Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinis
© Darin et al; licensee BioMed Central Ltd. 2003
Received: 20 August 2003
Accepted: 04 November 2003
Published: 04 November 2003
Mycoplasma 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.
Here 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.
While 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.
The mollicutes (lat., molis, soft; cutis, skin) or mycoplasmas, with over 100 different species, are the smallest self-replicating organisms known at present and constitute a distinct class within the prokaryotes characterized by their lack of a rigid cell wall. They can be classified into fermentative strains, which gain energy by fermentation of carbohydrates and non-fermentative strains that are unable to metabolize carbohydrates via glycolysis. The mycoplasmas are extra cellular parasites usually attached to the external surface of cells, but can also penetrate these . In humans, M. pneumoniae is a frequent cause of respiratory infections, and is at the origin of approximately 20% of all community-acquired pneumonias . The mycoplasmas may also lead to genitourinary and neonatal infections . In addition, mycoplasmas have been implicated in the pathogenesis of AIDS  and rheumatoid arthritis , although their precise contribution is still under debate. Being 'minimal cells', mycoplasma have also been used to investigate the machinery of self-replicating organisms .
Beside health problems, mycoplasma contamination constitutes one frequent problem when studying cultured cells (estimated frequency varying from 5 to 35%). The strains M. hyorhinis, M. orale, M. arginini, M. fermetans, M. hominis and Acholeplasma laidlawii represent 90–95% of the contaminating isolates [7, 8]. Contamination is initially often difficult to detect because the contaminated culture grows well and appears normal by ordinary light microscopy. In addition, mycoplasma is highly contagious and can rapidly spread through the cell stocks. The possible consequences of mycoplasma infection for the host-cells are multiple and variable, ranging from no apparent effect to extensive changes with inhibition of cell proliferation, induction of apoptosis, induction of cytokines and oxidative radicals, and malignant transformation [9–12]. There is also a possibility that mycoplasmal biological activities may erroneously be interpreted as being of host origin .
The consequences of mycoplasma-contamination on the host cell metabolism are not well established. In mycoplasma-infected individuals, a decrease of cell respiration, dehydrogenase activities and ATP content have been described in tracheal cell explants and the cytochrome c oxidase (COX) activity has been claimed to be decreased in muscle tissue [14, 15]. In cultured cells, the consumption of the nutrients in the medium may affect the cell metabolism by interfering with deoxyribonucleic acid, ribonucleic acid and protein synthesis . The aim of this study was to evaluate the in vitro consequences of cultured skin fibroblasts contamination by one frequent mycoplasma strain, M. hyorhinis, on mitochondrial enzyme determination. In the course of this study, M. hyorhinis was quantified by a new and convenient approach using the assay of the phosphate acetyltransferase activity, absent from human cultured skin fibroblasts.
Respiratory chain enzyme activities in mycoplasma contaminated fibroblasts before and after antibiotic treatment. Values are means ± 1 SD of values obtained independently on 4 different fibroblast cultures. Data between brackets represent values for uninfected, Ciprofloxacin-naïve, fibroblasts (n > 400). NQR, NADH quinone oxidoreductase; SCCR, Succinate cytochrome c reductase; GPCCR, glycerol-3-phosphate cytochrome c reductase; QCCR, quinol cytochrome c reductase; COX, cytochrome c oxidase; CS, citrate synthase; SD, Standard deviation.
Fibroblasts heavily contaminated with M. hyorhinis
Fibroblasts after Ciprofloxacin treatment
Enzyme activity (nmol/min/mg protein)
(n = 4)
(n = 4)
20.2 ± 3.8
15.8 ± 0.8
22.0 ± 13.6
19.2 ± 3.0
7.8 ± 3.1
6.7 ± 1.8
70.9 ± 25.3
66.6 ± 15.3
71.1 ± 25.3
49.7 ± 8.0
31.1 ± 6.2
43.3 ± 7.7
18.8 ± 7.3
15.2 ± 4.2
Enzyme activty ratios
3.7 ± 1.14
2.7 ± 0.84 (3.1 ± 0.2)
9.3 ± 0.84
8.0 ± 3.0 (7.5 ± 0.4)
1.0 ± 0.21
0.8 ± 0.29 (0.7 ± 0.05)
3.7 ± 1.15
3.5 ± 0.94 (4.0 ± 0.2)
9.3 ± 1.5
10.5 ± 3.9 (11.7 ± 0.5)
2.6 ± 0.61
2.9 ± 0.64 (2.2 ± 0.1)
4.2 ± 2.0
3.6 ± 1.6 (2.8 ± 0.2)
6.5 ± 2.0
2.8 ± 0.43 (3.4 ± 0.6)
9.9 ± 2.4
7.6 ± 0.49 (10.1 ± 1.6)
1.6 ± 0.38
2.7 ± 0.61 (2.1 ± 0.4)
Phosphate acetyltransferase activity
Phosphate acetyltransferase activity in different mycoplasma species. Numbers are the mean values of triplicates. M. fermantans 1 and 2 represent two different batches. DTNB, dithionitrobenzoic acid.
Phosphate acetyltransferase activity (nmol DTNB reduced/min/mg protein)
M. fermantans 1
M. fermantans 2
The contamination by mycoplasmas is a frequent problem encountered when studying cultured cells. The mycoplasmas may have a myriad of different effects upon the infected host cells, which may moreover depends on the mycoplasma species. However, all the mollicutes examined so far have truncated respiratory systems, lacking a complete tricarboxylic acid cycle and having no quinones or cytochromes, ruling out oxidative phosphorylation as an ATP-generating mechanism . Our analysis of RC activity in four human skin fibroblast cell lines heavily contaminated by M. hyorhinis indicates a potential mild decrease of cell respiration and substrate oxidation in the mycoplasma contaminated cells, but no significant effect on any of the RC components. Slight decrease of cell respiration might result from a competition in the use of intermediate substrates resulting from proliferating mycoplasmas in the cell cytosol.
The studied mycoplasma, M. hyorhinis, is a fermentative mollicute usually inhabiting the nasal cavity of swine and is one of the most common cell culture contaminants encountered, probably originating from the bovine sera . The mechanisms of ATP-generation in the mollicutes are not fully understood, but among a number of potential energy-yielding mechanisms in mollicutes is the acetate kinase-ATP-generating pathway . In this pathway acetyl-phosphate needs to be produced through the action of phosphate acetyltransferase on acetyl-CoA:
Although these enzymes have been reported to be commonly found in both fermentative and non-fermentative mollicutes , we found phosphate acetyltransferase mostly active in the two fermentative species (M. hyorhinis and M. fermentans). The enzyme was indeed particularly active in M. hyorhinis with two major consequences. On one hand, the activity of this enzyme can be confused with the activity of the citrate synthase, if this latter activity is not carefully checked to be dependent on oxaloacetate. On the other hand, measurement of the phosphate acetyltransferase activity represents a quite sensitive, convenient, and costless tool to detect the presence of M. hyorhinis in biological samples, particularly in human cultured cells where it represents a common contaminant. Its routine measurement along with citrate synthase assay may prove to be a valuable supplement to the diagnostic arsenal of mycoplasma detection.
The above data indicate that M. hyorhinis results in a potential mild decrease of cell respiration and substrate oxidation in the mycoplasma contaminated cells, but does not have any significant effect on the RC components. With the noticeable exception of citrate synthase, our study indicates that contamination by M. hyorhinis should not influence routine diagnostic procedures used to detect mitochondrial defects in cultured skin fibroblasts.
Mycoplasma detection and species identification
As a consequence of reduced cell proliferation and increased cell mortality, mycoplasma contamination was suspected in four human fibroblast cell lines and detection initially performed with the fluorescent Hoechst 33258 stain using the Mycoplasma stain kit (Sigma-Aldrich Co. Ltd, Irvine, UK) according to the manufacturer's description. The identification of M. hyorhinis as the contaminating species was carried out using molecular typing by PCR-based methods [22, 23].
Fibroblast cultures were established from skin biopsies obtained from 3-year to 77-year old individuals for diagnostic purpose but no evidence of RC dysfunction. Cells were cultured in RPMI 1640 (Life technologies SARL, Cergy Pontoise, France) supplemented with glutamax (446 mg/l), 10% foetal calf serum, 100 μg/ml streptomycin, 100 IU/ml penicillin, 200 μM uridine and 2.5 mM sodium pyruvate, at 37°C under standard conditions . When indicated, cells were treated with 20 μg/ml Ciprofloxacin (Bayer, Leverkusen, Germany) for 14 days and culture medium changed every 3–4 day. Ciprofloxacin treatment is considered as a safe and effective method for elimination of cell culture mycoplasmas .
Cell respiration and enzyme activities
Polarographic measurements were performed on fresh cells before and after 1 week of the antibiotic treatment. Cell pellets frozen on the same occasion were thawed and used for spectrophotometric measurements, which were performed blindly. Intact fibroblast respiration and mitochondrial substrate oxidation with malate and glutamate and with succinate (Figure 1) were polarographically studied in cell suspensions of digitonin-permeabilized fibroblasts as previously described . The activity of the RC complexes, NADH-ubiquinone reductase (NQR, complex I), succinate-cytochrome c reductase (SCCR, complex II-III), glycerol-3-phosphate cytochrome c reductase, (GPCCR, glycerol-3-Phosphate dehydrogenase + complex III), decylubiquinol-cytochrome c reductase (QCCR, complex III), cytochrome c oxidase (COX, complex IV), mitochondrial ATP synthetase (ATPase, complex V) were spectrophotometrically measured on freeze-thaw permeabilized fibroblasts in cell suspension as previously described [26, 27]. The NQR, and ATPase activity were measured in 1 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 0.8 mM NADH, 1 mM KCN, 50 μM decylubiquinone for NQR assay, subsequently added with 3 μM rotenone, 5 mM MgCl2, 10 mM KCl, 2 mM PEP, 0.5 mM ATP, 4 IU lactic dehydrogenase and pyruvate kinase for ATPase assay. Cytochrome c reductase activities (SCCR, GCCR, QCCR) were measured in 1 ml of 10 mM KH2PO4 (pH 7.8), EDTA 2 mM in the presence of 1 mM KCN, 3 μM rotenone and 0.2 mM ATP. Succinate (10 mM), glycerol 3-phosphate (20 mM), decylubiquinol (50 μM) were used as substrates for detremining SCCR, GCCR and QCCR, respectively. COX activity was measured in 1 ml of 10 mM KH2PO4 (pH 6.5) in the presence of 2.5 mM lauryl-maltoside, and 10 μM reduced cytochrome c. Citrate synthase was spectrophotometrically measured at 412–600 nm in the presence of 0.1% Triton X-100 by following the appearance of the free SH-group of the released CoA-SH upon the addition of 10 mM oxaloacetate to a cell suspension to which 100 μM acetyl-CoA and 2 mM DTNB (Dithionitrobenzoic acid; Ellman's reagent) have been added . In order to discriminate between the phosphate-independent acetyl-CoA acetyltransferase and the phosphate-dependant phosphate acetyltransferase, the analysis was performed either in 10 mM KH2PO4 or in 10 mM MOPS (3-N-morpholino-propane-sulfonic acid) (pH 7.8). All measurements were carried out at 37°C and assay media supplemented with 1 mg/ml bovine serum albumin. All chemicals were analytical reagent grade from Sigma-Aldrich Chimie (Saint Quentin Fallavier; France). The protein content was measured using the Bradford method.
Student's T-test was performed using the Statview 5.0 software (SAS Institute Inc. USA).
This project was supported by Grants from the Wenner-Gren Foundations, the Swedish Association of Neurologically Disabled, the Swedish Medical Association and the Association Française contre l'Ataxie de Friedreich (AFAF)
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