The identification and location of succinyl residues and the characterization of the interior arabinan region allow for a model of the complete primary structure of Mycobacterium tuberculosis mycolyl arabinogalactan

Abstract

The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-alpha-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear alpha-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

FIGURE 1.

The endogenous arabinase releases oligo-arabinosides containing succinyl groups. A, preferred sites of action of the endogenous arabinase. When the GalNH₂ is present (dashed bond), the Ara₂₀ site is strongly preferred over the Ara₁₉ and Ara₁₈ site. B, MALDI-TOF mass spectrum of the arabinan fragments released from M. smegmatis mycolyl arabinogalactan between m/z 4000 and 4400. The non-succinylated and succinylated Ara₁₈ and Ara₁₉ oligo-arabinosides are labeled. Both M+Na⁺, and, at16 amu higher, M+ K⁺ ions are seen. C, MALDI-TOF mass spectrum of the non-galactosaminylated arabinan fragments released from M. tuberculosis mycolyl arabinogalactan between m/z 4000 and 4400. The non-succinylated and succinylated Ara₁₈ and Ara₁₉ oligo-arabinosides are labeled. D, MALDI-TOF mass spectrum of the galactosaminylated arabinan fragments released from M. tuberculosis mycolyl arabinogalactan between m/z 4700 and 4800 showing the major Ara₂₀GalNH₂ fragment (both as M+ Na⁺ and M+ K⁺) and the lack of any corresponding succinylated version.

FIGURE 2.

Identification of the succinyl residues in mycobacterial mAGP. A, structure of the dioctyl derivative of succinic acid and depiction of the origin of the ions at m/z 213 and 231. B, mass spectrum of standard succinyl dioctyl ester. The M+H ion at m/z 343 is formed in the e.i. mode in the ion trap of the mass spectrometer. C, mass spectrum of succinyl dioctyl ester released by octanolysis of mAGP from M. tuberculosis. D, mass spectrum of succinyl dioctyl ester released by octanolysis of mAGP from M. smegmatis. In C and D, the ion in the molecular weight region is less pronounced because of less material in the ion trap than in B.

FIGURE 3.

The one-dimensional NMR spectrum of the mixture of endogenous arabinase solubilized non-galactosaminylated oligo-arabinans from M. tuberculosis. The methylene hydrogens on the succinyl group (labeled b and c) are evident at 2.5 and 2.65 ppm as is the upfield arabinosyl ring proton (from succinyl substitution) at 4.92 (labeled a).

FIGURE 4.

Location of the succinyl group on O2 of an α-3,5-Araf residue by NMR. A, summary of the chemical shifts of the relevant nuclei. B, HSQC two-dimensional NMR spectrum between 3.5 and 5.5 ppm (¹H) and 58 and 89 ppm (¹³C). C, HSQC two-dimensional NMR spectrum between 2.2 and 2.8 ppm (¹H) and 25 and 38 ppm (¹³C). D, gHMBC two-dimensional NMR between 2.2 and 5.5 ppm (¹H) and 166 and 184 ppm (¹³C). E, one-dimensional TOCSY from 2.5 to 5.5 ppm with a mixing time of a 150 ms. F, one-dimensional TOCSY from 2.5 to 5.5 ppm with a mixing time of 30 ms.

FIGURE 5.

Location of the succinyl groups in mycobacterial arabinan by mass spectrometry. A, full mass spectrum of the non-galactosaminylated arabinan enzyme-released fragments from M. tuberculosis mAGP after per-O-acetylation. The succinylated version of each fragment is indicated with arrows. The peaks marked with asterisks are of unknown origin. B, plot of % succinylation (as revealed by the M+Na⁺ ions of the non-succinylated oligo-arabinan enzyme products and the succinylated versions at 58 amu higher m/z values) for Ara_7–8 and Ara_18–20. The different degree of succinylation of the two classes of oligo-arabinosides combined with the NMR data shows that the major succinyl group is on the interior α-3,5-branched arabinosyl residue. C, major (dark square) and minor (gray circles) positions of succinylation revealed by mass spectrometry and NMR analyses together (see text for further information).

FIGURE 6.

The MALDI-TOF mass spectrum of the higher molecular weight arabinan fragments released from M. tuberculosis mycolyl arabinogalactan. A, galactosaminylated fraction, which eluted first on the Biogel P2 column. B, non-galactosaminylated fraction which eluted later on the Biogel P2 column. The series of Ara₂₀GalNH₂ to Ara₃₀GalNH₂ is readily apparent in A and Ara₁₈-Ara₃₀ in B; the succinylated oligo-arabinans in B are also apparent (not marked).

FIGURE 7.

The structure of the mycolyl arabinogalactan and its attachment to peptidoglycan. The key features include the 31-unit long arabinan (red) of which two-thirds are substituted with mycolyl residues, one-third with succinyl and one-third with glucosaminosyl residues. Three chains are present per galactan (blue) chain. The GalNH₂ and the succinyl residues are shown in green. The remaining features and ambiguities of the structure are presented under “Discussion.”