A Major Cell Wall Lipopeptide of Mycobacterium avium subspecies paratuberculosis

John T. Belisle,Delphi Chatterjee,Sebabrata Mahapatra,Sukantha Chandrasekaran,Michael R. McNeil,Julia M. Inamine,Torsten M. Eckstein,Christopher D. Rithner,Philip W. Ryan

doi:10.1074/jbc.m512465200

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne disease in cattle and other ruminants, is proposed to be at least one of the causes of Crohn disease in humans. MAP and Mycobacterium avium subspecies avium, a closely related opportunistic environmental bacterium, share 95% of their genes and exhibit homologies of more than 99% between these genes. The identification of molecules specific for MAP is essential for understanding its pathogenicity and for development of useful diagnostic tools. The application of gas chromatography, mass spectrometry, and nuclear magnetic resonance led to the structural identification of a major cell wall lipopeptide of MAP, termed Para-LP-01, defined as C20 fatty acyl-D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester. Variations of this lipopeptide with different fatty acyl moieties (C16 fatty acyl through C17, C18, C19, C21 to C22) were also identified. Besides the specificity of this lipopeptide for MAP, the presence of an N-Me-L-valine represents the first reported N-methylated amino acid within an immunogenic lipopeptide of mycobacteria. Sera from animals with Johne disease, but not sera from uninfected cattle, reacted with this lipopeptide, indicating potential biological importance.

Highlights

One of the causes of Crohn disease in humans (2)
Lipidomics is a promising area of study because members of the family Mycobacteriaceae contain large numbers of complex lipids in their cell wall, and we recently identified several cell envelope and culture filtrate lipids present in Mycobacterium avium subspecies paratuberculosis (MAP) strain K-10 but absent from MAA strain 2151.3 Here we report the chemical structure and seroreactivity of Para-LP-01, a major cell wall-associated MAPspecific lipopeptide
A lipid designated Para-LP-01 was recently identified in a lipidomic analysis of MAP strain K-10 in comparison to MAA strain 2151.3 It was selected for further characterization because it is a major cell wall-associated lipid, was well isolated when the total lipids were separated by two-dimensional thin layer chromatography (TLC) using an apolar solvent system, and was present only in MAP (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Chemical Reagents—All chemical reagents were of the highest grade from Sigma unless otherwise specified. Extraction of Total Lipids and Isolation of Lipopeptide Para-LP-01— Total lipids were extracted from lyophilized cells with chloroform/ methanol (2:1) (30 ml/g dried cells) at 55 °C for 3 h. Partial Hydrolysis of Lipopeptide Para-LP-01—Hydrolysis of the lipid was performed with 6 N HCl at 110 °C for 15, 40, 90, and 180 min. MALDI-TOF—Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was performed in the Macromolecular Resources facility, Colorado State University, with an Ultraflex MALDI/TOF/TOF (Bruker Daltonics, Billerica, MA) with 2,5 dihydrobenzoic acid and NaI as a matrix. Lipid Para-LP-01 and its deuteropermethylated derivative were suspended in methanol, 0.1% trifluoric acid at a concentration of 1 mg/ml. Wells were washed five times with 200 ␮l of blocking buffer, and 100 ␮l of the secondary antibody (sheep anti-bovine IgG coupled to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX)) diluted 1:2000 was added and incubated for 2 h. After 5 min the reaction was stopped with 100 ␮l of 2 N sulfuric acid and the A450 was determined with a plate reader model 680 (Bio-Rad)

RESULTS

Fatty acyl

DISCUSSION