Abstract
Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.
Highlights
Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity
We investigated whether KanPIM2, KanLM or KanLAM may be involved in the apoptosis-inducing activity of M. kansasii
Extensive studies performed on LAM from M. smegmatis, M. tuberculosis, or from M. bovis BCG have shown that important biological effects depend on the degree and chemical structure of capping functions and other substituents on the arabinan moiety of LAM
Summary
Strain and Culture Conditions—M. kansasii PHRI 901 was isolated from a human immunodeficiency virus-positive patient. The 31P spectra of both compounds were acquired with a spectral width of 16,233 Hz collected in 16,384 data points Both experiments were recorded using a composite pulse decoupling during acquisition using the GARP sequence at the carbon or phosphorus frequency [27]. Oligosaccharides generated by either mild hydrolysis or acetolysis were reduced using NaBH4 in 0.05 M NH4OH for 4 h and per-methylated prior analysis by GC/MS on a WCOT fused silica 30-m ϫ 0.25-mm inner diameter column (chrompack). Matrix-assisted Laser Desorption Ionization-Time of Flight MS—The molecular mass of the per-acetylated oligosaccharides was measured by MALDI-MS on a Vision 2000 time-of-flight instrument (Finnigan Mat) equipped with a 337-nm UV laser; 1 l of sample at a concentration of 100 pmol/l was mixed with an equal volume of matrix (2,5-dihydroxybenzoic acid dissolved in methanol/water, 70:30) and allowed to crystallize. The membrane was subsequently incubated with anti-rabbit antibodies conjugated to alkaline phosphatase (1:5000 dilution; Roche Applied Science)
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