Novel synthetic lipopeptides derived from Mycoplasma hyorhinis upregulate calpastatin in SH-SY5Y neuroblastoma cells and induce a neuroprotective effect against amyloid-ß-peptide toxicity.

Previously, we showed that contamination of SH-SY5Y neuroblastoma cells by Mycoplasma hyorhinis strains NDMh and MCLD leads to increased levels of calpastatin (the endogenous, specific inhibitor of the Ca2+-dependent protease calpain), resulting in inhibition of calpain activation. We have found that the increased calpastatin level is promoted by the lipoprotein fraction (MhLpp) of the mycoplasmal membrane. Here, we present MhLpp-based novel synthetic lipopeptides that induce upregulation of calpastatin in SH-SY5Y neuroblastoma cells, leading to protection of the treated cells against Ca2+/amyloid-ß-peptide toxicity. These lipopeptides present a new class of promising agents against calpain-induced cell toxicity.

Mycoplasma gallisepticum inactivated by targeting the hydrophobic domain of the membrane preserves surface lipoproteins and induces a strong immune response.

An innovative approach for inactivation of Mycoplasma gallisepticum using the hydrophobic photoinduced alkylating probe 1, 5-iodonaphthylazide (INA) is described. Treatment of washed M. gallisepticum mid-exponential culture (0.2 mg cell protein /mL) with INA followed by irradiation with far-ultraviolet light (310-380 nm) completely abolished viability. Transmission electron microscopy showed that the majority of the inactivated M. gallisepticum were comparable in size to intact cells, but that part of the INA-treated M. gallisepticum preparation also contained low density cells and membrane vesicles. Confocal microscopy revealed that untreated M. gallisepticum cells were internalized by chicken red blood cells (c-RBCs), whereas the INA-inactivated cells remained attached to the outer surface of the c-RBCs. INA treatment of M. gallisepticum resulted in a complete inactivation of F0F1 -ATPase and of the L-arginine uptake system, but the cytoplasmatic soluble NADH2 dehydrogenase was only partially affected. Western blot analysis of the lipoprotein fraction showed that the INA-treated M. gallisepticum retained their lipoproteins. Following subcutaneous injection of M. gallisepticum INA-bacterin, 100% and 68.8% of chickens were positive by the rapid serum agglutination test and enzyme-linked immunosorbent assay respectively, 2 weeks post-injection. These data suggest that the photoinducible alkylating agent INA inactivates M. gallisepticum but preserves its surface lipoproteins and thus has the potential to be used as a general approach for the inactivation of mycoplasmas for vaccine development.

Mycoplasma hyorhinis induces proinflammatory responses in mice lymphocytes.

Mycoplasmas are frequent contaminants of cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction, and metabolic pathways. Mycoplasma hyorhinis, the major contaminant of tissue cultures, has been implicated in a variety of diseases in swine. Most human and animal mycoplasmas remain attached to the surface of epithelial cells. Nonetheless, we have recently shown that M. hyorhinis is able to invade nonphagocytic melanoma cells. In the present study, we show by confocal laser scanning microscopy, that by exposing mice splenocytes to intact M. hyorhinis, intracellular mycoplasmas were detected. Mycoplasmal components were not detected within splenocytes after exposure to heat inactivated M. hyorhinis or to a purified M. hyorhinis lipoprotein (LPP) fraction. However, incubation of the splenocytes with intact M. hyorhinis cells, heat inactivated cells or M. hyorhinis LPP fraction induced accelerated cell proliferation and the secretion of interferon gamma and interleukin 17. Thus, M. hyorhinis and its LPPs can be added to the list of infectious agents causing direct stimulation of proinflammatory responses by mammalian lymphocytes.

Cardiolipin synthetase is involved in antagonistic interaction (reverse CAMP phenomenon) of Mycoplasma species with Staphylococcus aureus beta-hemolysis.

Mycoplasma hyorhinis has been implicated in a variety of swine diseases. However, little is known about the hemolytic capabilities of Mycoplasma species in general or M. hyorhinis in particular. In this study, we show that M. hyorhinis possesses beta-hemolytic activity which may be involved in the invasion process. M. hyorhinis also possesses antagonistic cooperativity (reverse CAMP phenomenon) with Staphylococcus aureus beta-hemolysis, resulting in the protection of erythrocytes from the beta-hemolytic activity of S. aureus (reverse CAMP). The reversed CAMP phenomenon has been attributed to phospholipase D (PLD) activity. In silico analysis of the M. hyorhinis genome revealed the absence of the pld gene but the presence of the cls gene encoding cardiolipin synthetase, which contains two PLD active domains. The transformation of Mycoplasma gallisepticum that has neither the cls gene nor the reverse CAMP phenomenon with the cls gene from M. hyorhinis resulted in the reverse CAMP phenomenon, suggesting for the first time that reverse CAMP can be induced by cardiolipin synthetase.

Reconstitution of an active arginine deiminase pathway in Mycoplasma pneumoniae M129.

Some species of the genus Mycoplasma code for the arginine deiminase pathway (ADI), which enables these bacteria to produce ATP from arginine by the successive reaction of three enzymes: arginine deiminase (ArcA), ornithine carbamoyltransferase (ArcB), and carbamate kinase (ArcC). It so far appears that independently isolated strains of Mycoplasma pneumoniae encode an almost identical truncated version of the ADI pathway in which the proteins ArcA and ArcB have lost their original enzymatic activities due to the deletion of significant regions of these proteins. To study the consequences of a functional ADI pathway, M. pneumoniae M129 was successfully transformed with the cloned functional arcA, arcB, and arcC genes from Mycoplasma fermentans. Enzymatic tests showed that while the M. pneumoniae ArcAB and ArcABC transformants possess functional arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase, they were unable to grow on arginine as the sole energy source. Nevertheless, infection of a lung epithelial cell line, A549, with the M. pneumoniae transformants showed that almost 100% of the infected host cells were nonviable, while most of the lung cells infected with nontransformed M. pneumoniae were viable under the same experimental conditions.

The phospholipid profile of mycoplasmas.

The de novo synthesized polar lipids of Mycoplasma species are rather simple, comprising primarily of the acidic glycerophospholipids PG and CL. In addition, when grown in a medium containing serum, significant amounts of PC and SPM are incorporated into the mycoplasma cell membrane although these lipids are very uncommon in wall-covered bacteria. The exogenous lipids are either incorporated unchanged or the PC incorporated is modified by a deacylation-acylation enzymatic cycle to form disaturated PC. Although their small genome, in some Mycoplasma species, other genes involved in lipid biosynthesis were detected, resulting in the synthesis of a variety of glycolipis, phosphoglycolipids and ether lipids. We suggest that analyses and comparisons of mycoplasma polar lipids may serve as a novel and useful tool for classification. Nonetheless, to evaluate the importance of polar lipids in mycoplasma, further systematic and extensive studies on more Mycoplasma species are needed. While studies are needed to elucidate the role of lipids in the mechanisms governing the interaction of mycoplasmas with host eukaryotic cells, the finding that a terminal phosphocholine containing glycolipids of M. fermentans serves both as a major immune determinants and as a trigger of the inflammatory responses, and the findings that the fusogenicity of M. fermentans with host cells is markedly stimulated by lyso-ether lipids, are important steps toward understanding the molecular mechanisms of M. fermentans pathogenicity.

Phospholipase A and glycerophosphodiesterase activities in the cell membrane of Mycoplasma hyorhinis.

Mycoplasma hyorhinis, the major contaminant of tissue cultures, has been implicated in a variety of diseases in swine. Most human and animal mycoplasmas remain attached to the surface of epithelial cells. Nonetheless, we have recently shown that M. hyorhinis is able to invade and survive within nonphagocytic melanoma cells. The invasion process may require the damaging of the host cell membrane by either chemical, physical or enzymatic means. In this study, we show that M. hyorhinis membranes possess a nonspecific phospholipase A (PLA) activity capable of hydrolyzing both position 1 and position 2 of 1-acyl-2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminododecanoyl) phosphatidylcholine. In silico analysis of the M. hyorhinis genome shows that the PLA of M. hyorhinis shares no homology to described phospholipases. The PLA activity of M. hyorhinis was neither stimulated by Ca (2+) nor inhibited by EGTA and had a broad pH spectrum. Mycoplasma hyorhinis also possess a potent glycerophosphodiesterase (GPD), which apparently cleaves the glycerophosphodiester formed by PLA to yield glycerol-3-phosphate. Possible roles of PLA and GPD in invading host eukaryotic cells and in forming mediators upon the interaction of M. hyorhinis with eukaryotic cells are suggested.

Calpastatin upregulation in Mycoplasma hyorhinis-infected cells is promoted by the mycoplasma lipoproteins via the NF-κB pathway.

Mycoplasma hyorhinis frequently contaminates cultured cells, with effects on synthetic and metabolic pathways. We demonstrated for the first time that contamination of cells by a strain of M. hyorhinis (NDMh) results in increased levels of calpastatin (the endogenous inhibitor of the ubiquitous Ca(2+) -dependent protease calpain). We now show that the calpastatin upregulation by NDMh in neuroblastoma SH-SY5Y cells resides in the NDMh lipoprotein fraction (LPP), via the NF-κB transcription pathway. NF-κB activation requires dissociation of the cytoplasmic NF-κB/IκB complex followed by NF-κB translocation to the nucleus. NDMh-LPP induced translocation of the NF-κB RelA subunit to the nucleus and upregulated calpastatin. RelA translocation and calpastatin elevation were prevented when dissociation of the NF-κB/IκB complex was inhibited either by transfection with the non-phosphorylatable IκB mutant ΔNIκBα, or by using PS1145, an inhibitor of the IκB kinase (IKK complex). Increased calpastatin levels attenuate calpain-related amyloid-ß-peptide and Ca(2+) -toxicity (these are central to the pathogenesis of Alzheimer's Disease). LPP-induced elevation of calpastatin provides an example of effects on non-inflammatory intracellular proteins, the outcome being significant alterations in host cell functions. Since calpastatin level is important in the control of calpain activity, mycoplasmal LPP may be of interest in treating some pathological processes involving excessive calpain activation.

Adhesion and biofilm formation of Mycoplasma pneumoniae on an abiotic surface.

We demonstrated that when M. pneumoniae was grown on an abiotic surface of either glass or polystyrene with a serum-containing medium, the bacteria adhered to the surface and formed highly differentiated volcano-like biofilm structures. As adherence to the surface and/or biofilm formation was totally inhibited by anti-P1 polyclonal monospecific antibodies, we suggest that the adherence of M. pneumoniae to the abiotic surface and/or biofilm formation is associated with P1, the major tip organelle protein of this organism. Furthermore, adherence and/or biofilm formation was markedly inhibited by treating the serum component of the growth medium with neuraminidase or by growing the bacteria in the presence of sialyllactose, suggesting that the initial step in the adherence to and/or biofilm formation by M. pneumoniae on an abiotic surface is the interaction of the bacterium through its tip organelle with sialic acid residues of serum glycoproteins.

Genome analysis of a Mycoplasma hyorhinis strain derived from a primary human melanoma cell line.

The complete genome of Mycoplasma hyorhinis strain MCLD has been sequenced and annotated. This genome differs by the inversion of a 14.4-kb and a 3.7-kb fragment and the deletion of a 9.9-kb fragment from M. hyorhinis strain HUB-1, isolated from swine respiratory tract. The genome revealed 778 coding sequences (CDSs), with a limited number of vlp genes encoding variable surface lipoproteins.

Neuroprotective effects of Mycoplasma hyorhinis against amyloid-ß-peptide toxicity in SH-SY5Y human neuroblastoma cells are mediated by calpastatin upregulation in the mycoplasma-infected cells.

Mycoplasmas are frequent contaminants of cell cultures. Contamination leads to altered synthetic and metabolic pathways. We have found that contamination of neuroblastoma SH-SY5Y cells by a strain of Mycoplasma hyorhinis derived from SH-SY5Y cell culture (NDMh) leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease, calpain) in NDMh-infected cells. We have now examined effects of amyloid-ß-peptide (Aß) (central to the pathogenesis of Alzheimer's disease) on uncontaminated (clean) and NDMh-infected SH-SY5Y cells. Aß was toxic to clean cells, resulting in necrotic cell damage. Aß treatment led to activation of calpain and enhanced proteolysis, cell swelling, cell membrane permeability to propidium iodide (PI) (without nuclear apoptotic changes), and diminished mitochondrial enzyme activity (XTT reduction). Aß-toxicity was attenuated in the high calpastatin-containing NDMh-infected cells, as shown by inhibition of calpain activation and activity, no membrane permeability, normal cell morphology, and maintenance of mitochondrial enzyme activity (similar to attenuation of Aß-toxicity in non-infected cells overexpressing calpastatin following calpastatin-plasmid introduction into the cells). By contrast, staurosporine affected both clean and infected cells, causing apoptotic damage (cell shrinkage, nuclear apoptotic alterations, caspase-3 activation and caspase-promoted proteolysis, without PI permeability, and without effect on XTT reduction). The results indicate that mycoplasma protects the cells against certain types of insults involving calpain. The ratio of calpastatin to calpain is an important factor in the control of calpain activity. Exogenous pharmacological means, including calpastatin-based inhibitors, have been considered for therapy of various diseases in which calpain is implicated. Mycoplasmas provide the first naturally occurring biological system that upregulates the endogenous calpain inhibitor, and thus may be of interest in devising treatments for some disorders, such as neurodegenerative diseases.

Genomic features and insights into the biology of Mycoplasma fermentans.

We present the complete genomic sequence of Mycoplasma fermentans, an organism suggested to be associated with the pathogenesis of rheumatoid arthritis in humans. The genome is composed of 977,524 bp and has a mean G+C content of 26.95 mol%. There are 835 predicted protein-coding sequences and a mean coding density of 87.6 %. Functions have been assigned to 58.8 % of the predicted protein-coding sequences, while 18.4 % of the proteins are conserved hypothetical proteins and 22.8 % are hypothetical proteins. In addition, there are two complete rRNA operons and 36 tRNA coding sequences. The largest gene families are the ABC transporter family (42 members), and the functionally heterogeneous group of lipoproteins (28 members), which encode the characteristic prokaryotic cysteine 'lipobox'. Protein secretion occurs through a pathway consisting of SecA, SecD, SecE, SecG, SecY and YidC. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The genes encoding DnaK-DnaJ-GrpE and Tig, forming the putative complex of chaperones, are intact, providing the only known control over protein folding. Eighteen nucleases and 17 proteases and peptidases were detected as well as three genes for the thioredoxin-thioreductase system. Overall, this study presents insights into the physiology of M. fermentans, and provides several examples of the genetic basis of systems that might function as virulence factors in this organism.

Mycoplasma hyorhinis upregulates calpastatin and inhibits calpain-dependent proteolysis in SH-SY5Y neuroblastoma cells.

Mycoplasmas often contaminate cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction and metabolic pathways. Mycoplasmal contamination is often unnoticed, so that mycoplasma-induced alterations in cell functions may not be appreciated, unless specifically studied. Here, we show for the first time that contamination of SH-SY5Y cells by Mycoplasma hyorhinis leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease calpain), resulting in inhibition of Ca(2+)-induced calpain activation and inhibition of calpain-promoted proteolysis in the mycoplasmal-infected cells. Calpain activity is recovered upon calpastatin removal from extracts of contaminated cells. The calpain-calpastatin system has been implicated in a variety of physiological and pathological processes (signal transduction, motility, cell cycle, cell differentiation, membrane damage and apoptosis). Because the ratio of calpastatin to calpain is an important factor in the control of calpain activity within the cell, the elevated calpastatin may protect the mycoplasma-infected cells against certain types of damage (e.g. caused by high Ca(2+)). Thus, our results are important for studies on the modulation of host cells by mycoplasmas, and relevant to the pathobiology of processes involving mycoplasmal infections. The mycoplasma-infected cells provide a system for identifying factors that participate in the regulation of cellular calpastatin.

Invasion of melanoma cells by Mycoplasma hyorhinis: enhancement by protease treatment.

Mycoplasma hyorhinis (strain MCLD) was recently isolated from a melanoma cell culture. Growth of MCLD was considerably improved by 24 serial passages in a modified Hayflick's mycoplasma medium. Transmission electron microscopy showed that MCLD exhibits a polymorphic appearance, with ovoid or elongated cells frequently harboring an electron-dense core at one of the poles. Adherence of M. hyorhinis to melanoma cells followed saturation kinetics. Furthermore, although M. hyorhinis has been considered to remain attached to the surface of the host cells, we show for the first time, qualitatively by confocal laser scanning microscopy and quantitatively by a gentamicin resistance assay, that MCLD is able to invade melanoma cells. The ingested mycoplasmas were randomly distributed in the cytoplasm, tending to concentrate near the plasma membrane. Both adherence to and invasion of melanoma cells by M. hyorhinis strain MCLD were dramatically enhanced by mild proteolytic digestion with proteinase K (2.5 microg/mg cell protein for 2.5 min at 37 degrees C) that affected the surface-exposed proteins of this organism, mainly the major 47-kDa lipoprotein. We suggest that the intracellular location of M. hyorhinis strain MCLD is a privileged niche, which may explain the survival of M. hyorhinis in tissue cultures. The enhanced binding to and invasion of melanoma cells by protease treatment may be due to either the activation or the enhanced exposure of an adhesin(s) on the mycoplasmal cell surface.

Binding of IgA by Mycoplasma penetrans.

The current study shows that Mycoplasma penetrans strain GTU binds human serum immunoglobulin A (IgA) and secretory IgA but not IgG. Binding of IgA was associated almost exclusively with the lipoprotein fraction obtained by Triton X-114 fractionation of isolated M. penetrans membranes. Western immunoblot analysis of isolated membranes of M. penetrans strain GTU and of the Triton X-114 fraction showed that the major IgA-binding component was a lipoprotein with a molecular mass of 38 kDa, one of the major lipoproteins of this organism. The authors suggest that the high IgA-binding capacity of M. penetrans strain GTU may serve as a defense mechanism, conferring on this microorganism the ability to evade clearance mediated by specific IgA antibodies.

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.

Alpha-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen.

Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with epsilon-aminocaproic acid. The eluted approximately 50-kDa protein was identified by mass spectrometric techniques as alpha-enolase. The possibility that alpha-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-alpha-enolase antiserum, which showed that alpha-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-alpha-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that mycoplasmal surface alpha-enolase is a major Plg binding protein of M. fermentans.

Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells.

In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluorescent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.