Strains Of Mycoplasma Hyorhinis Research Articles

Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists

Calpain (intracellular Ca2+-dependent protease) and calpastatin (calpain specific endogenous inhibitor) are widely distributed in biological systems, and have been implicated in many cellular physiological and pathological processes. Calpastatin level is of central importance to the control of calpain activity. We demonstrated for the first time that calpastatin is overexpressed in mycoplasma-contaminated cultured cells (SH-SY5Y cells that are infected by a strain of Mycoplasma hyorhinis (NDMh)). We have found that the calpastatin-upregulating activity resides in the mycoplasmal membrane lipoproteins, and is associated with NF-κB activation. Calpain-promoted proteolysis is attenuated in the NDMh lipoprotein-treated cells. Here we show that the NDMh lipoproteins promoted an increase in calpastatin in SH-SY5Y cells via the TLR2/TAK1/NF-κB pathway. The synthetic mycoplasmal lipopeptide MALP-2 and the bacterial lipopeptide PAM3CSK4 (TLR2 agonists) also promoted calpastatin upregulation. LPS (TLR4 agonist) activated NF-κB without calpastatin increase in the cell. In contrast, lipoteichoic acid (TLR2 agonist) upregulated calpastatin not via NF-κB activation, but via the MEK1/ELK1 pathway. Zymosan and peptidoglycan, TLR2 agonists that lack lipids, did not induce calpastatin upregulation. Cell treatment with a calpastatin-upregulating agonist (lipoteichoic acid) led to the attenuation of Ca2+-promoted calpain activity, whereas agonists that do not upregulate calpastatin (LPS, Zymosan) were ineffective. Overall, the results indicate that in these non-immune cells, calpastatin is upregulated by TLR2-agonists containing lipids, with more than one downstream pathway involved. Such agonists may be useful for studying mechanisms and factors involved in calpastatin regulation. In addition, suitable TLR2 agonists may be of interest in devising treatments for pathological processes involving excessive calpain activation.

Neuroprotective effects of Mycoplasma hyorhinis against amyloid-β-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-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.

Macrolides and Lincomycin Susceptibility of Mycoplasma hyorhinis and Variable Mutation of Domain II and V in 23S Ribosomal RNA

A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.

Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.

Macrolide susceptibility of Mycoplasma hyorhinis isolated from piglets

Twenty strains of Mycoplasma hyorhinis were investigated for their in vitro susceptibilities to 15 antimicrobial agents by broth and agar dilution methods. Two of the 20 field strains showed low susceptibility to 14- and 16-membered macrolide antimicrobial agents tested. The two field strains were considered inducibly resistant to macrolides.

Detection of mycoplasma contamination through modulation (stimulation or inhibition) of thymidine incorporation by unstimulated mouse spleen cells

In the present report we describe a rapid ans sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture supernatants to modulate [ 3H]TdR incorporation by unstimulated mouse splenocytes. Several mycoplasma species ( Mycoplasma orale, culturable and non-culturable strains of Mycoplasma hyorhinis) inhibited [ 3H]TdR incorporation and permitted the detection of some contaminated cell cultures that would otherwise have escaped detection in assays measuring [ 3H]TdR incorporation by mitogen-stimulated splenocytes. On the other hand, several other mycoplasma species ( Mycoplasma arginini, Mycoplasma hominis) strongly enhanced [ 3H]TdR incorporation by unstimulated splenocytes. This enhancement was optimally detectable on day 2 after initiation of the cultures. The sensitivity of the assay was determined for a mycoplasma species (culturable M. hyorhinis) that inhibited as well as for one ( M. arginini) that enhanced [ 3H]TdR incorporation. In both cases, the sensitivity was such that 1−3× 10 2 mycoplasma colony-forming units (CFU) could be detected.

Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.

Identification and characterization of proteinase K-resistant proteins in members of the class Mollicutes

Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.

Survival of Mycoplasma hyorhinis in trypsin solutions

The survival of four strains of Mycoplasma hyorhinis in stock solutions of trypsin was tested at 22, 4 and −15°C. Low (10 4–10 5 cfu/ml) and high (10 6–10 7 cfu/ml) initial concentrations of each strain were used, each was tested three times. A regular decrease of low and high concentrations (1 log in 10 and 20 min, respectively) was seen at 22°C. At 4°C the low concentrations showed a reduction of about 1 log/h, while apart from one strain high concentrations hardly decreased during the first 6 h and the survival time ranged from 24 to more than 30 h at the end of which there was a reduction of 4 logs. At −15°C low concentrations survived up to 1 week in only one of the three tests, high concentrations survived for more than 12 weeks (reduction 3 logs). These latter results suggest that mycoplasmas may be present in trypsin as clumps, which deteriorate very slowly. A study was also performed to compare the sensitivity of different cultural procedures for detecting mycoplasmas.

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.

Serological Comparison of Type Strains of Porcine, Bovine, and Ovine Mycoplasmas with Atypical Colony Morphology

The type strains of Mycoplasma hyopneumoniae, M. flocculare, M. dispar, and M. ovipneumoniae, all characterized by nipple-less colonies on solid media, were compared serologically. By indirect hemagglutination and by complement fixation tests they were found to constitute a related group. By crossed immunoelectrophoresis a sharing of common antigens was demonstrated, whereas no cross reactivity was noted by the metabolism inhibition test. The type strains of Mycoplasma hyorhinis and Mycoplasma bovirhinis were included in the study for comparison. Although some cross reaction was noted, they appeared just moderately related to the nipple-less group as well as to each other.

Some characteristics of mycoplasma virus Hr 1, isolated from and infecting Mycoplasma hyorhinis. Brief report.

Mycoplasma virus Hr 1 is a short tailed bacteriophage with a polyhedral head about 34 nm across and a tail about 14 nm long. It produces plaques on some strains of Mycoplasma hyorhinis.

The isolation of a ‘non-cultivable’ strain of Mycoplasma hyorhinis from a mammalian live virus vaccine

The isolation of a ‘non-cultivable’ strain of Mycoplasma hyorhinis from a mammalian live virus vaccine

Covert infection of a mouse mammary tumor cell line with Mycoplasma hyorhinis: cosedimentation with mouse mammary tumor virus in sucrose density gradients.

Supernatant fluids from cultures of a mouse mammary tumor (MT) cell line were found to produce a specific cell detachment effect when inoculated into HeLa cells. The cell detachment factor (CDG) responsible for this effect was examined. Repeated attempts to cultivate this CDF in bacteriologic, fungal, and mycoplasma media were unsuccessful. However, using the DNA fluorochrome staining technique and specific immunofluorescent staining procedures, the CDF was identified positively as a noncultivable strain of Mycoplasma hyorhinis. It was also noted that this CDF could be labeled with [3H]uridine in MT cell cultures, concentrated, and banded at a density of 1.18 g/cm3 when centrifuged to equilibrium in a 20 to 60% sucrose gradient. Using a multiple antibiotic treatment regimen, the MT cells were "cured" of the M. hyorhinis contaminant. Re-infection of these cells with an exogenous strain of M. hyorhinis resulted in the same cell detachment effect, and this strain when labeled with [3H]uridine also sedimented at a density of 1.18 g/cm3. The salient feature of these studies is that M. hyorhinis sediments at the same density of mouse mammary tumor virus (MMTV) in sucrose density gradients. This was demonstrated by sucrose density gradient analyses of a purified sample of MMTV, assaying for reverse transcriptase activity, and a [3H]uridine labeled sample of the M. hyorhinis present in the MT cell cultures.

Isolation of Mycoplasma hyorhinis and Mycoplasma fermentans from cell cultures

A mycoplasma medium with boiled blood and sucrose (Herderschee medium), a commercially available (Difco) PPLO medium and an enriched trypticase soy broth medium were tested for their suitability for isolation and subcultivation of a fastidious strain of Mycoplasma hyorhinis (DBS 1050) and a M. fermentans strain (IM 1) isolated on Herderschee medium. The mycoplasma strains were maintained in VERO-cell cultures, for which M. fermentans proved to be very pathogenic. It was therefore, stored at −70°C after two passages in the cell culture. M. hyorhinis was inoculated in various VERO-cell passages and subsequently propagated in the cells. On Herderschee and Difco media M. hyorhinis strain DBS 1050 could always be isolated from the cell cultures one to four days after trypsinization of the cells. Recovery succeeded better from frozen (−70°C) cell suspensions than from supernatant cell culture medium, and also better after direct inoculation on agar medium than after inoculation in broth. Primary isolations on agar medium could nearly always be subcultivated. The concentration of organisms detected on agar medium at primary isolation and the facility of subsequent subcultivation in mycoplasma medium seemed to depend partly on the number of mycoplasma passages in the cell culture, and partly on the subline of the cell culture used for inoculation. M. fermentans strain IM 1 could be isolated on Herderschee medium and on Difco medium with sucrose from supernatant cell culture medium as well as from frozen cell suspensions and equally well from one to seven days after trypsinization of the cell culture, not just after trypsinization. Both mycoplasma strains were only detected after anaerobic incubation (5% CO2 in N2). They could be cultivated as well when 2·5% instead of 10% (w/v) yeast extract was incorporated in the media. The yeast extract could be boiled and heated up to 120°C without impairing its growth promoting quality. Therefore, nicotinamide adenine dinucleotide is dispensable for these two strains. Neither mycoplasma strain could be cultivated on enriched trypticase soy broth medium. The implication of these observations when testing cell cultures and live virus vaccines for contamination with mycoplasma are discussed.

Cultivation of formerly noncultivable strains ofMycoplasma hyorhinis

Growth on axenic agar medium is one of several characters by which mycoplasmas are defined. In apparent contradiction of the definition, DBS 1050 and other noncultivable strains ofMycoplasma hyorhinis do not grow on axenic medium but grow in cell culture. Our results show that BHK-21 cell extracts support DBS 1050 growth in appropriate medium. An inhibition assay, based on a virus neutralization format, shows that a variety of common medium ingredients inhibit DBS 1050 growth. The most potent activity was found in yeast extract. All other noncultivable strains ofM. hyorhinis tested have a yeast extract sensitivity, while cultivable strains do not. The apparent cell dependence of DBS 1050 can be attributed to growth inhibition due to factors present in a wide variety of peptones and extracts commonly used in medium; preferential growth in cell cultures is due to the absence of effective levels of these factors. Data are not available to determine if cell cultures provide growth factors not found in standard medium. The infraspecific taxon,M. hyorhinis cultivar α, is proposed for formerly noncultivable strains ofM. hyorhinis.

Evaluation of Some Serological Techniques for The Identification of Mycoplasma Hyorhinis and Mycoplasma Suipneumoniae

Eighteen strains of Mycoplasma hyorhinis and a strain of Mycoplasma suipneumoniae were tested in 4 serological tests, i. e., disc growth inhibition, metabolic inhibition, indirect haemagglutination and indirect epi-immunofluorescence. Only with immunofluorescence could all tested strains of M. hyorhinis be shown; no cross-reactions between M. hyorhinis and M. suipneumoniae could be detected. The other tests failed in many cases to identify strains of the same species, and they gave cross-reactions between M. hyorhinis and M. suipneumoniae.

The antigenic differentiation of seven strains of Mycoplasma hyorhinis by growth-inhibition, metabolism-inhibition, latex-agglutination, and polyacrylamide-gel-electrophoresis tests.

SUMMARY Seven strains of Mycoplasma hyorhinis obtained from different geographic areas were compared by growth-inhibition, metabolism-inhibition and latex-agglutination tests. Although serologically interrelated, the strains showed differences ranging from slight to profound, and therefore division into subtypes was suggested. Statistical analysis by the coefficient of concordance and by the rank correlation coefficient supported the finding that all three sero-logical methods are probably equally suitable for serological differentiation. Polyacrylamide gel electrophoresis revealed no appreciable differences among the strains.

The Ultrastructure of Mycoplasma hyorhinis in Broth Culture

Summary A pathogenic strain of Mycoplasma hyorhinis in the logarithmic phase of growth in broth culture was examined by electron microscopy and found to have a structure typical of the genus. The organism reproduced by binary fission and by budding to form elementary bodies.

Identification of Mycoplasma Hyorhinis

When fresh unheated horse serum was used in mycoplasma agar medium, it was possible to type both laboratory and freshly isolated strains of Mycoplasma hyorhinis using the disk growth inhibition test. Fifteen freshly isolated strains of M. hyorhinis , consisting of ten serum resistant and five serum sensitive strains, could be typed using this method. When commercial unheated horse serum was used in the agar medium only six of the 15 strains could be typed using the disk growth inhibition test. The combination of fresh horse serum in the agar medium and M. hyorhinis antiserum-impregnated disks appeared to have a synergistic effect and inhibited the growth of all M. hyorhinis cultures on agar medium. Quand on employait un sérum frais non chauffé de cheval dans un milieu de gélose contenant du mycoplasme, on pouvait typer les souches du laboratoire et celles fraîchement isolées de M. hyorhinis en employant la méthode du disque d’inhibition de la croissance. Quinze souches fraîchement isolées de M. hyorhinis , constituées par dix souches résistantes au sérum et cinq sensibles, pouvaient être typées en employant cette méthode. Quand on employait dans le milieu de gélose du sérum de cheval commercial non chauffé, on pouvait typer seulement six des quinze souches par la méthode du disque d’inhibition de croissance. La combinaison de sérum frais de cheval dans le milieu de gélose et de disques imprégnés d’antisérum contre M. hyorhinis semblait avoir un effet de synergie et inhibait la croissance de toutes les cultures de M. hyorhinis en milieu de gélose. Wenn frisches, nicht erhitztes Pferdeserum für Mycoplasmaagarkulturböden verwendet wurde, war es möglich, unter Verwendung des “Disk growth inhibition” -tests im Laboratorium gezogene und frisch kultivierte Stämme von Mycoplasma hyorhinis zu differenzieren. Mit dieser Methode konnten 15 frisch kultivierte Stämme von M. hyorhinis isoliert werden, die aus zehn serumresistenten und fünf auf Serum reagierenden Stämmen bestanden. Wenn industriel geliefertes, nichte erhitztes Pferdeserum im Agarkulturmedium verwendet wurde, konnten mit dieser Methode nur sechs von 15 Stämmen identifiziert werden. Die Kombinierung von frischem Pferdeserum im Nährboden mit Scheibchen, die mit M. hyorhinis-Antiserum imprägniert waren, schien eine synergistische Wirkung zu haben und das Wachstum aller M. hyorhinis -Kulturen im Agarnährboden zu verhindern. Cuando se utilizó suero de caballo, fresco y sin calentar, en un medio de agar mycoplasma, fué posible tipificar tanto las cepas de laboratorio como las aisladas recientemente de Mycoplasma hyorhinis , utilizando la prueba de inhibición del crecimiento en disco. Utilizando este método se pudieron tipificar quince cepas aisladas recientemente de M. hyorhinis , comprendiendo diez cepas resistentes al suero y cinco sensibles al suero. Cuando se utilizó suero de caballo comercial no calentado en el medio de agar sólo se pudieron tipificar seis de las quince cepas, usando el test de inhibición del crecimiento en disco. La combinación del suero de caballo fresco en el medio de agar y los discos impregnados de antisuero de M. hyorhinis pareció mostrar un efecto sinérgico e inhibió el crecimiento de todos los M. hyorhinis en cultivo en un medio de agar.