Box 1: The cell envelope of mycobacteria
The mycobacterial cell wall is a complex structure that is required for cell growth, resistance to antibiotics and virulence76, 105, 106. It is composed of three distinct macromolecules — peptidoglycan, arabinogalactan and mycolic acids — which are surrounded by a non-covalently linked outer capsule of proteins and polysaccharides23, 76, 105, 107 (see the figure). The high density of lipids in the cell wall prevents accurate Gram staining, and mycobacteria are known as acid-fast, as they can be stained by acid-fast dyes, such as Ziehl–Neelsen stain23. The cell wall is the most common target of antituberculosis drugs, and many compounds that are in clinical use or under development target enzymes that synthesize distinct layers of the cell wall108.
The peptidoglycan layer surrounds the plasma membrane and comprises long polymers of the repeating disaccharide N-acetyl glucosamine–N-acetyl muramic acid (NAG–NAM) that are linked via peptide bridges. The peptidoglycan precursor lipid II is generated in the cytoplasm18, 23 and is probably transported across the periplasm by the transmembrane protein MviN21. Unidentified hydrolases are required to open the peptidoglycan mesh for the insertion of new precursors18, which are added in an 'inside to outside' manner109. The penicillin-binding proteins (PBPs) PonA1 and PonA2 incorporate new subunits into the existing structure. Transpeptidases, such as PBPA, PBPB, LdtA and LdtB, crosslink the newly inserted material23. Compared with other model bacteria, such as Escherichia coli and Bacillus subtilis, mycobacterial peptidoglycan is heavily crosslinked. Up to 80% of the peptidoglycancontains non-traditional 3–3 peptide crosslinks instead of traditional 4–3 crosslinks72, 75. The peptidoglycan polymer also has modifications, such as glycolylation of NAM residues73, 110 and amidation of the D-Glu and meso- diaminopimelic acid (mDAP) residues of the peptide side chain72, 111 (see the figure). Amidation may mask recognition by the innate immune receptor nucleotide-binding oligomerization domain-containing 1 (NOD1), which has been observed in B. subtilis112. However, glycolylated NAM is efficiently recognized by NOD2 and induces the production of inflammatory cytokines in Mycobacterium tuberculosis-infected macrophages113, 114.
A layer of arabinogalactan surrounds the peptidoglycan layer (see the figure). Galactan comprises a repeating disaccharide unit of 6-D-Galfβ1–5-D-Galfβ and is synthesized by the galactofuranosyl transferases Glf, GlfT1 and GlfT2. Galactan is modified with long arabinan polymers that are synthesized by the successive actions of DprE1 and DprE2115 and the arabinofuranosyltransferasesAftA, EmbA, EmbB and AftB105. There is variability in arabinosylation, and some galactan chains remain free of arabinan116. Arabinan chain termini are branched, and this motif is generated by the actions of AftC105, AftD117 and Rv1459 (Ref. 105). Arabinan can also be further modified by the addition of succinyl or unusual non-N-acetylated galactosamine (GalN) moieties105. The GalN modifications are mostly present in pathogenic mycobacteria and may promote efficient infection, as has been observed in Francisella tularensis74.
Most arabinan is ligated with long-carbon-chain mycolic acids76, 77, 105, which form the characteristic thick waxy lipid coat of mycobacteria23, 118 and are major contributors to the impermeability of the cell wall23, 76, 77 and to virulence76, 100. Mycolic acids are formed from two fatty acids, a saturated shorter C20–C26 α-branch that is connected to a C60–C90 meromycolate branch. These branches are generated by the FASI and FASII complexes76, 77, 119. FASI products are transferred to FASII for extension by the combined action of 3-oxoacyl acyl carrier protein synthase 3 (FabH), acyl carrier protein AcpM and malonyl CoA-acyl carrier protein transacylase FabD76, 77. Mycolic acids are then processed and matured by a cascade of enzymes76, 77, 120, 121, which results in three distinct meromycolate variants: α-meroacids, methoxy-meroacids and keto-meroacids76, 77. All three variants are required for full virulence during infection and have varying levels of saturation, cyclopropanation and oxygenation76, 77. The inner membrane transporter MmpL shuttles mycolic acids to the cell surface122, 123, 124, where the FbpABCD (also known as antigen 85) complex ligates the mycolic acid monomers to arabinogalactan76, 77, 105. Although the coordination of mycolic acid and arabinogalactan synthesis and insertion in the cell wall remains unknown, recent data suggest that these macromolecules are produced and exported at the cell pole19, 125, where new cell growth occurs in mycobacteria.
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
- Karen J. Kieser &
- Eric J. Rubin
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
- Eric J. Rubin
Competing interests statement
The authors declare no competing interests.
Karen J. Kieser
Karen J. Kieser is a Ph.D. student in the Program in Biological Sciences in Public Health at Harvard University, Cambridge, Massachusetts, USA. Her thesis work investigates the enzymatic regulation of cell elongation and division in mycobacteria.
Eric J. Rubin
Eric J. Rubin is Professor of Immunology and Infectious Diseases at the Harvard School of Public Health, Boston, Massachusetts, USA. His laboratory uses genetic techniques and cell biology tools to probe fundamental processes of mycobacterial proliferation and pathogenesis.
- Non-traditional 3–3 peptide crosslinks
Peptide bonds between the third residues (meso-diaminopimelic acid (mDAP) in mycobacteria) of two peptide tails in peptidoglycan; a traditional crosslink is a peptide bond between the third and fourth residues (between DAP and D-Ala in mycobacteria) of two peptide tails.