Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures.

Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers.

[1]  L. Kremer,et al.  Toll-Like Receptor 2 (TLR2)-Dependent-Positive and TLR2-Independent-Negative Regulation of Proinflammatory Cytokines by Mycobacterial Lipomannans1 , 2004, The Journal of Immunology.

[2]  Harshini Mukundan,et al.  Optimizing a waveguide-based sandwich immunoassay for tumor biomarkers: evaluating fluorescent labels and functional surfaces. , 2009, Bioconjugate chemistry.

[3]  G. Puzo,et al.  Lipoarabinomannans: from structure to biosynthesis. , 2003, Biochimie.

[4]  Aaron S. Anderson,et al.  Waveguide-Based Biosensors for Pathogen Detection , 2009, Sensors.

[5]  B. Monsarrat,et al.  Structural characterization of Mycobacterium tuberculosis lipoarabinomannans by the combination of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 2001, Analytical chemistry.

[6]  P. Brennan,et al.  Lipoarabinomannan of Mycobacterium tuberculosis. Capping with mannosyl residues in some strains. , 1992, The Journal of biological chemistry.

[7]  K. Khoo,et al.  Structural definition of the non-reducing termini of mannose-capped LAM from Mycobacterium tuberculosis through selective enzymatic degradation and fast atom bombardment-mass spectrometry. , 1993, Glycobiology.

[8]  J. Lamb,et al.  The inhibitory effects of mycobacterial lipoarabinomannan and polysaccharides upon polyclonal and monoclonal human T cell proliferation. , 1988, Clinical and experimental immunology.

[9]  Alimuddin Zumla,et al.  Urine for the diagnosis of tuberculosis: current approaches, clinical applicability, and new developments , 2010, Current opinion in pulmonary medicine.

[10]  R. Modlin,et al.  Control of Mycobacterium tuberculosis through mammalian Toll-like receptors. , 2002, Current opinion in immunology.

[11]  Basil I. Swanson,et al.  Pathogen detection using single mode planar optical waveguides , 2005 .

[12]  G. Besra,et al.  Truncated Structural Variants of Lipoarabinomannan in Ethambutol Drug-resistant Strains of Mycobacterium smegmatis , 1996, The Journal of Biological Chemistry.

[13]  P. Brennan,et al.  Lipoarabinomannan of Mycobacterium: Mannose capping by a multifunctional terminal mannosyltransferase , 2008, Proceedings of the National Academy of Sciences.

[14]  D. Golenbock,et al.  Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis. , 1999, Journal of immunology.

[15]  P. Brennan.  Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. , 2003, Tuberculosis.

[16]  R. Chaisson,et al.  Diagnostic Accuracy of a Urine Lipoarabinomannan Test for Tuberculosis in Hospitalized Patients in a High HIV Prevalence Setting , 2009, Journal of acquired immune deficiency syndromes.

[17]  L. Sechi,et al.  Clinical Utility of a Commercial LAM-ELISA Assay for TB Diagnosis in HIV-Infected Patients Using Urine and Sputum Samples , 2010, PloS one.

[18]  P. Brennan,et al.  Structural features of the arabinan component of the lipoarabinomannan of Mycobacterium tuberculosis. , 1991, The Journal of biological chemistry.

[19]  K. M. Grace,et al.  Integrated optical biosensor for detection of multivalent proteins. , 1999, Optics letters.

[20]  G. Kaplan,et al.  Suppression of T-cell proliferation by Mycobacterium leprae and its products: the role of lipopolysaccharide. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Jim F Huggett,et al.  Low sensitivity of a urine LAM-ELISA in the diagnosis of pulmonary tuberculosis , 2009, BMC infectious diseases.

[22]  M. Rivière,et al.  Comparative structural study of the mannosylated-lipoarabinomannans from Mycobacterium bovis BCG vaccine strains: characterization and localization of succinates. , 1997, Glycobiology.

[23]  André Lopez,et al.  Highly ordered supra-molecular organization of the mycobacterial lipoarabinomannans in solution. Evidence of a relationship between supra-molecular organization and biological activity. , 2004, Journal of molecular biology.

[24]  S. Patil,et al.  Lipoarabinomannan antigen and anti-lipoarabinomannan antibody profile in the serum of patients with mycobacterial infections and their significance in disease process , 1995 .

[25]  Xia Jin,et al.  Proliferation, But Not Interleukin 2 Production, of Gag-Specific CD8+ T Cells Is Associated With Low HIV Viremia and High CD4 Counts in HIV-1-Infected Chinese Individuals , 2009, Journal of acquired immune deficiency syndromes.

[26]  G. Besra,et al.  Mycobacterium tuberculosis cell envelope. , 1996, Current topics in microbiology and immunology.

[27]  G. Besra,et al.  Structure, function and biogenesis of the mycobacterial cell wall. , 1997, Biochemical Society Transactions.

[28]  D. Golenbock,et al.  Mycobacterial lipoarabinomannan recognition requires a receptor that shares components of the endotoxin signaling system. , 1996, Journal of immunology.

[29]  P. Brennan,et al.  Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase , 2006, Proceedings of the National Academy of Sciences.

[30]  T. Yoshimura,et al.  Partition of amphiphilic molecules into phospholipid vesicles and human erythrocyte ghosts: measurements by ultraviolet difference spectroscopy. , 1984, Biochemistry.

[31]  C. Petzold,et al.  Structural characterization of lipoarabinomannans from Mycobacterium tuberculosis and Mycobacterium smegmatis by ESI mass spectrometry , 2005, Journal of the American Society for Mass Spectrometry.

[32]  P. Brennan,et al.  Lipoarabinomannan. Multiglycosylated form of the mycobacterial mannosylphosphatidylinositols. , 1992, The Journal of biological chemistry.

[33]  L. Sibley,et al.  Mycobacterial lipoarabinomannan inhibits gamma interferon-mediated activation of macrophages , 1988, Infection and immunity.

[34]  G. Rook,et al.  Lipoarabinomannan from Mycobacterium tuberculosis induces the production of tumour necrosis factor from human and murine macrophages. , 1989, Clinical and experimental immunology.

[35]  K. Khoo,et al.  Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects. , 1998, Glycobiology.