Synthesis in Normal and Lesch-Nyhan Fibroblasts Infected

Transcription

1 INFECTION AND IMMUNITY, Jan. 1983, p /83/ $02.00/0 Copyright ) 1983, American Society for Microbiology Vol. 39, No. 1 De Novo Purine Synthesis, Purine Salvage, and DNA Synthesis in Normal and Lesch-Nyhan Fibroblasts Infected with Mycoplasma pneumoniae SUSAN UPCHURCHt* AND MICHAEL G. GABRIDGE W. Alton Jones Cell Science Center, Lake Placid, New York Received 26 April 1982/Accepted 1 October 1982 The effects of Mycoplasma pneumoniae on host cell metabolism were studied by using two types of host cells, MRC-5 human lung fibroblasts, a normal cell line, and Lesch-Nyhan fibroblasts, a cell line deficient in hypoxanthine-guanine phosphoribosyl transferase (EC ). The susceptibilities of the two cell types were determined by infecting the cells with M. pneumoniae at different multiplicities of infection (MOI). Our data indicate that the Lesch-Nyhan cells were four times more susceptible to damage by M. pneumoniae than the MRC-5 cells. The effects of different MOIs (10 and 50) on de novo purine synthesis, DNA synthesis, and the development of a cytopathic effect were determined. In both cell types, the higher MOI inhibited de novo purine synthesis to a greater extent than the lower MOI. This correlated closely with the cytopathic effect which developed in the monolayers (i.e., the more the inhibition of de novo purine synthesis, the greater the cytopathic effect which developed). In the Lesch-Nyhan cells, DNA synthesis was completely inhibited by the high MOI, whereas in the MRC-5 cells, DNA synthesis was stimulated by the high MOI. In the MRC-5 cells infected with M. pneumoniae, purine salvage activity increased, as indicated by an increase in adenosine deaminase (EC ) activity. These data indicate that M. pneumoniae alters host cell metabolism, particularly the nucleic acid metabolic pathways. This may explain in part the mechanism of pathogenesis of M. pneumoniae infection. Mycoplasma pneumoniae causes a severe cytopathic effect (CPE) in human lung fibroblasts (12), as well as ciliostasis in hamster tracheal organ cultures (5). The mechanisms by which the mycoplasmas induce this damage are not well understood. Numerous factors must be considered in determining how a mycoplasma actually damages a host cell. These factors include the metabolic state of the mycoplasma (9), the metabolic state of the host (24), attachment of the mycoplasma to the host (4, 10, 16, 20), structural components and toxic factors of the mycoplasma (3, 7, 18), and the metabolic consequences of infection (6-11, 14, 19). Attachment of mycoplasmas to a sialoglycoprotein receptor on the host cell is one of the first steps involved (4, 10, 16, 20), but little is known about what happens to the host cell once the mycoplasmas are attached. Mycoplasma membranes (7, 10), cell extracts (3), and cell products, such as hydrogen peroxide (18), can cause cytotoxic and ciliostatic effects in a host cell. Much of the work done on this problem has t Present address: Department of Pathology, University of Texas Medical Branch, Galveston, TX involved the use of hamster tracheal organ cultures, in which a variety of metabolic and structural alterations have been found. These include decreases in dehydrogenase activity (7), ATP content (11), oxygen uptake (6), amino acid uptake, orotic acid uptake, galactose uptake, and protein and RNA synthesis (14) and alterations in membrane and ciliary structure (1, 2). In addition, the metabolic state of a mycoplasma is crucial in determining whether it can damage a cell (9). It has also been shown that adenine and related nucleic acid analogs can modulate such infections (8), but the mechanism by which this occurs is not yet clear. The human lung fibroblast model (12, 24) is a better system than explant cultures for determining the metabolic alterations which ensue from a mycoplasma infection. Monolayer cultures have the advantage that all of the cells in the cultures are genetically and morphologically homogeneous, so that each cell is affected in approximately the same manner. In hamster tracheal explant cultures, there are a variety of cell types, and of these types only the ciliated epithelial cells are thought to be susceptible to mycoplasmas. In this latter case, any metabolic

2 VOL. 39, 1983 determinations reflect the total cell population and not just the target cells. Previous work indicated that there is a close correlation between host cell nucleic acid metabolism and the ability of M. pneumoniae to cause a cytopathic effect (CPE) in host cells (24). In this study, we used normal human lung fibroblasts (MRC-5 cells) and Lesch-Nyhan fibroblasts, which are deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC ), to probe the effects of M. pneumoniae infection further (15, 23, 27). As the Lesch- Nyhan cells are HGPRT deficient, they lack the purine salvage pathway. Therefore, these cells must rely totally on de novo purine metabolism for their purines. This defect would seemingly cause Lesch-Nyhan cells to be more susceptible than normal cells to damage by M. pneumoniae if M. pneumoniae affects any of the pathways involved in nucleic acid metabolism. We examined the role of de novo purine metabolism, purine salvage, and DNA metabolism in these two cell lines to determine more precisely the effects of M. pneumoniae on host cell nucleic acid metabolism. Our data suggest that interference with de novo purine synthesis in host cells plays a critical role in the induction of cytotoxicity in M. pneumoniae-infected cells. MATERIALS AND METHODS Mycoplasmas. M. pneumoniae strain PI 1428 was furnished by J. Tully, National Institutes of Health. Mycoplasmas were grown in G199H medium and harvested with glass beads in Eagle basal medium (BME) supplemented with 10o fetal bovine serum (FBS), as previously described (24). For attachment studies, mycoplasmas were grown for three passages in complete G199H medium containing 5 FCi of 3Hlabeled amino acids (protein hydrolysate; Schwarz/ Mann) per ml. The labeled mycoplasmas were washed three times in complete phosphate-buffered saline and then harvested in BME supplemented with 10%o FBS. Viable counts were determined in duplicate on G200 agar plates (12). Fibroblast cultures. Cultivation of MRC-5 human embryo lung fibroblasts has been described previously (24). Lesch-Nyhan fibroblast cultures were obtained from the Institute for Medical Research, Camden, N.J. (repository no. GM0377), and were maintained in BME supplemented with 10% FBS (Biolabs) in the same manner as the MRC-5 cells. Cell cultures were monitored for mycoplasma contamination by direct culture and DNA-staining techniques. Both methods indicated that the cultures did not contain contaminating mycoplasmas. Both types of fibroblast cultures were prepared for experiments in the same way. The cells were seeded onto four 12-mm circular glass cover slips per 35-mm petri dish at a concentration of 1.0 x 105 cells per dish. The cover slip cultures were incubated for 48 h before infection. Replicates of eight cover slip cultures were used for each time or treatment data point. Infection of cultures. Infection of the cover slip METABOLISM IN M. PNEUMONIAE-INFECTED CELLS 165 cultures with M. pneumoniae has been described previously (24). The cover slip cultures were transferred to dishes containing 2.0 ml of M. pneumoniae (harvested during the exponential growth phase) in BME supplemented with FBS. This mixture was incubated statically at 37 C for 2 h to allow the mycoplasmas to attach to the fibroblasts. The cover slip cultures were then transferred to fresh BME supplemented with 10% FBS and incubated at 37 C in 5% CO2 until they were assayed for a CPE or for DNA or de novo purine synthesis. Determination of a CPE. The CPE which developed in the fibroblast cultures was measured quantitatively by using a leucine incorporation assay (24). At 72 h after the fibroblasts were infected with M. pneumoniae (when sufficient time had elapsed in order for the CPE to develop), the cover slip cultures were transferred to medium containing 1,uCi of [3H]leucine (120.1 Ci/mmol; New England Nuclear Corp.) per ml for 8 h. After this incubation with [3H]leucine, the cellular macromolecules were precipitated with 5% trichloroacetic acid (TCA) and washed. Each cover slip was assayed for radioactivity by using a Beckman model LS7500 liquid scintillation counter. This assay measured the leucine incorporated by the total cell population on the cover slip. The cultures which developed a CPE had suffered damage to the monolayer, with many cells dying and detaching from the cover slip. This assay corresponded directly to a count of cells remaining on each cover slip. From the amount of label incorporated, the degree of the CPE (or CPE ratio) could be determined. The CPE ratio is defined as follows: (counts of [3H]leucine incorporated per minute by infected fibroblasts/counts of [3H]leucine incorporated per minute by control fibroblasts) x 100. A high ratio indicates little CPE, whereas a low ratio indicates a pronounced CPE. Measurement of thymidine incorporation and de novo purine synthesis. De novo purine synthesis was measured by determining the incorporation of ['4C]formate into TCA-precipitable material in the fibroblasts. At different times after infection of the cover slip cultures (4, 24, 48, and 72 h), the cultures were transferred to fresh medium containing 5,uCi of ["C] sodium formate (52.7 mci/mmol; New England Nuclear Corp.) per ml. The cultures were incubated for 8 h in this medium; then they were precipitated with 5% TCA, washed three times in phosphate-buffered saline, and counted. As the mycoplasmas were unable to incorporate [14C]formate, no correction was necessary for the incorporation of label by the mycoplasmas in the infected fibroblasts. At the same time that the cells were labeled with the formate, duplicate cultures were trypsinized and counted with a hemacytometer to normalize the data to a single-cell basis. This alleviated the problem of differences in incorporation of the label due to a loss of cells from the infected monolayers Ṫhymidine incorporation was measured in a manner similar to the way in which de novo purine synthesis was measured. To measure thymidine incorporation, the incorporation of [3H]thymidine into TCA-precipitable material was followed. At the same times used for measuring de novo purine synthesis, duplicate cultures were transferred to fresh medium containing 1 FtCi of [3H]thymidine (20.0 Ci/mmol; New England Nuclear Corp.) per ml. The cultures were incubated

3 166 UPCHURCH AND GABRIDGE MOI FIG. 1. Effect of MOI on the development of a CPE in MRC-5 (@) and Lesch-Nyhan (0) cells. Fibroblast cultures were infected with increasing concentrations of M. pneumoniae strain PI The CPE was determined 72 h after infection by determining the number of cells remaining on the cover slip using a protein synthesis assay. Each value is the mean + standard error of the mean for eight replicates. for 2 h in this medium and then precipitated with 5% TCA, washed, and counted. To account for mycoplasma incorporation, duplicate cover slip cultures containing M. pneumoniae alone were also assayed. These were established in a manner which duplicated the number of mycoplasmas in the infected cultures as closely as possible. Attachment studies showed comparable attachments of M. pneumoniae to the monolayer cultures and the glass cover slips. The counts incorporated by the infected fibroblasts were corrected for the counts incorporated by the mycoplasmas alone. Again, as in measuring de novo purine synthesis, cell counts were determined for duplicate cultures with a hemacytometer to standardize data to a singlecell basis. Measurement of adenosine deaminase activity. Adenosine deaminase (EC ) was measured by a procedure adapted from the method of Van der Weyden et al. (25). Infected and control cells were washed twice in calcium- and magnesium-free phosphate-buffered saline and trypsinized. The cells were washed twice after trypsinization, counted, and suspended in 200 RI of a permeabilizing buffer (17) consisting of 1% Tween 80, 0.25 M sucrose, 0.05 M HEPES (N-2- hydroxyethylpiperazine-n'-2-ethanesulfonic acid) (ph 7.0), and 2 mm dithiothreitol. These cells were incubated at 25 C for 30 min to permeabilize their membranes, and 100,ul of reaction buffer consisting of 100 mm Tris (ph 7.4) and 1.8 x 105 nmol of ["C]adenosine (45.5 mci/mmol; New England Nuclear Corp.) per ml was then added. This mixture was incubated for 30 min at 37 C. The reaction was stopped by adding 100 p1 of cold absolute ethanol; 1 pul of each reaction mixture was then spotted onto polyethyleneiminecellulose thin-layer chromatography plates which were prespotted with 1 pul of 5 mm adenosine and 1 RI of 5 mm inosine. The thin-layer chromatography plates were developed by using ascending chromatography with 1.4 M lithium chloride (21). The spots were visualized with UV light and marked. Each spot was cut out and counted by using liquid scintillation counting to determine the amount of adenosine converted to inosine. Statistics. Statistical analyses were done by using the t test (26). INFECT. IMMUN. RESULTS Susceptibility of MRC-5 human lung fibroblasts and Lesch-Nyhan fibroblasts to M. pneumoniae infection. The responses of the two types of fibroblasts to different multiplicities of infection (MOI) by M. pneumoniae were studied. This experiment was performed to determine whether there was a difference in susceptibility between MRC-5 cells (with normal purine metabolism) and Lesch-Nyhan cells (heavily dependent on de novo purine synthesis) to mycoplasma infection. In this experiment, both types of fibroblasts were plated at a density of 1.0 x 105 cells per dish and grown as described above. At the time of infection, counts of both host cells and mycoplasma cells were made to establish the MOI. Fibroblast cell numbers were determined by direct counting with a hemacytometer, whereas mycoplasma cell numbers were determined by plate counts. Replicate dishes of each type of fibroblast were infected with serial twofold dilutions of M. pneumoniae. CPE ratios were determined as described above for each group at 72 h postinfection. Figure 1 shows that there was a significant difference (P < 0.005) in the responses of the two types of cells to mycoplasma infection. The Lesch-Nyhan cells were four times more susceptible to the mycoplasmas than the normal MRC-5 cells were. An MOI of less than 120 was needed to cause a CPE ratio of 50 (50% destruction of the monolayer) in the Lesch-Nyhan cells, whereas an MOI of 500 was needed to cause the equivalent CPE ratio in the MRC-5 cells. These MOIs were the total numbers of mycoplasmas to which each cell was exposed. The actual number of mycoplasmas which infected each cell was probably considerably less, since only 1 to 10% of mycoplasma cells normally attach. In addition, mycoplasmas can also attach to glass cover slips and exposed petri dishes, thus decreasing the number of mycoplasmas attaching to the fibroblasts by approximately 50%, as the cells are approximately 50% confluent. This alters the MOI necessary for a CPE ratio of 50 in Lesch- Nyhan cells to between 0.6 and 6 and the MOI TABLE 1. Attachment of M. pneumoniae to MRC-5 human lung fibroblasts and Lesch-Nyhan fibroblasts Mycoplasma attachment' Cell type % of cpm/cover slip MRC-5 MRC-5 76,418 ± 3,700b 100 ± 5 Lesch-Nyhan 56,373 ± 2, ± 5 a M. pneumoniae strain PI 1428 labeled with 3Hlabeled protein hydrolysate. bmean ± standard error of the mean for eight replicates.

4 VOL. 39, 1983 METABOLISM IN M. PNEUMONIAE-INFECTED CELLS or. tordro / -Nsr 75 if " A 50 c Time (hrs) FIG. 2. Comparison of the responses of de novo purine synthesis in MRC-5 human lung fibroblasts (0) and Lesch-Nyhan fibroblasts (L-N) (0) after infection with M. pneumoniae strain PI De novo purine synthesis was measured by determining the amount of ['4C]sodium formate incorporated into the cells during an 8-h labeling period beginning 4, 24, 48, or 72 h after infection of the cultures. Each value is the mean ± standard error of the mean for eight replicates. necessary for a CPE ratio of 50 in MRC-5 cells to between 2.5 and 25. One possibility which could account for the difference in the susceptibilities of the Lesch- Nyhan and MRC-5 fibroblasts to mycoplasma infection is a difference in the ability of the mycoplasma cells to attach to the different types of fibroblasts. To determine whether this was a factor, we measured the attachment of M. pneumoniae to the fibroblasts. Mycoplasma cells were labeled with 3H-labeled protein hydrolysate for three passages in complete G199H medium as described above, and 48-h cover slip cultures of both types of fibroblasts were transferred to 2.0 ml of the labeled mycoplasma suspension and incubated for 2 h to allow the mycoplasmas to attach to the monolayers. After this incubation, the cover slip cultures were washed three times in phosphate-buffered saline and counted. Table 1 shows that there was a significant difference (P < 0.005) in the attachment of M. pneumoniae to the two cell types, with the mycoplasmas attaching only 74% as well to the Lesch-Nyhan cells as to the MRC-5 cells. However, the Lesch-Nyhan cells were more susceptible to cytotoxic effects by the mycoplasmas than the MRC-5 cells, indicating that the differences in susceptibility were not due to attachment. Response of de novo purine synthesis in MRC-5 and Lesch-Nyhan fibroblasts to infection by M. pneumoniae. Lesch-Nyhan fibroblasts and MRC-5 fibroblasts were infected with M. pneumoniae at an MOI of 10. De novo purine synthesis was measured at 4, 24, 48, and 72 h postinfection as described above (Fig. 2). Immediately after infection (i.e., after 4 h), de novo purine synthesis was significantly inhibited in both cell types (P < 0.005), although synthesis was inhib Tine (hrs) FIG. 3. Effects of MOI on de novo purine synthesis in MRC-5 human lung fibroblasts. Fibroblast cultures were infected with high- and low-dose mycoplasma suspensions (MOIs of 10 [0] and 50 [I], respectively). De novo purine synthesis was measured at 4, 24, 48, and 72 h postinfection. Each value is the mean ± standard error of the mean for eight replicates. ited to a greater extent in the Lesch-Nyhan cells. At 24 h after infection, de novo purine synthesis remained low in both cell types (less than 25% of the control level of synthesis). By 48 h, this pattern began to change. De novo purine synthesis remained low in the normal MRC-5 fibroblasts (below 50% of the control levels at 48 and 72 h postinfection); however, de novo purine synthesis increased in the Lesch-Nyhan cells to almost 100% of the control level at 48 h postinfection and to 75% of the control level at 72 h postinfection. Effect of MOI on de novo purine synthesis in Lesch-Nyhan and MRC-5 fibroblasts. Since the MOI was critical in determining the degree of damage that the fibroblast monolayers sustained due to M. pneumoniae infection, we investigated the effect of the MOI on de novo purine synthesis in the fibroblasts. In this experiment, both MRC-5 and Lesch-Nyhan cells were infected with M. pneumoniae at MOIs of 10 and MI -. ContW Time (hrs) IMOI FIG. 4. Effects of MOI on de novo purine synthesis in Lesch-Nyhan fibroblasts. Fibroblast cultures were infected with M. pneumoniae strain PI 1428 at an MOI of 50 (0) or 10 (0). De novo purine synthesis was measured at 4, 24, 48, and 72 h postinfection. Each value is the mean ± standard error of the mean for eight replicates. I0

5 168 UPCHURCH AND GABRIDGE TABLE 2. Correlation of CPE ratio with the rate of de novo purine synthesis De novo purine synthesisa CPE Cell type MOI c' cof rtio CM controld MRC x 1o-3 49 ± 9 63 ± 7 MRC x 1O-4 25 ± 6 13 ± 4 Lesch-Nyhan x ± 9 20 ± 3 Lesch-Nyhan x ± 3 8 ± 2 a Mean ± standard error of the mean for eight replicates. b Determined 72 h after infection by measuring the rate of incorporation of [3H]leucine into monolayer cultures. c Determined 4 h after infection by measuring the rate of incorporation of [14C]sodium formate into cells. Values are counts of 14C incorporated per minute per fibroblast cell. d Values are percentages of the control fibroblast incorporation of [14C]sodium formate for each experiment and cell type. De novo purine synthesis was measured in these cultures at 4, 24, 48, and 72 h after infection. Figures 3 and 4 show the results for the MRC-5 and Lesch-Nyhan cells, respectively. At 4 h postinfection, de novo purine synthesis responded similarly in the two cell lines to the different MOIs. At the low MOI, de novo purine synthesis in the MRC-5 cells was 50% of the control rate, whereas at the high MOI it was 25% of the control rate. The Lesch-Nyhan fibroblasts responded like the MRC-5 cells, although de novo purine synthesis was inhibited to a greater degree at both MOIs in the Lesch-Nyhan cells than in the MRC-5 cells. At the low MOI, de novo purine synthesis was inhibited to 30% of the control rate, whereas at the high MOI it was inhibited to less than 20% of the control rate. These results at 4 h corresponded to the effects of different MOIs on the development of a CPE (the higher the MOI, the greater the damage that the fibroblasts sustained due to mycoplasma infection). De novo purine synthesis in the MRC-5 fibroblasts never recovered to the control rate in cells infected with M. pneumoniae at either MOI throughout the 72-h test period. The rate of de novo purine synthesis remained consistently lower in the cells infected at the high MOI at each point measured, indicating a greater degree of damage in these cells. The response of de novo purine synthesis in the Lesch-Nyhan cells followed a similar pattern. There was no significant difference (P > 0.05) in the rates of synthesis in the fibroblasts infected at the different MOIs at 24 h postinfection. By 48 and 72 h, de novo purine synthesis was inhibited to a greater extent in the cells infected at the higher MOI, as in the MRC-5 cells. Comparison of the rate of de novo purine synthesis with the development of a CPE. The importance of inhibition of de novo purine synthesis in the fibroblasts became evident when the degree of inhibition at 4 h postinfection was compared with the CPE which developed in the cultures. In this experiment, the fibroblasts (both Lesch-Nyhan and MRC-5) were infected with M. pneumoniae at two different MOIs. De novo purine synthesis was measured as usual 4 h postinfection. At 72 h postinfection, after the CPE had had a chance to develop, the CPE was measured (Table 2). We found that the inhibition of de novo purine synthesis in both cell types correlated closely with the CPE which developed in the cells: the greater the inhibition of de novo purine synthesis, the greater the CPE (i.e., a lower CPE ratio). Response of adenosine deaminase in MRC-5 fibroblasts to infection with M. pneumoniae. Since de novo purine synthesis was inhibited in the MRC-5 fibroblasts, we investigated the alterations in the purine salvage pathway of MRC-5 human lung fibroblasts due to M. pneumoniae infection. In this experiment, fibroblasts were seeded at a concentration of 1.0 x 105 cells per 100-mm tissue culture dish and grown for 48 h before infection. Because of the number of cells needed to measure a reaction, dishes were used rather than cover slip cultures. Each dish was infected with 10 ml of an M. pneumoniae suspension corresponding to an MOI of 50. After 2 h of incubation at 37 C, the mycoplasmas were aspirated off the fibroblasts and replaced with v x.0 b 6r INFECT. IMMUN Tine (hrs) FIG. 5. Response of adenosine deaminase in MRC- 5 human lung fibroblasts to infection by M. pneumoniae. Fibroblast cultures were infected with M. pneumoniae at an MOI of 50. Adenosine deaminase activity was measured in control (0) and infected (0) cultures at 4, 24, 48, and 72 h postinfection. Results are presented as percentages of adenosine converted to inosine per fibroblast cell. Each value is the mean ± standard error of the mean for three replicates.

6 VOL. 39, 1983 TABLE 3. Alterations in thymidine incorporation 4 h postinfection in MRC-5 and Lesch-Nyhan cells infected with M. pneumoniae Thymidine uptake' Cell type MOI cpmb %cof cpm ~~control" MRC x ± 32 MRC x ± 70 Lesch-Nyhan x ± 4 Lesch-Nyhan ± 4 a Mean ± standard error of the mean for eight replicates. b Determined 4 h after infection by measuring the rate of incorporation of [3H]thymidine into cells. Values are expressed as the counts of [3H]thymidine incorporated per minute per fibroblast cell. I Values are percentages of the control fibroblast incorporation of [3H]thymidine for each experiment and each cell type. fresh BME supplemented with 10% FBS. At 4, 24, 48, and 72 h postinfection, the monolayers were trypsinized, and adenosine deaminase activity was determined as described above (Fig. 5). The conversion of adenosine to inosine is presented as percent conversion to inosine per fibroblast. By this assay, M. pneumoniae demonstrated negligible activity. Shortly after infection, there were essentially no differences in adenosine deaminase activity between control and infected cells (P > 0.05). By 24 h, however, adenosine deaminase activity had increased in the infected cells and decreased in the control cells. This trend continued through the 48- and 72-h measurements. In the infected cells, adenosine deaminase activity increased nearly linearly throughout the time of infection. Conversely, adenosine deaminase activity in control cells decreased throughout the experiment. This indicates that in addition to the changes observed in de novo purine metabolism, changes in the purine salvage pathway occurred in the M. pneumoniae-infected fibroblasts. Alterations in DNA synthesis in fibroblasts infected with M. pneumoniae. Because of these changes that occurred in the de novo purine synthesis and purine salvage pathways in the infected fibroblasts, we examined the effect of infection on overall DNA synthesis in fibroblasts. Both MRC-5 and Lesch-Nyhan cells were infected with M. pneumoniae at two different MOIs. At the same time, cover slips which did not have any fibroblasts were exposed to the same concentrations of mycoplasmas. Attachment experiments revealed that the numbers of mycoplasmas attached to the cover slips were similar to the numbers attached to the monolayer cultures. DNA synthesis was measured as described above 4 h postinfection. The label METABOLISM IN M. PNEUMONJAE-INFECTED CELLS 169 incorporated by the infected fibroblasts was corrected for the label incorporated by the mycoplasmas alone. As DNA synthesis was measured 4 h after attachment, there was little loss in mycoplasma viability in BME supplemented with FBS. Table 3 shows that in the MRC-5 cells infected at a low MOI, there was essentially no change in DNA metabolism compared with uninfected cells. However, in the cells infected with M. pneumoniae at a high MOI, there was a significant (P < 0.005) change. The rate of DNA synthesis in these cells was more than 400% greater than the rate of synthesis in the control cells. This represented a dramatic change in nucleic acid metabolism considering that de novo purine synthesis was inhibited in these cells. DNA synthesis in the Lesch-Nyhan cells showed a different response. In cells infected at a low MOI, DNA synthesis was inhibited by almost 90%, whereas in cells infected at a high MOI, DNA synthesis was completely inhibited. This difference in the responses of DNA synthesis in the Lesch-Nyhan cells and the MRC-5 cells was almost certainly due to the lack of a purine salvage pathway in the Lesch-Nyhan cells. In these cells, when de novo purine synthesis was inhibited, the cells had no source of purines since they could not salvage them. Therefore, DNA synthesis was inhibited. Conversely, in the MRC-5 cells, when de novo purine synthesis was inhibited, these cells could still salvage purines and thereby maintain DNA synthesis. DISCUSSION Mutant cell lines with losses of specific enzymes and pathways offer an advantage in the study of host-parasite interactions. Such cell lines can be used to determine whether particular metabolic reactions are important in the infection process. We used Lesch-Nyhan fibroblasts, which are HGPRT deficient, to help probe the role of nucleic acid metabolism in M. pneumoniae infection. These cells lack a purine salvage pathway due to their deficiency of HGPRT and thus are dependent on de novo purine synthesis as their sole purine source. Therefore, any alterations which occur in de novo purine synthesis in infected mutant cells should be especially pronounced compared with normal cells having both de novo and salvage pathways. When we compared the susceptibilities of the Lesch-Nyhan cells and normal MRC-5 human lung fibroblasts to M. pneumoniae infection, we found that the mutant cells were four times more susceptible to cytotoxic effects by the mycoplasmas than the normal cells were. This difference

7 170 UPCHURCH AND GABRIDGE could not be attributed to differences in mycoplasma attachment, since the mycoplasmas attached to the normal MRC-5 cells with slightly greater efficiency. This indicated that more mycoplasmas infected the normal cells, but caused less damage to them than to the Lesch-Nyhan cells. Since the plating efficiency and growth of the Lesch-Nyhan cells were comparable to the plating efficiency and growth of the MRC-5 cells, this difference in susceptibility is apparently related to the enzyme deficiency in the Lesch- Nyhan cells. However, additional undetected differences between the cell types also may have affected the susceptibility of the two cell types. A measurement of the rate of de novo purine synthesis in the cells indicated that M. pneumoniae inhibited de novo purine synthesis in both cell types. The degree of inhibition was greater for the Lesch-Nyhan cells than for the MRC-5 cells. This corresponds to the greater sensitivity of the Lesch-Nyhan cells to mycoplasmas. The inhibition of de novo purine synthesis also corresponds to the greater sensitivity of the Lesch- Nyhan cells since they have no way to compensate for the decrease in purine synthesis through purine salvage, in contrast to the normal MRC-5 cells. The degree of inhibition of de novo purine synthesis in both cell types corresponded directly to the severity of the CPE which developed in the infected cells. The CPE, which was characterized by a rounding up and release of cells from the monolayer, was significantly greater in cells infected at a higher MOI. This is consistent with our previous findings, which indicated that the host cell metabolism at the time of infection and within the first few hours after infection was critical in establishing whether a CPE developed in the monolayer (24). With this in mind, any changes which occur in the host cells after M. pneumoniae infection probably need to be initiated during that critical 2- to 4-h period immediately after infection in order to induce a CPE in the monolayer. Therefore, the degree of inhibition of de novo purine synthesis observed 4 h after infection is probably indicative of the severity of damage which the fibroblasts will sustain due to the infection. Since these changes were noted in de novo purine synthesis, we also determined what changes occurred in purine salvage in normal MRC-5 cells. Measurements of adenosine deaminase, an enzyme important in this pathway, showed significantly higher levels of activity in the infected cells. These alterations in enzymatic activity were detected by 24 h postinfection. Since both de novo purine synthesis and purine salvage are important in subsequent DNA synthesis, we determined the levels of thymidine incorporation 4 h postinfection. This was possi- INFECT. IMMUN. ble early after infection due to the similarity of attachment of the mycoplasmas to monolayers and to glass cover slips. Later determinations were not possible because of the difficulty in distinguishing thymidine incorporation by an unknown number of mycoplasmas from incorporation by fibroblasts. M. pneumoniae does not survive in BME supplemented with 10% FBS for extended periods of time. Therefore, the label incorporated by mycoplasmas growing on monolayers does not necessarily correspond to the label incorporated by mycoplasmas maintained on cover slips in this medium. Thymidine phosphorylase activity in the mycoplasmas (13) did not appear to be a problem when this method was used. We found that in the normal MRC-5 fibroblasts infected at a low MOI, no change in thymidine incorporation occurred. However, in cells infected at a high MOI, a significant increase in thymidine incorporation was observed. This occurred even though the rate of de novo purine synthesis decreased in these cells. In contrast to the MRC-5 cells, thymidine incorporation and, thus, DNA synthesis were inhibited in Lesch-Nyhan cells infected with M. pneumoniae. Cells infected at a low MOI showed a decrease in DNA synthesis of almost 90%. Cells infected at a high MOI showed complete inhibition of DNA synthesis. This difference in the responses of the Lesch-Nyhan cells and the MRC-5 cells may be due to the lack of a purine salvage pathway in Lesch-Nyhan cells, since inhibition of de novo purine synthesis causes inhibition of DNA synthesis. In this study we demonstrate that nucleic acid metabolism may play an important role in the pathogenesis of M. pneumoniae infection. Previous work has shown that nucleic acids are a factor in the host-parasite interaction between M. pneumoniae and hamster tracheal organ cultures, as well as in other mycoplasma infections (19). Hu et al. (14) demonstrated a decrease in RNA synthesis in tracheal rings infected with M. pneumoniae. The work of these authors suggested a role of the mycoplasmas at the transcriptional or translational level of host cell metabolism, rather than in causing alterations in the transport of precursor molecules. Gabridge and Barden-Stahl (8) found that small amounts of adenine added to the medium alleviated the ciliostatic effects of M. pneumoniae on hamster tracheal organ cultures. These authors also showed that when the tracheal rings were prelabeled with [14C]adenine, the label could later be recovered in the mycoplasmas. Since mycoplasmas are known to require preformed nucleic acids for growth (22), these alterations of nucleic acid metabolism in host cells infected with M. pneumoniae are probably a reflection of that requirement.

8 VOL. 39, 1983 Exactly how the mycoplasmas cause these changes in the host cells remains to be determined. The mechanism of pathogenesis of M. pneumoniae infections is a complicated problem. There are many factors involved in this dynamic interaction, including attachment, host cell metabolism, mycoplasma metabolism, and external factors, such as medium and serum. Therefore, it is unlikely that any one factor can be considered solely responsible for the development of a CPE in cells infected with M. pneumoniae. However, a change in de novo purine synthesis and the corresponding changes in nucleic acid metabolism are rapid and quantitatively significant. As such, they should be considered as some of the major factors in delineating the mechanism of pathogenesis in this type of infection. ACKNOWLEDGMENT This work was supported in part by Public Health Service grant Al from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Carson, J. L., A. M. 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