activation factor P48 in Mycoplasma species

Microbial Pathogenesis 1999; 27: 145–153

Article available online at http://www.idealibrary.com on

Article No. 1999.0293

MICROBIAL PATHOGENESIS

Expression of the monocytic differentiation/ activation factor P48 in Mycoplasma species Robert E. Hall∗, Daniel P. Kestler, Sujata Agarwal & Keith M. Goldstein Department of Medicine, Division of Hematology/Oncology, University of Tennessee Medical Center/ Graduate School of Medicine, Knoxville, TN, U.S.A. (Received February 4, 1999; accepted in revised form April 29, 1999)

P48 is a 48 kd monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukemia line. It induces differentiation of HL-60 promyelocytic leukemia cells along the monocytic pathway and production of IL1, TNF-a and IL6 in human monocytes and monocytic cell lines. Recently our laboratory isolated cDNA clones for P48 from Reh cells and genomic clones from Mycoplasma fermentans DNA and showed that P48 is a M. fermentans gene product. In this paper we report the analysis of P48 expression at the DNA, mRNA and protein levels in different Mycoplasma species. Polymerase Chain Reaction (PCR) analysis of extracted DNA using P48-specific oligonucleotide primers revealed P48 sequences in M. fermentans but not M. hominis, M. iowae, M. genitalium or M. capricolum. Southern analysis of Mycoplasma DNAs revealed hybridizing bands in M. fermentans and M. capricolum under low stringency, but only in M. fermentans under high stringency. Consistent with this, Northern blot studies revealed a single hybridizing transcript in M. fermentans but not in other Mycoplasma species tested. However, Western blot studies with anti-P48 antibodies revealed P48 antigenic material in M. fermentans, as well as M. hominis and M. iowae. These studies demonstrate that the gene for P48 is derived from M. fermentans or a closely related species and is absent in these other species tested. However, the P48 protein exhibits shared antigenic determinants among several Mycoplasma species which presently are of unknown function or significance. P48 is a Mycoplasma-derived immunomodulatory molecule which may be important in Mycoplasma pathophysiology and may be useful in understanding human haematopoietic differentiation and the control of cytokine biosynthesis.  1999 Academic Press Key words: Mycoplasma, modulin, differentiation factor.

Introduction A large number of cytokines and other factors have now been identified which modulate ∗ Author for correspondence: R. E. Hall, 410 Memorial Research Center, University of Tennessee Medical Center/ Graduate School of Medicine, Knoxville, TN 37920, U.S.A. 0882–4010/99/090145+09 $30.00/0

growth, differentiation and functional activity of haematopoietic and lymphoid cells. Cytokines [1–4] and vitamins [5, 6] are known to induce differentiation and activate leukocytes. Several microbial products collectively known as modulins (reviewed in reference 7) including LPS [8], TSST-1 [9], erythrogenic toxins A and C [10], and a variety of Mycoplasma products [11–15] have  1999 Academic Press

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been shown to activate leukocytes in vitro but have not been shown to induce differentiation. Mycoplasmas are members of the class Mollicutes, which are bacteria that lack a cell wall and are parasites in a wide range of hosts including tissue culture cell lines. These organisms have also been implicated as pathogens or co-pathogens in several human and animal diseases, in which host immune reactions are also postulated to play a role. Mycoplasmas are of further interest because they have the smallest genomes (about 600–1700 kb) of all known self-replicating organisms. Several years ago our laboratory identified and purified a 48 kd protein (termed P48) from human leukemic cell line conditioned media which induced human haematopoietic differentiation and activation of mononuclear phagocytes. The biochemical characteristics and biologic activities of this factor, which have been previously reported [16–20], include induction of monocytic differentiation and cytolytic activity in the HL-60 (human promyelocytic leukemia) cell line, growth inhibition toward a large panel of other human and mouse tumour cell lines, induction of secretion of IL-1 and TNF-a by human peripheral blood monocytes and induction of accumulation of mRNAs for several cytokines including IL1-a and -b, IL6 and TNF-a. Recently we cloned the gene encoding for this factor and demonstrated that it is a Mycoplasma fermentans gene product which is likely to be a lipoprotein [21]. P48 is an interesting molecule with immunomodulatory and haematopoietic differentiation activities and may be important in the pathophysiology of Mycoplasma infections. In this report we further characterize the expression of P48 in a panel of Mycoplasma species in order to determine whether expression is restricted to M. fermentans or present in other species. We show that of the species tested P48 DNA sequences of appreciable homology and P48 RNA transcript are present only in M. fermentans. However, P48 antigenic determinants which cross-react with anti-P48 antibodies are detected in other Mycoplasma species, the significance and function of which are presently unclear.

Results PCR analysis of P48 in Mycoplasma species Previous results from our laboratory demonstrated that P48 is a M. fermentans gene prod-

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uct and that the original detection of P48 protein in Reh cell line conditioned medium was probably the result of Mycoplasma contamination. In order to test for possible expression of P48 and P48 homologues in other Mycoplasma species, we used PCR to analyse genomic DNA from several other Mycoplasma species and cell lines using P48 specific primers BH1 and BH35 and Mycoplasma genus-specific rRNA primers [22]. For comparison, we chose five species of Mycoplasma from the three main phylogenetic clades: M. fermentans and M. hominis from the hominis clade, M. genitalium and M. iowae from the pneumoniae clade and M. capricolum from the spiroplasma/mycoides clade. Using P48-specific primers, we observed amplification of a prominent band at 0.68 kb with M. fermentans, but not with M. hominis, M. iowae, M. genitalium or M. capricolum genomic DNA template (Fig. 1). When these polymerase chain reaction (PCR) products were blotted onto nylon membranes and probed with 32P-labelled P48 cDNA probe, strong hybridization was observed for M. fermentans but not the others. In contrast, all Mycoplasma species genomic DNA tested revealed a strong 0.6 kb band after amplification with Mycoplasma genus-specific rRNA primers.

Southern analysis of Reh cell line and M. fermentans DNA We have previously shown that the Reh cell line in our laboratory contained Mycoplasma ribosomal RNA gene sequences detected by PCR analysis, whereas multiple other cell lines did not [21]. Interestingly, we did not detect Mycoplasma contamination of this cell line using commercially available Mycoplasma staining or probe hybridization methods. The cDNA for P48 was initially cloned from RNA extracted from Reh cells, and later shown to be 98.5% homologous to P48 gene cloned from M. fermentans genomic DNA, thus demonstrating that P48 was derived from M. fermentans or a closely related strain [21]. In order to further identify the Mycoplasma contaminating our Reh cells and to test the hypothesis that the P48 cDNA sequence cloned from Reh cells was identical to Mycoplasma fermentans, Southern blots were performed using genomic DNA extracted from these sources, digested with several restriction enzymes and probed with 32P-labelled P48 cDNA. We found closely similar banding patterns for StuI, HaeIII and BanI but a major

P48 monocytic differentiation factor in Mycoplasmas

Figure 1. Polymerase chain reaction (PCR) analysis of the P48 gene in Mycoplasmas species. Panel A: ethidium bromide stained PCR products from 0.8 lg genomic DNA template analysed on 1.5% agarose gel. Lanes 7–11 are PCR products using Mycoplasma genus specific rRNA primers [22], and lanes 2–6 are products using P48-specific primers BH1 and BH35. Lanes: 1, UX174 HaeIII-digested DNA markers; 2 and 7, M. fermentans; 3 and 8, M. iowae; 4 and 9, M. hominis; 5 and 10, M. genitalium; 6 and 11, M. capricolum. DNA marker sizes in kilobase pairs (kbp) are indicated at the left margin. Panel B: Southern analysis of the gel in Panel A hybridized with 32P-labelled P48 cDNA probe. The lane assignments are the same as in Panel A.

difference in band size with XbaI and a modest difference with ClaI (Fig. 2). This suggested that there was a high degree of genetic similarity, with possible polymorphisms (RFLPs), between P48 gene sequences in M. fermentans and Reh.

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Figure 2. Southern analysis of Mycoplasma fermentans and Reh cell line. DNA samples were digested with various restriction endonucleases and probed with 32 P-labelled P48 cDNA. Lanes 1–5 are M. fermentans DNA (2 lg lane−1), and lanes 6–10 are Reh DNA (5 lg lane−1). Restriction endonucleases are: lanes 1 and 7, StuI; 2 and 8, HaeIII; 3 and 9, ClaI; 4 and 10, BanI; 5 and 6, XbaI. DNA molecular weight markers in kbp are shown at the right margin.

hybridized with the P48 cDNA probe. Under low stringency (3× SSC wash at 56°C) a band from M. capricolum hybridized weakly, and this was released under higher stringency. Ethidium bromide staining revealed that all lanes were loaded with approximately equal amounts of DNA, and DNA from all Mycoplasma species tested hybridized strongly with the Mycoplasma 16S ribosomal cDNA probe (not shown). We previously reported that a computer search in the GenBank for DNA sequence homologies with the P48 gene revealed no homologies greater than 60% over 208 bp, including the complete genome of M. genitalium [21].

Western blot analysis of Mycoplasma species reveals anti-P48 cross-reacting material in several species

Southern analysis of Mycoplasma species Polymerase chain reaction studies here and reverse transcriptase (RT)-PCR previously reported [21] support M. fermentans or a closely related species as the source of the P48 gene. As described above, no PCR-amplified P48 DNA sequences were detected in other Mycoplasma species tested. In order to confirm and extend these observations, we compared Southern blots of restriction endonuclease digested DNA from various Mycoplasma species (Fig. 3). Using EcoRIdigested DNA, under moderately stringent hybridization (56°C, 6× SSC) and wash (56°C, 0.2× SSC) conditions, only M. fermentans DNA

The above studies revealed the presence of the P48 gene in M. fermentans with minimally homologous DNA sequences in M. capricolum and no significant hybridization with DNA from other Mycoplasma species tested. In order to examine for the presence of P48 antigenic protein determinants conserved across various species, we performed Western blot analysis with these different Mycoplasma species. In these studies we used mouse antibodies against purified recombinant P48 expressed in Escherichia coli, which is expected to provide little or no posttranslational modification of P48, thus producing antibodies primarily or exclusively

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Figure 3. Southern analysis of Mycoplasma genomic DNA probed with P48 cDNA. DNA (3 lg) was digested with EcoRI and submitted to Southern analysis with 32P-labelled P48 cDNA probe. Lanes are: 1, M. fermentans; 2, M. hominis; 3, M. iowae; 4, M. genitalium; 5, M. capricolum. The blots were hybridized under moderate stringency (56°C, 6× SSC, 16 h) and washed up to 56°C, 0.2× SSC. DNA markers in kbp are indicated at the right margin.

against the polypeptide backbone of the molecule. In order to control for specificity, parallel blots were performed using pre-immune sera from the same animal later immunized with rP48. As shown in Fig. 4, M. fermentans yielded the strongest reaction of those tested, with M. hominis and M. iowae also clearly reacting with mouse anti-P48 antiserum but not control preimmune serum. In data not shown, we found similar patterns of reactivity with rabbit antiP48 antiserum, and in addition there was weak reactivity with other species including M. genitalium and M. capricolum.

Northern analysis of Mycoplasma species In order to further compare nucleic acids from M. fermentans and other Mycoplasma species, Northern blots were performed using total cellular RNA extracted from Mycoplasmas and probed with 32P-labelled P48 cDNA (Fig. 5). A single 1.0 kb transcript was observed in RNA from M. fermentans, but no hybridizing transcript was observed in RNA from other Mycoplasmas tested. In agreement with these data, we previously reported that RT-PCR analysis revealed P48 mRNA in M. fermentans but not in M. hominis, M. iowae, M. synoviae and M. lipophilum [21].

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Figure 4. Western blot analysis of P48 protein in Mycoplasma species. Approximately equivalent amounts of Mycoplasma cell lysates based on Ponceau S staining of SDS-PAGE gels (represent 0.2–0.6 ml cultures) were electrophoresed on 0.1% SDS-12% PAGE gels and then submitted to Western blot analysis with pre-immune mouse serum control (lanes 1–7) or mouse anti-rP48 antiserum (lanes 9–15). Lanes are: 1 and 15, 0.3 lg purified recombinant P48 (rP48); 2 and 14, 0.6 lg partially-purified P48 from Reh cell tissue culture medium [21]; 3 and 13, M. iowae; 4 and 12, M. hominis; 5 and 11, M. genitalium; 6 and 10, M. fermentans; 7 and 9, M. capricolum. Lane 8 contains prestained protein markers transferred to the blot, with molecular weights (Mr) in kd indicated at the right margin. P48 in Reh cell tissue culture medium (lane 4) is detected as a Mr 48 kd band, whereas recombinant P48 (lane 15) is detected as a Mr 18 kd band [21].

Taken together with Southern and Western blot data, this strongly suggested that the P48 gene is present in the genome and is expressed only in M. fermentans, but that anti-P48 cross-reacting protein antigens were present in other Mycoplasmas.

Discussion Our laboratory has previously identified, purified and extensively characterized a 48 kd protein termed P48, which possesses a number of monocytic differentiation and activation properties [16–20]. P48 was originally identified in the conditioned media of the Reh human leukemic cell line, and later shown to be present associated with cell membranes [19]. Recently we reported the cDNA and genomic cloning of P48 and showed that P48 represents a gene product of a Mycoplasma which had contaminated our culture of the Reh cell line [21].

P48 monocytic differentiation factor in Mycoplasmas

Figure 5. Northern analysis of RNA from Mycoplasma species. In Panel A, the blot was probed with 32Plabelled P48 cDNA, and in Panel B the same blot was probed with 32P-labelled Mycoplasma 16S rRNA cDNA probe. Lanes (7.5 lg RNA each) are: 1, M. fermentans; 2, M. capricolum; 3, M. genitalium; 4, M. hominis; 5, M. iowae. Mycoplasma rRNA positions are shown at the right margin in Svedberg units (S). The film was overexposed in order to test for possible P48 transcripts in all lanes.

The deduced amino acid sequence of P48 was found to have a typical signal sequence of a lipoprotein and a characteristic signal peptidase II cleavage site suggesting that P48 is a lipoprotein. In the current study we report PCR, Southern blot and Northern blot analyses of several Mycoplasma species with P48 specific primers and cDNA probe. These studies revealed significant homologous nucleic acid sequences in M. fermentans but not with other Mycoplasma species examined, suggesting that the P48 gene is restricted to very few (possibly only one) Mycoplasma species. Southern analysis of Reh and M. fermentans DNAs probed with P48 cDNA showed close similarity but not identical banding patterns, with some differences in restriction fragment sizes between the two DNAs (Fig. 2, XbaI and ClaI digests). This suggests the presence of restriction fragment length polymorphisms (RFLPs) and possible strain differences between the Mycoplasma infecting our Reh cell line and M. fermentans. Restriction fragment length polymorphisms have been previously reported for other genes in several other Mycoplasma species, the most studied being 16S ribosomal RNA genes [29, 30]. An alternative explanation for the

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differences observed in restriction fragments is that the DNAs may have differential methylation of cytosine residues, but this would appear to be unlikely since Mycoplasma DNA is characteristically AT rich [31]. Mycoplasma species were also examined for P48 protein using Western blots, and a 48 kd protein was found to react strongly with antiP48 antibodies in M. fermentans. Interestingly, several other Mycoplasma species had anti-P48 cross-reacting antigen(s). Since the anti-P48 antiserum used was raised against recombinant P48 produced in E. coli, the antibodies are likely to recognize P48 peptide determinants rather than post-translational modifications such as carbohydrate or lipid moieties. Thus, shared P48 protein epitopes are likely to be present in several Mycoplasma species. Consistent with these observations, antigenic epitopes are known to be as small as 7–10 amino acid residues long [32], and the degenerate amino acid code could permit amino acids homologies without significant homologies at the DNA level. Other investigators have reported other Mycoplasma proteins with shared epitopes across species and minimal or no homology at the DNA level [33, 34]. A large number of laboratories have reported that several Mycoplasma membrane proteins exhibit immunomodulatory activities toward human leukocytes. These include induction of the cytokines IL-1, TNFa and IL-6 [12–15], expression of MHC antigens [35], stimulation of oxidative radicals [36, 37], and macrophage activation [38]. Very few of these molecules have been purified or cloned and remain biochemically poorly characterized. It may be that some of these immunomodulatory activities in partially-purified Mycoplasma membrane preparations are due to P48 or antigenically crossreacting molecules. It is likely that immunomodulatory activities of these and other Mycoplasma molecules are important in the pathogenesis of Mycoplasma infections. Our studies have shown that M. fermentans, or a closely related species, appears to be the source of the P48 gene and protein, and that other Mycoplasma species tested to date do not have significant homologous DNA sequences. However, other Mycoplasma species do appear to have shared protein epitopes which are currently of unknown significance or biologic activity. Of further interest are several studies by others which have implicated M. fermentans in the morbidity, and also possibly in

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the pathogenesis, of the Acquired Immunodeficiency Syndrome and rheumatoid arthritis [39–41]. Detailed characterization of immunomodulatory Mycoplasma proteins such as P48, and their genes, should lead to better understanding of the pathogenesis of Mycoplasma infections and mechanisms controlling cytokine expression and immunomodulation in the infected host.

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P48 forward (40-mer) primer, nucleotides 85–124 (relative to ATG start codon at nt 1; reference 21) (5′GAT GAA TTC AAT AAT TCA TTC AAA GAG AAA GAT ATT AGT A 3′); BH35, P48 reverse (21-mer) primer spanning nucleotides 745–765 (5′ AAC ATT ATT AAT AAC AGT GTT 3′). Mycoplasma genus specific rRNA primers [22] used in this work were: GP01, forward (22mer): 5′ ACT CCT ACG GGA GGC AGC AGT 3′; MGSO, reverse (27-mer): 5′ TGC ACC ATC TGT CAC TCT GTT AAC CTC 3′.

Materials and methods Reagents Tissue culture media, antibiotics and Trizol RNA extraction reagents were obtained from GIBCO/ BRL (Grand Island, NY, U.S.A.), cDNA synthesis reagents were purchased from Promega (Madison, WI, U.S.A.), and PCR reagents were from GIBCO and Perkin-Elmer (Foster City, CA, U.S.A.).

Cells and cell lines The Reh (human pre-B leukemia) cell line was maintained in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 lg ml−1 streptomycin. This cell line has been maintained in our lab for several years and was recently shown to be contaminated with Mycoplasma [21].

RNA extraction RNA was extracted from cells and Mycoplasmas using the guanidinium isothiocyanate/acid phenol-chloroform method [23], with all solutions made in diethylpyrocarbonate-treated water. The extracted RNA was dissolved in 0.2 M Na-Acetate (pH 6.0) and precipitated with 2.5 volumes of absolute ethanol at −80°C, washed several times with cold 80% ethanol, dried at room temperature, and dissolved in 1 mM DTT containing 1 U ll−1 placental RNAse inhibitor (Promega).

PCR

Mycoplasma cells (M. iowae 695, M. fermentans PG18, M. hominis PG21, M. genitalium G37 and M. capricolum subsp. capricolum Calif. kld.) were a kind gift of Dr Joseph G. Tully, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research and Development Center, Frederick, MD, U.S.A. Mycoplasma synoviae and M. lipophilum were obtained from the American Type Culture Collection, Rockville, MD, U.S.A.

Polymerase chain reaction reaction mixtures (25 ll) contained 0.8 lg genomic DNA, 0.8 lM each of forward (BH1) and reverse (BH35) primers, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.25 mM dNTPs and 1.3 U Taq DNA polymerase. All dilutions were made in sterile water. The amplification cycle profiles for PCR consisted of 25 cycles as previously described [20]. Polymerase chain reaction reactions with Mycoplasma-specific primers were run under the same reaction conditions. Polymerase chain reaction products (10 ll aliquots) were electrophoreses on 1 or 1.5% agarose gels in TBE or TAE buffer [24], stained with ethidium bromide, and visualized under UV light. HaeIII restriction endonuclease-digested UX174 replicative from DNA (GIBCO/BRL) was used as DNA size markers.

Oligonucleotide primers

DNA extraction and Southern analysis

Oligonucleotide primers used in these studies were synthesized on an Applied Biosystems Model 391 DNA synthesizer as follows: BH1,

Mammalian cell DNA was extracted using perchlorate-chloroform [25], and Mycoplasma DNA was extracted using SDS-phenol/proteinase K

Mycoplasma cultures

P48 monocytic differentiation factor in Mycoplasmas

[26]. Restriction endonuclease digestion was performed under standard conditions [24]. Restriction endonuclease-digested DNA samples or PCR amplification products were electrophoresed on 0.8% (genomic DNA) or 1–1.5% (PCR products) agarose gels which were then submitted to Southern blot analysis as previously described [21]. Unless stated otherwise in figure legends, hybridizations were performed for 16 h at 65°C in 6× SSC containing 5× Denhardt’s solution, 150 lg ml−1 sonicated herring sperm DNA, 0.1% sodium pyrophosphate, and 106 cpm ml−1 32P-labelled heatdenatured cDNA probe. The probe for the P48 gene was the 0.6 kbp EcoRI excised fragment of P48 cDNA Clone 22 [21], and for the Mycoplasma rRNA gene was a PCR-amplified 0.6 kbp fragment excised after agarose electrophoresis [21]. Probes were labelled using random primer methods in the presence of 32P-dATP and 32PdCTP (specific activity 3000 Ci mM−1; ICN, Irvine, CA, U.S.A.) to a specific activity >108 cpm lg−1 DNA [24]. Unless stated otherwise in figure legends, the membranes were washed to a stringency of 0.2× SSC at 65°C. Membranes were then exposed to XAR or Biomax MR-1 film (Eastman Kodak, Rochester, NY, U.S.A.) with Lightning Plus intensifying screens (Dupont, Wilmington, DE, U.S.A.) at −80°C and developed.

Northern analysis Procedures were performed as previously described [21]. Hybridization was carried out at 41°C for 16 h with 32P-labelled heat denatured cDNA probe (106 cpm ml−1, specific activity >108 cpm lg−1 DNA) in prehybridization buffer containing 10% dextran sulfate and 50% formamide, followed by washing once (15 min, room temperature) in 3× SSC and then further washing up to high stringency (0.1× SSC, 41°C). The blots were then exposed to film and developed as described above.

Mouse anti-recombinant P48 (rP48) antiserum Recombinant P48 in the pET-15b expression vector was purified from bacterial cell lysates by extraction of rP48 containing inclusion bodies with 6 M guanidine-HCl with slow renaturation

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by successive dialysis as described for recombinant interleukin-6 [27]. The solubilized rP48 was submitted to chromatography over a 1.5×50 cm column of Sephadex G75, and the peak of activity was applied to a 1.5×6 cm DEAE-Sepharose CL6B column in 20 mM TrisHCl (pH 8.0), 5% glycerol. The flow-through was then applied to a 1.5×6 cm column of CMSepharose CL6B and eluted with a 0–0.6 M NaCl gradient in 20 mM Tris-HCl (pH 8.0), 5% glycerol, where it eluted at 0.25–0.45 M NaCl. Preimmune blood for control serum was obtained from Balb/C mice which were then immunized i.p. with 3 lg of rP48 in Complete Freund’s Adjuvant, followed 10–14 days later by three 10 lg i.p. boosts in Incomplete Freund’s Adjuvant and bleeding for antiserum 10–14 days after the last boost.

Western blots Proteins from Mycoplasma cell lysates were electrophoresed on 0.1% SDS–12% PAGE gels [28] and then transferred at room temperature onto 0.2 l nitrocellulose (Micron Separation Incorporated, Westboro, MA, U.S.A.) for 16 h at 125 mA in transfer buffer containing 20 mM Tris base, 150 mM glycine, 20% v/v methanol, pH 8.0. Filters were blocked 90 min with 5% dry milk in TBST buffer (10 mM Tris-HCl, pH 8.3, 150 mM NaCl, 0.05% Tween 20) followed by two washes (5 min each) with TBST. Filters were rocked (90 min, room temperature) with 1:400 mouse anti-rP48 antiserum or pre-immune serum from the same mouse, washed three times in TBST, rocked (1 h, room temperature) with 1: 5000 alkaline phosphatase-conjugated goat antimouse Fc-IgG (Promega), and washed three times in TBST. Filters were developed using the Protoblot AP System (Promega) with BCIP (5bromo-4-chloro-3-indolyl phosphate) and nitro blue tetrazolium in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2 according to the manufacturer’s instructions.

Acknowledgements The authors would like to thank Dr Joseph G. Tully for kindly providing some of the Mycoplasma cells used in this study and for helpful discussions and suggestions. This work was supported by NIH grants CA-58205 and CA-72591.

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