Purification of phenolic glycolipid from mycobacterium tuberculosis

Vigil, Scott Brandon, author
Hesser, Danny C., author
Dobos, Karen, author
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Mycobacterium tuberculosis (Mtb) is a very successful human pathogen, infecting one-third of the world's population. Although originally thought to have little to no genetic variation, Mtb has been recently shown to be distinguished to clades based on geographical distribution. Further, members of specific clades (and/or subclades) have been shown to possess differences in virulence. Several clades from Asia, Africa, and India were found to express phenolic glycolipid (PGL). PGL is absent in many North American and European clades due to gene deletion. The West Beijing strains of Mtb, HN878, W4, and W451, contain PGL as part of their cellular envelope. These strains demonstrated reduced production of important immune-mediating cytokines including tumor necrosis factor alpha and interleukin 12. Subsequent evidence demonstrated this was due to PGL. Thus, it is believed that PGL works in concert with other factors to enhance the virulence of Mtb. Purification of PGL from these strains will permit further studies on the molecular interactions of PGL during infection with Mtb. In our laboratory, we attempted to isolate PGL from Mtb strain HN878. Initially, a lipid extraction was performed with 1:2 chloroform: methanol. This extract was then analyzed via preparatory and analytical Thin-Layer Chromatography (TLC) plates with a 90:10 and 95:5 chloroform: methanol developing solution. These TLC plates were then sprayed with CuSO4 and α-naphthol for sugar and carbon compound detection. Ultra-violet light was used to view and outline what is believed to be the major PGL band. This band was scraped off the TLC and will be extracted with diethyl ether to purify PGL. Once purified, we will perform studies with naïve and Mtb-infected macrophages to assess the role of PGL in the pro-inflammatory response during infection.
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thin-layer chromatography
Mycobacterium tuberculosis
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