This article was downloaded by: [The Aga Khan University] On: 02 December 2014, At: 22:19 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lncn20
Mycoplasma Pneumoniae Thymidine Phosphorylase a
b
Liya Wang , Sebastian R. Schmidl & Jörg Stülke
b
a
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Center, Uppsala, Sweden b
Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, Göttingen, Germany Published online: 18 Jun 2014.
To cite this article: Liya Wang, Sebastian R. Schmidl & Jörg Stülke (2014) Mycoplasma Pneumoniae Thymidine Phosphorylase, Nucleosides, Nucleotides and Nucleic Acids, 33:4-6, 296-304, DOI: 10.1080/15257770.2013.853783 To link to this article: http://dx.doi.org/10.1080/15257770.2013.853783
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Nucleosides, Nucleotides and Nucleic Acids, 33:296–304, 2014 C Taylor and Francis Group, LLC Copyright ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2013.853783
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
MYCOPLASMA PNEUMONIAE THYMIDINE PHOSPHORYLASE
2 ¨ Stulke ¨ Liya Wang,1 Sebastian R. Schmidl,2 and Jorg 1 Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Center, Uppsala, Sweden 2 Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, G¨ottingen, Germany
2
Mycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection with Mycoplasmas compromises the efficacy of anticancer and antiviral nucleoside analog-based drugs due to the presence of Mycoplasma thymidine phosphorylase (TP). In this study, a TP-deficient strain of Mpn was generated in order to study the effect of Mpn TP in the metabolism of nucleoside analogs. Deficiency in TP activity led to increased uptake and incorporation of radiolabeled deoxyuridine and uracil but thymidine uptake was not affected. The activities of enzymes in the salvage of thymidine and deoxyuridine, e.g., thymidine kinase and uracil phosphoribosyltransferase were upregulated in the TP-deficient mutant, which may explain the increased uptake of deoxyuridine and uracil. Thirty FDA-approved anticancer and antiviral nucleoside and nucleobase analogs were used to screen their inhibitory activity toward the TP mutant and the wild type strain. Seven analogs were found to inhibit strongly the growth of both wild type and TP mutant. Differences in the inhibitory effect of several purine analogs between the two strains were observed. Further study is needed in order to understand the mechanism of inhibition caused by these analogs. Our results indicated that TP is not an essential gene for Mpn survival and TP deficiency affects other enzymes in Mpn nucleotide metabolism, and suggested that Mycoplasma nucleotide biosynthesis pathway enzymes are potential targets for future development of antibiotics.
Keywords Mycoplasma pneumonia; thymidine phosphorylase; thymidine kinase; nucleoside and nucleobase analogs; growth inhibition
INTRODUCTION Mycoplasmas are wall-less gram-positive bacteria that are pathogens to humans, animals, and plants, etc. Mycoplasma pneumoniae (Mpn) is a Received 30 August 2013; accepted 6 October 2013. This work was supported by a grant from the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning. Address correspondence to Liya Wang, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Center, Box 575, SE-751 23, Uppsala, Sweden. E-mail: [email protected]
296
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
Mycoplasma Pneumoniae Thymidine Phosphorylase
297
human pathogen and causes upper and lower respiratory tract infections and accounts for approximately 30% cases of community-acquired pneumonia. Mpn is also associated with many extra-pulmonary diseases, such as asthma and rheumatoid arthritis.[1,2] Epidemics of Mpn infections have been reported in Scandinavian countries and other European countries during 2010–2012.[3] Macrolide-resistant Mpn strains have emerged worldwide and been spreading in Europe, USA, and Asia, especially in Japan and China with >90% isolates that are macrolide resistant.[4] Therefore, new antibiotics are needed. Mycoplasmas can also grow in close contact with human cells without apparent pathology and have been detected/isolated from bone marrow of leukemia patients, and from ovarian and cervical cancer tissues. In particular Mycoplasma hyorhinis (M. hyorhinis) was found at unusually high frequency in cancer tissues. Chronic infection by Mycoplasma species may increase host cell vulnerability to transform and promote tumor growth.[5–7] Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine (dT), deoxyuridine (dU), and uridine, and plays an important role in maintaining the plasma level of dT and dU. TP deficiency in human is associated with mitochondrial neurointestinal encephalomyopathy.[8] TP is also known as the only endothelial mitogenic and angiogenic factor and plays an important role in cancer cell migration and metastasis, and activation of nucleoside and nucleobase-based anticancer drugs.[9] Recent studies have shown that Mycoplasma infection in cancer tissues or cells compromises the efficacy of nucleoside analog-based drugs due to the presence of Mycoplasma TP.[10–12] M. hyorhinis TP was characterized recently and it was shown that this enzyme is capable of degrading dT, dU, and uridine in addition to several nucleoside analogs used in anticancer and antiviral therapy, such as trifluorothymidine and 5-fluorodeoxyuridine, and the presence of M. hyorhinis in cultured cancer cells greatly reduces the efficacy of nucleoside analogs.[12] Mpn TP, encoded by the deoA gene (MPN064), consists of 421 amino acid and shows 41% sequence identity to M. hyorhinis TP and 33% sequence identity to human TP. The amino acid residues made up the phosphate-binding site and dT-binding site are highly conserved among these protein sequences,[12] suggesting that Mpn TP should have similar enzymatic properties to human and M. hyorhinis TP. In this study, Mpn TP (deoA)-deficient mutant was generated and the metabolism of dT, dU, and Ura was studied in the TP-deficient mutant and compared with the wild type. The activities of enzymes in the salvage of dT, dU, and Ura were determined. In addition, 30 FDA-approved anticancer nucleoside and nucleobase prodrugs were used to screen for their inhibitory effect on Mpn growth and several potent growth inhibitors were found.
298
L. Wang et al.
MATERIALS AND METHODS Nucleosides
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
[3H]-thymidine ([3H]-dT, 20 Ci/mmol) and uracil [3H]-Ura (29 Ci/mmol) were purchased from PerkinElmer (Upplands V¨asby, Sweden). [3H]-deoxyuridine ([3H]-dU, 27 Ci/mmol) was from Moravek Biochemicals, Inc. (Brea, CA). Non-radiolabeled nucleosides were from Sigma-Aldrich (St. Louis, MO). Nucleoside and nucleobase analogs library was kindly provided by Prof. P¨ar Nordlund, from the Karolinska Institute, Stockholm, Sweden. Generation of Mpn TP-Deficient Mutant Mpn TP (MPN624, gene name: deoA) gene was disrupted by using a global transposon mutagenesis method essentially as described.[13,14] Positive clones were isolated on agar-broth medium plates and cultured in Hayflick’s medium. DNA was extracted and used as template in PCR reactions using primers specific for Mpn TP in combination with transposon-specific primers to ensure that insertion site was only at the TP gene. The TP-deficient mutant strain was viable and the growth was apparently normal in Hayflick’s medium. The culture was then expanded and total protein was extracted as previously described.[14] Mycoplasma Culture and Inhibition Studies Mpn laboratory strain M129 (wild type) and the TP-deficient strain were cultured at 37◦ C in CO2 incubator using 75 cm2 tissue culture flask containing 50 ml Hayflick’s medium and harvested at Day 4 when the medium color change was observed.[14] Total protein was extracted[15] and used to determine TP, thymidine kinase (TK), and uracil phosphoribosyltransferase (UPRT) activity. Uptake of radiolabeled dT, dU, and Ura was performed essentially as described.[14] Total uptake is the percent of radioactivity recovered in the cells divided by total radioactivity added to the growth medium. Percent of acid insoluble (radioactivity found in DNA and RNA) was also calculated.[14] These experiments were done more than three times and data are given as mean ± SD. Growth inhibition of the wild type and TP-deficient strains was performed in 96-well plates as previously described.[16] MIC90 is the minimal concentration required to produce 90% inhibition. Enzyme Assays TK and TP activities were determined using [3H]-dT as the radiolabeled substrate essentially as described.[14] UPRT activity was determined using the DE-81 filter paper assay with [3H]-Ura as substrate, similar to the method
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
Mycoplasma Pneumoniae Thymidine Phosphorylase
299
described for hypoxanthine guanine phosphoribosyltranferase.[17] Briefly, the reaction mixture contained 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2 , 5 mM DTT, 15 mM NaF, 1 mM phosphoribosyl pyrophosphate, and various concentrations of [3H]-Ura in a total of 50 μl. The reaction was initiated by the addition of Mycoplasma extracts, at 0, 5, 10, and 15 minute intervals, 10 μl reaction mixture was withdrawn, spotted onto the DE-81 filter paper and dried. The unreacted substrate was washed out and the products were eluted in 0.5 ml 0.1 M HCl/0.2 M KCl, mixed with scintillation fluid and counted in a liquid scintillation counter. All enzyme activities were expressed as the formation of product (nmol) per minute and per mg protein. All assays were done in triplicate and the data were presented as mean ± SD. Statistical Analysis Student’s t-test was used for statistical analysis. P value 500 >500 >500 >500 15.6 7.8 31.2 >500 >500 >500 0.2 >500 >500 31.2 31.2 >500 1.9
>500 >500 >500 7.8 2.4 7.8 >500 >500 >500 >500 25 1.8 31.2 >500 >500 >500 >500 15.6 7.8 62.5 >500 >500 >500 0.2 >500 >500 31.2 31.2 >500 1.9
Growth inhibition was performed in 96-well plates in the presence of analogs (two-fold series dilution with growth medium) with each concentration in 3–4 replica. The experiments were repeated once with the same results. MIC = minimal concentrations of the compound that produced 90% inhibition. ∗ data from Ref. [16].
TP mutant growth with MIC90 value of 0.2 μg/ml. Gemcitabine, trifluorothymidine, and dipyridamole had an MIC90 value of ∼2 μg/ml. Antiviral dT analogs, e.g., azidothymidine and stavudine were also strong inhibitors of Mpn growth with MIC90 of 7.8 μg/ml. 5-halogenated pyrimidine analogs, e.g., 5-fluorodeoxyuridine, 5-iododeoxyuridine, 5-fluorouracil, and 5-fluorocytosine also inhibited Mpn growth but with higher MIC90 values. Interestingly, purine analogs ribavirin, pentoxifylline, acyclovir, and gancyclovir inhibited the wild type but not the TP mutant growth. Mycophenolate mofetil inhibited the TP mutant growth stronger than the wild type. Adefovir depivoxil inhibited the TP mutant but not the wild type growth (Table 3).
302
L. Wang et al.
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
DISCUSSION Mycoplasmas lack the de novo synthesis of purine and pyrimidine bases, and therefore, they solely rely on the salvage of nucleosides and bases from the growth medium or their host for the biosynthesis of DNA and RNA precursors. In this study, we generated a TP-deficient Mpn mutant and showed that this TP mutant is viable. Mpn TP is apparently responsible for the degradation of dT and dU present in the growth medium since both dT and dU were stable in the TP mutant culture medium. Interestingly, lacking of TP activity led to increased uptake and metabolism of dU, dT, or Ura and the induction of TK and UPRT activity. Many anticancer drugs are nucleoside and nucleobase analogs, which either target the nucleotide synthesis pathway or once metabolized in the cells the active metabolites interfere with DNA or RNA synthesis and thus cause cell death. Cellular TP, in this aspect, plays a dual role: inhibition of TP activity can abrogate the tumorigenesis and metastatic properties of TP. On the other hand, many anticancer drugs, such as capecitabine, require the activation by TP in order to exert their cytotoxic effect.[11] The presence of Mycoplasma TP in the vicinity of cancer cells may not only contribute to tumorigenesis and metastatic development of tumor cells but also compromise efficacies of those analogs that are substrates or inhibitors of Mycoplasma TP. Therefore, it is important to study the effect of Mycoplasma infection on the metabolism and stability of current nucleoside analog-based prodrugs, as was shown by recent study with M. hyorhinis TP.[12] These results will benefit cancer patients in choosing correct therapeutic regime and also development of new drugs. Our screening of 30 FDA-approved nucleoside and nucleobase analogs revealed several analogs that are potent inhibitors of Mycoplasma growth, particularly the anticancer drugs 6-thioguanine, trifluorothymidine, dipyridamole, and gemcitabine. The mechanism of the observed growth inhibition by these analogs is most likely inhibition of target enzymes in the metabolism of these drugs, e.g., TK, or incorporation of the active metabolites, e.g., analog triphosphates into DNA or RNA leading to cell death.[16] Although at this moment there is no explanation why some of the purine analogs inhibited the wild type but not the TP mutant or vice versa. It is possible that these purine analogs or their metabolites act as substrates and/or inhibitors of TP and thus affected pyrimidine metabolism, as was shown with human uridine phosphorylase 1, which was inhibited by the purine analog vidarabine.[18] Another possible mechanism is that TP deficiency may have altered regulation of enzymes in purine metabolism, such as deoxyadenosine kinase or hypoxanthine guanine phosphoribosyltransferase. Further studies are needed in order to clarify this discrepancy. In conclusion, TP mutant Mpn was viable and deficiency in TP activity led to increased activities of enzymes in the pyrimidine salvage, e.g., TK
Mycoplasma Pneumoniae Thymidine Phosphorylase
303
and UPRT. Several nucleoside and nucleobase analogs showed potent inhibitory effects on Mpn growth. Further investigation of the mechanism of action of these drugs may lead to development of specific antibiotics against Mycoplasma or other bacterial infection.
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
REFERENCES 1. Razin, S.; Yogev, D.; Naot, Y. Molecular biology and pathogenicity of Mycoplasmas. Microbiol. Mol. Biol. Rev. 1998, 62, 1094–1156. 2. Waites, K.B.; Talkington, D.F. Mycoplasma pneumoniae and its role as a human pathogen. Clin. Microbiol. Rev. 2004, 17, 697–728. 3. Lenglet, A.; Herrado, Z.; Magiorakos, A.; Leitmeyer, K.; Coulombier, D. Surveillance status and recent data for Mycoplasma pneumoniae infection in the European union and European economic area, January 2012. Euro. Surveill 2012, 17, 2–7. 4. Bebear, C.; Pereyre, S.; Peuchant, O. Mycoplasma pneumoniae: susceptibility and resistance to antibiotics. Future Microbiol. 2011, 6, 423–431. 5. Tsai, S.; Waer, D.J.; Shih, J.W.; Lo, S.C. Mycoplasmas and oncogenesis: persistant infection and multistage malignant transformation. Proc. Natl. Acad. Sci. USA 1995, 92, 10197– 10201. 6. Yu, E.J.; Rha, S.Y.; Kim, T.S.; Chung, H.C.; Oh, B.K.; Ang, W.I.; Noh, S.H.; Jeung, H.C. Angiogenic factor thymidine phosphorylase increases cancer cell invasion activity in patients with gastric adenocarcinoma. Mol. Cancer Res. 2008, 6, 1554–1566. 7. Namiki, K.; Goodison, S.; Porvasnik, S.; Allan, R.W.; Iczkowski, K.A.; Urbanek, C.; Reyes, L.; Sakamoto, N.; Rosser, C.J. Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells. PLoS ONE 2009, 4, e6872. 8. Nishino, I.; Spinazzola, A.; Papadimitriou, A.; Hammans, S.; Steiner, I.; Hahn, C.; Connolly, A.; Verloes, A.; Guimaraes, J.; Maillard, I.; Hamano, H.; Donati, M.; Semrad, C.; Russell, J.; Andreu, A.; Hadjigeorgiou, G.; Vu, T.; Tadesse, S.; Nygaard, T.; Nonaka, I.; Hirano, I.; Bonilla, E.; Rowland, L.; DiMauro, S.; Hirano, M. Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations. Ann. Neurol. 2000, 47, 792–800. 9. Akiyama, S.; Furukawa, T.; Sumizawa, T.; Takebayashi, Y.; Nakajima, Y.; Shimaoka, S.; Haraguchi, M. The role of thymidine phosphorylase, an angiogenic enzyme, in tumor progression. Cancer Sci. 2004, 95, 851–857. 10. Bronckaers, A.; Balzarini, J.; Liekens, S. The cytostatic activity of pyrimidine nucleosides is strongly modulated by Mycoplasma hyorhinis infection: implications for cancer therapy. Biochem. Pharmacol. 2008, 76, 188–197. 11. Bronckaers, A.; Gago, F.; Balzarini, J.; Liekens, S. The dual role of thymidine phosphorylase in cancer development and chemotherapy. Med. Res. Rev. 2009, 29, 903–953. 12. Vande Voorde, J.; Gago, F.; Vrancken, K.; Liekens, S.; Balzarini, J. Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues. Biochem. J. 2012, 445, 113–123. 13. Halbedel, S.; St¨ulke, J. Dual phosphorylation of Mycoplasma pneumoniae HPr by enzyme I and HPr kinase suggests an extended phosphoryl group susceptibility of HPr. FEMS Microbiol. Lett. 2004, 247, 193–198. ¨ 14. Wang, L.; Hames, C.; Schmidt, S.; Stulke, J. Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae. Mol. Microbiol. 2010, 77, 1502–1511. 15. Wang, L.; Westberg, J.; B¨olske, G.; Eriksson, S. Novel deoxynucleoside-phosphorylating enzymes in Mycoplasmas: evidence for efficient utilization of deoxynucleosides. Mol. Microbiol. 2001, 42, 1065–1073. 16. Sun, R.; Wang, L. Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs. BMC Microbiol. 2013, 13, 184.
304
L. Wang et al.
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
17. Welin, M.; Egeblad, L.; Johansson, A.; Stenmark, P.; Wang, L.; Flodin, S.; Nyman, T.; Tr´esaugues, L.; Kotenyova, T.; Johansson, I.; Eriksson, S.; Eklund, H.; Nordlund, P. Structural and function studies of the human phosphoribosyltransferase domain containing protein 1. FEBS J. 2010, 277, 4920–4930. 18. Egeblad, L.; Welin, M.; Flodin, S.; Gr¨aslund, S.; Wang, L.; Balzarini, J.; Eriksson, S.; Nordlund, P. Pan-pathway based interaction profiling of FDA-approved nucleoside and nucleobase analogs with enzymes of the human nucleotide metabolism. PLoS ONE 2012, 7, e37724.
Nucleosides, Nucleotides and Nucleic Acids Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lncn20
Mycoplasma Pneumoniae Thymidine Phosphorylase a
b
Liya Wang , Sebastian R. Schmidl & Jörg Stülke
b
a
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Center, Uppsala, Sweden b
Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, Göttingen, Germany Published online: 18 Jun 2014.
To cite this article: Liya Wang, Sebastian R. Schmidl & Jörg Stülke (2014) Mycoplasma Pneumoniae Thymidine Phosphorylase, Nucleosides, Nucleotides and Nucleic Acids, 33:4-6, 296-304, DOI: 10.1080/15257770.2013.853783 To link to this article: http://dx.doi.org/10.1080/15257770.2013.853783
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Nucleosides, Nucleotides and Nucleic Acids, 33:296–304, 2014 C Taylor and Francis Group, LLC Copyright ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2013.853783
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
MYCOPLASMA PNEUMONIAE THYMIDINE PHOSPHORYLASE
2 ¨ Stulke ¨ Liya Wang,1 Sebastian R. Schmidl,2 and Jorg 1 Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Center, Uppsala, Sweden 2 Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, G¨ottingen, Germany
2
Mycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection with Mycoplasmas compromises the efficacy of anticancer and antiviral nucleoside analog-based drugs due to the presence of Mycoplasma thymidine phosphorylase (TP). In this study, a TP-deficient strain of Mpn was generated in order to study the effect of Mpn TP in the metabolism of nucleoside analogs. Deficiency in TP activity led to increased uptake and incorporation of radiolabeled deoxyuridine and uracil but thymidine uptake was not affected. The activities of enzymes in the salvage of thymidine and deoxyuridine, e.g., thymidine kinase and uracil phosphoribosyltransferase were upregulated in the TP-deficient mutant, which may explain the increased uptake of deoxyuridine and uracil. Thirty FDA-approved anticancer and antiviral nucleoside and nucleobase analogs were used to screen their inhibitory activity toward the TP mutant and the wild type strain. Seven analogs were found to inhibit strongly the growth of both wild type and TP mutant. Differences in the inhibitory effect of several purine analogs between the two strains were observed. Further study is needed in order to understand the mechanism of inhibition caused by these analogs. Our results indicated that TP is not an essential gene for Mpn survival and TP deficiency affects other enzymes in Mpn nucleotide metabolism, and suggested that Mycoplasma nucleotide biosynthesis pathway enzymes are potential targets for future development of antibiotics.
Keywords Mycoplasma pneumonia; thymidine phosphorylase; thymidine kinase; nucleoside and nucleobase analogs; growth inhibition
INTRODUCTION Mycoplasmas are wall-less gram-positive bacteria that are pathogens to humans, animals, and plants, etc. Mycoplasma pneumoniae (Mpn) is a Received 30 August 2013; accepted 6 October 2013. This work was supported by a grant from the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning. Address correspondence to Liya Wang, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Center, Box 575, SE-751 23, Uppsala, Sweden. E-mail: [email protected]
296
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
Mycoplasma Pneumoniae Thymidine Phosphorylase
297
human pathogen and causes upper and lower respiratory tract infections and accounts for approximately 30% cases of community-acquired pneumonia. Mpn is also associated with many extra-pulmonary diseases, such as asthma and rheumatoid arthritis.[1,2] Epidemics of Mpn infections have been reported in Scandinavian countries and other European countries during 2010–2012.[3] Macrolide-resistant Mpn strains have emerged worldwide and been spreading in Europe, USA, and Asia, especially in Japan and China with >90% isolates that are macrolide resistant.[4] Therefore, new antibiotics are needed. Mycoplasmas can also grow in close contact with human cells without apparent pathology and have been detected/isolated from bone marrow of leukemia patients, and from ovarian and cervical cancer tissues. In particular Mycoplasma hyorhinis (M. hyorhinis) was found at unusually high frequency in cancer tissues. Chronic infection by Mycoplasma species may increase host cell vulnerability to transform and promote tumor growth.[5–7] Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine (dT), deoxyuridine (dU), and uridine, and plays an important role in maintaining the plasma level of dT and dU. TP deficiency in human is associated with mitochondrial neurointestinal encephalomyopathy.[8] TP is also known as the only endothelial mitogenic and angiogenic factor and plays an important role in cancer cell migration and metastasis, and activation of nucleoside and nucleobase-based anticancer drugs.[9] Recent studies have shown that Mycoplasma infection in cancer tissues or cells compromises the efficacy of nucleoside analog-based drugs due to the presence of Mycoplasma TP.[10–12] M. hyorhinis TP was characterized recently and it was shown that this enzyme is capable of degrading dT, dU, and uridine in addition to several nucleoside analogs used in anticancer and antiviral therapy, such as trifluorothymidine and 5-fluorodeoxyuridine, and the presence of M. hyorhinis in cultured cancer cells greatly reduces the efficacy of nucleoside analogs.[12] Mpn TP, encoded by the deoA gene (MPN064), consists of 421 amino acid and shows 41% sequence identity to M. hyorhinis TP and 33% sequence identity to human TP. The amino acid residues made up the phosphate-binding site and dT-binding site are highly conserved among these protein sequences,[12] suggesting that Mpn TP should have similar enzymatic properties to human and M. hyorhinis TP. In this study, Mpn TP (deoA)-deficient mutant was generated and the metabolism of dT, dU, and Ura was studied in the TP-deficient mutant and compared with the wild type. The activities of enzymes in the salvage of dT, dU, and Ura were determined. In addition, 30 FDA-approved anticancer nucleoside and nucleobase prodrugs were used to screen for their inhibitory effect on Mpn growth and several potent growth inhibitors were found.
298
L. Wang et al.
MATERIALS AND METHODS Nucleosides
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
[3H]-thymidine ([3H]-dT, 20 Ci/mmol) and uracil [3H]-Ura (29 Ci/mmol) were purchased from PerkinElmer (Upplands V¨asby, Sweden). [3H]-deoxyuridine ([3H]-dU, 27 Ci/mmol) was from Moravek Biochemicals, Inc. (Brea, CA). Non-radiolabeled nucleosides were from Sigma-Aldrich (St. Louis, MO). Nucleoside and nucleobase analogs library was kindly provided by Prof. P¨ar Nordlund, from the Karolinska Institute, Stockholm, Sweden. Generation of Mpn TP-Deficient Mutant Mpn TP (MPN624, gene name: deoA) gene was disrupted by using a global transposon mutagenesis method essentially as described.[13,14] Positive clones were isolated on agar-broth medium plates and cultured in Hayflick’s medium. DNA was extracted and used as template in PCR reactions using primers specific for Mpn TP in combination with transposon-specific primers to ensure that insertion site was only at the TP gene. The TP-deficient mutant strain was viable and the growth was apparently normal in Hayflick’s medium. The culture was then expanded and total protein was extracted as previously described.[14] Mycoplasma Culture and Inhibition Studies Mpn laboratory strain M129 (wild type) and the TP-deficient strain were cultured at 37◦ C in CO2 incubator using 75 cm2 tissue culture flask containing 50 ml Hayflick’s medium and harvested at Day 4 when the medium color change was observed.[14] Total protein was extracted[15] and used to determine TP, thymidine kinase (TK), and uracil phosphoribosyltransferase (UPRT) activity. Uptake of radiolabeled dT, dU, and Ura was performed essentially as described.[14] Total uptake is the percent of radioactivity recovered in the cells divided by total radioactivity added to the growth medium. Percent of acid insoluble (radioactivity found in DNA and RNA) was also calculated.[14] These experiments were done more than three times and data are given as mean ± SD. Growth inhibition of the wild type and TP-deficient strains was performed in 96-well plates as previously described.[16] MIC90 is the minimal concentration required to produce 90% inhibition. Enzyme Assays TK and TP activities were determined using [3H]-dT as the radiolabeled substrate essentially as described.[14] UPRT activity was determined using the DE-81 filter paper assay with [3H]-Ura as substrate, similar to the method
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
Mycoplasma Pneumoniae Thymidine Phosphorylase
299
described for hypoxanthine guanine phosphoribosyltranferase.[17] Briefly, the reaction mixture contained 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2 , 5 mM DTT, 15 mM NaF, 1 mM phosphoribosyl pyrophosphate, and various concentrations of [3H]-Ura in a total of 50 μl. The reaction was initiated by the addition of Mycoplasma extracts, at 0, 5, 10, and 15 minute intervals, 10 μl reaction mixture was withdrawn, spotted onto the DE-81 filter paper and dried. The unreacted substrate was washed out and the products were eluted in 0.5 ml 0.1 M HCl/0.2 M KCl, mixed with scintillation fluid and counted in a liquid scintillation counter. All enzyme activities were expressed as the formation of product (nmol) per minute and per mg protein. All assays were done in triplicate and the data were presented as mean ± SD. Statistical Analysis Student’s t-test was used for statistical analysis. P value 500 >500 >500 >500 15.6 7.8 31.2 >500 >500 >500 0.2 >500 >500 31.2 31.2 >500 1.9
>500 >500 >500 7.8 2.4 7.8 >500 >500 >500 >500 25 1.8 31.2 >500 >500 >500 >500 15.6 7.8 62.5 >500 >500 >500 0.2 >500 >500 31.2 31.2 >500 1.9
Growth inhibition was performed in 96-well plates in the presence of analogs (two-fold series dilution with growth medium) with each concentration in 3–4 replica. The experiments were repeated once with the same results. MIC = minimal concentrations of the compound that produced 90% inhibition. ∗ data from Ref. [16].
TP mutant growth with MIC90 value of 0.2 μg/ml. Gemcitabine, trifluorothymidine, and dipyridamole had an MIC90 value of ∼2 μg/ml. Antiviral dT analogs, e.g., azidothymidine and stavudine were also strong inhibitors of Mpn growth with MIC90 of 7.8 μg/ml. 5-halogenated pyrimidine analogs, e.g., 5-fluorodeoxyuridine, 5-iododeoxyuridine, 5-fluorouracil, and 5-fluorocytosine also inhibited Mpn growth but with higher MIC90 values. Interestingly, purine analogs ribavirin, pentoxifylline, acyclovir, and gancyclovir inhibited the wild type but not the TP mutant growth. Mycophenolate mofetil inhibited the TP mutant growth stronger than the wild type. Adefovir depivoxil inhibited the TP mutant but not the wild type growth (Table 3).
302
L. Wang et al.
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
DISCUSSION Mycoplasmas lack the de novo synthesis of purine and pyrimidine bases, and therefore, they solely rely on the salvage of nucleosides and bases from the growth medium or their host for the biosynthesis of DNA and RNA precursors. In this study, we generated a TP-deficient Mpn mutant and showed that this TP mutant is viable. Mpn TP is apparently responsible for the degradation of dT and dU present in the growth medium since both dT and dU were stable in the TP mutant culture medium. Interestingly, lacking of TP activity led to increased uptake and metabolism of dU, dT, or Ura and the induction of TK and UPRT activity. Many anticancer drugs are nucleoside and nucleobase analogs, which either target the nucleotide synthesis pathway or once metabolized in the cells the active metabolites interfere with DNA or RNA synthesis and thus cause cell death. Cellular TP, in this aspect, plays a dual role: inhibition of TP activity can abrogate the tumorigenesis and metastatic properties of TP. On the other hand, many anticancer drugs, such as capecitabine, require the activation by TP in order to exert their cytotoxic effect.[11] The presence of Mycoplasma TP in the vicinity of cancer cells may not only contribute to tumorigenesis and metastatic development of tumor cells but also compromise efficacies of those analogs that are substrates or inhibitors of Mycoplasma TP. Therefore, it is important to study the effect of Mycoplasma infection on the metabolism and stability of current nucleoside analog-based prodrugs, as was shown by recent study with M. hyorhinis TP.[12] These results will benefit cancer patients in choosing correct therapeutic regime and also development of new drugs. Our screening of 30 FDA-approved nucleoside and nucleobase analogs revealed several analogs that are potent inhibitors of Mycoplasma growth, particularly the anticancer drugs 6-thioguanine, trifluorothymidine, dipyridamole, and gemcitabine. The mechanism of the observed growth inhibition by these analogs is most likely inhibition of target enzymes in the metabolism of these drugs, e.g., TK, or incorporation of the active metabolites, e.g., analog triphosphates into DNA or RNA leading to cell death.[16] Although at this moment there is no explanation why some of the purine analogs inhibited the wild type but not the TP mutant or vice versa. It is possible that these purine analogs or their metabolites act as substrates and/or inhibitors of TP and thus affected pyrimidine metabolism, as was shown with human uridine phosphorylase 1, which was inhibited by the purine analog vidarabine.[18] Another possible mechanism is that TP deficiency may have altered regulation of enzymes in purine metabolism, such as deoxyadenosine kinase or hypoxanthine guanine phosphoribosyltransferase. Further studies are needed in order to clarify this discrepancy. In conclusion, TP mutant Mpn was viable and deficiency in TP activity led to increased activities of enzymes in the pyrimidine salvage, e.g., TK
Mycoplasma Pneumoniae Thymidine Phosphorylase
303
and UPRT. Several nucleoside and nucleobase analogs showed potent inhibitory effects on Mpn growth. Further investigation of the mechanism of action of these drugs may lead to development of specific antibiotics against Mycoplasma or other bacterial infection.
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
REFERENCES 1. Razin, S.; Yogev, D.; Naot, Y. Molecular biology and pathogenicity of Mycoplasmas. Microbiol. Mol. Biol. Rev. 1998, 62, 1094–1156. 2. Waites, K.B.; Talkington, D.F. Mycoplasma pneumoniae and its role as a human pathogen. Clin. Microbiol. Rev. 2004, 17, 697–728. 3. Lenglet, A.; Herrado, Z.; Magiorakos, A.; Leitmeyer, K.; Coulombier, D. Surveillance status and recent data for Mycoplasma pneumoniae infection in the European union and European economic area, January 2012. Euro. Surveill 2012, 17, 2–7. 4. Bebear, C.; Pereyre, S.; Peuchant, O. Mycoplasma pneumoniae: susceptibility and resistance to antibiotics. Future Microbiol. 2011, 6, 423–431. 5. Tsai, S.; Waer, D.J.; Shih, J.W.; Lo, S.C. Mycoplasmas and oncogenesis: persistant infection and multistage malignant transformation. Proc. Natl. Acad. Sci. USA 1995, 92, 10197– 10201. 6. Yu, E.J.; Rha, S.Y.; Kim, T.S.; Chung, H.C.; Oh, B.K.; Ang, W.I.; Noh, S.H.; Jeung, H.C. Angiogenic factor thymidine phosphorylase increases cancer cell invasion activity in patients with gastric adenocarcinoma. Mol. Cancer Res. 2008, 6, 1554–1566. 7. Namiki, K.; Goodison, S.; Porvasnik, S.; Allan, R.W.; Iczkowski, K.A.; Urbanek, C.; Reyes, L.; Sakamoto, N.; Rosser, C.J. Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells. PLoS ONE 2009, 4, e6872. 8. Nishino, I.; Spinazzola, A.; Papadimitriou, A.; Hammans, S.; Steiner, I.; Hahn, C.; Connolly, A.; Verloes, A.; Guimaraes, J.; Maillard, I.; Hamano, H.; Donati, M.; Semrad, C.; Russell, J.; Andreu, A.; Hadjigeorgiou, G.; Vu, T.; Tadesse, S.; Nygaard, T.; Nonaka, I.; Hirano, I.; Bonilla, E.; Rowland, L.; DiMauro, S.; Hirano, M. Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations. Ann. Neurol. 2000, 47, 792–800. 9. Akiyama, S.; Furukawa, T.; Sumizawa, T.; Takebayashi, Y.; Nakajima, Y.; Shimaoka, S.; Haraguchi, M. The role of thymidine phosphorylase, an angiogenic enzyme, in tumor progression. Cancer Sci. 2004, 95, 851–857. 10. Bronckaers, A.; Balzarini, J.; Liekens, S. The cytostatic activity of pyrimidine nucleosides is strongly modulated by Mycoplasma hyorhinis infection: implications for cancer therapy. Biochem. Pharmacol. 2008, 76, 188–197. 11. Bronckaers, A.; Gago, F.; Balzarini, J.; Liekens, S. The dual role of thymidine phosphorylase in cancer development and chemotherapy. Med. Res. Rev. 2009, 29, 903–953. 12. Vande Voorde, J.; Gago, F.; Vrancken, K.; Liekens, S.; Balzarini, J. Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues. Biochem. J. 2012, 445, 113–123. 13. Halbedel, S.; St¨ulke, J. Dual phosphorylation of Mycoplasma pneumoniae HPr by enzyme I and HPr kinase suggests an extended phosphoryl group susceptibility of HPr. FEMS Microbiol. Lett. 2004, 247, 193–198. ¨ 14. Wang, L.; Hames, C.; Schmidt, S.; Stulke, J. Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae. Mol. Microbiol. 2010, 77, 1502–1511. 15. Wang, L.; Westberg, J.; B¨olske, G.; Eriksson, S. Novel deoxynucleoside-phosphorylating enzymes in Mycoplasmas: evidence for efficient utilization of deoxynucleosides. Mol. Microbiol. 2001, 42, 1065–1073. 16. Sun, R.; Wang, L. Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs. BMC Microbiol. 2013, 13, 184.
304
L. Wang et al.
Downloaded by [The Aga Khan University] at 22:19 02 December 2014
17. Welin, M.; Egeblad, L.; Johansson, A.; Stenmark, P.; Wang, L.; Flodin, S.; Nyman, T.; Tr´esaugues, L.; Kotenyova, T.; Johansson, I.; Eriksson, S.; Eklund, H.; Nordlund, P. Structural and function studies of the human phosphoribosyltransferase domain containing protein 1. FEBS J. 2010, 277, 4920–4930. 18. Egeblad, L.; Welin, M.; Flodin, S.; Gr¨aslund, S.; Wang, L.; Balzarini, J.; Eriksson, S.; Nordlund, P. Pan-pathway based interaction profiling of FDA-approved nucleoside and nucleobase analogs with enzymes of the human nucleotide metabolism. PLoS ONE 2012, 7, e37724.
