Collected Thoughts on Mycobacterial Lipoarabinomannan, a Cell Envelope Lipoglycan

Abstract

The presence of lipoarabinomannan (LAM) in the Mycobacterium tuberculosis (Mtb) cell envelope was first reported close to 100 years ago. Since then, numerous studies have been dedicated to the isolation, purification, structural definition, and elucidation of the biological properties of Mtb LAM. In this review, we present a brief historical perspective on the discovery of Mtb LAM and the herculean efforts devoted to structurally characterizing the molecule because of its unique structural and biological features. The significance of LAM remains high to this date, mainly due to its distinct immunological properties in conjunction with its role as a biomarker for diagnostic tests due to its identification in urine, and thus can serve as a point-of-care diagnostic test for tuberculosis (TB). In recent decades, LAM has been thoroughly studied and massive amounts of information on this intriguing molecule are now available. In this review, we give the readers a historical perspective and an update on the current knowledge of LAM with information on the inherent carbohydrate composition, which is unique due to the often puzzling sugar residues that are specifically found on LAM. We then guide the readers through the complex and myriad immunological outcomes, which are strictly dependent on LAM's chemical structure. Furthermore, we present issues that remain unresolved and represent the immediate future of LAM research. Addressing the chemistry, functions, and roles of LAM will lead to innovative ways to manipulate the processes that involve this controversial and fascinating biomolecule.

Keywords: Mycobacterium tuberculosis; lipoarabinomannan; lipoglycan; mycobacteria; tuberculosis.

Figure 1

Schematic Representation of the evolution of Mtb LAM structural studies. Approximate timeline for last 100 years starting in 1925.

Figure 2

Schematic representation of ManLAM from Mtb. The antiLAM monoclonal antibodies (mAbs) CS-35, A194-01, and CHCS9-08 are shown to react with the LAM arabinan terminal arrangements (Ara₄ and Ara₆, respectively). These three mAbs are widely used in immunoassays for TB diagnosis. The cartoon is based on a screening of 12 synthetic arabinan glycoconjugates by Dr. Todd Lowary [40]. In order to identify which motifs of LAM are being recognized by anti-LAM antibodies, an exhaustive digestion of LAM with commercially available α-mannosidase (derived from Jack Beans) is performed. This digestion removes the mannose caps of LAM (depicted as grey hexagons). An additional digestion using an endo-arabinanase (in-house isolated from Cellulomonas) releases arabinan fragments from the D-arabinan domain, mainly Ara₄, Ara₅, and Ara₆ fragments. These enzymatic digestions allowed the identification of the Ara₄ and Ara₆ motifs as the ones being recognized by monoclonal antibodies against LAM and were used in the development of TB diagnosis downstream.

Figure 3

Dominant arabinan termini of Mtb lipoarabinomannan from culture. Only non-reducing ends are shown. The terminal structures evolved after extensive enzymatic degradation of LAM followed by liquid chromatography with tandem mass spectrometry (LC/MS-MS) analyses [41]. Abbreviations: Ara = D-Arabiniose, Man = D-Mannose, Suc = Succinates.

Figure 4

MALDI-TOF analysis of LAM from Mtb. The mass spectrometry (MS) was done on Bruker ultrafleXtreme matrix-assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS), indicating a molecular mass for ManLAM of approximately 15 kDa. Heterogeneity in mass of 162 m/z corresponds to a hexose and 100 m/z corresponds to succinates.