The OtsAB Pathway Is Essential for Trehalose Biosynthesis in Mycobacterium tuberculosis

The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from α-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose. The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from α-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose. The non-reducing disaccharide trehalose (α-d-glucopyranosyl-(1, 1)-α-d-glucopyranoside) is found in bacteria, yeast, fungi, plants, and invertebrates, but not in mammalian cells (reviewed in Ref. 1Elbein A.D. Pan Y.T. Pastuszak I. Carroll D. Glycobiology. 2003; 13: 17R-27RCrossref PubMed Scopus (1443) Google Scholar). It can serve as a carbon source, as a storage carbohydrate, and as a stress protectant. Trehalose can function as a compatible solute to stabilize cells during osmotic stress, and its accumulation has been widely implicated in preserving cell viability during exposure to a range of environmental stresses, including heat shock, dehydration, and hypoxia (2Arguelles J.C. Arch. Microbiol. 2000; 174: 217-224Crossref PubMed Scopus (334) Google Scholar, 3Alvarez-Peral F.J. Zaragoza O. Pedreno Y. Arguelles J.C. Microbiology (Read.). 2002; 148: 2599-2606Crossref PubMed Scopus (157) Google Scholar, 4Chen Q. Haddad G.G. J. Exp. Biol. 2004; 207: 3125-3129Crossref PubMed Scopus (211) Google Scholar). In mycobacteria and related actinomycetes, trehalose is also a major structural constituent of cell wall glycolipids and acts as a carrier for mycolic acids during biosynthesis of the cell wall (5Daffe M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar, 6Ryll R. Kumazawa Y. Yano I. Microbiol. Immunol. 2001; 45: 801-811Crossref PubMed Scopus (130) Google Scholar). We are interested in analyzing the role of trehalose in the pathogenesis of Mycobacterium tuberculosis. Current models of tuberculosis envisage the contribution of at least two phenotypic forms of the bacteria: an actively replicating form involved in the initial establishment of infection and during active disease and a non-replicating form involved in latent infection and, in persistence, during chemotherapy. Current drugs act primarily on replicating bacteria, and a key challenge for improved tuberculosis control is to develop novel compounds that are equally active against the non-replicating populations. It is anticipated that these would allow shortening of the conventional six-month treatment regimen, as well as providing preventive therapy for individuals who are carrying a latent infection. We hypothesized that the dual role of trehalose, in biogenesis of the cell wall of replicating bacteria and as a stress protectant in non-replicating bacteria, would make its biosynthesis an appropriate target for this new class of drug. However, we have reported previously (7De Smet K.A. Weston A. Brown I.N. Young D.B. Robertson B.D. Microbiology (Read.). 2000; 146: 199-208Crossref PubMed Scopus (209) Google Scholar) that three independent pathways are available for trehalose biosynthesis in M. tuberculosis: the OtsAB pathway (which utilizes glucose and glucose-6-phosphate), the TreYZ pathway (which makes trehalose from glycogen), and the TreS enzyme (which can convert maltose to trehalose). If each of these pathways is able to generate the trehalose pool required for growth and survival of M. tuberculosis, this would obviously compromise the activity of a drug targeting any single enzyme. In addition, genome sequence analysis has identified two open reading frames in M. tuberculosis with homology to the otsB gene that encodes the trehalose-6-phosphate phosphatase required for the OtsAB pathway (8Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry C.E. II I Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6557) Google Scholar). Interestingly, one of the homologues (otsB1, Rv2006) is a member of a regulon that is induced by exposure to hypoxia or to low concentrations of nitric oxide and is associated with non-replicating survival of M. tuberculosis (9Voskuil M.I. Schnappinger D. Visconti K.C. Harrell M.I. Dolganov G.M. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (775) Google Scholar). Again, the existence of alternative branching pathways is an important consideration in the assessment of potential drug targeting. Recent reports have analyzed the role of these three pathways for trehalose biosynthesis in the fast growing saprophytic mycobacterium Mycobacterium smegmatis (10Klutts S. Pastuszak I. Edavana V.K. Thampi P. Pan Y.T. Abraham E.C. Carroll J.D. Elbein A.D. J. Biol. Chem. 2003; 278: 2093-2100Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 11Pan Y.T. Carroll J.D. Elbein A.D. Eur. J. Biochem. 2002; 269: 6091-6100Crossref PubMed Scopus (47) Google Scholar, 12Pan Y.T. Koroth Edavana V. Jourdian W.J. Edmondson R. Carroll J.D. Pastuszak I. Elbein A.D. Eur. J. Biochem. 2004; 271: 4259-4269Crossref PubMed Scopus (76) Google Scholar, 13Woodruff P.J. Carlson B.L. Siridechadilok B. Pratt M.R. Senaratne R.H. Mougous J.D. Riley L.W. Williams S.J. Bertozzi C.R. J. Biol. Chem. 2004; 279: 28835-28843Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and in the taxonomically related Corynebacterium glutamicum (14Tzvetkov M. Klopprogge C. Zelder O. Liebl W. Microbiology (Read.). 2003; 149: 1659-1673Crossref PubMed Scopus (80) Google Scholar, 15Wolf A. Kramer R. Morbach S. Mol. Microbiol. 2003; 49: 1119-1134Crossref PubMed Scopus (152) Google Scholar). Mutagenesis studies indicated that the three pathways are functionally redundant in M. smegmatis (13Woodruff P.J. Carlson B.L. Siridechadilok B. Pratt M.R. Senaratne R.H. Mougous J.D. Riley L.W. Williams S.J. Bertozzi C.R. J. Biol. Chem. 2004; 279: 28835-28843Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Single mutations targeting each of the pathways generated no apparent phenotypic defects in the resulting clones as assessed by growth or by glycolipid content. A triple mutant, with all three pathways inactivated, was dependent on the provision of exogenous trehalose for growth and was defective in stationary phase survival and in exposure to elevated temperature (13Woodruff P.J. Carlson B.L. Siridechadilok B. Pratt M.R. Senaratne R.H. Mougous J.D. Riley L.W. Williams S.J. Bertozzi C.R. J. Biol. Chem. 2004; 279: 28835-28843Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). A different hierarchy of trehalose biosynthesis pathways was found in C. glutamicum. Again, mutation of all three pathways was accompanied by a marked growth defect, but in C. glutamicum this was also observed in the case of an otsA-treY double knockout. Analysis of the effect of different combinations of mutations on trehalose content and bacterial survival following osmotic stress led to the conclusion that TreYZ represents the major pathway for trehalose biosynthesis in C. glutamicum, with OtsAB playing a minor accessory role, and TreS contributing to trehalose degradation through its ability to catalyze the interchange of trehalose and maltose (14Tzvetkov M. Klopprogge C. Zelder O. Liebl W. Microbiology (Read.). 2003; 149: 1659-1673Crossref PubMed Scopus (80) Google Scholar, 15Wolf A. Kramer R. Morbach S. Mol. Microbiol. 2003; 49: 1119-1134Crossref PubMed Scopus (152) Google Scholar). To date, the only information on the contribution of the various pathways to trehalose anabolism in M. tuberculosis comes from a transposon site hybridization mutagenesis study carried out by Sassetti, Boyd, and Mol. Microbiol. 2003; PubMed Scopus Google Scholar). that into the and associated with growth defects in vitro and that into treS significantly The aim of the present study was to the role of each of the three trehalose biosynthesis pathways in M. tuberculosis, during growth in laboratory and during infection in a mouse model. In to the results with M. smegmatis and with C. glutamicum, studies that OtsAB is the pathway in M. tuberculosis. The mutagenesis results are with studies that trehalose-6-phosphate phosphatase activity is associated with only one of the homologues in M. tuberculosis V.K. Pastuszak I. Carroll J.D. Thampi P. Abraham E.C. Elbein A.D. Arch. Biochem. 2004; PubMed Scopus Google Scholar) and this enzyme as an target for drug of and was the and in I and the to the of the with the indicated in the and either of the in the which as a T. P. P. M. J. Brown I.N. Young D.B. Med. 2001; PubMed Scopus Google Scholar). into and on and by and of was to of a into M. tuberculosis and M. as by T. P. P. M. J. Brown I.N. Young D.B. Med. 2001; PubMed Scopus Google Scholar). on with and was by and not for and gene and either of the for are gene and either of the for are in a new To of the a was by a of the gene at the site L. S. A. PubMed Scopus Google Scholar). the gene the sequence by and was into II and with to the gene a that the this was into the L. R. J. Young D.B. Microbiology (Read.). 2002; 148: PubMed Scopus Google Scholar). was and into M. tuberculosis as but Deletion of the of the gene with the was as In Analysis of of of in the with single cell The of mycobacteria has been I. V. K. B. S. T. N. A. 1998; PubMed Google Scholar, C. 2000; Full Text PDF PubMed Scopus Google and the of single was by a conventional and for all The of was by as by M.A. Young D.B. J. 2004; PubMed Scopus Google Scholar). The was from each on and and in and To in the of of and the of at The from each infection assessed for survival and of Trehalose OtsB2 and TreS the for indicated in I and with and for into into the and and the from in found these and all carried out phase at induced with The was in of and the to an The was to the the was against and stored at The analyzed on and with of TreS for analysis from a (7De Smet K.A. Weston A. Brown I.N. Young D.B. Robertson B.D. Microbiology (Read.). 2000; 146: 199-208Crossref PubMed Scopus (209) Google Scholar) and acid and studies of of and found that the at a with an apparent of that TreS was these OtsB2 was against to free and was the with as the activity was in a of trehalose-6-phosphate and of enzyme. was in a at for of and and the at was to determine the of free which was in the range of M. M. Elbein A.D. J. PubMed Google Scholar). was by the trehalose-6-phosphate with of glucose-6-phosphate, or from was by the in the with or A of and was as was and at on a at a of with a triple including for maltose and trehalose and TreS enzyme was and and of M. tuberculosis with in Trehalose and analysis the of three potential pathways for biosynthesis of trehalose in M. tuberculosis (7De Smet K.A. Weston A. Brown I.N. Young D.B. Robertson B.D. Microbiology (Read.). 2000; 146: 199-208Crossref PubMed Scopus (209) Google Scholar). To contribution to we from the three pathways for for and on either of the in the and into M. tuberculosis in which a double had in of the gene by the identified by against the on the and for and the of to the anticipated of for of which a on the required was by and not The and clones no growth but the in and on by the To the for the to an we generated a of M. tuberculosis carrying a of the gene into the site on the of this with the in double in which the had the of the that to the is of the that this gene has an essential role for in vitro of M. tuberculosis. role by the and the of phenotype for the is with the that the of functional trehalose-6-phosphate phosphatase activity V.K. Pastuszak I. Carroll J.D. Thampi P. Abraham E.C. Elbein A.D. Arch. Biochem. 2004; PubMed Scopus Google Scholar). We the with two of the M. tuberculosis M. and M. the M. to a of M. that a functional gene as a of a T. Eiglmeier K. J.C. N. M. S. S. C. C. S. Harris B. R. J. R. L. Parkhill J. Barrell B.G. S.T. 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Again, we to generate clones in M. was not but in to M. tuberculosis, we also to generate clones in the M. to for on The in M. the double the M. tuberculosis in no growth phenotype in of Trehalose in a of tuberculosis of each of the three trehalose biosynthesis pathways and for ability to disease in mice. by of bacteria, and the of was in the and from and infection The bacterial in the was at all time with in and at and the and bacterial to the at the late of infection the a but in in the The for all was observed in a independent analyzed by the time to death for in the different the had a marked with of the with the with the which had all by survival time of The a in to with a survival time of The no from the results that the growth associated with the mutation observed during in vitro culture is also during infection and a in phenotype for the mutation during infection in the and of Trehalose trehalose biosynthesis in M. tuberculosis and to for drug OtsB2 and TreS into and with an The by The trehalose-6-phosphate phosphatase by the gene was analyzed in a for high throughput screening. The enzyme was with a range of to determine its no of was from any trehalose-6-phosphate by the of and to the with trehalose-6-phosphate as The was found to the was by or by of in the of The was to of M. tuberculosis trehalose-6-phosphate phosphatase in a new of for trehalose-6-phosphate in a new We observed previously (7De Smet K.A. Weston A. Brown I.N. Young D.B. Robertson B.D. 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The aim of the present study was to generate in each of these pathways to determine whether or not they are functionally redundant for growth and survival in vitro and during infection. results a of the OtsAB pathway, which trehalose by of glucose and Deletion of the gene in marked defects in growth of M. tuberculosis in vitro and in and the functional by to strictly essential for The of exogenous trehalose not the The absence of any apparent phenotypic associated with that the TreYZ pathway, which has the potential of trehalose from α-1,4-linked glucose polymers, is not essential for M. tuberculosis these survival of with a suggests that interconversion trehalose and maltose plays a role during M. tuberculosis infection. results with are with from high throughput transposon mutagenesis Mol. Microbiol. 2003; PubMed Scopus Google Scholar, S. A. 2003; PubMed Scopus Google Scholar). into or had no for growth in vitro or in a mouse S. A. 2003; PubMed Scopus Google Scholar). growth defects associated with into the and and significantly growth was for into treS Mol. Microbiol. 2003; PubMed Scopus Google Scholar). It is not whether the transposon or whether enzyme activity that the ability to the of the in the of it may that growth in a pool provides a of to allow the to with It is that of the is to a there is no that is of an is an of and the gene which encodes a A.D. M. J. PubMed Google Scholar). the OtsAB pathway represents the for trehalose biosynthesis found in a range of bacteria, plants, and invertebrates, its in M. tuberculosis is in to in related bacterial The TreYZ pathway in C. glutamicum (14Tzvetkov M. Klopprogge C. Zelder O. Liebl W. Microbiology (Read.). 2003; 149: 1659-1673Crossref PubMed Scopus (80) Google and the three pathways are functionally redundant in M. smegmatis (13Woodruff P.J. Carlson B.L. Siridechadilok B. Pratt M.R. Senaratne R.H. Mougous J.D. Riley L.W. Williams S.J. Bertozzi C.R. J. Biol. Chem. 2004; 279: 28835-28843Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In Mycobacterium and treS are OtsAB the only pathway for trehalose biosynthesis S.T. Eiglmeier K. Parkhill J. N. Garnier T. Churcher C. Harris D. K. Basham D. Brown D. Chillingworth T. Connor R. Davies Devlin K. S. Feltwell T. A. Hamlin N. Holroyd S. Hornsby T. Jagels K. C. J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.A. Rutter S. Seeger K. S. M. Skelton J. Squares R. Squares S. K. Taylor K. Whitehead S. Barrell B.G. Nature. 2001; PubMed Scopus Google Scholar). The for in the hierarchy of trehalose biosynthesis pathways is but may reflect in and in for the free sugar in the as well as in the cell A was observed M. tuberculosis and the related M. in which we to was also the case for an M. with a mutation in the TreYZ pathway, as well as for M. in which the and are Again, this may reflect in the two M. has in with M. tuberculosis, including a mutation in that the of the into the acid T. Eiglmeier K. J.C. N. M. S. S. C. C. S. Harris B. R. J. R. L. Parkhill J. Barrell B.G. S.T. Gordon S.V. S. A. 2003; PubMed Scopus Google Scholar). they in a pathway, there was a in phenotypic the of the and otsB in M. tuberculosis. The phenotype of the that the phosphatase function of at by the conclusion from studies that open reading annotated as in the is in this V.K. Pastuszak I. Carroll J.D. Thampi P. Abraham E.C. Elbein A.D. Arch. Biochem. 2004; PubMed Scopus Google Scholar). acts as an important for control in it the in through the of and its accumulation is to the M.A. R. C. PubMed Scopus Google Scholar). function in mycobacteria the of the mutation in to the mutation observed in of the OtsB2 as an target for drug and we have for the and of the which are to provide a for an The is for trehalose-6-phosphate and not act on any of the sugar that an would not with mammalian The high throughput of potential A key in the of OtsB2 as a target is the role of trehalose biosynthesis in the survival of non-replicating during infection. results show that the OtsAB pathway is important for initial of M. tuberculosis in mice, they not allow to determine whether survival of with the OtsAB is a of this in initial growth or additional in bacterial To for drugs that are to act against the M. tuberculosis in there is a to develop that allow to at different stages of the infection are in to this in which is the control of a C. J. N. R. D. B. and B. D. In to the the growth with during the initial by a late in bacterial and, in The TreS requirement for infection with M. tuberculosis could reflect either a for the of additional trehalose or, conversely, for of stored trehalose into maltose and into glucose. studies that provide the major for of M. tuberculosis. required for from are in and are essential for the virulence of M. tuberculosis in the mouse S. A. 2003; PubMed Scopus Google Scholar, J. R. L. B. I. J.D. S. A. 2003; PubMed Scopus Google Scholar, J.D. K. A. B. D. J.C. Nature. 2000; PubMed Scopus Google Scholar). It is that trehalose provides an additional of glucose during infection. In trehalose can to glucose by a but no has been identified in the genome of M. tuberculosis (8Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry C.E. II I Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6557) Google Scholar). M. tuberculosis have a of trehalose which can convert trehalose to glucose and in F. P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google this is for growth in vitro or during infection in Mol. Microbiol. 2003; PubMed Scopus Google Scholar, S. A. 2003; PubMed Scopus Google Scholar). The ability of TreS to and (7De Smet K.A. Weston A. Brown I.N. Young D.B. Robertson B.D. Microbiology (Read.). 2000; 146: 199-208Crossref PubMed Scopus (209) Google Scholar, 12Pan Y.T. Koroth Edavana V. Jourdian W.J. Edmondson R. Carroll J.D. Pastuszak I. Elbein A.D. Eur. J. Biochem. 2004; 271: 4259-4269Crossref PubMed Scopus (76) Google Scholar) may provide an important pathway for the of glucose from Pan Y.T. Koroth Edavana V. Jourdian W.J. Edmondson R. Carroll J.D. Pastuszak I. Elbein A.D. Eur. J. Biochem. 2004; 271: 4259-4269Crossref PubMed Scopus (76) Google Scholar) have an alternative pathway for of glucose. have found that M. smegmatis TreS glucose as a of a to trehalose from maltose, the of maltose to two of one of which is to glucose to In we have that OtsAB is the dominant pathway for trehalose biosynthesis in M. tuberculosis and that its by either of the two alternative The trehalose-6-phosphate phosphatase by the gene represents an and tractable drug