Production and Purification of Phosphatidylinositol Mannosides from Mycobacterium smegmatis Biomass

Abstract

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac₁ PIM₂ and Ac₁ PIM₄ , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc² 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac₁ PIM₂ and 1.8 mg of Ac₁ PIM₄ . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc² 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc² 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.

Keywords: cell membrane; glycolipids; mycobacteria; phosphatidylinositol mannosides (PIMs); purification.

Figure 1:

Analysis of fractions eluted from the first silica gel column. A, staining with Primuline Spray Reagent; and B, staining with Orcinol Spray Reagent. Controls for Ac₁PIM₂ and Ac₁PIM₄ are also shown. The use of the controls facilitates the identification of the PIMs spots.

Figure 2:

HPTLC analysis of the fractions eluted from the HPLC column. A, staining with Orcinol Spray Reagent; and B, staining with Primuline Spray Reagent. Controls for Ac₁PIM₂ and Ac₁PIM₄ were also applied on the HPTLC plate.

Figure 3:

HPTLC analysis of fractions collected from the silica gel column used for purification of Ac₁PIM₄. A, staining with Orcinol Spray Reagent and B, staining with Primuline Spray Reagent. Fractions 12 to 16 were pooled. Controls for Ac₁PIM₂ and Ac₁PIM₄ are also shown

Figure 4:

HPTLC analysis of fractions collected from the silica gel column used for purification of Ac₁PIM₂. A, staining with Orcinol Spray Reagent and B, staining with Primuline Spray Reagent. Fractions 11 to 13 were pooled. Control for Ac₁PIM₂ + PI was also applied.

Figure 5:

Structures of Ac₁PIM₂ (A) and Ac₁PIM₄ (B) from M. smegmatis mc²155 ΔPimE. Mannose residues are labeled with numbers 1 to 4 and acyl chains are represented by R₁ and R₂ for palmitic acid and tuberculostearic acid, respectively.

Figure 6:

¹H-NMR spectra of the purified preparations of Ac₁PIM₄ (A) and Ac₁PIM₂ (B). Numbers 1 to 4 identify the anomeric protons of the mannosyl groups depicted in Figure 5; -CH₂- corresponds to methylene groups in acyl chains; α corresponds to terminal methyl groups of acyl chains; β identifies the inner methyl in tuberculostearic acid; and the asterisk corresponds to the proton bound to carbon-2 of the glycerol residue. NMR spectra were acquired at 800 MHz and 25⁰C; samples were dissolved in CDCl₃/CD₃OD/D₂O (70:30:2; v/v/v).

Figure 7:

Organization scheme for the purification of PIMs from 6 × 60 g of biomass.