Abstract
The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear (alpha1-->6)-Manp mannan domain, which is further substituted by an (alpha1-->5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-(alpha1-->2)-Manp-(alpha1--> dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-alpha production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-alpha-inducing activity. This conclusively demonstrates that a linear (alpha1-->6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity.
Highlights
The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium
A lipoglycan with a SDS-PAGE migration similar to that of M. tuberculosis LAM was purified from T. paurometabola (Fig. 1A)
coupled to laser-induced fluorescence (CE-laser-induced fluorescence (LIF)) analysis of the total acid hydrolyzed lipoglycan showed that it contained Ara and Man, in a ratio of 1:1.7
Summary
TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-␣ when tested with either human or murine monocyte/macrophage cell lines. We established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-␣inducing activity This conclusively demonstrates that a linear (␣136)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity. Unlike the previously mentioned members the genus Tsukamurella is in its infancy, whereas the type strain, Tsukamurella paurometabola, has had a long taxonomical history, with names including Corynebacterium paurometabolum [4], Gordona aurantiaca [5], and Rhodococcus aurantiacus [6] This taxonomical puzzle was resolved, when in 1988 Collins et al [7] showed that the 16 S RNAs of R. aurantiacus and C. paurometabolum were 99% homologous.
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