Mycoplasma Hyorhinis Research Articles

Efficient treatment of Mycoplasma hyorhinis contamination in MCF-7 breast cancer cells with doxycyclin

Mycoplasma contamination of continuous cell cultures is one of the major problems in cell culture technology. Only a limited number of compounds are active against those organisms. We first identified the presence of Mycoplasma hyorhinis in our MCF-7 breast cancer cell line. We then show that in these cells, doxycyclin was as effective as Plasmocin against this mycoplasma.

Effect of storage conditions on detection of mycoplasma in biopharmaceutical products

Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples. Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 10(1) CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial titers (10(1) and 10(6) CFU/ml) after storage at 4 degrees C and -30 degrees C were analyzed. During storage, viable organisms were found with little or no reduction in titers after storage for 8 wk at -30 degrees C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred after 4 wk storage for high-dose cultures (10(6) CFU/ml) at 4 degrees C. The titers of viable organisms were diminished over 8 wk at 4 degrees C under aerobic and anaerobic conditions.

Mycoplasmal membrane protein p37 promotes malignant changes in mammalian cells

Evidence of Mycoplasma hyorhinis infection in human gastric cancer tissues has been found in previous work. In this study, we demonstrate that the expression of p37, a membrane lipoprotein of M. hyorhinis, in mammalian cells induces antisenescence, enhances clonogenicity in soft agar, and co-operates with human epidermal growth factor receptor-related 2 to inhibit cell adhesion. Conversely, truncated p37 protein, with the first 28 amino acids deleted from its N terminal, promotes cell senescence. Taken together, our findings suggest that p37 promotes malignant changes in mammalian cells. With the identification of this molecular component, which is responsible for mycoplasma malignancy-promoting activity, it is possible that a better understanding of the relationship between M. hyorhinis infection and human gastric cancers will lead to novel diagnostics and therapeutics.

Epidemiological Investigation of the Prevalence and Features of Postweaning Multisystemic Wasting Syndrome in Japan

To investigate the prevalence and features of postweaning multisystemic wasting syndrome (PMWS) in Japan, an epidemiological study was conducted in 692 weaned pigs with various clinical signs, commonly including wasting or weight loss, collected from 129 swine farms between 2000 and 2003. The presence of PMWS was diagnosed by the detection of characteristic histological lesions and moderate to large amounts of porcine circovirus type 2 (PCV2) antigen within the lesions in multiple lymphoid tissues. Postweaning multisystemic wasting syndrome was positive in 23.4% of pigs (162/692) over the course of the study, and occurred in 50.4% of the farms (65/129). Mortality in 30-120-day-old pigs in the farms positive for PMWS varied from 0.1 to 32.0%. No significant difference in mortality was seen between PMWS-positive and -negative farms (P = 0.1). However, mortality was significantly higher in the PMWS-positive farms where PMWS was diagnosed in more than 50% of the pigs examined compared to farms negative for PMWS (P = 0.02). These findings indicate that PMWS has spread widely in Japan. Moreover it may exist in variable forms in swine farms, including an epidemic form or a subtle endemic or sporadic form. A case-control study suggested that risk factors for the occurrence of PMWS include porcine reproductive and respiratory syndrome (PRRS) pneumonias and Mycoplasma hyorhinis infection.

Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

A Multiplex PCR to Identify Porcine Mycoplasmas Present in Broth Cultures

Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.

Mycoplasma hyorhinis in Taiwan: Diagnosis and isolation of swine pneumonia pathogen

This study attempted to determine whether one multiplex polymerase chain reaction (PCR) is an effective adjunct method for diagnosing Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infection, and whether M. hyorhinis should be considered as an enzootic pneumonia or porcine respiratory disease complex pathogen in Taiwan. To our knowledge, this study is the first to isolate and identify M. hyorhinis as a porcine pathogen in Taiwan. A novel isolation method and a multiplex PCR test were applied to detect and isolate M. hyorhinis. The correlation of M. hyorhinis with swine pneumonia was also examined using a challenge test. Based on weight, 18 pigs were assigned to three groups and housed throughout the study in a specific-pathogen-free (SPF) facility and provided with aseptic feed and water. Groups 1 ( n = 6) and 2 ( n = 6) were challenged with 5 mL M. hyorhinis culture via tracheal intubation on day 1. The M. hyorhinis strains ATIT-1, -3, and-7 were used to infect group 1 and the strain ATCC 27717 was used for group 2. Culture medium was replaced by phosphate-buffered saline in group 3 ( n = 6). All pigs were slaughtered on day 28, and their lungs were removed for examination of lesions. Of the six pigs in group 1 challenged with wild-type strains, two had typical mycoplasma pneumonia lesions. No gross lung lesions were observed in groups 2 and 3. Although further examination is necessary to confirm that wild-type strains can cause pneumonia, it appears that M. hyopneumoniae is no longer the only mycoplasma pathogen implicated in the diagnosis of swine enzootic pneumonia (SEP).

Lipoprotein p37 from Mycoplasma hyorhinis inhibiting mammalian cell adhesion

p37 protein is a membrane lipoprotein of Mycoplasma hyorhinis, and our previous work showed that there was high ratio of M. hyorhinis infection in human gastric carcinoma. To investigate the possible functions of p37 in cancer development, the nucleotide sequence of p37 gene was modified and expressed well in transfected cells. We found that p37 localized at the Golgi apparatus and could be secreted out of the cell. Human gastric cancer cells AGS, after being transfected with the p37 gene, were smaller, more spherical and easy to detach from each other. Their adhesion to matrix was also diminished and cytoskeleton in these stable p37 AGS cell was rearranged and transcription co-factor beta-actin was transferred to nucleolus with down-regulation of ICAM-1 and integrin beta1. These findings will be helpful for us to elucidate the effects of p37 on eukaryotic cells as well as to better understand the potential relationship between cancer and mycoplasma infection.

Impact of Mycoplasma hyorhinis infection on L-arginine metabolism: Differential regulation of the human and murine iNOS gene

Infection with mycoplasma is a common problem in cell cultures, with Mycoplasma hyorhinis being the predominant species. Here we investigate the effect of M. hyorhinis infection on L-arginine metabolism, with focus on iNOS-mediated NO synthesis in murine keratinocytes and the human colon cancer cell line DLD-1. iNOS and arginase are L-arginine-metabolizing enzymes involved in the regulation of inflammatory processes, with NO contributing to innate immunity. In murine cells, M. hyorhinis infection enhances cytokine-induced iNOS expression and augments iNOS activity, whereas in the absence of cytokines it causes de novo induction of iNOS mRNA without subsequent translation into iNOS protein. In turn, arginase-1 mRNA expression is diminished in M. hyorhinis-infected murine keratinocytes, resulting in decreased arginase activity. One of the underlying upstream mechanisms is NF-kappaB activation. In contrast, in human cells neither iNOS mRNA nor protein expression is affected by M. hyorhinis infection, but NO synthesis is enhanced, which may be caused by increased L-arginine import. This demonstrates that infection with M. hyorhinis leads to different effects on gene regulation of the murine and human iNOS gene. Our study underlines the importance of routine checking of cell cultures for mycoplasma contamination, particularly in studies on NO-mediated effects or inflammatory processes.

P37 induces tumor invasiveness

Previous studies have shown a statistically significant correlation between human carcinomas and monoclonal antibody detection of a Mycoplasma hyorhinis-encoded protein known as p37. A potential mechanism of p37 is that it might promote invasion and metastasis. Recombinant p37 enhanced the invasiveness of two prostate carcinoma and two melanoma cell lines in a dose-dependent manner in vitro, but did not have a significant effect on tumor cell growth. Furthermore, the increased binding to cell surfaces and the enhanced invasive potential of cancer cells from exposure to p37 could be completely reversed by preincubation of the cancer cells with an anti-p37 monoclonal antibody. Sequence comparisons, followed by three-dimensional molecular modeling, revealed a region of similarity between p37 and influenza hemagglutinin A, a sialic acid-binding protein that plays a critical role in viral entry. Binding of p37 to prostate carcinoma cells was found to be at least partially sialic acid dependent because neuraminidase treatment decreased this binding. Taken together, these observations suggest that M. hyorhinis can infect humans and may facilitate tumor invasiveness via p37. These results further suggest that p37 may be a molecular target for cancer therapy.

Deconstructing PTI-1: PT1-1 is a truncated, but not mutated, form of translation elongation factor 1A1, eEF1A1

The prostate tumor-inducing gene 1 ( PTI-1) transcript is detected in various human carcinoma cells. PTI-1 is reported to consist of a 5′ untranslated region (5′ UTR) homologous to mycoplasma 23S rRNA and a coding region corresponding to a truncated and mutated form of the translation elongation factor 1A, eEF1A. We have found that the PTI-1 transcript may encode a truncated, but not mutated, form of the human isoform eEF1A1. Additionally, the 5′ UTR sequence of PTI-1 from genomic DNA of different cell lines and blood samples varies from the original sequence. This 5′-UTR region of PTI-1 presents a fusion of E. coli and Mycoplasma hyorhinis 23S rRNA. We have overexpressed the potential PTI-1 protein in E. coli and various human cell lines. The resulting protein could be detected by western blotting using anti-eEF1A antibodies. However, we were unable to detect the PTI-1 protein in LNCaP cell extracts. The potential roles of the PTI-1 protein in carcinogenesis and the origin of the PTI-1 gene in the human genome are discussed.

Impact of Mycoplasma hyorhinis infection on l-arginine metabolism: differential regulation of the human and murine iNOS gene

Infection with mycoplasma is a common problem in cell cultures, with Mycoplasma hyorhinis being the predominant species. Here we investigate the effect of M. hyorhinis infection on L-arginine metabolism, with focus on iNOS-mediated NO synthesis in murine keratinocytes and the human colon cancer cell line DLD-1. iNOS and arginase are L-arginine-metabolizing enzymes involved in the regulation of inflammatory processes, with NO contributing to innate immunity. In murine cells, M. hyorhinis infection enhances cytokine-induced iNOS expression and augments iNOS activity, whereas in the absence of cytokines it causes de novo induction of iNOS mRNA without subsequent translation into iNOS protein. In turn, arginase-1 mRNA expression is diminished in M. hyorhinis-infected murine keratinocytes, resulting in decreased arginase activity. One of the underlying upstream mechanisms is NF-kappaB activation. In contrast, in human cells neither iNOS mRNA nor protein expression is affected by M. hyorhinis infection, but NO synthesis is enhanced, which may be caused by increased L-arginine import. This demonstrates that infection with M. hyorhinis leads to different effects on gene regulation of the murine and human iNOS gene. Our study underlines the importance of routine checking of cell cultures for mycoplasma contamination, particularly in studies on NO-mediated effects or inflammatory processes.

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.

Recognition of Mycoplasma hyorhinis by CD99-Fc molecule.

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.

Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

The pyruvate dehydrogenase complex of Mycoplasma hyopneumoniae contains a novel lipoyl domain arrangement

The genes encoding the pyruvate dehydrogenase (PDH) complex ( pdhA, pdhB, pdhC and pdhD) from Mycoplasma hyopneumoniae have been cloned and sequenced. The genes are arranged into two operons, designated pdhAB and pdhCD, which are not found together in the chromosome. The pdhA, pdhB, pdhC and pdhD genes encode proteins of predicted molecular masses of 44.2 kDa (pyruvate dehydrogenase major subunit; E1α), 36.6 kDa (pyruvate dehydrogenase minor subunit; E1β), 33.1 kDa (dihydrolipoyl acetyltransferase; E2) and 66.3 kDa (dihydrolipoyl dehydrogenase; E3), respectively. Sequence analysis of the pdhCD operon revealed the presence of a lipoyl-binding domain in pdhD but not in pdhC. The lipoyl domain is believed to act as a “swinging arm” that spans the gaps between the catalytic domains of each of the subunits. Portions of the N-terminal regions of pdhA and pdhD were expressed as 6×His-tag fusion proteins in Escherichia coli and purified by nickel affinity chromatography. The purified proteins were used to raise antibodies in rabbits, and Western blot analysis was performed with the polyclonal rabbit antiserum. Both the pdhA and pdhD genes were expressed among various strains of M. hyopneumoniae as well as the porcine mycoplasmas, Mycoplasma hyorhinis and Mycoplasma flocculare. Southern hybridisation analysis using probes from pdhA and pdhD detected one copy of each gene in the chromosome of M. hyopneumoniae. Since previous studies have shown pyruvate dehydrogenase activity in M. hyopneumoniae [J. Gen. Microbiol. 134 (1988) 791], it appears likely that a functional lipoyl-binding domain in the N terminus of PdhC is not an absolute prerequisite for pyruvate dehydrogenase enzyme activity. We hypothesise that the lipoyl-binding domain of PdhD is performing the enzymatic function normally attributed to the PdhC lipoyl-binding domain in other organisms. Searches of pyruvate dehydrogenase gene sequences derived from other Mycoplasma species showed that a putative lipoyl domain was absent in the pdhC gene from Mycoplasma pulmonis. However, like other bacterial species, pdhC gene sequences from Mycoplasma capricolum, Mycoplasma genitalium and Mycoplasma pneumoniae contain a putative lipoyl domain.

Characterization of gentamicin-resistant Mycoplasma hyorhinis

A study was conducted to determine if a gentamicin-resistant strain of mycoplasma could be developed for use in validating current mycoplasma detection methods for biologic product harvest cell culture fluid (CCF) containing gentamicin. A strain of gentamicin-resistant Mycoplasma hyorhinis was isolated and characterized. The study showed that this organism was similar to the wild-type strain in all ways examined except gentamicin resistance. Both strains of mycoplasma (the gentamicin resistant and the wild-type) exhibited comparable growth patterns and showed 100% homology based on DNA sequencing and analysis of a 464-bp PCR product. Also, analysis using species-specific antisera identified both strains as M. hyorhinis. Two commonly used lot release mycoplasma detection methods (culture and DNAF) consistently detected mycoplasmas in spiked biologic product harvest CCF containing gentamicin but not in unspiked samples. This study demonstrates the first isolation and characterization of a gentamicin-resistant M. hyorhinis that can be used to validate mycoplasma detection methods for biologic product harvest CCF containing gentamicin.

Protein P37 of mycoplasma hyorhinis induces secretion of TNF-α from human peripheral blood mononuclear cells

High mycoplasmal infection ratio in gastric cancer tissues suggests a possible association between mycoplasma infection and tumorigenesis. Because TNF-α plays an important role in carcinogenesis caused by microbes infection and P37 is a major immunogen of mycoplasma hyorhinis (M. hyor.), investigating whether P37 could induce expression and secretion of TNF-α will be very significant to elucidate the possible molecular mechanism of gastric carcinogenesis involved with M. hyor. At the present study, we cloned full gene of p37 by PCR and mutated the 7 codes of TGA into TGG firstly, then expressed the P37 protein successfully with pGEX-4T-1 vector in E. coli, which was verified with Western blot. By RT-PCR and sensitive L929 cell toxic assay, we found that P37 protein could induce expression and secretion of TNF-α from human peripheral blood mononuclear cells, and the inducing activity of P37 could be dramatically blocked by McAb PD4. These results suggest that the induction of TNF-α secretion by P37 probably plays an important role in diseases caused by M. hyor. infection and needs to be further investigated.

Infection by Mycoplasma hyorhinis strongly enhances uptake of antisense oligonucleotides: a reassessment of receptor-mediated endocytosis in the HepG2 cell line.

This paper shows that the approximately 66 kDa band, previously isolated from the HepG2 cell line as an oligonucleotide (ON) plasma membrane 'receptor', is induced by Mycoplasma infection. Moreover, this band has been identified as the invariant membrane protein of Mycoplasma hyorhinis, p70, based on ribosomal DNA sequencing combined with ON ligand blotting after p70 immunoprecipitation by a monoclonal antibody. Whereas antibiotic treatment of infected HepG2 cells strongly decreased ON capture, as measured by a biochemical assay, conversely, deliberate infection of HeLa cells with M.hyorhinis dramatically promoted ON uptake but did not affect receptor-mediated endocytosis of transferrin. This was confirmed by confocal microscopy of infected HepG2 cells, which also showed an indistinguishable labelling pattern after exposure of living cells to fluorescent ON and after p70 immunolabelling in permeabilised fixed cells. We propose that ON binds to p70 on M.hyorhinis attached at the cell surface, after which the complex is internalised by 'piggy-back' endocytosis.

Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinis

BackgroundMycoplasma contaminations are a recurrent problem in the use of cultured cells, including human cells, especially as it has been shown to impede cell cycle, triggering cell death under various conditions. More specific consequences on cell metabolism are poorly known.ResultsHere we report the lack of significant consequence of a heavy contamination by the frequently encountered mycoplasma strain, M. hyorhinis, on the determination of respiratory chain activities, but the potential interference when assaying citrate synthase. Contamination by M. hyorhinis was detected by fluorescent imaging and further quantified by the determination of the mycoplasma-specific phosphate acetyltransferase activity. Noticeably, this latter activity was not found equally distributed in various mycoplasma types, being exceptionally high in M. hyorhinis.ConclusionWhile we observed a trend for respiration reduction in heavily contaminated cells, no significant and specific targeting of any respiratory chain components could be identified. This suggested a potential interference with cell metabolism rather than direct interaction with respiratory chain components.