Interaction of Mycoplasma pneumoniae with HeLa Cells

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The susceptibility of HeLa cells to Mycoplasma pneumoniae-induced injury was ... synthesis, which declined very gradually over 7 days in infected HeLa cells ...
INFECTION AND IMMUNITY, Aug. 1988, p. 2054-2059

Vol. 56, No. 8

0019-9567/88/082054-05$02.00/0 Copyright © 1988, American Society for Microbiology

Interaction of Mycoplasma pneumoniae with HeLa Cells DUNCAN C. KRAUSE* AND YI-YWAN CHEN

Department

of Microbiology,

803 Biological Sciences Building, University of Georgia, Athens, Georgia 30602 Received 14 March 1988/Accepted 26 April 1988

The susceptibility of HeLa cells to Mycoplasma pneumoniae-induced injury was examined. Infections were initiated with relatively low mycoplasma doses, carried out in a culture medium incapable of supporting M. pneumoniae replication in the absence of host cells, and monitored for up to 10 days. Under these conditions, a time- and dose-dependent decline in the number of viable host cells compared with that of uninfected controls was observed. The effect of M. pneumoniae infection on host cell macromolecular synthesis was also evaluated. At high doses of infection, synthesis of both protein and RNA declined rapidly relative to that in control cells. At lower doses there was a biphasic response in protein synthesis, which was substantially lower than that in the uninfected control by day 1 postinfection, returned to control levels by day 4 postinfection, and was again less than that in control cells by day 7 postinfection. In contrast, no transient recovery was observed in RNA synthesis, which declined very gradually over 7 days in infected HeLa cells compared with that in uninfected control cells. The ability of HeLa cells to support the proliferation of M. pneumoniae under these experimental conditions was demonstrated by quantitation of mycoplasma CFU in the nonpermissive medium in the presence or absence of HeLa cells. A negligible increase in the number of M. pneumoniae was observed over 4 days when HeLa cells were absent, while CFU increased by almost 20-fold when M. pneumoniae was cultured in the presence of HeLa cells. The susceptibility and response in macromolecular synthesis in M. pneumoniae-infected HeLa cells differed from that recently described for a nontransformed culture of hamster trachea epithelial cells under the same experimental conditions (Y.-Y. Chen and D. C. Krause, Infect. Immun. 56:570-576, 1988), underscoring the importance of the choice of host cell for in vitro modeling of M. pneumoniae pathogenesis.

Mycoplasma pneumoniae is a pathogen of the human respiratory tract. This procaryote causes tracheobronchitis and pneumonia, with the highest incidence of disease occurring in older children and young adults. Within this age group M. pneumoniae is the leading cause of pneumonia (14), though the most common manifestation of infection is a nonspecific and commonly misdiagnosed tracheobronchitis (15). In vivo and in vitro studies using a hamster model (10, 13, 16, 25, 26) and microscopic examination of clinical specimens (11) indicate that adherent mycoplasmas damage the tracheal epithelium, leading to ciliostasis and exfoliation. The first critical step in pathogenesis is the adherence of the invading mycoplasmas to the tracheal epithelium. The pivotal role of cytadsorption is reflected by the observation that nonadhering M. pneumoniae variants are avirulent upon intranasal inoculation in the hamster model (22, 30), and reacquisition of the capacity for cytadsorbtion reconfers virulence (29, 31). While adherence clearly provides a means of resisting mucociliary clearance, it is apparent that adherence also permits the intimate association between host and pathogen that is crucial for nutrient acquisition (8). Metabolic and ultrastructural changes in tracheal tissue upon exposure to M. pneumoniae have been addressed extensively. Collier et al. (12) and Collier and Baseman (10) described a gradual decrease in ciliary activity during infection, accompanied eventually by exfoliation. Freeze-fracture electron microscopy reveals alterations in the distribution of host cell membrane particles, including a necklace structure that encircles the cilia at the base of the shaft (5). In vitro observations of ciliostasis correlate well with clinical findings that mucociliary clearance in the respiratory tract can remain depressed for extended periods following M. pneu*

moniae infection (4). Cytopathological changes are accompanied at the biochemical level by a transient rise and then decline in galactose uptake and utilization, reductions in the uptake of precursors for macromolecular synthesis, alterations in RNA and protein synthesis (25, 26), decreases in the level of intracellular ATP (18), and changes in nucleotide content (35). While the manifestations of host cell dysfunction during M. pneumoniae infection have been thoroughly described, albeit under variable experimental conditions, the mechanism(s) whereby the mycoplasmas effect this outcome is poorly understood. Whether the adherence event itself contributes to cell injury is not clear. Gabridge et al. reported that membrane preparations from virulent M. pneumoniae trigger ciliostasis in hamster tracheal rings in organ culture (17). More recently, Chandler and Barile have described a protein-rich extract of M. pneumoniae that appears to be membrane derived and that likewise causes ciliostasis in tracheal rings in vitro (6). Both the biochemical basis and the physiological significance of these observations remain unclear, however. Hydrogen peroxide (9) and superoxide (33) are normal by-products of M. pneumoniae metabolism, and the accumulation of these reactive metabolites in the microenvironment surrounding mycoplasma-bound host cells may lead to peroxidative damage to the tracheal epithelium (9), a contention supported by recent studies by Almagor et al. (13). These investigators have shown that M. pneumoniae infection leads to an inhibition of host cell catalase activity, rendering the cells more susceptible to peroxidative damage, which can be detected on the basis of the accumulation of products of lipid peroxidation. Their studies were performed with high doses of mycoplasmas, which led to toxicity within a 24-h period. It is not clear, however, whether their experimental conditions can be equated with those found in

Corresponding author. 2054

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a natural infection. On the other hand, M. pneumoniae is a strict parasite, requiring exogenous nucleic acid precursors, cholesterol, and certain fatty acids in addition to glucose as a carbon and energy source. During infection, these nutrients must be acquired from the host cell or the extracellular milieu. The contribution of nutrient acquisition by M. pneumoniae to host cell injury, however, remains unclear. Recent studies in our laboratory into the interaction between M. pneumoniae and epithelial host cells have focused on the development of an in vitro model system that might permit distinguishing between peroxidative and nonperoxidative cell injury. We recently described the interaction between M. pneumoniae and hamster trachea epithelial (HTE) cells at low to moderate multiplicities of infection (MOIs) over extended periods and with culture conditions nonpermissive for mycoplasma growth in the absence of host cells (8). In the present study, we compare and contrast these results with findings obtained with HeLa cells, a transformed cell line of human origin. MATERIALS AND METHODS Host cell culture conditions. HeLa cells, kindly provided by Kathy Spindler, Department of Genetics, University of Georgia, Athens, were cultured in minimum essential medium (Eagle salts with glutamine and sodium bicarbonate; Sigma Chemical Co., St. Louis, Mo.) supplemented with 10% fetal bovine serum (MEM-FBS; Sigma) in a humidified atmosphere of 95% air and 5% CO2. Hoechst stain 33258 was used to test periodically for mycoplasma contamination of the HeLa cells by the method of Chen (7). M. pneumoniae cultures. M. pneumoniae M-129, broth passage 18, was used for these studies. The virulence of this strain, originally cultured from a clinical specimen, has been previously established (22). Log-phase cultures grown in Hayflick medium (23) at 37°C in 75-cm2 tissue culture dishes were washed once with sterile phosphate-buffered saline (PBS) (pH 7.2), harvested in fresh Hayflick medium, and passed through a 25-gauge needle eight times to disaggregate the mycoplasmas. The suspension was passed through a sterile filter (pore size, 1.2 ,um) to remove the larger remaining aggregates and was then used to infect cell cultures. This step was necessary to maintain consistency in the size of CFU because of a tendency of M. pneumoniae to aggregate. CFU were determined on PPLO agar plates (32). HeLa cell infections. Forty-eight hours before being infected, HeLa cells were seeded in 24-well microdilution plates at a density of 2.5 x 104 cells per well and incubated as described above. At the time of infection, the HeLa cells were near confluency. The culture medium was replaced with 1 ml of fresh MEM-FBS which had been preequilibrated to the proper pH and temperature. Test samples received 125 RI of the M. pneumoniae suspension in Hayflick medium, while the control wells received 125 RI of sterile Hayflick medium. The mycoplasmas were allowed to attach during a 2-h incubation at 37°C with 5% C02, after which the medium was removed and replaced with fresh MEM-FBS which contained no Hayflick medium. MOI, or the number of mycoplasmas added per host cell to initiate the infection, was established by diluting and plating the mycoplasma inoculum prior to infection and counting the number of host cells present per sample with a hemacytometer. It is important to note that, because only about 5% of the mycoplasmas initially added remained in the wells when the culture medium was changed at the end of the initial 2-h incubation, the MOIs reported are approximately 20-fold higher than

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they would be if determined at the end of the initial incubation (data not shown). Evaluation of host cell viability. On days 4, 7, and 10 postinfection, samples were assayed for viability by trypan blue exclusion. Samples not assayed at these time points had their old culture medium replaced with fresh MEM-FBS. Trypan blue staining was carried out as follows. The old medium was removed from each well, transferred to individual culture tubes, and replaced with PBS containing 0.02% EDTA and 0.05% trypsin to release the cells. The resulting cell suspensions were transferred to their respective culture tubes, which contained the old culture medium. Trypan blue in Hanks balanced salts solution was added to a final concentration of 0.04%, and the culture was incubated for 5 min. The stained and unstained cells were then counted with the aid of a microscope and a hemacytometer. All samples were tested in quadruplicate. Host cell macromolecular synthesis during infection. To investigate further the manifestations of M. pneumoniae infection of HeLa cells in vitro, the levels of macromolecular synthesis in infected and control cells were compared. Host cell protein synthesis was evaluated as follows. HeLa cells seeded in 24-well culture dishes containing 12-mm sterile glass cover slips were infected with M. pneumoniae as described above. At the indicated time points, the old culture medium was replaced with 1 ml of fresh MEM (no FBS) containing erythromycin (80 ,ug/ml; Sigma) and incubated for 2 h at 37°C in 5% CO2. A U-14C-amino acid solution (1.81 mCi/mg; ICN Radiochemicals, Irvine, Calif.) was added to a final concentration of 1 ,uCi per well. The labeling medium was removed after a 3-h pulse at 37°C in 5% C02, 1 ml of MEM-FBS with erythromycin was added, and the incubation was continued for 1 h. The cover slips were washed once with PBS and treated sequentially with 10% trichloroacetic acid (TCA) for 20 min at 4°C, 5% TCA for 10 min at 4°C, and 95% ethanol at room temperature (21, 28). The cover slips were then air dried and analyzed for radioactivity. No significant accumulation of radioactivity was detectable in HeLa cell-free controls containing only M. pneumoniae and erythromycin. All samples were tested in

triplicate.

In a similar fashion, the effect of M. pneumoniae infection on RNA synthesis in HeLa cells was evaluated. This assay exploited the ability of eucaryotic cells to utilize orotic acid as a precursor for RNA synthesis and the inability of M. pneumoniae to do so (25, 27). HeLa cells were seeded in 24-well culture plates as described above for protein synthesis assays but with no cover slips in the wells. At selected time points, the culture medium was replaced with 1 ml of

fresh, preequilibrated MEM (no FBS) containing 1 ,uCi of [6-'4C]orotic acid (56 mCi/mmol; ICN). The cultures were incubated for 4 h as described above, after which the labeling medium was removed, fresh MEM-FBS was added, and the incubation was continued for another 5 h. The wells were then washed with PBS, scraped gently into 1 ml of PBS, and transferred to 25-mm-diameter filters (pore size, 0.22 p.m) in a vacuum manifold (Millipore Corp., Bedford, Mass.). Highmolecular-weight nucleic acids were precipitated with the sequential addition of 2 ml of cold 10% TCA, 2 ml of cold 5% TCA, and 2 ml of 95% ethanol. The filters were then air dried and analyzed for radioactivity. Background controls included samples to which were added 1 ,ug of actinomycin D (Sigma) per well during the pulse and chase to inhibit all RNA synthesis. All samples were tested in triplicate. M. pneumoniae growth under experimental culture conditions. To evaluate the extent of mycoplasma growth in the

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INFECT. IMMUN.

TABLE 1. Protein synthesis in HeLa cells during M. pneumoniae infection Disintegrations per minutea Infected cells at MOI of:

Day postinfection

1 2 4 7

Uninfected cells 4,000

400

40

1,847 ± 132 (46%) 2,331 ± 622 (33%) 3,945 ± 733 (24%) 15,536 + 3,586 (34%)

3,536 ± 629 (88%) 6,187 ± 114 (88%) 16,199 ± 561 (98%) 35,308 + 3,085 (76%)

2,891 ± 482 (72%) 6,050 ± 605 (86%) 16,466 ± 601 (100%) 37,311 ± 3,260 (80%)

4,013 7,006 16,527 46,162

± 227 ± 1,014 ± 2,028 ± 3,265

a Incorporation of 14C-amino acids into TCA-precipitable material; mean ± standard deviation of three samples and percent of control value (disintegrations per minute for infected cells/disintegrations per minute for uninfected control cells) x 100%.

presence or absence of HeLa cells in the culture conditions described, and thus indirectly to investigate the M. pneumoniae requirement for host HeLa cells as a source of nutrients, viable mycoplasmas were quantitated over a 4-day incubation in MEM-FBS with or without HeLa cells. HeLa cells were seeded in 24-well culture dishes as described above. Cell-free control wells received only growth medium. After 48 h, the culture medium was replaced with fresh MEM-FBS, and M. pneumoniae suspensions in Hayflick medium were added and incubated for 2 h at 37°C in 5% Co2. Prior to infection, a portion of the inoculum was also serially diluted and plated for titration. At the end of the incubation, the medium was removed and replaced with fresh, preequilibrated MEM-FBS. At time zero (immediately after the medium was replaced) or at day 4, the growth medium was removed and transferred to separate culture tubes. The cell monolayers were trypsinized briefly, releasing the cells and mycoplasmas into suspension, and these suspensions were transferred to the culture tubes which contained the previously removed growth medium. The samples were serially diluted in Hayflick medium and plated on PPLO agar plates (32). RESULTS M. pneumoniae toxicity for HeLa cells. M. pneumoniae attached to HeLa cells in a time- and temperature-dependent fashion (data not shown). Maximum adherence occurred within 2 h, with approximately 5% of the cells in the inoculum remaining adherent. The specificity of attachment was verified by pretreatment of the cells with neuraminidase, which decreased adherence by approximately 65%, a level of inhibition comparable to that previously described for tracheal rings in organ culture (30). When HeLa cells were infected with a high mycoplasma dose (4,000 CFU added per cell initially, with approximately 5% or 200 adherent mycoplasmas per cell remaining when the culture medium was changed after 2 h), there was a dramatic decrease in the number of viable host cells to less than 30% of uninfected control values by day 4 postinfection (Fig. 1). With lower MOIs, the time- and dose-dependent nature of M. pneumoniae toxicity for HeLa cells was apparent. At an initial MOI of only 40 (or approximately two adherent mycoplasmas remaining per cell), the number of viable HeLa cells decreased by almost 40% relative to control values by day 10 postinfection. Macromolecular synthesis during M. pneumoniae infection. On day 1 postinfection, the level of protein synthesis in HeLa cells infected with M. pneumoniae at a high MOI was only 46% of that observed in uninfected control cells (Table 1). Protein synthesis in infected cells never returned to control levels through the duration of the infection, remain-

ing approximately 25 to 35% of that detected in control cells. Protein synthesis in host cells infected with lower mycoplasma doses was approximately 12 to 25% lower than that in uninfected controls at day 1 postinfection. However, a transient recovery was consistently observed over the next 3 days, as the levels of TCA-precipitable radioactivity were comparable for infected and uninfected HeLa cells. By day 7 postinfection, the level of protein synthesis in infected HeLa cells was again lower than that in control cells. In similar experiments, in which protein synthesis was monitored beyond day 7, the decline in protein synthesis in infected HeLa cells compared with that in uninfected control cells continued (data not shown). The level of RNA synthesis in HeLa cells infected with a high dose of M. pneumoniae was substantially lower by day 1 postinfection than was that observed in uninfected cells (Table 2). RNA synthesis in infected HeLa cells remained low compared with that in uninfected control cells throughout the infection, in a manner similar to that described above for protein synthesis in HeLa cells at a comparable MOI. On the other hand, at lower MOIs, the levels of RNA synthesis were comparable to control levels through day 2 postinfection, in contrast to what was described above for protein synthesis. By day 4 postinfection, slightly lower levels of RNA synthesis were observed in infected cells compared with those in uninfected control cells. The changes in RNA Day post-infection

100 -TI

1 80

-E

107

PERCENT 60 F

LL

CONTROLa

40 F

201

OL

L,

J

.

I

M01-4000 M01-400

I.

M01-40

FIG. 1. Viability of HeLa cells infected with M. pneumoniae. Values represent the mean and standard deviation of four samples. aPercent control = (viable cells in infection group/viable cells in control group) x 100%.

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TABLE 2. RNA synthesis in HeLa cells during M. pneumoniae infection Disintegrations per minutea Day

Ifed

postinfection 6,600

1 2 4 7

190 393 536 2,358

± 43 (33%) + 132 (34%) ± 86 (25%) ± 332 (43%)

necte cells at 660

572 1,126 1,804 4,785

± ± ± ±

MOI

of

42 (99%) 54 (96%) 66 (83%) 445 (86%)

66

577 1,265 2,070 5,118

± ± ± ±

11 (100%) 58 (108%) 169 (95%) 523 (92%)

Uninfected cells

576 1,172 2,185 5,546

± ± ± ±

24 21 37 122

a Incorporation of [14C]orotic acid into TCA-precipitable material; mean ± standard deviation of three samples and percent of control value (disintegrations per minute for infected cells/disintegrations per minute for uninfected control cells) x 100o.

synthesis (Table 2) correlated well temporally with the decline in the number of viable host cells (Fig. 1). Host cell support of mycoplasma growth. The culture medium used in this study (MEM-FBS) was previously shown to be incapable of supporting M. pneumoniae growth (8). The toxicity of M. pneumoniae for HeLa cells in this culture medium suggested that the host cells were supporting mycoplasma proliferation. This possibility was examined indirectly by quantitating mycoplasma CFUs over a 4-day culture in MEM-FBS in the presence or absence of HeLa cells. The initial inoculum was 1.28 x 108 CFU per sample. In the absence of HeLa cells there was only a slight increase between day 0 and day 4 in mycoplasma CFU when incubated in MEM-FBS (from 4.51 x 106 + 0.29 x 106 CFU to 6.80 x 106 + 0.31 x 106 CFU). In contrast, mycoplasma CFU increased almost 20-fold when cultured in the same medium in the presence of HeLa cells (from 4.02 x 106 ± 0.6 x 106 CFU to 70.70 x 106 ± 8.60 x 106 CFU), suggesting that M. pneumoniae acquires vital nutrients from the host cells. DISCUSSION The study described here, which evaluated the interaction of M. pneumoniae with HeLa cells at low MOIs in a nonpermissive culture medium, was initiated in parallel with a similar study (8) using HTE cells (20). Our intention was to work out the experimental parameters with the more convenient HeLa cell line and then apply those parameters to the HTE cells, which we feel provide a more accurate model for the pathogenesis of M. pneumoniae infections. In the process of developing the experimental system, we observed significant differences both in the sensitivities of the two cell types to M. pneumoniae-induced injury and in the manifestations of this toxicity at the molecular level. High doses of M. pneumoniae resulted in a decrease of more than 70% in the number of viable HeLa cells (Fig. 1), while similar mycoplasma doses consistently resulted in only a 45 to 50% decrease in the number of viable HTE cells under the same culture conditions (8). Furthermore, M. pneumoniae infection under nonpermissive conditions led to a dose-dependent decline in protein synthesis in HeLa cells in this study but led to a dose-dependent rise in HTE cells in the other study (8). It seems that there are at least three possible explanations for the differences in sensitivity between the two cell types. In the first place, the different degrees of sensitivity observed in HTE and HeLa cells may merely reflect differences in the number of receptor sites for mycoplasma adherence to these two cell types. While adherence assays indicated that M. pneumoniae attaches at comparable levels to HTE and HeLa cells (data not shown), such assays must be interpreted cautiously, due to mycoplasma adherence to exposed plastic or glass surfaces.

On the other hand, Hoffmann et al. have described greater sensitivity to killing by hydrogen peroxide for human fibroblasts compared with fibroblasts from hamsters (24). Hence, HeLa cells, which are of human origin, may be less resistant than HTE cells to the oxidative stress resulting from mycoplasma generation of hydrogen peroxide and superoxide. In the studies by Hoffmann et al., sensitivity correlated with the number of DNA strand breaks in the peroxide-treated cells but not with the ability of the cells to destroy the hydrogen peroxide. Finally, HTE cells exhibit cell-cell contact inhibition of growth and hence stop proliferating once monolayer confluency is reached. HeLa cells are transformed, and by definition exhibit no such contact inhibition. Hence, differences in the metabolic activities of the host cell types could account for the observed differences in susceptibility to M. pneumoniae toxicity, a hypothesis supported by the observation by Upchurch and Gabridge (34) that actively growing human lung fibroblasts are more susceptible to M. pneumoniae pathogenesis than are resting (e.g., serum-deprived) fibroblasts. The ability of HeLa cells to support M. pneumoniae growth under nonpermissive conditions was demonstrated here. Whether the mycoplasmas are utilizing HeLa cell metabolic by-products accumulating in the extracellular milieu or are actively obtaining nutrients to the detriment of the host cell cannot be determined from these studies. That the presence of M. pneumoniae is detrimental is clear; however, the toxicity may not necessarily be the direct result of parasitism. For example, if the more metabolically active HeLa cells were better able to support mycoplasma growth, this might result in higher levels of hydrogen peroxide and superoxide generation by M. pneumoniae, resulting in greater peroxidative damage. This interpretation is consistent with the observation that the decline in the number of viable host cells and the alterations in protein synthesis in infected HeLa cells are comparable to those we described when HTE cells were infected in a growth medium permissive for M. pneumoniae growth (8). On the other hand, Gabridge and Stahl have provided evidence that purine acquisition by M. pneumoniae is detrimental to the host cells, as exogenous adenine can provide some protection against ciliostasis in M. pneumoniae-infected tracheal rings in a nonpermissive medium (19). The transient recovery in protein synthesis is noteworthy. In their original description of alterations in macromolecular synthesis in M. pneumoniae-infected tracheal rings, Hu et al. (25) reported a rapid and significant decline in protein synthesis with time. Their studies were performed with a culture medium permissive for M. pneumoniae growth, while a nonpermissive medium was utilized in the present study. The latter should more closely mimic the necessity in

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natural infections for mycoplasma nutrient acquisition from host cells. The use of a nonpermissive medium for extended periods in the current study may have permitted detection of the transient recovery in protein synthesis described here. This also prompts speculation as to whether the initial (day 1 postinfection) and secondary (day 7 postinfection) inhibition of host cell protein synthesis reflects different mechanisms of toxicity. One possible explanation is that the initial decline in protein synthesis reflects the membrane toxicity originally described by Gabridge et al. (17) and confirmed by Chandler and Barile (6). The delayed inhibition in macromolecular synthesis may then be a consequence of peroxidative damage, parasitism, or both. In summary, HeLa cells exhibited significant sensitivity to M. pneumoniae infection even at very low MOIs and in a culture medium incapable of supporting M. pneumoniae growth in the absence of host cells. It is possible to monitor the interaction between host and parasite for as long as 10 days following infection under these conditions, perhaps permitting elucidation of the mechanism(s) contributing to the demise of the host cells. It is noteworthy that both the degree of sensitivity and the alterations in host cell metabolism during infection can vary, depending on the cell type and the culture conditions. This underscores the need both for cautious interpretation of experimental findings from in vitro models and for parallel in vivo studies to establish the physiological significance of those in vitro experimental observations. We anticipate that the described differences between HTE and HeLa cells in their interaction with M. pneumoniae will contribute to a better understanding of the molecular nature of those interactions.

ACKNOWLEDGMENTS This research was supported by a grant from the American Lung Association. We thank Cathy Gutfreund for her technical assistance and Pat Bates for typing the manuscript.

1.

2. 3.

4. 5.

6. 7. 8. 9.

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ide dismutases. Proc. Fed. Eur. Biochem. Soc. Symp. 62:49-56. 34. Upchurch, S., and M. G. Gabridge. 1981. Role of host cell metabolism in the pathogenesis of Mycoplasma pneumoniae infection. Infect. Immun. 31:174-181. 35. Upchurch, S., and M. G. Gabridge. 1982. Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae. Infect. Immun. 38:631-636.