Search

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-β-peptide toxicity. These lipopeptides present a new class of promising agents against calpain-induced cell toxicity.

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.

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.

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.

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-β-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.

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.

We present the complete genomic sequence ofMycoplasma 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 ofM. fermentans, and provides several examples of the genetic basis of systems that might function as virulence factors in this organism.

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-β-peptide (Aβ) (central to the pathogenesis of Alzheimer's disease) on uncontaminated (clean) and NDMh-infected SH-SY5Y cells. Aβ was toxic to clean cells, resulting in necrotic cell damage. Aβ 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). Aβ-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 Aβ-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.

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.

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 Ca2+-dependent protease calpain), resulting in inhibition of Ca2+-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 Ca2+). 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.

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 μg/mg cell protein for 2.5 min at 37°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.

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.

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.

The choline-containing phosphoglycolipid, MfGL-II, is the major polar lipid of Mycoplasma fermentans PG18. Anti-MfGL-II antisera raised in rabbits using the purified MfGL-II as an immunogen were employed in immunogold electron microscopic and immunofluorescence studies showing that MfGL-II is uniformly distributed and exposed on the cell surface of M. fermentans cells. The specificity of the antibodies was determined by immunostaining of lipid extracts separated by thin layer chromatography. The antibodies recognize lipids specific to M. fermentans but did not cross-react with lipid extracts of M. penetrans, M. capricolum, M. gallisepticum or Acholeplasma laidlawii. As phosphocholine almost completely abolished antibody interaction with MfGL-II in an ELISA assay it is suggested that the anti-MfGL-II repertoire is composed primarily of anti-phosphocholine antibodies. The anti-MfGL-II antisera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGL-II plays a major role in M. fermentans-host cell interaction.

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).

Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37°C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans . The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with β-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.

The human CD99 protein is expressed on many cell types and is mostly abundant on lymphocytes and on several tumors. Different functions were attributed to the CD99 receptor, including adhesion, apoptosis and activation. However, until now the only ligand suggested to be recognized by CD99 was CD99 itself. In order to identify possible new CD99 ligands we constructed a CD99 protein fused to human IgG1. Surprisingly, a pronounced specific staining of melanoma cell lines that were infected with mycoplasmas was observed whereas clean cells were not recognized. Staining was specific, as other fusion proteins did not recognize the mycoplasma-infected cells. Sequencing of the 23s-16s region revealed that the contaminating agent is Mycoplasma hyorhinis. The CD99 interaction with M. hyorhinis was direct since it was blocked by anti-CD99 monoclonal antibody and by M. hyorhinis. It was also strain-specific as other mycoplasmas were not recognized. Our results show that CD99 interacts with a novel ligand of M. hyorhinis.

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.

The mycoplasmas form a large group of prokaryotic microorganisms with over 190 species distinguished from ordinary bacteria by their small size, minute genome, and total lack of a cell wall. Owing to their limited biosynthetic capabilities, most mycoplasmas are parasites exhibiting strict host and tissue specificities. The aim of this review is to collate present knowledge on the strategies employed by mycoplasmas while interacting with their host eukaryotic cells. Prominant among these strategies is the adherence of mycoplasma to host cells, identifying the mycoplasmal adhesins as well as the mammalian membrane receptors; the invasion of mycoplasmas into host cells including studies on the role of mycoplasmal surface molecules and signaling mechanisms in the invasion; the fusion of mycoplasmas with host cells, a novel process that raises intriguing questions of how microinjection of mycoplasma components into eukaryotic cells subvert and damage the host cells. The observations of diverse interactions of mycoplasmas with cells of the immune system and their immunomodulatory effects and the discovery of genetic systems that enable mycoplasmas to rapidly change their surface antigenic composition have been important developments in mycoplasma research over the past decade, showing that mycoplasmas possess an impressive capability of maintaining a dynamic surface architecture that is antigenically and functionally versatile, contributing to the capability of the mycoplasmas to adapt to a large range of habitats and cause diseases that are often chronic in nature.

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.

Choline-containing lipids were identified and characterized in the cell membrane of Mycoplasma fermentans and were shown to participate in the adhesion to the surface of eukaryotic cells, to stimulate mycoplasma fusion with eukaryotic cells, and to induce cytokine secretion by cells of the immune system. These findings suggest that choline-containing lipids are important mediators of tissue pathology in the infectious process caused by M. fermentans.

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.

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.

Mycoplasma fermentans seems to be involved in several pathogenic conditions in humans, and is among other things capable of fusing with T-cells and lymphocytes. The choline-containing phosphoglycolipid 6'-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-alpha-D-glucopyranosyl-(1'-->3)-1,2-diacylglycerol (MfGL-II) in the membrane of M. fermentans has been suggested to enhance the fusion process, and the characteristics of MfGL-II were therefore investigated. When a cell culture ages the fraction of MfGL-II increases, and the fraction of the other major membrane lipid, phosphatidylglycerol (PtdGro), decreases concomitantly. Swelling experiments showed that the permeability and osmotic fragility are markedly reduced in aged cells. MfGL-II is selectively released into the surrounding medium when aged M. fermentans cells are incubated in buffer containing EDTA. The physico-chemical properties of MfGL-II were studied by NMR spectroscopy and differential scanning calorimetry, and they can explain the biochemical results. The temperature for the transition between gel and lamellar liquid crystalline (Lalpha) phases is 35-45 degrees C higher for MfGL-II than for PtdGro, which most probably gives rise to the reduced permeability in aged cells. At high water contents MfGL-II forms an Lalpha phase and isotropic aggregates which were interpreted to be vesicles with a radius of approximately 450 A. It is proposed that MfGL-II forms vesicles in the surrounding medium when it is released from the cell membrane. Neither EDTA nor Ca2+ ions have a significant influence on the aggregate structures formed by MfGL-II. Our results indicate that MfGL-II has no fusogenic properties. It is more probable that a recently identified lysolipid in the M. fermentans membrane acts as a fusogen.

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 ≈ 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.

Summary: 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 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.

Mycoplasmas are minute wall-less bacterial parasites that exhibit strict host and tissue specificities. They enter, multiply and survive within the host for extended periods by circumventing host defenses. Their intimate interaction with eukaryotic cells, and in some cases the subsequent invasion into or fusion with these cells, mediates cell damage. Mycoplasmas also modulate the activity of host cells by a variety of direct mechanisms and/or indirectly by cytokine-mediated effects.

The partially purified 57-kDa protein of Spiroplasma melliferum was autophosphorylated when incubated with ATP in the presence of ZnCl2. Autophosphorylation was also apparent by showing the in situ phosphorylation of the 57-kDa protein band separated by polyacrylamide gel electrophoresis under nondenaturing conditions. The autophosphorylation was affected neither by the pH of the reaction mixture nor by the presence of NaF. The steady state level of the phosphorylated 57-kDa protein remained constant for up to 15 min, suggesting the absence of a phosphoprotein phosphatase activity in the preparation. As the initial phosphorylation rate did not decrease upon a 100-fold dilution of the 57-kDa protein under constant substrate concentration, it is suggested that the autophosphorylation is an intramolecular process.

Spiroplasma melliferum cells utilize arginine via the arginine dihydrolase pathway. L-Arginine uptake by intact cells was a saturable process both as a function of time and arginine concentration (Km = 40 μM). Uptake was not affected by pH in the range pH 5.0-8.0, or by L-citrulline, D-arginine, L-histidine or L-canavanine at concentrations tenfold higher than that of L-arginine. In contrast, L-arginine uptake was markedly inhibited by L-ornithine and partially inhibited by L-lysine. Uptake was neither affected by protonophores nor by cation ionophores, but was inhibited by protease treatment or by the sulfhydryl reagents p-chloromercuribenzoate or N-ethylmaleimide. Sealed membrane vesicles prepared by fusing isolated S. melliferum membranes with asolectin-cholesterol vesicles catalysed a rapid exchange (t1/2 = 1 min) between arginine and ornithine. Exchange did not require ATP and could be demonstrated in both directions, i.e. with either arginine or ornithine trapped within vesicles. These observations suggest that the driving forces for arginine uptake by whole cells are the concentration gradients of arginine and ornithine formed by arginine metabolism.

The major unidentified polar lipid (compound X), recently demonstrated in the cell membrane of Mycoplasma fermentans, was purified by preparative silicic acid column chromatography. Chemical analyses of acid-hydrolyzed compound X revealed that, in addition to fatty acids, it contains glycerol, choline and phosphate in a molar ratio of approximately 1:1:2, and an amino acid that has a retention time similar to that of homoserine. The methylated fatty acid fraction of compound X was subjected to gas-liquid chromatography and revealed methyl palmitate and methyl stearate in a 4.6:1 molar ratio. The structure of compound X was further analyzed by combining mass spectrometry, 31P-NMR and 1H-NMR. The positive and negative fast atom bombardment spectra showed a major component of M(r) 1048 and a minor component of M(r) 1076. Two different phosphate groups were identified in each of the components by 31P-NMR. Fast atom bombardment, tandem mass spectrometry, negative and positive chemical ionization mass spectrometry together with mass spectra analyses of the water-soluble and ether-soluble products obtained by methanolysis has shown that, in addition to palmitic and stearic acid residues, the presence of glycerol, ribitol, cholinephosphate and homoserinephosphate residues. It is suggested that the apparent structure of compound X is either a phosphatidylcholine attached via a phosphotriester bond to a ribitolphosphohomoserine moiety or a phosphatidylhomoserine attached via a phosphotriester bond to a ribitolphosphocholine moiety. The major molecular species is the dipalmitoylderivative (M(r) 1048), whereas the minor molecular species is a stearoyl palmitoyl derivative (M(r) 1076).

Mycoplasmas are the smallest and simplest self-replicating bacteria [1]. These microorganisms lack a peptidoglycan based rigid cell wall and thus are not susceptible to antibiotics, such as penicillin and its analogues, which are effective against most bacterial contaminants of cell cultures. The trivial name mycoplasma encompasses all species included in the class Mollicutes: i.e. the genera Mycoplasma, Acholeplasma, Spiroplasma, Anaeroplasma and Ureaplasma. Because mycoplasmas have an extremely small genome (0.58– 2.20 Mb compared with the 4.64 Mb of Escherichia coli), these organisms have limited metabolic options for replication and survival. The smallest genome of a self-replicating organism known at present is the genome of Mycoplasma genitalium (0.58 Mb; Ref. 2). Comparative genomic studies suggested that the genome of this organism still carries almost double the number of genes included in the minimal gene set essential for cellular function [3]. Owing to their limited biosynthetic capabilities, most mycoplasmas are parasites, exhibiting strict host and tissue specificities [4]. The aim of this review is to collate present knowledge on the strategies employed by mycoplasmas while interacting with tissue culture cells. Prominent among these strategies is the adherence of mycoplasmas to host cells, the invasion of mycoplasmas into host cells and the fusion of mycoplasmas with host cells. We shall discuss the intriguing questions of how a mycoplasma infecting tissue culture cells subvert and damage the host cells by mediating transformation of the cells, affecting the signal-transduction pathways and the metabolism of immune and nonimmune cells. We shall also present and discuss the common procedures for isolation, identification and eradication of a mycoplasma contamination of tissue cultures.

Objective Mycoplasma salivarium is a human oral potential pathogen that preferentially resides in dental plaques and gingival sulci. It has been suggested that this organism may play an etiological role in inflammatory processes in the oral cavity. The aim of this work was to determine whether M. salivarium possesses a potent oxidant scavenging capacity (OSC). Design The OSC of M. salivarium was quantified by a highly sensitive luminal-dependent chemiluminescence assay in the presence of cocktails that induced a constant flux of luminescence resulting from the generation of peroxide, hydroxyl radical (cocktail A) and NO, superoxide and peroxynitrites (cocktail B). Results M. salivarium markedly reduced oxidative stress by scavenging both free reactive oxygen and nitrogen species. The OSC of M. salivarium was much higher than that of other Mycoplasma species. Most of M. salivarium OSC was confined to the cytosolic fraction and was markedly increased in the presence of tannic acid, red blood cells or mucin. The cytosolic OSC of M. salivarium was heat stable and not affected by sodium azide or prolonged proteolysis. However, it was markedly decreased upon dialysis, suggesting that the major reducing activity is not enzymatic but rather, a low molecular weight compound(s). Conclusions The ability of M. salivarium to scavenge oxidants may play a role in the survival and pathogenicity of this microorganism. The enhanced OSC of M. salivarium in the presence of tannic acid, red blood cells or mucin might have a significant importance to assess complex interactions with polyphenols from nutrients, salivary proteins and red blood cells extravasated from injured capillaries during infection and inflammation in oral tissues.

Thede novosynthesized polar lipids ofMycoplasmaspecies 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 someMycoplasmaspecies, 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 moreMycoplasmaspecies 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 ofM. fermentansserves both as a major immune determinants and as a trigger of the inflammatory responses, and the findings that the fusogenicity ofM. fermentanswith host cells is markedly stimulated by lyso-ether lipids, are important steps toward understanding the molecular mechanisms ofM. fermentanspathogenicity.

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-β-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.

Marked cell swelling followed by lysis was observed when Spiroplasma floricola cells were incubated in iso-osmotic solutions of NaCl, KCI, choline chloride or sorbitol in the absence of an energy source. In the presence of an energy source the cells did not swell suggesting that S. floricola relies on an energy-dependent mechanism(s) for cell volume regulation. An ammonium chloride dilution procedure was utilized to generate a pH gradient (inside acid) across the cell membrane of S. floricola cells. The addition of NaCl resulted in an intracellular alkalization suggesting the presence of a Na+/H+ exchange activity. In 22Na+-loaded cells, glucose-dependent 22Na+ extrusion was observed at acidic pH in both the presence and absence of Na+ ions. The extrusion was completely inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μM) and partially inhibited by dicyclohexylcarbodiimide (DCCD, 100 μM) indicating that in S. floricola, Na+ movement is driven by the electrochemical gradient of H+ via a Na+/H+ antiporter. The specific ATPase activity of S. floricola membranes was at least twofold higher than that described in other mollicutes. Activity was Mg2+-dependent over the pH range (6.5-8.5) tested, but was very little affected by Na+ (up to 100 mM). DCCD (25 μM) markedly inhibited both membrane-bound and solubilized ATPase activity, whereas orthovanadate (50 μM) had only a small inhibitory effect. The properties of the enzyme are consistent with a F0F1-ATPase. It is suggested that the enzyme operates in the direction of hydrolysing ATP formed by glycolysis leading to the generation of a δpH, which is the major driving force for the Na+/H+ antiport activity.

Mycoplasma infection can substantially affect the biological properties of cells in vitro. We have devised a method for the selective killing of mycoplasmas, e.g., A. laidlawii, M. fermentans M. hyorhinis and M. arginini , from experimentally infected cell cultures. This approach is based on the differential binding of the lipophilic fluorescent probe Merocyanine 540 followed by illumination with visible light. The efficiency of the procedure depends on the Merocyanine 540 concentration, the intensity of illumination, and the presence of oxygen in the medium. When A. laidlawii contaminated correal endothelial cell cultures were treated simultaneously with Merocyanine 540 and DNA-binding fluorochrome Hoechst 33258 and then illuminated, a significant degree of eradication was observed, even after one cycle of treatment. This combined treatment is therefore recommended as an effective method of purging mycoplasmas from contaminated cultures.

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.

Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells. The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S. floricola membranes because of lipid mixing upon fusion. The progression of S. floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles. Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle. Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures. Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle. Nonetheless, upon aging of S. floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed. A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected. The possible influence of these changes on the fusogenic activity of the cells is discussed.

Characteristics of the fusion of Mycoplasma fermentans (incognitus strain) with cultured lymphocytes were investigated. The rate and extent of fusion were monitored continuously in an assay that measured lipid mixing on the basis of dequenching of a fluorescent probe, octadecylrhodamine (R18), incorporated into mycoplasmas. Fusion of M. fermentans was detected with CD4+ (Molt-3) cells, CD4- (12E1) cells, and primary peripheral-blood lymphocytes. The level of fusion was relatively low (8%-12%). Detection of a similarly low level of fusion by fluorescence microscopy suggested the involvement of a specific lymphocyte subpopulation. After a short lag period, fusion at 37 degrees C proceeded exponentially for approximately 30 minutes and was virtually complete at 60 minutes. Throughout the process, lymphocytes remained intact. Fusion was stimulated by CaCl2 but not by MgCl2; its inhibition by antisera to M. fermentans and by pretreatment of M. fermentans with proteolytic enzymes implied that the mycoplasmas possessed a proteinase-sensitive receptor involved in fusion. Mycoplasmas were rendered nonfusogenic by treatment with the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone; 5 microM) but were unaffected by treatment with chlorpromazine (10 microM) or DCCD (dicyclohexylcarbodiimide; 50 microM); these findings suggested that a proton gradient across the cell membrane is required for fusion.

Mycoplasma infection of cell cultures is widespread and has major detrimental effects on cellular physiology and metabolism. Since cell culture is used extensively, both in research and in industrial production processes, questions of primary concern arise, such as: how can mycoplasma contamination be detected; what are the effects of such contamination on cellular functions; what methods are available for eliminating contamination?

The nucleotide sequence of a 7.8 kbp DNA fragment from the genome of Mycoplasma gallisepticum has been determined. The fragment contains a cluster of nine tightly linked genes coding for the subunits of the M. gallisepticum ATPase. The gene order is I (I-subunit), B (a-subunit), E (c-subunit), F (b-subunit), H (delta-subunit), A (alpha-subunit), G (gamma-subunit), D (beta-subunit) and C (epsilon-subunit). Two open reading frames were identified in the flanking regions; one (ORFU), preceding the I gene, encodes at least 110 amino acids and the other (ORFS), following the C gene, encodes at least 90 amino acids. The deduced amino acid sequences of the various subunits are presented and discussed with regard to the structure, function and differing sensitivity of the M. gallisepticum enzyme to dicyclohexylcarbodiimide and aurovertin. The alpha- and beta-subunits of the F1 portion are well conserved (51% and 65% identity with those of Escherichia coli), whereas the gamma-, delta- and epsilon-subunits, as well as the F0-subunits, show a low percentage identity. Nonetheless, the secondary structure of the F0-subunits show a high degree of similarity to the corresponding subunits of E. coli. Two very strong potential amphipathic alpha-helices are predicted in the delta-subunit and the N-terminus of the b-subunit contains two hydrophobic helical stretches. The possible roles of these structural properties in the close association of the F1 and F0 multisubunit complexes among mycoplasmas are discussed.

Covalent modification ofSpiroplasma floricola membrane proteins by fatty acids was determined by in vivo labeling of the cells with radioactive fatty acids followed by separation on one-dimensional SDS-polyacrylamide gels and visualization by autoradiography. Approximately 25 different proteins were found to be labeled with [3H]-palmitate, whereas almost none were labeled with [3H]-oleate. The radioactivity could not be removed from the palmitoylated membrane proteins by boiling in SDS or by exhaustive extraction with chloroform-methanol (2∶1). Nevertheless, treating the palmitoylated proteins with a 0.1N KOH solution removed approximately 70% of the bound [3H]-palmitate. The major protein-bound fatty acid species were identified, following their release from the protein by chemical cleavage, as palmitic acid and stearic acid (83% and 7.5%, respectively).

Poly(ethylene glycol) (PEG 8000) can induce cell-cell fusion of Mycoplasma capricolum cells, and it can promote the formation of intergeneric hybrids of various Mycoplasma, Acholeplasma and Spiroplasma species. The extent of fusion was quantitatively evaluated by following the dequenching of octadecylrhodamine fluorescent label incorporated into donor cell membranes after their incubation with recepient cells. The results of dequenching experiments were confirmed by electron microscopy, as well as by angle light-scattering measurements. Fusion appeared to require the presence of Mg 2+ , but was completely inhibited by either 0.1% glutaraldehyde or 100 μM chlorpromazine, and was partially suppressed by proteolytic enzymes, carbonyl cyanide- m -chlorophenylhydrazone, or thiol reagents.

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.

We have recently found that membranes of Spiroplasma spp. strain MQ-1 (hereafter referred to as MQ-1) induce both tumor necrosis factor alpha (TNF alpha) secretion by bone marrow macrophages and blast transformation of lymphocytes via a mechanism different from that operated by bacterial lipopolysaccharide (LPS). This report presents evidence indicating that the MQ-1-derived membrane component(s) which activates bone marrow macrophages to secrete TNF alpha is, at least in part, protein. This conclusion is supported by our findings that TNF alpha secretion was reduced following exposure of MQ-1 membranes to elevated temperatures, extreme acidic pH treatment and incubation with protease K or pronase. Furthermore, following lipid extraction of MQ-1 membranes, most of both induction of TNF alpha secretion and blast transformation activities appeared in the 'protein' fraction. When membranes were chromatographed on a phenyl-Sepharose column, two major peaks were obtained, one containing most of the TNF alpha induction activity and the other the mitogenic activity. Neither peak coeluted with the peak of bulk membrane lipids. The possibility that the spiroplasma membrane component inducing TNF alpha secretion is acylated protein is discussed.

The permeability properties of Mycoplasma gallisepticum cells treated with a purified preparation of tetanolysin were investigated by determining the initial swelling rates of cells suspended in an isoosmotic solution of electrolytes or nonelectrolytes. The swelling, initiated by the tetanolysin, depended on the tetanolysin concentration and was markedly affected by the molecular size of the various osmotic stabilizers utilized. Thus, the initial swelling rates in an isoosmotic solution of monosaccharides were much higher than those in isoosmotic solutions of di-, tri-, or tetrasaccharides. Cell swelling induced by tetanolysin was much lower with energy-depleted M. gallisepticum cells, with arsenate-treated cells, or when the membrane potential (delta psi) was collapsed by valinomycin (10 microM) plus KCl (100 mM). Swelling was not affected by the proton-conducting ionophore carbonyl cyanide-m-chlorophenylhydrazone (1 to 10 microM) or by nigericin (5 microM). These results support the concept that the damage induced by tetanolysin is due to the formation of water-filled pores within the membranes of energized M. gallisepticum cells. Such pores allow the diffusion of hydrophilic molecules into the cells and may vary in size, depending on the tetanolysin concentration utilized.