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.
Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA. Harshini@lanl.gov
Harshini
H
Mukundan
Dominique N
DN
Price
Matthew
M
Goertz
Ramakrishnan
R
Parthasarathi
Gabriel A
GA
Montaño
Sandeep
S
Kumar
Matthew R
MR
Scholfield
Aaron S
AS
Anderson
S
S
Gnanakaran
Srinivas
S
Iyer
Jurgen
J
Schmidt
Basil I
BI
Swanson
D000818:Animals; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D016339:Spectrometry, Mass, Fast Atom Bombardment
D008051:Lipid Bilayers; D008070:Lipopolysaccharides; C050016:lipoarabinomannan; D008358:Mannose