NAD is a necessary cofactor present in all living cells. Some bacteria cannot synthesize NAD and must use the salvage pathway to import niacin or nicotinamide riboside via substrate importers NiaX and PnuC, respectively. Although homologues of these two importers and their substrates have been identified in other organisms, limited data exist in , specifically, on its effect on overall virulence. Here, we sought to characterize the substrate specificity of NiaX and PnuC in TIGR4 and the contribution of these proteins to virulence of the pathogen. Although binding affinity of each importer for nicotinamide mononucleotide may overlap, we found NiaX to specifically import nicotinamide and nicotinic acid, and PnuC to be primarily responsible for nicotinamide riboside import. Furthermore, a mutant is completely attenuated during both intranasal and intratracheal infections in mice. Taken together, these findings underscore the importance of substrate salvage in pneumococcal pathogenesis and indicate that PnuC could potentially be a viable small-molecule therapeutic target to alleviate disease progression in the host.


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  1. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. (1990). Basic local alignment search toolJ Mol Biol 215403410 [View Article][PubMed]. [Google Scholar]
  2. Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Res 2533893402 [View Article][PubMed]. [Google Scholar]
  3. Braun M., Bungert S., Friedrich T. (1998). Characterization of the overproduced NADH dehydrogenase fragment of the NADH : ubiquinone oxidoreductase (complex I) from Escherichia coliBiochemistry 3718611867 [View Article][PubMed]. [Google Scholar]
  4. Brown K.D., Maqsood S., Huang J.Y., Pan Y., Harkcom W., Li W., Sauve A., Verdin E., Jaffrey S.R. (2014). Activation of SIRT3 by the NAD? precursor nicotinamide riboside protects from noise-induced hearing lossCell Metab 2010591068 [View Article][PubMed]. [Google Scholar]
  5. Cantó C., Houtkooper R.H., Pirinen E., Youn D.Y., Oosterveer M.H., Cen Y., Fernandez-Marcos P.J., Yamamoto H., Andreux P.A., other authors. (2012). The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesityCell Metab 15838847 [View Article][PubMed]. [Google Scholar]
  6. Chalkiadaki A., Guarente L. (2012). Sirtuins mediate mammalian metabolic responses to nutrient availabilityNat Rev Endocrinol 8287296 [View Article][PubMed]. [Google Scholar]
  7. Chandler J.L., Gholson R.K. (1972). De novo biosynthesis of nicotinamide adenine dinucleotide in Escherichia coli: excretion of quinolinic acid by mutants lacking quinolinate phosphoribosyl transferaseJ Bacteriol 11198102[PubMed]. [Google Scholar]
  8. Chewapreecha C., Marttinen P., Croucher N.J., Salter S.J., Harris S.R., Mather A.E., Hanage W.P., Goldblatt D., Nosten F.H., other authors. (2014). Comprehensive identification of single nucleotide polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genesPLoS Genet 10e1004547 [View Article][PubMed]. [Google Scholar]
  9. Chiarugi A., Dölle C., Felici R., Ziegler M. (2012). The NAD metabolome – a key determinant of cancer cell biologyNat Rev Cancer 12741752 [View Article][PubMed]. [Google Scholar]
  10. Frederick D.W., Davis J.G., Dávila A. Jr, Agarwal B., Michan S., Puchowicz M.A., Nakamaru-Ogiso E., Baur J.A. (2015). Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolismJ Biol Chem 29015461558 [View Article][PubMed]. [Google Scholar]
  11. Friedrich T. (1998). The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coliBiochim Biophys Acta 1364134146 [View Article][PubMed]. [Google Scholar]
  12. Granok A.B., Parsonage D., Ross R.P., Caparon M.G. (2000). The RofA binding site in Streptococcus pyogenes is utilized in multiple transcriptional pathwaysJ Bacteriol 18215291540 [View Article][PubMed]. [Google Scholar]
  13. Grose J.H., Bergthorsson U., Xu Y., Sterneckert J., Khodaverdian B., Roth J.R. (2005). Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella entericaJ Bacteriol 18745214530 [View Article][PubMed]. [Google Scholar]
  14. Herbert M., Sauer E., Smethurst G., Kraiss A., Hilpert A.K., Reidl J. (2003). Nicotinamide ribosyl uptake mutants in Haemophilus influenzaeInfect Immun 7153985401 [View Article][PubMed]. [Google Scholar]
  15. Horton R.M., Cai Z.L., Ho S.N., Pease L.R. (1990). Gene splicing by overlap extension: tailor-made genes using the polymerase chain reactionBiotechniques 8528535[PubMed]. [Google Scholar]
  16. Huang N., Sorci L., Zhang X., Brautigam C.A., Li X., Raffaelli N., Magni G., Grishin N.V., Osterman A.L., Zhang H. (2008). Bifunctional NMN adenylyltransferase/ADP-ribose pyrophosphatase: structure and function in bacterial NAD metabolismStructure 16196209 [View Article][PubMed]. [Google Scholar]
  17. Huang N., De Ingeniis J., Galeazzi L., Mancini C., Korostelev Y.D., Rakhmaninova A.B., Gelfand M.S., Rodionov D.A., Raffaelli N., Zhang H. (2009). Structure and function of an ADP-ribose-dependent transcriptional regulator of NAD metabolismStructure 17939951 [View Article][PubMed]. [Google Scholar]
  18. Ishino Y., Shinagawa H., Makino K., Tsunasawa S., Sakiyama F., Nakata A. (1986). Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coliMol Gen Genet 20417 [View Article][PubMed]. [Google Scholar]
  19. Jaehme M., Guskov A., Slotboom D.J. (2014). Crystal structure of the vitamin B3 transporter PnuC, a full-length SWEET homologNat Struct Mol Biol 2110131015 [View Article][PubMed]. [Google Scholar]
  20. Jurtshuk P. Jr (1996). Bacterial Metabolism. In Medical Microbiology, 4th edn, chapter 4. Edited by S. Baron.Galveston, TXUniversity of Texas Medical Branch. [Google Scholar]
  21. Kemmer G., Reilly T.J., Schmidt-Brauns J., Zlotnik G.W., Green B.A., Fiske M.J., Herbert M., Kraiss A., Schlör S., other authors. (2001). NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzaeJ Bacteriol 18339743981 [View Article][PubMed]. [Google Scholar]
  22. Khan N.A., Auranen M., Paetau I., Pirinen E., Euro L., Forsström S., Pasila L., Velagapudi V., Carroll C.J., other authors. (2014). Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3EMBO Mol Med 6721731[PubMed]. [Google Scholar]
  23. Kotrbova-Kozak A., Kotrba P., Inui M., Sajdok J., Yukawa H. (2007). Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum RAppl Microbiol Biotechnol 7613471356 [View Article][PubMed]. [Google Scholar]
  24. Kurnasov O.V., Polanuyer B.M., Ananta S., Sloutsky R., Tam A., Gerdes S.Y., Osterman A.L. (2002). Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesisJ Bacteriol 18469066917 [View Article][PubMed]. [Google Scholar]
  25. Lacks S., Hotchkiss R.D. (1960). A study of the genetic material determining an enzyme activity in PneumococcusBiochim Biophys Acta 39508518 [View Article][PubMed]. [Google Scholar]
  26. Luong T.T., Kim E.H., Bak J.P., Nguyen C.T., Choi S., Briles D.E., Pyo S., Rhee D.K. (2015). Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniaeInfect Immun 83108119 [View Article][PubMed]. [Google Scholar]
  27. Nobelmann B., Lengeler J.W. (1996). Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism. In J Bacteriol 17867906795[PubMed]. [Google Scholar]
  28. Patel M.S., Nemeria N.S., Furey W., Jordan F. (2014). The pyruvate dehydrogenase complexes: structure-based function and regulationJ Biol Chem 2891661516623 [View Article][PubMed]. [Google Scholar]
  29. Rodionov D.A., Li X., Rodionova I.A., Yang C., Sorci L., Dervyn E., Martynowski D., Zhang H., Gelfand M.S., Osterman A.L. (2008). Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulonNucleic Acids Res 3620322046 [View Article][PubMed]. [Google Scholar]
  30. Rodionov D.A., Hebbeln P., Eudes A., ter Beek J., Rodionova I.A., Erkens G.B., Slotboom D.J., Gelfand M.S., Osterman A.L., other authors. (2009). A novel class of modular transporters for vitamins in prokaryotesJ Bacteriol 1914251 [View Article][PubMed]. [Google Scholar]
  31. Satoh M.S., Lindahl T. (1992). Role of poly(ADP-ribose) formation in DNA repairNature 356356358 [View Article][PubMed]. [Google Scholar]
  32. Sauer E., Merdanovic M., Mortimer A.P., Bringmann G., Reidl J. (2004). PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzaeAntimicrob Agents Chemother 4845324541 [View Article][PubMed]. [Google Scholar]
  33. Schmidt-Brauns J., Herbert M., Kemmer G., Kraiss A., Schlör S., Reidl J. (2001). Is a NAD pyrophosphatase activity necessary for Haemophilus influenzae type b multiplication in the blood stream?Int J Med Microbiol 291219225 [View Article][PubMed]. [Google Scholar]
  34. Singh S.K., Kurnasov O.V., Chen B., Robinson H., Grishin N.V., Osterman A.L., Zhang H. (2002). Crystal structure of Haemophilus influenzae NadR protein. A bifunctional enzyme endowed with NMN adenyltransferase and ribosylnicotinamide kinase activitiesJ Biol Chem 2773329133299 [View Article][PubMed]. [Google Scholar]
  35. Sorci L., Martynowski D., Rodionov D.A., Eyobo Y., Zogaj X., Klose K.E., Nikolaev E.V., Magni G., Zhang H., Osterman A.L. (2009). Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensisProc Natl Acad Sci U S A 10630833088 [View Article][PubMed]. [Google Scholar]
  36. Sorci L., Blaby I.K., Rodionova I.A., De Ingeniis J., Tkachenko S., de Crécy-Lagard V., Osterman A.L. (2013). Quinolinate salvage and insights for targeting NAD biosynthesis in group A streptococciJ Bacteriol 195726732 [View Article][PubMed]. [Google Scholar]
  37. Spector M.P., Hill J.M., Holley E.A., Foster J.W. (1985). Genetic characterization of pyridine nucleotide uptake mutants of Salmonella typhimuriumJ Gen Microbiol 13113131322[PubMed]. [Google Scholar]
  38. Temple L., Sage A., Christie G.E., Phibbs P.V. Jr (1994). Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1J Bacteriol 17647004709[PubMed]. [Google Scholar]
  39. ter Beek J., Duurkens R.H., Erkens G.B., Slotboom D.J. (2011). Quaternary structure and functional unit of energy coupling factor (ECF)-type transportersJ Biol Chem 28654715475 [View Article][PubMed]. [Google Scholar]
  40. Wilkinson A., Day J., Bowater R. (2001). Bacterial DNA ligasesMol Microbiol 4012411248 [View Article][PubMed]. [Google Scholar]
  41. Zhu N., Olivera B.M., Roth J.R. (1988). Identification of a repressor gene involved in the regulation of NAD de novo biosynthesis in Salmonella typhimuriumJ Bacteriol 170117125. [Google Scholar]

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