Transporters Involved in the Biogenesis and Functionalization of the Mycobacterial Cell Envelope

Abstract

The biology of mycobacteria is dominated by a complex cell envelope of unique composition and structure and of exceptionally low permeability. This cell envelope is the basis of many of the pathogenic features of mycobacteria and the site of susceptibility and resistance to many antibiotics and host defense mechanisms. This review is focused on the transporters that assemble and functionalize this complex structure. It highlights both the progress and the limits of our understanding of how (lipo)polysaccharides, (glyco)lipids, and other bacterial secretion products are translocated across the different layers of the cell envelope to their final extra-cytoplasmic location. It further describes some of the unique strategies evolved by mycobacteria to import nutrients and other products through this highly impermeable barrier.

Figure 1:. The cell envelope of Mycobacterium tuberculosis.

Cryo-electron micrograph of vitreous cryosections (35 nm) of M. bovis BCG and schematic representation of the cell envelope of Mtb. The overall schematic and individual structures are not drawn to scale. Inner membrane proteins are not shown for the sake of clarity. The most recent model of Mtb AG indicates that it contains about 80 glycosyl residues distributed between a galactan domain made of ~ 23 Galf residues and two arabinan domains each containing about 26 Araf residues. The lipid component of LM and LAM is a mannosylated phosphatidyl-myo-inositol moiety that serves to anchor the lipoglycans in the inner and outer membranes of the cell envelope. Extending from this anchor and common to LM and LAM is a linear α(1→6)-linked mannan backbone made up of 20-25 Manp residues elaborated by single Manp units which are α(1→2)-linked. In LAM, a single D-arabinan chain consisting of ~ 60 Araf residues is further attached to the mannan backbone. The arabinan domains of both AG and LAM is made of stretches of α(1→5)-linked Araf residues with precisely positioned α(3→5)-branch sites. Minor covalent modifications, including galactosamine (GalN) substituents and succinyl residues, may modify some of the Araf residues of LAM and AG. Mono-, di- and tri-mannoside caps modify the arabinan termini of Mtb LAM. AG, arabinogalactan; PG, peptidoglycan; SL, sulfolipids (SL); DAT, 2,3-diacyltrehaloses; PAT, polyacyltrehaloses; PGL, phenolic glycolipids; PI, phosphatidyl-myo-inositol; PIM, phosphatidylinositol mannosides; PDIM, phthiocerol dimycocerosates; TDM, trehalose dimycolates; TMM, trehalose monomycolates; LAM, lipoarabinomannan; LM, lipomannan.

Figure 2:. Structures of predominant forms of mycolic acids found in M. tuberculosis and M. smegmatis.

α, methoxy and keto-mycolates are found in Mtb. α, α’ and epoxy-mycolates are found in M. smegmatis. Mycobacterial mycolic acids contain 60 to 90 carbon atoms of which 42 to 62 are in the meromycolate chain and 20-26 in the α-branch. The shorter α’ mycolates contain 60-62 carbon atoms.

Figure 3:. Export and recycling of mycolic acid-containing cell envelope constituents.

FAS-I, FAS-II, Pks13, FadD32, SAM-dependent mycolic acid (MA) methyltransferases and TmaT are components of the enzymatic machinery required for the intracytoplasmic synthesis of TMM and its acetylation prior to export. LpqN is a lipoprotein thought to serve as a periplasmic chaperone in the MmpL3- and MmpL11-dependent translocation pathways. LpqY-SugABC is an ABC-importer involved in the recycling of trehalose. Mce1 is thought to participate in the recycling of mycolic acids. Whether MmpL11 translocates wax ester mycolates across the plasma membrane under their free form or on a carrier molecule remains to be defined. Note that MmpL11 is also thought to be involved in the export of long-chain TAG in Mtb [see Fig. 6]. AcTMM, acetylated TMM; AG-bound MA, arabinogalactan-bound mycolic acids. See text for details.

Figure 4:. Organization of the gene clusters involved in the biosynthesis and export of acyltrehaloses, glycopeptidolipids, phthiocerol dimycocerosates and phenolic glycolipids.

Genes involved in the translocation of cell envelope constituents are represented as green arrows; genes involved in their biosynthesis as black arrows. Accessory genes, gap, sap and mmpS are in pink. fadD genes encode acyl-AMP ligases; papA, chp and pE genes encode acyltransferases; pks genes encode the polyketide synthases responsible for the elongation of the multimethyl-branched fatty acyl chains esterifying the listed lipids. Other biosynthetic genes are as follows: LOS (the Mtb canettii locus is shown): wbbL2, rhamnosyltransferase; mcan_15441, methyltransferase; mcan_15451, putative glycosyltransferase; mcan_15471, glycosyltransferase. GPL (the M. smegmatis mc²155 locus is represented): rfbA, alpha-D-hexose-1-phosphate-thymidylyl-transferase; rmlB, UDP-hexose-4-epimerase; gtf genes encode glycosyltransferases; rmt genes and fmt encode methyltransferases; atf1 encodes an acetyltransferase; mbtB encodes a protein of unknown function; mps genes encode non-ribosomal protein synthases. PDIM/PGL (the Mtb H37Rv locus is represented): the clusters of genes encompassing fadD26-ppsE and pks1-Rv2949c are responsible for the synthesis of phthiocerol and phenolphthiocerol; the cluster encompassing papA5-fadD28 is involved in mycocerosic acid synthesis and transfer; Rv2951c, Rv2952 and Rv2953 are involved in the modification of the lipid moiety of PDIM and PGL; and the cluster encompassing Rv2954c-Rv2959c is responsible for the formation of the glycosidic domain of PGL.

Figure 5:. Proposed pathways for outer membrane (glyco)lipid translocation in mycobacteria.

See text for details. The major sulfolipid SL-I (2,3,6,6’-tetraacyl α-α’-trehalose-2’-sulfate) is represented. In SL-I, trehalose is sulfated at the 2’ position and esterified with palmitic acid and the multimethyl-branched phthioceranic and hydroxyphthioceranic acids. SL₁₂₇₈ is the diacylated SL precursor synthesized in the cytoplasm. The MmpL8 transporter represented here is that of Mtb, involved in the export of sulfolipids, and is not to be confused with the MmpL8 transporter from M. abscessus which is involved in the synthesis and export of diacylated glucose-based lipids (see text for details). In DAT (2,3-di-O-acyltrehalose), trehalose is esterified with palmitic acid and the multimethyl-branched mycosanoic acid. In PAT, trehalose is esterified with palmitic acid and the multimethyl-branched mycolipenic acids. TMP is a diacyltrehalose precursor of TPP carrying one phleic acyl chain and one C₁₄-C₁₉ fatty acyl chain; TMP is synthesized in the cytoplasm and is thought to be the TPP biosynthetic precursor translocated across the plasma membrane. MmpL10s refers to the MmpL10 protein from M. smegmatis. In the structure of LOS of Mtb Canettii represented here, R = Acetyl. In the structures of the phthiocerol dimycocerosates (PDIM) and phenolic glycolipids (PGL) of Mtb presented herein, p, p’=3-5; n, n’=16-18; m1 (PDIM) = 20-22; m2 (PGL) =15-17; R= CH₂-CH₃ or CH₃. One of the most abundant forms of GPLs found in M. smegmatis is shown. It contains a 2-methoxy C28 fatty acyl residue, a di-O-acetyl 6-deoxytalosyl residue attached to the allo-threonine residue, and a 3,4 di-O-methyl rhamnosyl residue attached to the alaninol residue.

Figure 6:. Proposed export pathways for glycerolipids and lipoglycans.

The transporters proposed to participate in the translocation of phospholipids, triglycerides (TAG), apolar forms of phosphatidylinositol mannosides (phosphatidylinositol di- or tri-mannosides), lipomannan (LM) and lipoarabinomannan (LAM) are shown. See text for details. The structure of LAM from Mtb is presented on the righthand side with a delineation of its PIM anchor, the LM moiety, and its mannan and arabinomannan domains (the two latter being found in the capsular material of Mtb). Ac₁PIM₂, triacylated form of PIM₂; Ac₁PIM₆, triacylated form of PIM₆. MV, membrane vesicle. In TAG, n = 6 -18.

Figure 7.. Structures of E. coli AcrB and M. smegmatis MmpL3.

A. Structure of an asymmetric AcrB trimer in a ligand-bound state (PDB ID: 3AOD). Each AcrB protomer adopts a specific conformation: Loose (L, green), Tight (T, teal) and Open (O, pink). Rifampin bound to the L and T protomers is shown in red. The Deep binding site of the T protomer is discussed in the text. B. AcrB monomer (PDB ID: 1IWG) with R1 and R2 transmembrane repeats shown in teal and yellow, respectively. The TM2 and TM8 helices responsible for coupling of substrate efflux with proton translocation are shown in blue and orange, respectively. C. Structure of a MmpL3 monomer (PDB ID: 6AJF) with the R1 and R2 repeats and TM2 and TM8 in the transmembrane domain highlighted. Coloring of the TM helices is same as in the panel B. Two molecules of 6-n-dodecyl-α, α-trehalose bound to the periplasmic porter domain of MmpL3 are shown in green. D. Periplasmic view of the conserved Asp407, Asp408 and Lys940 residues of TM4 and TM10 in the L/T protomer of AcrB (PDB ID: 2GIF). Essential residues are labeled. E. Periplasmic view of the conserved residues in the O protomer of AcrB (PDB ID: 2GIF). F. Periplasmic view of the essential residues in TM4 and TM10 of MmpL3 (PDB ID: 6AJF).

Figure 8.. Nutrient uptake mediated by outer membrane proteins in M. tuberculosis.

The proteins CpnT, SpmT and PPE51 are localized in the OM and take up small, hydrophilic nutrients. The surface-localized proteins PPE36 and PPE62 mediate heme uptake across the OM. The periplasmic DppA protein binds heme and transports it to the Dpp importer located in the IM of Mtb.