Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthethic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from Mycobacterium tuberculosis and Mycobacterium leprae enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM₂) and the hexamannoside (PIM₆). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GCMS) analyses, the additional C16 fatty acyl substituent on PIM₂ and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM₂ may constitute a key regulatory step in their biosynthesis.

Globally, tuberculosis is slowly declining each year and it is estimated that 37 million lives were saved between 2000 and 2013 through effective diagnosis and treatment. Currently, diagnosis relies on demonstration of the bacteria, Mycobacterium tuberculosis (Mtb), in clinical specimens by serial sputum microscopy, culture and molecular testing. Commercial immunoassay lateral flow kits developed to detect Mtb lipoglycan lipoarabinomannan (LAM) in urine as a marker of active TB exhibit poor sensitivity, especially in immunocompetent individuals, perhaps due to low abundance of the analyte. Our present study was designed to develop methods to validate the presence of LAM in a quantitative fashion in human urine samples obtained from culture-confirmed TB patients. Herein we describe, a consolidated approach for isolating LAM from the urine and quantifying D-arabinose as a proxy for LAM, using Gas Chromatography/Mass Spectrometry. 298 urine samples obtained from a repository were rigorously analyzed and shown to contain varying amounts of LAM-equivalent ranging between ~10–40 ng/mL. To further substantiate that D-arabinose detected in the samples originated from LAM, tuberculostearic acid, the unique 10-methyloctadecanoic acid present at the phosphatidylinositol end of LAM was also analyzed in a set of samples and found to be present confirming that the D-arabinose was indeed derived from LAM. Among the 144 samples from culture-negative TB suspects, 30 showed presence of D-arabinose suggesting another source of the analyte, such as disseminated TB or from non-tuberculosis mycobacterium. Our work validates that LAM is present in the urine samples of culture-positive patients in small but readily detectable amounts. The study further substantiates LAM in urine as a powerful biomarker for active tuberculosis.

In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure–function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal β-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α−arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.