Deciphering sulfoglycolipids of Mycobacterium tuberculosis

Abstract

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways. For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways. Sulfoglycolipids (SGLs) were originally discovered in 1959 by Middlebrook et al. (1Middlebrook G. Coleman C. Schaefer W.B. Sulfolipid from virulent tubercle bacilli.Proc. Natl. Acad. Sci. U S A. 1959; 45: 1801-1804Crossref PubMed Google Scholar) in human (H37Rv) and bovine (Vallée) virulent strains of Mycobacterium tuberculosis. Almost 2 decades later, Goren et al. (2Goren M.B. Brokl O. Schaefer W.B. Lipids of putative relevance to virulence in Mycobacterium tuberculosis: correlation of virulence with elaboration of sulfatides and strongly acidic lipids.Infect. Immun. 1974; 9: 142-149Crossref PubMed Google Scholar) showed that this family of lipids was specific to M. tuberculosis and proposed that their amount correlated with the virulence of the M. tuberculosis strain in the guinea pig model of infection. Sulfolipids consist of multi-acylated forms of trehalose sulfate that differ by the number, structure, and location of acyl moieties (3Goren M.B. Brokl O. Das B.C. Lederer E. Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. Nature of the acyl substituents.Biochemistry. 1971; 10: 72-81Crossref PubMed Scopus (63) Google Scholar). The major sulfolipid, SL-I, was characterized as 2,3,6,6′-tetraacyl-α-α’-trehalose-2′-sulfate acylated by two hydroxyphthioceranates (HPA), one phthioceranate (PA), and one palmitate (C16) or stearate (C18) (Fig. 1A). HPA are complex dextrorotatory fatty acids specific to the Mycobacterium genus and contain one hydroxyl group and several methyl groups arranged in a 2,4,6 pattern. PA are HPA analogs devoid of the hydroxyl group (3Goren M.B. Brokl O. Das B.C. Lederer E. Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. Nature of the acyl substituents.Biochemistry. 1971; 10: 72-81Crossref PubMed Scopus (63) Google Scholar, 4Goren M.B. Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. I. Purification and properties.Biochim. Biophys. Acta. 1970; 210: 116-126Crossref PubMed Scopus (92) Google Scholar, 5Goren M.B. Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. II. Structural studies.Biochim. Biophys. Acta. 1970; 210: 127-138Crossref PubMed Scopus (71) Google Scholar). Three other minor tetra-acylated forms of sulfolipids, differing from SL-I by the fatty acyl composition, namely, SL-I′, SL-II, and SL-II′, and one tri-acylated form, SL-III, have been described previously (Fig. 1B) (6Goren M.B. Mycobacterial fatty acid esters of sugars and sulfosugars.in: Hanahan D.J. Handbook of Lipid Research. Plenum Press, NY1990: 363-461Crossref Google Scholar). Later on, a compound called SL-IV and tentatively assigned to a 2,3-diacyltrehalose-2′-sulfate was isolated from clinical isolates of M. tuberculosis (7Daffe M. Papa F. Laszlo A. David H.L. Glycolipids of recent clinical isolates of Mycobacterium tuberculosis: chemical characterization and immunoreactivity.J. Gen. Microbiol. 1989; 135: 2759-2766PubMed Google Scholar) and proposed to correspond to the more polar uncharacterized sulfatide observed by Goren et al. (8Goren M.B. Das B.C. Brokl O. Sulfatide III of Mycobacterium tuberculosis, Strain H37Rv.New J. Chem. 1978; 2: 379-384Google Scholar) in the M. tuberculosis H37Rv strain. However, this compound was subsequently found to lack a sulfate ester group (9Lemassu A. Laneelle M.A. Daffe M. Revised structure of a trehalose-containing immunoreactive glycolipid of Mycobacterium tuberculosis.FEMS Microbiol. Lett. 1991; 62: 171-175Crossref PubMed Scopus (45) Google Scholar, 10Baer H.H. The structure of an antigenic glycolipid (SL-IV) from Mycobacterium tuberculosis.Carbohydr. Res. 1993; 240: 1-22Crossref PubMed Scopus (15) Google Scholar), requiring its classification as a sulfatide to be revised (11Cruaud P. Yamashita J.T. Casabona N.M. Papa F. David H.L. Evaluation of a novel 2,3-diacyl-trehalose-2’-sulphate (SL-IV) antigen for case finding and diagnosis of leprosy and tuberculosis.Res. Microbiol. 1990; 141: 679-694Crossref PubMed Scopus (45) Google Scholar). Nevertheless, the presence of di-acylated sulfolipids in the cell envelope of M. tuberculosis was definitively proven by three recent studies. Using Fourier transform ion cyclotron resonance MS to identify sulfated metabolites by virtue of their metabolic labeling with a stable sulfur isotope, Mougous et al. (12Mougous J.D. Leavell M.D. Senaratne of sulfated metabolites in with a and Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar) showed of several new sulfated in M. tuberculosis, di-acylated the role of the in et al. P. M.B. C. G. The role of MmpL8 in sulfatide and virulence of Mycobacterium Chem. PubMed Scopus Google Scholar) characterized polar sulfated in the knockout strain and characterized as di-acylated we di-acylated sulfolipids, SGLs to the of acyl the of the fatty which be or oxophthioceranoic as new by to M. F. G. J. G. G. sulfoglycolipids are novel with Mycobacterium PubMed Scopus Google Scholar). The structure of was for the first by a of MS and M. F. G. J. G. G. sulfoglycolipids are novel with Mycobacterium PubMed Scopus Google Scholar). In the we reevaluated the structure of SGLs from the reference strain M. tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains that have been described to intracellularly accumulate P. M.B. C. G. The role of MmpL8 in sulfatide and virulence of Mycobacterium Chem. PubMed Scopus Google Scholar, Mougous J.D. Leavell M.D. MmpL8 for biosynthesis and Mycobacterium tuberculosis Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar), by MALDI-time-of-flight and and two-dimensional The of SGLs in a clinical strain was with that in M. tuberculosis H37Rv, and the of the presence of SGLs in was also are in the of possible SGL biosynthetic pathways. M. tuberculosis H37Rv clinical M. tuberculosis I. M. M. J. C. of Mycobacterium tuberculosis insights tuberculosis of other and for a PubMed Scopus Google Scholar), M. tuberculosis strain Mougous J.D. Leavell M.D. MmpL8 for biosynthesis and Mycobacterium tuberculosis Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar), and M. tuberculosis H37Rv strain P. M.B. C. G. The role of MmpL8 in sulfatide and virulence of Mycobacterium Chem. PubMed Scopus Google Scholar) were at as were 4 from the and by in a for 2 at were in a and The which the was and further and were and The was in a of and to by the of at The was at for to an and an of the was a 2 with of and and and and and and and and and a of and was further by methyl a by with 2 of to with 2 of to 2 with 2 of and and with 2 of to of was by with trehalose was from and was further by with a by with of two with 2 of and two with 2 of and two with 2 of and two with 2 of and with 2 of to and three with 2 of to of was from to trehalose was found in to was further by a three with 2 of to to and to of was by with Purification was first by a of or was to Purification was also by in and was as previously further in a of and to by the of at The was at for to an and an The was and with a and with of to and in to to and to and were and to was from of the Mycobacterium M. tuberculosis Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium and Mycobacterium and as described previously for M. tuberculosis H37Rv For an was as previously and by for SGL was an (C18) and lipids were for a of at a of an in the of in with of in and of in of was in and were and their were by in of was at for in The was by the of 2 of of the the was with and three with The was in of to by was with a model with the was by with an at of with a of Glycolipids were in the or as in the from to were to the The acid or acid was at a of in In a 4 of glycolipid in and 4 of the were with a and of the was the The were at two with SGLs were by ion cyclotron resonance MS a with a and an ion were in a of and and at a of 2 The was to and the was to and were and The were in the with in the and was with a of lipids with The was and the was in the of were with an with an SGLs were in and in at chemical are in from the of and the of and and were as previously M. C. M. G. G. of the from Mycobacterium and to and Chem. PubMed Scopus Google Scholar). The first of this was to a to and SGL acyl the tri-, and di-acylated SGL forms and from M. tuberculosis H37Rv The was by the three acyl forms were in the (Fig. M. F. G. J. G. G. sulfoglycolipids are novel with Mycobacterium PubMed Scopus Google Scholar). was further a acid by of were from from and and from and However, to as by (Fig. and which the presence of The were by and their were by and The in was by two at and was assigned as a trehalose The compound in was characterized by the major ion at to trehalose M. J. Structural of a novel family of from Mycobacterium PubMed Scopus Google Scholar). contain lipids contain a that was from by M. F. G. J. G. G. sulfoglycolipids are novel with Mycobacterium PubMed Scopus Google Scholar). of M. tuberculosis H37Rv of of which the of the SGL acyl of was and from to of was this (Fig. of SGLs (Fig. to be the of for the in of the different SGL for the (Fig. a of from to with two major at and The of from one to the The that HPA to However, this major of of were also For the major ion at one at (Fig. which was tentatively assigned as one acid with and one acid The of this of in the of (Fig. and in with those found in the by Goren (6Goren M.B. Mycobacterial fatty acid esters of sugars and sulfosugars.in: Hanahan D.J. Handbook of Lipid Research. Plenum Press, NY1990: 363-461Crossref Google Scholar), in which and are the major PA from confirm this an was by analysis, the was and the was in the of The ion at showed only one at with the of for an one or one of the ion at an a for this by of of an at in with the presence of the of Nevertheless, bearing PA of with the of of the different acyl forms one or at the of the acylated and one one or one at the in a new The of (Fig. showed a of with a major ion at The to the by two in and with from to or from to the we that the major of to bearing two HPA and one a minor of at 2 (Fig. was observed and tentatively assigned to one PA of an was in this for the of for to for from to or from to The at and to bearing three HPA and one In this two minor of at 2 and 4 were (Fig. as by and were tentatively assigned to the two one and one and to one two and one was to fatty acids of acyl of SGL. at were assigned to contain a of one the at to a major with one and a minor with one the ion of at in showed at and to of and and at from the of (Fig. of at and and at and from and with and the (Fig. at and to the of or were also observed (Fig. showed to or trehalose bearing were to at at for (Fig. or at at for (Fig. from the of that fatty acyl were the the that the sulfate the of the different fatty acids positions 2 and of the be from with fatty acids we observed that in the in to the described from the of the fatty acid in the only For the at showed the of or with at and (Fig. However, of or was observed for or for those at that the fatty acids are the HPA are the of the to and this the of of we the in at and (Fig. of the of at showed to HPA and fatty acids and The major HPA were only at as of at were also only to SGL acylated by one and two or one also to SGL acylated by one one and one For was the major HPA with and in to the fatty acids and also a of at different forms one one one one or one for the major in at and (Fig. we we observed the ion in the as those observed for In and to and acylated by an the major HPA were at with of to and to in possible for acyl For and only the forms we different acyl forms one one one two one one or three The described that the a complex of We the of this by (C18) by (Fig. of in was as to the different acyl from the polar to the The first SGL were observed in and and to unexpected at and and were assigned by to SGL esterified by fatty acids and one as well as a compound at assigned by to a previously esterified by a in the only to at and to or at or were in we observed from the of that the of the in to a of characterized by with at from to were assigned by to one and one to We HPA from those as or with with with with to were observed from to SGLs were in strains other M. tuberculosis H37Rv, from the tuberculosis as Mycobacterium or M. tuberculosis or as Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium and Mycobacterium For the was and by for SGL of strains was found to The of SGLs in M. tuberculosis been correlated with a in the J. J. in the to of Mycobacterium tuberculosis PubMed Scopus Google Scholar, F. J. C. M. in the for the of that of in Mycobacterium tuberculosis PubMed Scopus Google Scholar). are in with those of studies that SGLs are specific from M. tuberculosis G. Goren M.B. the of the Mycobacterium tuberculosis sulfatides in Google Scholar, M. I. M. of an and for in the of its in Mycobacterium tuberculosis Microbiol. Lett. PubMed Google Scholar). M. tuberculosis H37Rv a strain, one that the acyl be a of the of this strain for the SGL acyl of a clinical to the I. M. M. J. C. of Mycobacterium tuberculosis insights tuberculosis of other and for a PubMed Scopus Google Scholar) was also The complete SGL was first by of the (Fig. the previously characterized M. tuberculosis H37Rv at was and the to were with those of the results of the M. tuberculosis H37Rv M. F. G. J. G. G. sulfoglycolipids are novel with Mycobacterium PubMed Scopus Google Scholar). the different acyl forms were and by (Fig. In the major SGL the as the described in M. tuberculosis H37Rv. The MS of (Fig. was to that of M. tuberculosis H37Rv for from to were observed (Fig. two of one family with two and one HPA and one family with one and two as by The (Fig. a of from to with and as previously observed for M. tuberculosis H37Rv The new the of an family of mono-acylated SGL observed in the from to (Fig. was by analysis, which that SGLs are acylated by one to in this at and assigned by to SGLs esterified by fatty acids one and two were also observed (Fig. M. tuberculosis mmpL8 knockout H37Rv and have been to a in and an of P. M.B. C. G. The role of MmpL8 in sulfatide and virulence of Mycobacterium Chem. PubMed Scopus Google Scholar, Mougous J.D. Leavell M.D. MmpL8 for biosynthesis and Mycobacterium tuberculosis Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar). We from strains for the by et al. P. M.B. C. G. The role of MmpL8 in sulfatide and virulence of Mycobacterium Chem. PubMed Scopus Google and for the by et al. Mougous J.D. Leavell M.D. MmpL8 for biosynthesis and Mycobacterium tuberculosis Natl. Acad. Sci. U S A. PubMed Scopus Google of strains a (Fig. However, their from that of the M. tuberculosis H37Rv wild-type strain (Fig. by the presence of a of with the with a of 2 (Fig. and be to the presence of i) a ii) a PA of an as a fatty acid devoid of hydroxyl group PA of and of one results in a of or a of an the fatty The first was by which at of a the two we first an by of which at and the was in with the of for an an the ion to the presence of a the acyl group oxophthioceranoic acyl with a of the presence of a PA the of confirm this and were to in the presence of the that only a group and an group be to the we this to the one of those a PA or an was observed with M. tuberculosis the at in was to The of the at the of one as observed for the strain (Fig. The fatty acid of the at was by and were at and in to the presence of and that the esterified by one and by one of and be from the M. tuberculosis H37Rv mmpL8 and to of was from of M. tuberculosis H37Rv only to of and to of were and also unexpected their were that of the SGL in the strain However, were at their by and their for studies be the major were observed at and and were esterified by one and two fatty acids as by analysis, are esterified by one or by two and one that an bearing two simple fatty acids be in of M. tuberculosis H37Rv of observed in the in an esterified by two or by one the structure from that of the as from and the presence of an acyl group be from chemical as the with the of a The chemical are by the M. J. G. antigen from Mycobacterium structure of a novel PubMed Scopus Google Scholar). as previously M. F. G. J. G. G. sulfoglycolipids are novel with Mycobacterium PubMed Scopus Google Scholar), the chemical of and at and that fatty acyl are and of and For of at with for in with the presence of a fatty acid of as previously described (8Goren M.B. Das B.C. Brokl O. Sulfatide III of Mycobacterium tuberculosis, Strain H37Rv.New J. Chem. 1978; 2: 379-384Google Scholar). For of at with for in with the presence of fatty acid of In the case of chemical that are acylated and of the and of the sulfated and was further by of which showed at assigned as a of the at to the for the In to the the of SGL to M. F. G. J. G. G. sulfoglycolipids are novel with Mycobacterium PubMed Scopus Google Scholar), we to their and structural The first of this was to and SGL acyl and this two were i) to of the from the the an and ii) to as and from SGL Goren et al. previously characterized SGL of which were tetra-acylated SL-I′, SL-II, and and one was tri-acylated (3Goren M.B. Brokl O. Das B.C. Lederer E. Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. Nature of the acyl substituents.Biochemistry. 1971; 10: 72-81Crossref PubMed Scopus (63) Google Scholar, 4Goren M.B. Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. I. Purification and properties.Biochim. Biophys. Acta. 1970; 210: 116-126Crossref PubMed Scopus (92) Google Scholar, 5Goren M.B. Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. II. Structural studies.Biochim. Biophys. Acta. 1970; 210: 127-138Crossref PubMed Scopus (71) Google Scholar, M.B. Mycobacterial fatty acid esters of sugars and sulfosugars.in: Hanahan D.J. Handbook of Lipid Research. Plenum Press, NY1990: 363-461Crossref Google Scholar). an chemical and MS and that i) identification of ii) of the acylated positions the trehalose identification of the acyl and and the that the of SL-I acylated by one or and that the by one PA (6Goren M.B. Mycobacterial fatty acid esters of sugars and sulfosugars.in: Hanahan D.J. Handbook of Lipid Research. Plenum Press, NY1990: 363-461Crossref Google Scholar). In this that the major was acylated the and by one PA and two was acylated by two PA and one and was acylated by three was also described as acylated by three HPA the and The tri-acylated was to be acylated the and by two In this we a in which we first the different acyl forms by and We of the structural described by Goren et as the presence of the the presence in of the fatty acids the and of the and the that the HPA for the first we a complete of acyl form, and in to the of we never observed the of the We the presence of this acyl form, at We showed that one and one HPA the and in with the by Goren et that one and two HPA the and the of a biosynthetic acyl forms and that three that the of by one However, Goren et al. found that the of SL-I major tetra-acylated acylated by one or one and two was by one in minor abundance in from the of one PA Goren et al. described SL-I to be acylated by HPA from to Using we a of the different SGL acyl We that HPA in from to of the the of PA We that was of of one PA and one was in of with three with one and with two to be the of for the of the different SGL we observed that this the from one to the were the for the of the The of SGL been to the of as methyl M. Leavell M.D. Mougous J.D. of Mycobacterium tuberculosis virulence lipids metabolic Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar), which be by the a that to were as cell Nevertheless, the major SGL the for the different SGL acyl forms. of the by to only SGL HPA or PA also to previously uncharacterized M. tuberculosis H37Rv SGL esterified by simple fatty acids only, and bearing a was more in the clinical strain, and a complete family of acylated by one HPA of was The identification of this mono-acylated was as the first to be a the of the sulfated trehalose (Fig. and the that the HPA been to sulfated trehalose P. M. Leavell M.D. and are for the biosynthesis of the Mycobacterium tuberculosis virulence Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar). the biosynthetic pathway of SGLs from an of SGLs produced by Our that of by the the first in SGL J.D. Senaratne and structure of the that PubMed Scopus Google Scholar). or stearate acyl from and HPA are by 2 The Mycobacterium tuberculosis the for the and fatty acids for Chem. PubMed Scopus Google Scholar) in with the P. and of fatty acids as in PubMed Scopus Google Scholar, J. of of Mycobacterium tuberculosis with to be in and with PubMed Scopus Google Scholar). two one and with the other P. M. Leavell M.D. and are for the biosynthesis of the Mycobacterium tuberculosis virulence Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar, A. are for biosynthesis in Mycobacterium PubMed Scopus Google Scholar), sulfated trehalose and further by a fatty acid the of The MmpL8 to be a described as a role in the of the forms from the the cell envelope P. M.B. C. G. The role of MmpL8 in sulfatide and virulence of Mycobacterium Chem. PubMed Scopus Google Scholar, Mougous J.D. Leavell M.D. MmpL8 for biosynthesis and Mycobacterium tuberculosis Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar). characterization of the showed that of was and that the strongly that and of SGLs are and that the in SGL biosynthesis be the of the of P. M.B. C. G. The role of MmpL8 in sulfatide and virulence of Mycobacterium Chem. PubMed Scopus Google Scholar). we that the was to with a structure different from that of the strain. the fatty acid was only was in a different of the sulfated of the of the that be the cell by MmpL8 in to be the (Fig. finding was that of the SGLs a fatty acid a of the hydroxyl a that in the strongly that MmpL8 in with in the elaboration of the fatty are proposed for that from fatty acid to the and positions of the trehalose intracellularly or metabolites from Mycobacterium tuberculosis: and Daffe M. The Mycobacterial Press, Google Scholar, M. The Daffe M. The Mycobacterial Press, Google Scholar). In the first be from in the and be to the of the cell by In the an to of to the of the cell by In results the of and to observed for the of of and positions of trehalose by a found to be with the cell M. The antigen a major of Mycobacterium PubMed Google Scholar). The of HPA HPA from i) the of the hydroxyl group PA or ii) an by In the the by of with the acyl the first be to the hydroxyl acid the hydroxyl group in the The Mycobacterium tuberculosis the for the and fatty acids for Chem. PubMed Scopus Google Scholar). Our this The of in of the mmpL8 that only of the by of with the acyl be to the hydroxyl The E. III and for M. tuberculosis and for We are also to P. and C. for for the and J. for and with trehalose hydroxyphthioceranoic acid oxophthioceranoic acyl acid acid with methyl trehalose