Characterization of a truncated lipoarabinomannan from the Actinomycete Turicella otitidis

Abstract

Lipoarabinomannan (LAM) lipoglycans have been characterized from a range of mycolic acid-containing actinomycetes and from the amycolate actinomycete Amycolatopsis sulphurea. To further understand the structural diversity of this family, we have characterized the lipoglycan of the otic commensal Turicella otitidis. T. otitidis LAM (TotLAM) has been determined to consist of a mannosyl phosphatidylinositol anchor unit carrying an (alpha 1-->6)-linked mannan core and substituted with terminal-arabinosyl branches. Thus, TotLAM has a novel truncated LAM structure. Using the human monocytic THP-1 cell line, it was found that TotLAM exhibited only minimal ability to induce tumor necrosis factor alpha. These findings contribute further to our understanding of actinomycete LAM diversity and allow further speculation as to the correlation between LAM structure and the immunomodulatory activities of these lipoglycans.

FIG. 1.

Purification of the lipoglycan fraction from T. otitidis. (A) Crude phenol extract was loaded onto the HIC column and washed through until fraction 11 to remove hydrophilic contaminant material. Gradient elution with increasing concentrations of propanol (15 to 65% [vol/vol]; fractions 11 to 40) was used to recover one peak of interest. Fractions from 41 onwards were eluted with 65% (vol/vol) propanol. Fractions (4 ml) were monitored by assay for carbohydrate (♦). (B) Gel filtration analysis in DOC buffer of the HIC-purified T. otitidis lipoglycan. Fractions of interest (I) were identified by SDS-PAGE analysis and pooled. Other fractions analyzed (II) did not contain lipoglycan. AU, arbitrary units.

FIG. 2.

SDS-PAGE analysis of T. otitidis and related M. bovis BCG lipoglycans. The gel was stained with a silver stain containing periodic acid. Lane 1, lipoglycan-containing fraction from T. otitidis; lane 2, LM and ManLAM from M. bovis BCG.

FIG. 3.

Expanded regions of the 1D ¹H spectrum (δ ¹H, 3.65 to 5.30) (A) and of 2D ¹H-¹³C HMQC spectrum (δ ¹H, 3.65 to 5.30; and δ ¹³C, 60 to 115) (B) in D₂O at 313 K of the TotLAM. I, t-α-Araf; II, t-α-Araf; III, 2,6-α-Manp.

FIG. 4.

CE-LIF electropherograms of monosaccharide and oligosaccharide APTS derivatives resulting from partial acid hydrolysis of TotLAM. Peaks labeled with asterisks arise from the reagent.

FIG. 5.

Structural model of TotLAM. From the molecular mass given by MALDI-MS, n could be estimated to 28.

FIG. 6.

Expanded regions of the 1D ³¹P spectrum (δ ³¹P, −2 to 7) (A) and 1D ¹H spectrum (δ ¹H, 5.3 to 3.0) and 2D ¹H-³¹P HMQC-HOHAHA spectrum (δ ³¹P, 0.7 to 4.0; δ ¹H, 5.3 to 3.0) (B) of the TotLAM in Me₂SO-d₆ at 343 K. Numerals alone correspond to the proton number of the myo-inositol units, and numerals following the letter G correspond to the proton number of the glycerol units.

FIG. 7.

TNF-α production by THP-1 cells in response to various stimuli. ManLAM, LM, ReqLAM, and TotLAM were tested at 10 (black bars) and 20 (white bars) μg/ml. ManLAM and LM were from M. bovis BCG, and ReqLAM was from R. equi. /, medium alone.