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Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species*

Open AccessPublished:November 15, 2012DOI:https://doi.org/10.1074/jbc.M112.419754
      Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.

      Introduction

      Mycobacteria represent a group of ubiquitous and diverse bacterial species, many of which can establish chronic infections in humans (
      • Grange J.
      ). An estimated one-third of the world's population is infected by Mycobacterium tuberculosis, the etiologic agent of human tuberculosis (TB),
      The abbreviations used are: TB
      tuberculosis
      FM
      free mycolic acid
      OADC
      oleic acid/albumin/dextrose/catalase
      TDM
      trehalose dimycolate
      TMM
      trehalose monomycolate
      TDMH
      TDM hydrolase
      mAGP
      mycolyl arabinogalactan
      NA
      nucleic acid
      PIM
      phosphatidylinositol myo-mannoside.
      which causes about 1.7 million deaths worldwide every year (
      • Dye C.
      • Lönnroth K.
      • Jaramillo E.
      • Williams B.G.
      • Raviglione M.
      Trends in tuberculosis incidence and their determinants in 134 countries.
      ). A distinct taxonomical classification for mycobacteria historically originated from their unique cellular morphology and acid-fastness, both directly attributed to their thick, hydrophobic, and atypically structured envelope (
      • Shinnick T.M.
      • Good R.C.
      Mycobacterial taxonomy.
      ,
      • Adams K.N.
      • Takaki K.
      • Connolly L.E.
      • Wiedenhoft H.
      • Winglee K.
      • Humbert O.
      • Edelstein P.H.
      • Cosma C.L.
      • Ramakrishnan L.
      Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism.
      ,
      • Ehrt S.
      • Schnappinger D.
      Mycobacterial survival strategies in the phagosome. Defence against host stresses.
      ,
      • Brennan P.J.
      • Nikaido H.
      The envelope of mycobacteria.
      ). The hydrophobicity of the envelope and consequently its low permeability to hydrophilic solutes contribute to a high level of intrinsic drug tolerance in mycobacteria (
      • Nikaido H.
      • Jarlier V.
      Permeability of the mycobacterial cell wall.
      ). Not surprisingly, the envelope of mycobacteria has long remained a subject of intense investigation, yet its architectural organization remains to be fully understood.
      The mycobacterial cell envelope is broadly stratified into a plasma membrane made of phospholipids, a core cell wall complex of covalently linked mycolyl-arabinogalactan-peptidoglycan (mAGP), and a membrane-like outer layer (
      • Brennan P.J.
      • Nikaido H.
      The envelope of mycobacteria.
      ,
      • Niederweis M.
      • Danilchanka O.
      • Huff J.
      • Hoffmann C.
      • Engelhardt H.
      Mycobacterial outer membranes: in search of proteins.
      ,
      • Hoffmann C.
      • Leis A.
      • Niederweis M.
      • Plitzko J.M.
      • Engelhardt H.
      Disclosure of the mycobacterial outer membrane. Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.
      ). The lipid bilayer in the membrane-like outer layer, also referred to as mycomembrane, is formed by mycolyl chains of mAGP and hydrocarbon chains of the outermost glycolipids, noncovalently associated with the cell wall core of the envelope (
      • Brennan P.J.
      • Nikaido H.
      The envelope of mycobacteria.
      ,
      • Hoffmann C.
      • Leis A.
      • Niederweis M.
      • Plitzko J.M.
      • Engelhardt H.
      Disclosure of the mycobacterial outer membrane. Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.
      ). The outermost lipids, which vary across mycobacterial species, are extractable to a detectable level without breaching envelope integrity, although genetic dispensability of only a few of these lipids is determined (
      • Ortalo-Magné A.
      • Lemassu A.
      • Lanéelle M.A.
      • Bardou F.
      • Silve G.
      • Gounon P.
      • Marchal G.
      • Daffé M.
      Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species.
      ).
      The mycolyl esters of trehalose, trehalose monomycolates (TMM) and trehalose 6,6′-dimycolate (TDM) are among the most conserved and abundant noncovalently associated lipids of the mycobacterial envelope. The nascent mycolyl chains, synthesized in the cytoplasm by fatty-acid synthase systems I and II, are esterified to trehalose to produce TMM, which is subsequently transported across the membrane by MmpL3 (
      • Takayama K.
      • Wang C.
      • Besra G.S.
      Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis.
      ,
      • Grzegorzewicz A.E.
      • Pham H.
      • Gundi V.A.
      • Scherman M.S.
      • North E.J.
      • Hess T.
      • Jones V.
      • Gruppo V.
      • Born S.E.
      • Korduláková J.
      • Chavadi S.S.
      • Morisseau C.
      • Lenaerts A.J.
      • Lee R.E.
      • McNeil M.R.
      • Jackson M.
      Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane.
      ). The abundant pool of TMM in the envelope is then utilized as a universal mycolyl donor by secreted mycolyltransferases, antigen 85 complex (Ag85A, Ag85B, and Ag85C), toward the constitutive synthesis of mAGP and TDM (
      • Takayama K.
      • Wang C.
      • Besra G.S.
      Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis.
      ,
      • Belisle J.T.
      • Vissa V.D.
      • Sievert T.
      • Takayama K.
      • Brennan P.J.
      • Besra G.S.
      Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis.
      ,
      • Puech V.
      • Guilhot C.
      • Perez E.
      • Tropis M.
      • Armitige L.Y.
      • Gicquel B.
      • Daffé M.
      Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis.
      ,
      • Jackson M.
      • Raynaud C.
      • Lanéelle M.A.
      • Guilhot C.
      • Laurent-Winter C.
      • Ensergueix D.
      • Gicquel B.
      • Daffé M.
      Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope.
      ). In addition, TMM is also used by the Ag85 complex to synthesize glucose monomycolate, which like other noncovalently linked mycolyl glycolipids, such as glycerol monomycolate and diarabino-dimycolyl glycerol, is produced under specific growth conditions depending on the type of carbon source in the medium (
      • Hattori Y.
      • Matsunaga I.
      • Komori T.
      • Urakawa T.
      • Nakamura T.
      • Fujiwara N.
      • Hiromatsu K.
      • Harashima H.
      • Sugita M.
      Glycerol monomycolate, a latent tuberculosis-associated mycobacterial lipid, induces eosinophilic hypersensitivity responses in guinea pigs.
      ,
      • Matsunaga I.
      • Naka T.
      • Talekar R.S.
      • McConnell M.J.
      • Katoh K.
      • Nakao H.
      • Otsuka A.
      • Behar S.M.
      • Yano I.
      • Moody D.B.
      • Sugita M.
      Mycolyltransferase-mediated glycolipid exchange in Mycobacteria.
      ,
      • Rombouts Y.
      • Brust B.
      • Ojha A.K.
      • Maes E.
      • Coddeville B.
      • Elass-Rochard E.
      • Kremer L.
      • Guerardel Y.
      Exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to mycolyl-arabinogalactan-peptidoglycan metabolism.
      ).
      The essential role of TMM as a precursor of mAGP, the core cell wall component, makes it an indispensable lipid for mycobacteria, as recently validated by the viability loss due to specific inhibition of its MmpL3 transporter (
      • Grzegorzewicz A.E.
      • Pham H.
      • Gundi V.A.
      • Scherman M.S.
      • North E.J.
      • Hess T.
      • Jones V.
      • Gruppo V.
      • Born S.E.
      • Korduláková J.
      • Chavadi S.S.
      • Morisseau C.
      • Lenaerts A.J.
      • Lee R.E.
      • McNeil M.R.
      • Jackson M.
      Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane.
      ,
      • Tahlan K.
      • Wilson R.
      • Kastrinsky D.B.
      • Arora K.
      • Nair V.
      • Fischer E.
      • Barnes S.W.
      • Walker J.R.
      • Alland D.
      • Barry 3rd, C.E.
      • Boshoff H.I.
      SQ109 targets MmpL3, a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis.
      ). Dispensability of TDM in the envelope, however, remains unclear, although it can be extracted from the bacilli in petroleum ether without any effect on their viability (
      • Noll H.
      • Bloch H.
      • Asselineau J.
      • Lederer E.
      The chemical structure of the cord factor of Mycobacterium tuberculosis.
      ), but the extent of TDM depletion from the ether-treated bacteria remained unclear in these studies. Furthermore, TDM cannot be genetically depleted from mycobacteria due to the functional redundancies in the Ag85A, Ag85B, and Ag85C (
      • Belisle J.T.
      • Vissa V.D.
      • Sievert T.
      • Takayama K.
      • Brennan P.J.
      • Besra G.S.
      Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis.
      ). A mutation in ag85A leads to partial depletion of TDM in Mycobacterium smegmatis, and inactivation or inhibition of Ag85C in M. tuberculosis leads to partial loss in mycolyl content of mAGP as well as the synthesis of TDM (
      • Puech V.
      • Guilhot C.
      • Perez E.
      • Tropis M.
      • Armitige L.Y.
      • Gicquel B.
      • Daffé M.
      Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis.
      ,
      • Warrier T.
      • Tropis M.
      • Werngren J.
      • Diehl A.
      • Gengenbacher M.
      • Schlegel B.
      • Schade M.
      • Oschkinat H.
      • Daffe M.
      • Hoffner S.
      • Eddine A.N.
      • Kaufmann S.H.
      Antigen 85C inhibition restricts Mycobacterium tuberculosis growth through disruption of cord factor biosynthesis.
      ). Moreover, the partial loss in the mycolyl contents of cell wall components in the ag85A mutant could be complemented by expression of Ag85B and Ag85C (
      • Puech V.
      • Guilhot C.
      • Perez E.
      • Tropis M.
      • Armitige L.Y.
      • Gicquel B.
      • Daffé M.
      Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis.
      ). Although any of the three mycolyltransferases can be inactivated individually without affecting cellular viability, simultaneous inactivation of all three genes has not been possible. It is also noteworthy that their simultaneous intracellular depletion using antisense oligonucleotides severely retards the growth of M. tuberculosis (
      • Harth G.
      • Zamecnik P.C.
      • Tabatadze D.
      • Pierson K.
      • Horwitz M.A.
      Hairpin extensions enhance the efficacy of mycolyl transferase-specific antisense oligonucleotides targeting Mycobacterium tuberculosis.
      ).
      TDM is studied in M. tuberculosis as one of the most potent immunomodulatory and granulomatogenic surface glycolipids (
      • Hunter R.L.
      • Olsen M.R.
      • Jagannath C.
      • Actor J.K.
      Multiple roles of cord factor in the pathogenesis of primary, secondary, and cavitary tuberculosis, including a revised description of the pathology of secondary disease.
      ,
      • Geisel R.E.
      • Sakamoto K.
      • Russell D.G.
      • Rhoades E.R.
      In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin is due principally to trehalose mycolates.
      ), although it is highly abundant in at least nine other species of pathogenic and nonpathogenic mycobacteria analyzed so far (
      • Ortalo-Magné A.
      • Lemassu A.
      • Lanéelle M.A.
      • Bardou F.
      • Silve G.
      • Gounon P.
      • Marchal G.
      • Daffé M.
      Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species.
      ,
      • Fujita Y.
      • Naka T.
      • McNeil M.R.
      • Yano I.
      Intact molecular characterization of cord factor (trehalose 6,6′-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry.
      ,
      • Kai M.
      • Fujita Y.
      • Maeda Y.
      • Nakata N.
      • Izumi S.
      • Yano I.
      • Makino M.
      Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae.
      ). Its constitutive abundance in nonpathogenic mycobacterial species raises the possibility that TDM and other noncovalently linked mycolyl-glycolipids could have crucial structural contributions in the integrity of the mycobacterial envelope. Recently, we identified Msmeg_1529, an M. smegmatis enzyme of the serine esterase superfamily that can hydrolyze purified TDM from various mycobacterial species, including M. tuberculosis in vitro (
      • Ojha A.K.
      • Trivelli X.
      • Guerardel Y.
      • Kremer L.
      • Hatfull G.F.
      Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms.
      ). This allowed us to question whether exogenous exposure to the purified recombinant esterase, henceforth called TDMH, may impact the integrity of the mycobacterial envelope. Here, we show that exposure to TDMH triggers an immediate release of free mycolic acids (FM) from noncovalently associated mycolyl-containing glycolipids, ultimately leading to rapid and extensive lysis of pathogenic species, such as M. tuberculosis, Mycobacterium bovis, and Mycobacterium marinum, as well as to a lesser extent of M. smegmatis and Mycobacterium avium. Although these findings highlight the structural contribution and importance of mycolyl glycolipids in the outer envelope of mycobacteria, they also open up new possibilities of improved detection and clearance of mycobacterial infections.

      DISCUSSION

      In this study, we report an unusual and unexpected consequence of mycobacterial lysis upon exposure to a cutinase-like serine esterase. The enzyme can hydrolyze purified TDM in vitro and sequentially deplete TMM and TDM from the envelope of the exposed cells. The early depletion of TMM and TDM indicates that these lipids are both exposed on the bacterial surface and therefore are among the first molecules encountered by the enzyme. Loss of trehalose mycolates could itself cause osmotic lysis and/or create openings for the enzyme to access the inner components and breach envelope integrity. Either of these situations therefore reveals a structural role of the glycolipids in maintaining the integrity of the mycobacterial envelope. Involvement of TMM is a more complex scenario to imagine because of the dynamic range of intermediate conformations it could assume during synthesis of terminal mycolyl esters, although a molecular subspecies of the glycolipid with a dedicated structural role cannot be ruled out. The role of TDM, however, is highly plausible because of its large abundance and its stable terminal structure as an end product. Given that the outermost layer of the envelope assumes a membrane-like configuration (
      • Hoffmann C.
      • Leis A.
      • Niederweis M.
      • Plitzko J.M.
      • Engelhardt H.
      Disclosure of the mycobacterial outer membrane. Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.
      ), TDM could likely be organized in a bilayer configuration constituting the outer leaflet of the mycomembrane, with trehalose as the polar head group at the environmental interface and mycolic acids forming the hydrophobic tail. The 60–90-carbon mycolic acid could either be in an extended conformation to span the entire thickness (∼8 nm) of the mycomembrane or in a folded conformation to fit into the outer leaflet of the bilayer (
      • Niederweis M.
      • Danilchanka O.
      • Huff J.
      • Hoffmann C.
      • Engelhardt H.
      Mycobacterial outer membranes: in search of proteins.
      ,
      • Hoffmann C.
      • Leis A.
      • Niederweis M.
      • Plitzko J.M.
      • Engelhardt H.
      Disclosure of the mycobacterial outer membrane. Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.
      ). The folded mycolic acid chain, presumably formed through interaction of the keto or methoxy groups with the lipid head group (
      • Niederweis M.
      • Danilchanka O.
      • Huff J.
      • Hoffmann C.
      • Engelhardt H.
      Mycobacterial outer membranes: in search of proteins.
      ), is the likely conformation. This is extrapolated from the biophysical analysis of mycolic acid monolayers as well as from the visual evidence of hydrophobic interface in the cryo-electron microscopy of the outer membrane (
      • Hoffmann C.
      • Leis A.
      • Niederweis M.
      • Plitzko J.M.
      • Engelhardt H.
      Disclosure of the mycobacterial outer membrane. Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.
      ,
      • Villeneuve M.
      • Kawai M.
      • Kanashima H.
      • Watanabe M.
      • Minnikin D.E.
      • Nakahara H.
      Temperature dependence of the Langmuir monolayer packing of mycolic acids from Mycobacterium tuberculosis.
      ).
      The likely integration of TDM in the outer membrane, its limited extractability from the intact envelope and disintegration of the envelope upon its depletion together raise a strong possibility that TDM, and therefore the outer membrane, could be indispensable for the overall integrity of mycobacteria. Moreover, this is consistent with the cryo-EM visualization of the dissolved inner membrane in the region where the outer membrane lipids are removed by detergent extraction (
      • Hoffmann C.
      • Leis A.
      • Niederweis M.
      • Plitzko J.M.
      • Engelhardt H.
      Disclosure of the mycobacterial outer membrane. Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.
      ,
      • Portevin D.
      • De Sousa-D'Auria C.
      • Houssin C.
      • Grimaldi C.
      • Chami M.
      • Daffé M.
      • Guilhot C.
      A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.
      ). The indispensability of TDM is also suggested by growth inhibition of M. tuberculosis in the presence of a chemical inhibitor of Ag-85C that reduces the level of TDM without affecting the levels of mAGP and TMM (
      • Warrier T.
      • Tropis M.
      • Werngren J.
      • Diehl A.
      • Gengenbacher M.
      • Schlegel B.
      • Schade M.
      • Oschkinat H.
      • Daffe M.
      • Hoffner S.
      • Eddine A.N.
      • Kaufmann S.H.
      Antigen 85C inhibition restricts Mycobacterium tuberculosis growth through disruption of cord factor biosynthesis.
      ).
      Given the detrimental consequences of TDMH exposure, mycobacteria must have a mechanism to regulate its physiological activity. Regulation of TDMH is indeed manifested by a growth phase-dependent induction of its activity toward production of free mycolic acids during biofilm maturation of M. smegmatis (
      • Ojha A.K.
      • Trivelli X.
      • Guerardel Y.
      • Kremer L.
      • Hatfull G.F.
      Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms.
      ,
      • Ojha A.K.
      • Baughn A.D.
      • Sambandan D.
      • Hsu T.
      • Trivelli X.
      • Guerardel Y.
      • Alahari A.
      • Kremer L.
      • Jacobs Jr., W.R.
      • Hatfull G.F.
      Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.
      ). Furthermore, delayed depletion of TDM upon TDMH exposure also suggests a two-tier regulatory mechanism in which replenishment of the substrate could potentially salvage a dysfunctional regulation of tdmh expression. Although TDM hydrolase in other mycobacteria remains to be discovered, antibodies against some of the cutinase-like esterases, including the closest homologue of TDMH (Rv3452), are produced during active infection of M. tuberculosis in humans, indicating their expression during TB pathogenesis (
      • Brust B.
      • Lecoufle M.
      • Tuaillon E.
      • Dedieu L.
      • Canaan S.
      • Valverde V.
      • Kremer L.
      Mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis.
      ).
      In summary, TDMH solves a long standing challenge of exogenously breaching the envelope of mycobacteria. The improved frequency of positive detection upon TDMH treatment of samples with small number of bacilli highlights the potential of the enzyme in elevating the sensitivity of TB diagnosis, particularly in paucibacillary infections.

      Acknowledgments

      We thankfully acknowledge the gifts of M. marinum from Pamela Small and M. avium from Delphi Chatterjee, statistical analysis of the results by Abdus Wahed, and critical comments on the manuscript by Graham Hatfull. We also acknowledge excellent technical support from Kathleen Kulka.

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