for prolonged periods of time, apparently due to a potent antioxidant activity detected in M. penetrans.

The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans.

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.

Role of Mycoplasma penetrans endonuclease P40 as a potential pathogenic determinant.

Recently, we reported the purification to homogeneity and characterization of Ca(2+)- and Mg(2+)-dependent endonuclease P40 produced by Mycoplasma penetrans (M. Bendjennat, A. Blanchard, M. Loutfi, L. Montagnier, and E. Bahraoui, J. Bacteriol. 179; 2210-2220, 1997), a mycoplasma which was isolated for the first time from the urine of human immunodeficiency virus-infected patients. To evaluate how this nuclease could interact with host cells, we tested its effect on CEM and Molt-4 lymphocytic cell lines and on peripheral blood mononuclear cells. We observed that 10(-7) to 10(-9) M P40 is able to mediate a cytotoxic effect. We found that 100% of cells were killed after 24 h of incubation with 10(-7) M P40 while only 40% cytotoxicity was obtained after 72 h of incubation with 10(-9) M P40. Phase-contrast microscopy observations of P40-treated cells revealed morphological changes, including pronounced blebbing of the plasma membrane and cytoplasmic shrinkage characteristic of programmed cell death, which is in agreement with the internucleosomal fragmentation of P40-treated cell DNA as shown by agarose gel electrophoresis. We showed that (125)I-radiolabeled or fluorescein isothiocyanate-labeled P40 was able to bind specifically in a dose-dependent manner to the cell membrane of CEM cells, which suggested that the cytotoxicity of P40 endonuclease was mediated by its interaction with the cell surface receptor(s). The concentration of unlabeled P40 required to inhibit by 50% the formation of (125)I-P40-CEM complexes was about 3 x 10(-9) M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca(2+) dependent. Our results suggest that the cytotoxicity of M. penetrans observed in vitro is mediated at least partially by secreted P40, which, after interaction with host cells, can induce an apoptosis-like death. These results strongly suggest a major role of mycoplasmal nucleases as potential pathogenic determinants.