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 Ac₁PIM₂ and Ac₁PIM₄, which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc²155 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 Ac₁PIM₂ and 1.8 mg of Ac₁PIM₄. This is the first time the purification of phosphatidylinositol tetramannoside is reported.

Basic Protocol 1:

Growth of M. smegmatis mc²155 ΔPimE

Basic Protocol 2:

Extraction of lipids from M. smegmatis mc²155 ΔPimE

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

INTRODUCTION:

Tuberculosis (TB) is the top cause of death by infection. Recently, the World Health Organization set 2035 as the target deadline to reduce by 95% the number of human deaths caused by TB (Floyd et al., 2018). Since long ago, it is known that this disease is caused by the pathogen Mycobacterium tuberculosis, but the therapeutics currently available to fight the infection have many limitations. Therefore, it is urgent to direct research efforts to the development of new drugs (Tiberi et al., 2018). Phosphatidylinositol mannosides (PIMs) together with the precursor, phosphatidylinositol (PI), contribute up to 56% of phospholipids in mycobacteria cell wall and play a crucial role in permeability and pathogenicity (Dulberger, Rubin &, Boutte, 2020; Kalscheuer et al., 2019). These features, along with the absence of PIMs in humans, make the enzymes responsible for their synthesis attractive drug targets to treat TB. The PIMs components have in their core a phosphatidylinositol moiety, which carries 1 to 6 mannose residues and up to 4 acyl groups. The biosynthetic pathway from PIM₁ to PIM₆ is proposed to comprise six steps; the mannosyltransferases catalyzing the first two steps (PI to PIM₁, PimA; and PIM₁ to PIM₂, PimB) are known. In addition, the genes encoding the acyltransferase PatA, which acylates PIM₂ to Ac₁PIM₂, and PimE, the enzyme that catalyzes the conversion of Ac₁PIM₄ into Ac₁PIM₅, have been discovered (Korduláková et al., 2002, 2003; Morita et al., 2006; Schaeffer et al., 1999). However, the genes encoding the enzymes PimC, PimD and PimG, producing Ac₁PIM₃, Ac₁PIM₄, and Ac₁PIM₆, respectively, and the second acyltransferase remain elusive. The biochemical characterization of the enzymes involved in the biosynthesis of PIMs is not trivial since PIMs are not commercially available and considerable amounts of substrates are required. Consequently, chemical synthesis (Boonyarattanakalin, Liu, Michieletti, Lepenies, & Seeberger, 2008) or the purification of PIMs from mycobacterial biomass are the only strategies to obtain pure PIMs. Procedures to extract and purify PIMs (i.e., the different acylated forms of PIM₂ and PIM₆), from the cell wall of mycobacteria have been described in the literature (Gilleron et al., 2001; Khoo et al., 1995; Pirson et al., 2015; Rahlwes, Puffal, & Morita, 2019). Herein, protocols for production, purification, and quantification of Ac₁PIM₂ and Ac₁PIM₄, from M. smegmatis mc²155 ΔPimE are proposed. Basic protocol 1 describes the growth of M. smegmatis mc²155 ΔPimE, which accumulates Ac₁PIM₂ and Ac₁PIM₄. Basic protocol 2 describes an efficient and reliable method for extraction of phospholipids from the cellular membrane of M. smegmatis mc²155 ΔPimE. Basic protocol 3 refers to treatment of the crude lipid extract for removal of major contaminants. Basic protocol 4 includes a series of chromatographic procedures for isolation of the target PIMs, i.e., Ac₁PIM₂ and Ac₁PIM₄. Basic Protocol 5 considers two methods for quantification of the purified compounds. These protocols were optimized in our laboratory to allow for medium scale purification of PIMs. Importantly, the final product was free from the contaminating precursor, PI. Detailed information on starting biomass and final yield of PIMs species is provided. This is the first report on the purification of a phosphatidylinositol tetramannoside.

CAUTION 1:

M. smegmatis mc²155 ΔPimE is a Biosafety Level 1 (BSL-1) organism. Such organisms are not known to consistently cause disease in healthy adult humans and are of minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these organisms.

CAUTION 2:

Extraction, purification, TLC analysis and silica gel chromatography steps should be performed in a fume hood.

BASIC PROTOCOL 1

Basic protocol title:

Growth of M. smegmatis mc²155 ΔPimE.

Introductory paragraph:

We used biomass of Mycobacterium smegmatis mc²155 ΔPimE to purify PIMs (i.e., Ac₁PIM₂ and Ac₁PIM₄). Mycobacterium tuberculosis is a virulent strain and biosafety levels 2 or 3 laboratories are required to work with this organism. Therefore, Mycobacterium smegmatis is generally used as a model organism to study tuberculosis. We selected Mycobacterium smegmatis mc²155 ΔPimE as this strain lacks the gene encoding the enzyme PimE that catalyzes the conversion of Ac₁PIM₄ into Ac₁PIM₅, and, therefore, accumulates mainly Ac₁PIM₂ and Ac₁PIM₄ (Morita et al., 2006).

Cells were routinely grown in 2 L flasks at 37°C in the presence of oxygen. Growth in Middlebrook® 7H9 medium was compared with growth in LB medium to determine which method led to higher amounts of PIMs. Although the cell content of PIMs was similar, the LB medium was selected since it resulted in higher yields of biomass: circa 8 g of wet biomass per liter of LB medium compared to 6 g in Middlebrook® 7H9 medium. In addition, we examined the cultivation in LB medium of M. smegmatis mc²155 ΔPimE in a 60 L fermentation vessel, under three aeration conditions, to evaluate PIMs accumulation. The air flow rate was kept at either 0, 3, or 5 L/min. According to our results, the presence of oxygen is essential for the accumulation of PIMs. However, air flow rate at 3 or 5 L/min led to no significant differences in the amounts of PIMs present in the membrane. From 60 g of wet cells grown with an air flow rate of 3 L/min and following the protocol described here, we isolated 1.0 mg of Ac₁PIM₄, compared to 1.8 mg when cells were grown in flasks. Therefore, we recommend using flask cultures and LB medium.

Materials:

Glycerol stock solution of Mycobacterium smegmatis mc²155 ΔPimE (see Support Protocol 1).The strain was obtained from the team of Dr. Taroh Kinoshita, Osaka University, Japan. The construction of the strain was reported by Morita et al., 2006.

Middlebrook® 7H9 liquid medium (see recipe in Reagents and Solutions)

Kanamycin solution 50 mg/mL (see recipe in Reagents and Solutions)

LB medium (see recipe in Reagents and Solutions)

50 mM Tris-HCl pH 7.0 (see recipe in Reagents and Solutions)

Tween-80 (Sigma-Aldrich, cat. no. P4780–500ML)

Autoclave

50 mL sterile Erlenmeyer flask

37°C shaking incubator (Aralab Agitorb 200, or equivalent)

2 L sterile Erlenmeyer flask

500 mL centrifuge tubes

50 mL Falcon® tubes

Refrigerated centrifuge (capable of centrifuging at 10 000 × g and 4°C, Eppendorf 5804 R, or equivalent)

Spectrophotometer (visible light range; Beckman Coulter DU-800, or equivalent)

Cuvettes

Protocol steps with step annotations:

• 1Glycerol stock cultures were prepared as described in supporting protocols and frozen at −80°C (see Support Protocol).
• 2Transfer 500 μL of a glycerol stock culture to a 50 mL sterile Erlenmeyer flask containing 20 mL of sterile Middlebrook® 7H9 liquid medium supplemented with kanamycin to a final concentration of 40 μg/mL.
• 3Incubate the culture for 24 hours at 37°C and 180 rpm.
• 4Transfer 20 mL of the resulting cell culture to a 2 L sterile Erlenmeyer flask containing one liter of LB medium supplemented with 40 μg/mL of kanamycin. Measurement of the optical density at 600 nm was not used since cells aggregate during growth.
• 5Incubate for 48 hours at 37°C and 180 rpm.

Cells should reach the stationary phase (see Figure 1, Supplemental Materials). As the content of PIMs appears to be independent of the growth phase (our unpublished data), harvesting cells during the stationary phase leads to the recovery of higher biomass when compared to the exponential phase.

• 6Transfer the culture into the centrifugation tubes and centrifuge the cell suspension 30 minutes at 6 000 × g, 4°C.
• 7Discard the supernatant and recover the cell pellet into a Falcon® tube.
• 8Suspend the cell pellet in circa 20 mL of 50 mM Tris-HCl pH 7.0.
• 9Centrifuge 30 minutes at 4 700 × g, 4°C, to remove residual culture medium.
• 10Discard the supernatant solution, recover the cell pellet (around 8 g of wet biomass per liter of LB medium) and freeze at −80°C until further use.

BASIC PROTOCOL 2

Basic protocol title:

Extraction of lipids from M. smegmatis mc²155 ΔPimE.

Introductory paragraph:

PIMs present in the cellular membrane must be extracted from the biomass of M. smegmatis mc²155 ΔPimE. The method described in this section was adapted from Bligh & Dyer (1959) for total lipid extraction; it relies on the use of chloroform/methanol, which allows for solubilization of nonpolar molecules as well as some amphiphilic molecules, such as PIMs.

Materials:

CHCl₃/CH₃OH (1:1; v/v) [CHCl₃ (Honeywell, cat. no. 34854–2.5L), CH₃OH (Honeywell, cat. no. 34966–2.5L)]

Frozen cells of M. smegmatis mc²155 ΔPimE

Schott flasks

Magnetic stirring plate

Magnetic stirring bar

Glass centrifuge flasks (Sorvall Instruments DuPont, cat. no. 03163)

Refrigerated centrifuge (capable of centrifuging at 10 000 × g and 4°C, Beckman Avanti J-26 XPI, or equivalent)

Glass Pasteur pipettes

Nitrogen (N₂) stream

Protocol steps with step annotations:

• 1Add approximately 60 g of frozen cells to 100 mL of CHCl₃/CH₃OH (1:1; v/v) in a 250 mL Schott flask.

A lower quantity of frozen cells can be used if the volume of CHCl₃/CH₃OH (1:1; v/v) is adjusted to the new sample size. Make sure the cells do not thaw before adding the chloroform/methanol mixture.

• 2Incubate for 2 hours at room temperature with vigorous agitation using a magnetic stirring bar.
• 3Transfer the mixture into glass centrifuge flasks and centrifuge 30 minutes at 6 000 × g, 4°C.

After centrifugation, three layers are apparent: the top aqueous layer, the middle layer containing mostly cell debris and the bottom, yellow, chloroform layer containing the desired membrane lipids.

• 4Recover the bottom layer and discard the remaining ones.
• 5Dry completely the chloroform solution and methanol traces by using a N₂ stream.

This drying step takes around 1 hour, but it can be interrupted and the remaining solution stored at −20°C overnight. This step is crucial for the success of the next purification step. An efficient drying procedure ensures that the right proportion of chloroform is used and removes traces of methanol that can disrupt phase separation in the subsequent purification step.

BASIC PROTOCOL 3

Basic protocol title:

Treatment of the lipid extract for isolation of phospholipids.

Introductory paragraph:

This method involves a biphasic partitioning between chloroform and water: PIMs remain in the chloroform layer while more hydrophilic molecules are extracted by water. Next, hot acetone is used to solubilize contaminants. This results in an effective purification step since PIMs are insoluble in acetone.

Materials:

CHCl₃ (Honeywell, cat. no. 34854–2.5L)

Bi-distilled H₂O

Acetone (Merck Supelco, cat. no. 1.00014.2511)

CHCl₃/CH₃OH/H₂O (70:30:2; v/v/v) [CH₃OH (Honeywell, cat. no. 34966–2.5L)]

CHCl₃/CH₃OH/25% NH₃/H₂O (65:25:0.5:4; v/v/v/v) [25% NH₃ (Merck, cat. no. 1.05432.2500)]

CHCl₃/CH₃OH/25% NH₃/1M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v) [NH₄CH₃COO (Merck, cat. no. A7262–500G)]

Primuline Spray Reagent (see recipe in Reagents and Solutions)

Orcinol Spray Reagent (see recipe in Reagents and Solutions)

CHCl₃/CH₃OH (1:1; v/v)

Separatory funnel

Round bottom flask

Rotary evaporator with gas extraction connected to the fume hood

Glass centrifuge flasks (Sorvall Instruments DuPont, cat. no. 03163)

Refrigerated centrifuge (capable of centrifuging at 10 000 × g and 4°C, Beckman Avanti J-26 XPI, or equivalent)

50 mL Falcon® tubes

PTLC Silica gel 60 (glass plates 20 × 20 cm; Merck, cat. no. 1.05717.0001)

HPTLC silica gel 60 plates (glass plates 20 × 10 cm, Merck, cat. no. 1.05641.0001)

TLC chamber (22 × 21 × 10 cm)

Hair dryer

Ultraviolet lamp (Vilber lourmat, or equivalent)

Heat gun (Varitemp, max. temperature 400°C, or equivalent)

Nitrogen (N₂) stream

Protocol steps with step annotations:

• 1Solubilize completely the residue from step 5 (Basic protocol 2) with 100 mL of CHCl₃.
• 2Transfer the solubilized residue to a separatory funnel.
• 3Add 100 mL of bi-distilled H₂O to the separatory funnel, place the stopper, and mix vigorously.
• 4Release the internal pressure by inverting the separatory funnel and opening the valve; mix again. Repeat this procedure until no pressure is released upon opening the valve.
• 5Let the aqueous and chloroform layers separate overnight.

Due to the presence of amphiphilic molecules in the extract, separation of the phases is not immediate. If an emulsion is observed after 16 hours, add extra 25 mL fractions of water, mix vigorously after each addition, and allow 60 minutes for layer separation.

• 6Collect the bottom, yellow layer into a round bottom flask.
• 7Dry the collected layer by rotary evaporation.
• 8Once fully dried, add 100 mL of hot acetone (at approximately 50°C) and swirl the round bottom flask gently. The acetone should turn yellow, and a precipitate should be observed.

This is a purification step as PIMs are insoluble in acetone.

• 9Leave at 4°C overnight.
• 10Transfer the acetone solution, including the precipitate, into glass centrifuge tubes. Centrifuge 30 minutes at 7 000 × g, 4°C, discard the supernatant and solubilize the pellets with the minimal volume of CHCl₃/CH₃OH/H₂O (70:30:2; v/v/v). In parallel, solubilize the precipitate present in the round bottom flask with the minimal volume of the same solvent mixture. Combine the solubilized pellets into a glass flask, dry the solvent mixture by using a N₂ stream, and solubilize the residue with the same solvent mixture.

Preparation of PIM standards

• 11Apply 30 μL of the lipid extract (Basic protocol 3, step 10) on each lane (a total of 19 lanes) of a PTLC Silica gel 60 plate (the extracts should be spotted allowing for 1 cm in between spots and 1.5 cm from the bottom end of the plate).
• 12Develop the PTLC plate in a TLC chamber previously equilibrated with CHCl₃/CH₃OH/25% NH₄OH/H₂O (65:25:0.5:4; v/v/v/v). Allow the solvent front to reach the top. Remove the plate, dry with a hair dryer, and allow the plate to reach the room temperature in the fume hood. Develop the same plate again under the conditions described above (i.e., second run).

Add the developing solvent to the chamber until the solvent layer reaches 7 – 10 mm height. The gas atmosphere in the chamber must be in equilibrium with the solvent. Place a sheet of filter paper surrounding the walls of the chamber to accelerate equilibration and create a homogeneous atmosphere. Apply a little of silicon grease on the lid to ensure a tight sealing.

• 13Dry the plate, cut out the first lane (1.5 cm per 20 cm) and reveal the spots by spraying uniformly with the Primuline Spray Reagent followed by drying with a hair dryer. Expose to UV light and take a photograph. Next, spray uniformly the same plate with the Orcinol Spray Reagent. The staining recipes are described in Reagents and Solutions. Heat the plate by using a heat gun until spots are observed. Take a photograph immediately. Be aware that colors fade with time.

Primuline binds non-covalently to the fatty acid chains of lipids, fluorescing under UV light. This staining is non-destructive, hence the spots can be scraped and analyzed by mass spectrometry. With the Orcinol Spray Reagent, a chemical reaction occurs where the sugars (hexoses) are converted to hydroxymethylfurfural by reacting with 5-methylresorcinol. It gives a brown color to the sugar-moiety containing compounds; the method degrades the organic compounds. By using these two staining methods, we can distinguish PIMs from contaminants lacking hexose moieties (e.g., phospholipids, PI).

• 14Using the stained lane as guide, mark horizontal lines with a pencil at the level of the Ac₁PIM₂ and Ac₁PIM₄ spots (see Figure 2, Supplemental Materials); scrape off the silica gel areas containing the desired compounds. Recover the PIMs from the silica with CHCl₃/CH₃OH (1:1; v/v) and centrifuge to remove the silica. Confirm the compound identification of each spot by mass spectrometry. Use these solutions as standards in the subsequent TLC plates.

Analysis of the lipid extract

• 15Apply 1 to 2.5 μL of lipid extract (Basic protocol 3, step 10) on a HPTLC silica gel 60 plate; also apply the obtained PIM standards.
• 16Develop the HPTLC plate in a TLC chamber previously equilibrated with CHCl₃/CH₃OH/25% NH₃/1 M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v). Allow the solvent front to reach the top (takes about 35 min). Remove the plate, dry with a hair dryer, and visualize the PIMs as described in step 13 (Basic Protocol 3).
• 17Analyze the results and look for the presence of the desired PIMs.

BASIC PROTOCOL 4

Basic protocol title:

Isolation of phosphatidylinositol mannosides.

Introductory paragraph:

To isolate PIMs from remaining contaminants, several chromatographic steps are required. On the first silica gel column, cardiolipin and other unidentified lipids, are eluted by the first mobile phase, which contains a high proportion of chloroform (Pirson et al., 2015). The second mobile phase elutes primarily PIMs.

The second chromatographic step relies on HPLC to separate Ac₁PIM₂ from Ac₁PIM₄. A gradient with decreasing polarity results in the elution of Ac₁PIM₄ (higher mannose content corresponds to higher affinity to polar solvents), followed by Ac₁PIM₂. Generally, PI is the major contamination of the Ac₁PIM₄ preparation. On the other hand, an unknown lipid is present in the Ac₁PIM₂ fractions. Therefore, a third purification step on a silica gel column is needed. A single mobile phase is used to separate compounds according to their polarity: more hydrophobic components elute first, while PIMs elute in the later fractions. Purified fractions of Ac₁PIM₂ and Ac₁PIM₄ contain ammonium acetate that can be removed by several cycles of freeze-drying. Usually, three steps of lyophilization are needed for total removal.

Materials:

Silica gel chromatography for removal of major contaminants

CHCl₃ (Honeywell, cat. no. 34854–2.5L)

CHCl₃/CH₃OH (8:2; v/v) [CH₃OH (Honeywell, cat. no. 34966–2.5L)]

CHCl₃/CH₃OH (1:1; v/v)

CHCl₃/CH₃OH/25% NH₄OH /1 M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v) [25% NH₄OH (Merck, cat. no. 1.05432.2500), NH₄CH₃COO (Merck, cat. no. A7262–500G)]

Primuline Spray Reagent (see recipe in Reagents and Solutions)

Orcinol Spray Reagent (see recipe in Reagents and Solutions)

Silica gel 60 (0.040–0.063 mm) (230–400 mesh ASTM, Merck, Cat. no. 1.09385.1000)

Glass column 24 cm × 2.5 cm (inner diameter) fitted with a glass porous plate

PTFE membrane filter, 0.2 μm pore size (Whatman, Cat. No. 6720–5002)

20 mL glass vials

HPTLC silica gel 60 plates (glass plates 20 × 10 cm, Merck, cat. no. 1.05641.0001)

TLC chamber (22 × 21 × 10 cm)

Hair dryer

Ultraviolet lamp (Vilber lourmat, or equivalent)

Heat gun (Varitemp, max. temperature 400°C, or equivalent)

Nitrogen (N₂) stream

Analytical balance

High Performance Liquid Chromatography for isolation of phosphatidylinositol mannosides

CHCl₃/CH₃OH/H₂O (70:30:2; v/v/v) [CHCl₃ (Honeywell, cat. no. 34854–2.5L), CH₃OH (Honeywell, cat. no. 34966–2.5L)]

Eluent A, CHCl₃/CH₃OH/H₂O (240:1140:620; v/v/v) with 10 mM NH₄CH₃COO (see recipe in Reagents and Solutions)

Eluent B, CHCl₃/CH₃OH (7:3; v/v) with 50 mM NH₄CH₃COO (see recipe in Reagents and Solutions)

CHCl₃/CH₃OH/25% NH₄OH/1 M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v) [25% NH₄OH (Merck, cat. no. 1.05432.2500), NH₄CH₃COO (Merck, cat. no. A7262–500G)]

Primuline Spray Reagent (see recipe in Reagents and Solutions)

Orcinol Spray Reagent (see recipe in Reagents and Solutions)

PTFE membrane filter, 0.2 μm pore size (Whatman, Cat. No. 6720–5002)

Coral C18 global 100 Å 5 μm 250 × 21.2 mm semi-preparative HPLC column (imChem, cat. no. 250212–5-CO-C18G)

High performance liquid chromatography equipment (Waters 2535, or equivalent)

20 mL glass vials

HPTLC silica gel 60 plate (glass plates 20 × 10 cm, Merck, cat. no. 1.05641.0001)

TLC chamber (22 × 21 × 10 cm)

Hair dryer

Ultraviolet lamp (Vilber lourmat, or equivalent)

Heat gun (Varitemp, max. temperature 400°C, or equivalent)

Rotary evaporator or Nitrogen (N₂) stream

Analytical balance

Silica gel chromatography for purification of Ac₁PIM₂ and Ac₁PIM₄

CHCl₃ (Honeywell, cat. no. 34854–2.5L)

CHCl₃/CH₃OH/H₂O (70:30:2; v/v/v) [CH₃OH (Honeywell, cat. no. 34966–2.5L)]

CHCl₃/CH₃OH/25% NH₃ /1 M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v) [25% NH₃ (Merck, cat. no. 1.05432.2500), NH₄CH₃COO (Merck, cat. no. A7262–500G)]

Primuline Spray Reagent (see recipe in Reagents and Solutions)

Orcinol Spray Reagent (see recipe in Reagents and Solutions)

Silica gel 60 (0.040–0.063 mm) (230–400 mesh ASTM, Merck, Cat.no. 1.09385.1000)

Glass column (50 cm × 0.8 cm inner diameter) with a glass porous plate

1.5 mL microcentrifuge tubes

HPTLC silica gel 60 plates (Glass plates 20 × 10 cm, Merck, cat. no. 1.05641.0001)

TLC chamber (22 × 21 × 10 cm)

Hair dryer

Ultraviolet lamp (Vilber lourmat, or equivalent)

Heat gun (Varitemp, max. temperature 400°C, or equivalent)

Nitrogen (N₂) stream

Removal of ammonium acetate from the purified AcPIMs preparations

Milli-Q® H₂O

Liquid nitrogen

CDCl₃/CD₃OD/D₂O (70:30:2; v/v/v) [CDCl₃ (Eurisotop, cat. no. 865–49-6), CD₃OD (Eurisotop, cat. no. 811–98-3), D₂O (Cortecnet, cat. no. MBB50594V)]

Lyophilizer

NMR spectrometer (Bruker Avance 500 II, or equivalent)

Protocol steps with step annotations:

Silica gel chromatography for removal of major contaminants

• 1Weigh 26 g of silica gel 60 and transfer it to a 250 mL Schott bottle; add 90 mL of chloroform.

Always use freshly prepared and new resin.

• 2Allow the silica gel to equilibrate overnight.
• 3Take a glass column (24 cm length and 2.5 cm inner diameter) fitted with a glass porous plate at the bottom, fill it with the silica gel resin, and allow packing for around 20 min.

The final height of the resin in the column should be around 13–14 cm. Never let the top of the resin dry.

• 4Solubilize the residue resulting from step 10 (Basic Protocol 3) in 20 mL of CHCl₃, filter through a 0.2 μm pore size PTFE membrane, and load the extract onto the column.

The column has sufficient capacity to resolve up to 1.4 g of lipid extract (maximum sample volume of 20 mL) in a single run.

• 5Once the whole sample volume enters the resin, add 200 mL of CHCl₃/CH₃OH (8:2; v/v). The sample upload and chromatographic elution were carried out by gravity.
• 6Discard the first 200 mL of eluate.
• 7Add 240 mL of CHCl₃/CH₃OH (1:1; v/v) and collect 12 fractions of 20 mL each.

Analysis and treatment of the fractions collected

• 8Analyze the fractions eluted with CHCl₃/CH₃OH (1:1; v/v) on HPTLC by applying 2.5 μL of each fraction on a HPTLC plate.
• 9Develop the HPTLC plate in a TLC chamber prepared with CHCl₃/CH₃OH/25% NH₃/1M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v).
• 10Treat the plate using the Primuline and Orcinol Spray Reagents as described above (step 13, Basic Protocol 3).
• 11Analyze the results and pool together the fractions containing PIMs.

Figure 1 shows a representative HPTLC obtained with fractions collected during step 7. Fractions 4 to 12 were pooled together. The major components are Ac₁PIM₂, Ac₁PIM₄ and PI. Note that PI is not detected with the orcinol method. This step is very efficient for the removal of other contaminants.

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.

• 12Dry the pooled fraction with a N₂ stream and weigh.

High Performance Liquid Chromatography for isolation of phosphatidylinositol mannosides

• 13Solubilize the residue resulting from step 12 (Basic Protocol 4) in 4 mL of CHCl₃/CH₃OH/H₂O (70:30:2; v/v/v)

If small particles are observed in suspension, filter the sample through a PTFE membrane filter with 0.2 μm pore size. Up to 200 mg of residue can be injected in the HPLC column.

• 14After injection, HPLC run starts with the initial mobile phase corresponding to 85% of Eluent A and 15% of Eluent B. This mobile phase is provided during the initial 9 minutes of the run. From minute 10 to minute 60, the percentage of Eluent B is increased gradually from 15 to 20%. Finally, from minute 61 to minute 113, the percentage of Eluent B should be increased from 20 to 100%.
• 15Keep the flow rate at 10 mL/min throughout the run.
• 16Collect 30 fractions of 20 mL each into glass vials, starting at the time of injection (from 1 to 60 minutes).
• 17Continue collecting into a single flask until the percentage of B reaches 25% to make sure no desired compound is lost.

Analysis of the fractions collected

• 18Analyze the collected samples through HPTLC by applying 10 μL on each lane.
• 19Develop the HPTLC plate in a TLC chamber and stain with Primuline and Orcinol Spray Reagents, as described above.
• 20Pool fractions containing the same PIM, i.e., Ac₁PIM₂ and Ac₁PIM₄. Use a N₂ stream or a rotary evaporator to dry the samples and weigh the residues.

Figure 2 shows a representative TLC. Fractions 7 to 18 and 22 to 30 were pooled together to isolate Ac1PIM4 and Ac1PIM2, respectively. At this point, an aliquot of the pooled fractions was analyzed by ESI-MS and MS/MS in the negative mode. Ions at m/z of 1413.8 and m/z 1737.9 were identified in the samples containing Ac1PIM2 and Ac1PIM4, respectively, confirming the nature of the isolated compounds.

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.

Silica gel chromatography for purification of Ac₁PIM₂ and Ac₁PIM₄

• 21Add 45 mL of chloroform to 15 g of silica gel 60 in a Schott bottle.

Always use a new batch of resin.

• 22Allow the silica gel resin to equilibrate overnight.
• 23Pack a glass column (0.8 mm inner diameter, 50 cm height) with the silica gel resin.

The final height of the resin should be around 36–38 cm.

• 24Solubilize separately the residues resulting from step 20 (Basic Protocol 4) in 0.5 mL of CHCl₃/CH₃OH/H₂O (70:30:2; v/v/v).
• 25When the column is packed, apply 25 mL of CHCl₃/CH₃OH/25% NH₃/1 M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v).
• 26Load the Ac₁PIM₄–containing sample onto the column.
• 27When the sample is fully adsorbed, add 25 mL of CHCl₃/CH₃OH/25% NH₃/1M NH₄CH₃COO in water/H₂O (180:140:9:9:23; v/v/v/v/v)
• 28Discard the initial 5 mL eluate.
• 29Collect 24 fractions of 0.5 mL each into 1.5 mL disposable microcentrifuge tubes.

If the samples are intended for mass spectrometry analysis, use glass tubes instead of microcentrifuge tubes since plastic may release trace compounds in the presence of organic solvents.

Analysis of the fractions collected

• 30Analyze the fractions through HPTLC as described above by applying 2.5 μL of each fraction on each lane (Figure 3).
• 31Develop the HPTLC plate in a TLC chamber and stain with Primuline and Orcinol Spray Reagents, as described above.
• 32Pool together the fractions containing pure Ac₁PIM₄.
• 33Dry under a N₂ stream, solubilize the pooled fractions with 1–2 mL of CHCl₃/CH₃OH/H₂O (70:30:2; v/v/v) and analyze the fractions through HPTLC as described above by applying 1 μL of each pooled fraction. If the pooled fractions are not satisfactorily free of contaminants, repeat the procedure from step 21 (Basic Protocol 4). Dry the pure compound under a N₂ stream and store at -20°C until further use.
• 34For purification of the Ac₁PIM₂ sample obtained in step 20 (Basic protocol 4), follow the protocol proposed for Ac₁PIM₄ (steps 25 to 33, Basic protocol 4). HPTLC analysis of the eluates from the silica gel column is illustrated in Figure 4.

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.

Removal of ammonium acetate from the purified AcPIMs preparations

• 35Add 10 mL of Milli-Q® H₂O to the purified Ac₁PIM samples.
• 36Mix gently to dissolve ammonium acetate.

PIMs will not dissolve in water; a slightly turbid suspension will be observed.

• 37Freeze the sample in liquid nitrogen and lyophilize overnight. Repeat two or three times, the steps 35 to 37 for total removal of ammonium acetate.

Wear spectacles for eye protection or face screens.

• 38Solubilize the sample in 600 μL of CDCl₃/CD₃OD/D₂O (70:30:2; v/v/v) and analyze the sample through ¹H-NMR to assess the ammonium acetate content.

A singlet resonance at 1.9 ppm denotes the presence of ammonium acetate.

• 39If ammonium acetate remains, repeat the procedure from step 35 (Basic Protocol 4). When the sample is free of ammonium acetate, quantification of Ac₁PIMs can be performed either by proton NMR or the total phosphorus assay (Basic Protocol 5).

BASIC PROTOCOL 5

Basic protocol title:

Quantification of phosphatidylinositol mannosides.

Introductory paragraph:

Two quantification methods are described here: ¹H-NMR spectroscopy and assay for total phosphorus content. By using ¹H-NMR it is possible to assess both the purity and quantity of the desired PIMs. Moreover, this method provides information about the nature of contaminants and has the advantage of not destroying the sample but requires access to at least 500 MHz spectrometers. A capillary containing a solution of formate with known concentration is inserted in the NMR tube to be used as a concentration standard. Alternatively, quantification can be performed through determination of the total phosphorus content. This assay does not require expensive equipment but will lead to erroneous high values when the sample contains phosphorus components other than the target PIMs. In other words, a precise quantification can only be achieved with a pure sample of the desired compound. Briefly, this method implies destruction of the organic matter by acid treatment and complexation of the released inorganic phosphate with molybdate. Upon reduction with ascorbic acid, a blue color develops. The phosphorus content is determined from measurements of absorbance at 797 nm and comparison with a standard calibration curve.

Materials:

Quantification through ¹H-NMR

CDCl₃/CD₃OD/D₂O (70:30:2; v/v/v) [CDCl₃ (Eurisotop, cat. no. 865–49-6), CD₃OD (Eurisotop, cat. no. 811–98-3), D₂O (Cortecnet, cat. no. MBB50594V)]

5mm NMR tubes (Sigma-Aldrich, Norell, cat. no. NOR508UP7–5EA)

Capillary containing the formate standard [Sodium formate (Sigma-Aldrich, cat. no. S2140–100G)]

NMR spectrometer (Bruker Avance 500 II, or a higher magnetic field spectrometer)

NMR data analysis software (Bruker Topspin, or equivalent)

Total phosphorus quantification

Chromosulfuric acid solution (Merck, cat. no. 102499)

560 μg/mL Na₂HPO₄ · 2H₂O in H₂O (Merck, cat. no. 934 K12749180)

Milli-Q® H₂O

70% (v/v) HClO₄ (Merck, cat. no. 1005191001)

2.5% (w/v) (NH₄)₆Mo₇O₂₄ · 4H₂O in H₂O (Merck, cat. no. 431346–50G)

10% (w/v) Ascorbic acid in H₂O (Merck, cat. no. 1.00127.0100)

1.5 mL Microcentrifuge tubes

Silicon oil (Chem-Lab, cat. no. CL00.1918.1000) bath

Heating plate (must reach 180°C)

Hungate tubes (Bellco Glass, cat. no. BELC2047–16125)

Glass marbles

Boiling water bath

Cold water bath

96-well plates

96-well plate reader

Protocol steps with step annotations:

Quantification through ¹H-NMR

The molecular structures of Ac₁PIM₂ and Ac₁PIM₄ are depicted in Figure 5. The purity and quantity of the purified preparation of Ac₁PIMs can be easily assessed by ¹H-NMR, a non-destructive methodology. The analysis of the anomeric region of the spectrum (5.1 to 4.6 ppm) is particularly useful (Figure 6). Also, it provides a straightforward way to check whether PIMs species are contaminated with PI, as well as the degree of contamination.

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).

• 1Solubilize the PIM of interest in 600 μL of CDCl₃/CD₃OD/D₂O (70:30:2; v/v/v).
• 2Transfer the sample into a 5mm NMR tube.
• 3Acquire a ¹H-NMR spectrum.
• 4For quantification, a capillary (external diameter, 1.5 mm), containing a formate solution with known concentration (7 mM) is inserted into the NMR tube prepared in step 2.

Figure 6 shows NMR spectra of purified preparations of Ac₁PIM₂ and Ac₁PIM₄.

• 5Run a proton spectrum under fully relaxed conditions to allow quantification by comparison of the areas of the resonances due to the anomeric protons of the mannose residues and formate.

Contamination with PI can be judged from the intensity of the resonance labeled with an asterisk in Figure 6. This resonance corresponds to one proton from the glycerol moiety in PIMs, while the two resonances on the right side, labeled 1 and 2, correspond to two anomeric protons of two mannosyl groups. When PIMs are pure, the ratio between the glycerol resonance and the combined areas of 1 and 2, should be 1:2. A higher ratio denotes contamination with PI.

Total phosphorus quantification

This quantification protocol was adapted from Rouser et al. (1970) and relies on the degradation of the PIM through acid hydrolysis and complexation of the released inorganic phosphate with molybdate. Upon reduction of this complex with ascorbic acid, a blue color develops. Quantification is performed by measurement of the absorbance at 797 nm.

Preparation of the phosphate calibration curve:

• 6From a stock solution of 560 μg/mL of Na₂HPO₄·2H₂O, prepare the standards indicated in Table 1 (in duplicate) using disposable 1.5 mL microcentrifuge tubes.

Table 1:

Conditions for preparation of the phosphate standards to be used as a calibration curve in the total phosphorus quantification method.

Phosphate (nmol)	Na2HPO4.2H2O (μL)	70% (v/v) HClO4 (μL)	Milli-Q® H2O (μL)	2.5% (NH4)6Mo7O24.4H2O (μL)
0	0	125	825	125
3.15	1	125	824	125
6.29	2	125	823	125
12.58	4	125	821	125
22.02	7	125	818	125
31.46	10	125	815	125
47.19	15	125	810	125
62.92	20	125	805	125

Always add the HClO₄ solution before adding the (NH₄)₆Mo₇O₂₄·4H₂O solution since the complexation of molybdate with phosphate requires acidic conditions.

• 7Add 125 μL of a solution of ascorbic acid in water, 10% (w/v), prepared immediately before use and cold.
• 8Mix well.
• 9Heat in a boiling water bath for 10 minutes.
• 10Cool in cold water for 5 minutes.
• 11Transfer 200 μL of each solution into a 96-well plate and read absorbance at 797 nm.

Lipid digestion:

• 12Clean several Hungate tubes with chromosulfuric acid solution or DECONEX® 22 LIQ-x (phosphate-free detergent), overnight.

A careful cleaning of glassware is essential to remove any traces of phosphorus-containing compounds.

• 13Add a known amount of lipidic sample to a Hungate tube by using a Hamilton® syringe.

A linear correlation is observed up to 65 nmol of phosphate. If necessary, dilute the sample appropriately so that the final absorbance falls in the range zero to 1.0.

• 14Dry the sample completely.
• 15Add 125 μL of 70% (v/v) HClO₄ to the tube and cover it with a glass marble to allow reflux of the condensed water.
• 16Using a silicon oil bath, heat the samples at 180–190°C for 60 minutes. Organic matter will degrade and phosphorus present in the molecules will be released as inorganic phosphate. After 60 min heating, samples should be clear and colorless. If yellow, heat for a longer period.
• 17Allow the samples to cool down at room temperature.

Complexation with molybdenium:

• 18Add 825 μL of Milli-Q® H₂O and 125 μL of 2.5% (NH₄)₆MO₇O₂₄ · 4H₂O in water.
• 19Mix well.
• 20Incubate 10 minutes at room temperature to allow for the formation of the complex between phosphate and molybdate.

Reduction with ascorbic acid:

• 21Add 125 μL of a solution of 10% (w/v) ascorbic acid, freshly prepared and cold.
• 22Mix well.
• 23Heat in a boiling water bath for 10 minutes.

As the sample heats up, depending on the amount of phosphate, it should turn from yellow to blue. It is critical that the samples and the calibration standards are subjected to this procedure at the same time to minimize discrepancies.

• 24Cool in cold water for 5 minutes.
• 25Transfer 200 μL of each sample into a 96-well plate and read absorbance at 797 nm.

SUPPORT PROTOCOL 1

Support protocol title:

Glycerol stocks of M. smegmatis mc²155 ΔPimE.

Introductory paragraph:

For liquid culture and long-term storage of M. smegmatis mc²155 ΔPimE, glycerol stocks must be prepared. By freezing and storage at −80°C, the cells can be used for several months. Glycerol acts as a protectant of cells during freezing.

Materials:

Middlebrook 7H10 Agar (DifcoTM, cat. no. 262710)

Middlebrook® 7H9 liquid medium (DifcoTM, cat. no. 271310).

Kanamycin 50 mg/mL (Sigma-Aldrich, cat. no. K4000–5KG)

99% (v/v) glycerol

50 mL Erlenmeyer flask

37°C incubating shaker (Aralab Agitorb 200, or equivalent)

1.5 mL microcentrifuge tubes

1.5 mL cryovials

Protocol steps with step annotations:

1. Pick a single colony from a Middlebrook® 7H10 Agar plate with M. smegmatis mc²155 ΔPimE and insert it into 20 mL of Middlebrook 7H9 liquid medium containing 40 μg/mL of kanamycin in a sterile 50 mL Erlenmeyer flask.

2. Incubate for 48 hours at 37°C, 180 rpm until cells reach mid exponential phase.

3. Divide the volume of the culture into 750 μL aliquots and place in 1.5 mL sterile microcentrifuge tubes.

4. Centrifuge 5 minutes at 5 000 × g.

5. Discard the supernatant solution.

6. Add 750 μL of Middlebrook® 7H9 liquid medium to each aliquot and resuspend the pellet.

7. Add 250 μL of 99% (v/v) glycerol (sterile) and mix well. Transfer the cell suspension into cryovials.

8. Store at −80°C until needed.

REAGENTS AND SOLUTIONS:

Kanamycin 50 mg/mL

For 10 mL:

1. Weigh 0.5 g of kanamycin sulfate from Streptomyces kanamyceticus (Sigma-Aldrich, cat. no. K4000–5KG).

2. Dissolve in 5 to 7 mL of bi-distilled H₂O.

3. Correct final volume to 10 mL.

4. Filter in a sterile environment, using a sterile 0.2 μm pore size cellulose membrane syringe filter, and collect in sterile microcentrifuge tubes or vials.

5. Store at −20°C until needed.

Middlebrook® 7H10 Agar culture medium

For 100 mL:

1. Weigh 2.111 g of Middlebrook® 7H10 Agar (Difco™, cat.no. 262710).

2. Add 100 mL of bi-distilled water.

3. Add 0.556 mL of 99% glycerol (Gerbu, cat. no. 2006.1000) and mix.

4. Autoclave.

Middlebrook® 7H9 liquid culture medium

For 100 mL:

1. Weigh 0.522 g of Middlebrook® 7H9 (Difco™, cat. no. 271310).

2. Add 100 mL of bi-distilled water.

3. Add 0.222 mL of 99% glycerol and mix.

4. Autoclave.

LB culture medium

For 1 liter:

1. Add 10 g of Tryptone (Panreac Applichem, cat. no. A1553,0500), 10 g of NaCl (Honeywell, cat. no. 31434–1KG), and 5 g of yeast extract (Himedia, cat. no. RM027–500G) to approximately 700 mL of bi-distilled water. Mix well until fully dissolved.

2. Correct the final volume to 1 L.

3. Autoclave.

50 mM Tris-HCl pH 7.0

For 100 mL:

1. Weigh 0.606 g of tris(hydroxymethyl)aminomethane (Merck, cat. no. 1.08219.1000) and add approximately 50 mL of bi-distilled water.

2. Correct the pH to 7.0 by slowly adding droplets of 6 M HCl (Pronalab, cat.no 3174).

3. When at pH 7.0, adjust the final volume of the solution to 100 mL.

Primuline Spray Reagent

For 100 mL:

1. Weigh 5 mg of primuline (Sigma-Aldrich, cat. no. 206865–1G).

2. Dissolve in 20 mL of Milli-Q® water.

3. Add acetone (Merck, Supelco, cat. no. 1.00014.25.11) to a total volume of 100 mL.

4. Store in the dark or in an amber glass flask.

Orcinol Spray Reagent

For 90 mL:

1. Dissolve 180 mg of 5-methylresorcinol anhydrous (TCI, cat. no. M1235) in 5 mL of Milli-Q® water.

2. Add 75 mL of ethanol (absolute).

3. Place the solution on ice and slowly add 10 mL of concentrated (approx. 18 M) H₂SO₄ (Panreac, cat. no. 1310581212).

4. Store in the dark or in an amber glass flask.

Eluent A: CHCl₃/CH₃OH/H₂O (240:1140:620; v/v/v) with 10 mM NH₄CH₃COO

For 2 liters:

1. Take 240 mL of CHCl₃ (Honeywell, cat. no. 34854–2.5L).

2. Add 1140 mL of CH₃OH (Honeywell, cat. no. 34966–2.5L) and mix.

3. Add 620 mL of H₂O and mix.

4. Weigh 1.542 g of NH₄CH₃COO (Merck, cat. no. 939 CC622116) and add to the mixture.

Eluent B: CHCl₃/CH₃OH (7:3; v/v) with 50 mM NH₄CH₃COO

For 2 liters:

1. Mix 1400 mL of CHCl₃ (Honeywell, cat. no. 34854–2.5L) with 600 mL of CH₃OH (Honeywell, cat. no. 34966–2.5L).

2. Weigh 7.708 g of NH₄CH₃COO (Merck, cat. no. 939 CC622116) and add to the mixture (the ammonium acetate will take some time to dissolve. Mix well to speed up the process).

COMMENTARY:

Background Information:

In this work, extraction of PIMs was carried out from biomass of Mycobacterium smegmatis mc²155 ΔPimE. This strain has the advantage of accumulating relatively high levels of Ac₁PIM₂ and Ac₁PIM₄. Most of the methods reported in the literature for PIMs isolation use biomass of the pathogenic strains Mycobacterium tuberculosis (Pirson et al., 2015) and Mycobacterium bovis (Gilleron, Nigou, Cahuzac, & Puzo, 1999; Gilleron et al., 2001). However, a non-pathogenic strain, Mycobacterium smegmatis mc²155, was used by Rahlwes et al. (2019) in a study intended primarily to isolate lipomannan and lipoarabinomannan.

The method proposed in this article for extraction of glycolipids from biomass of M. smegmatis uses a mixture of chloroform and methanol and is based on the procedure described by Bligh & Dyer (1959), while the purification of PIMs was adapted from Gilleron et al. (2001) and Pirson et al. (2015). We performed a third chromatographic step that is essential to remove the strong contamination with phosphatidylinositol (in the case of Ac₁PIM₄) and unknown lipids (in the case of Ac₁PIM₂). Moreover, herein we provide detailed information for the successful extraction and purification of the target PIMs, i.e., Ac₁PIM₄ and Ac₁PIM₂. The former metabolite has been used for the characterization of PimE and the latter in the search for the gene encoding PimC (our unpublished work).

Critical Parameters:

Culture growth

Standard microbiologic sterile techniques are required for cultivation of M. smegmatis mc²155 ΔPimE (Basic Protocol 1).

Extraction and purification processes

Chloroform tends to degrade into hydrochloric acid. For all processes in the protocol described here, and before using chloroform, check the presence of hydrochloric acid by using a universal pH indicator paper.

The use of Silica gel 60 High Performance Thin Layer Chromatography (HPTLC) plates is crucial since lower resolution TLC plates do not resolve properly the different PIMs species.

Never reuse silica gel resin since the final preparation of the resin may contain fine silica gel particles, with a deleterious impact, namely on the quality of the NMR spectra.

Troubleshooting:

See Table 2 for troubleshooting recommendations.

Table 2:

List of common problems that may arise, possible causes and recommended solutions.

Problem	Possible Causes	Solution
General
Poor solubility of PIMs in CHCl3/CH3OH/H2O (70:30:2)	Due to the high volatility of chloroform its content in the solvent may vary with time	Always prepare a fresh solution with the correct proportion
Difficulty in adsorbing the sample on the HPTLC plate	Too concentrated sample was applied	Dilute the sample adequately
Spots on a HPTLC plate overlap	Too much sample was applied	Dilute sample or apply a lower volume
Spots barely visible on the HPTLC plate	Too little sample was applied	Concentrate sample or apply a larger volume
Distortion on the HPTLC due to differential compound migration	Deficient equilibration of the chamber atmosphere with the solvent	Prepare a new solvent solution and allow for proper equilibration
BASIC PROTOCOL 3
Cannot separate water and chloroform layers (Basic protocol 3, Step 5)	Poor solvent evaporation, especially methanol	Dry completely in a rotary evaporator and repeat the procedure
	Emulsion present resulting in a middle white layer	Add water fractions (e.g., 25 mL each) to destroy emulsion
BASIC PROTOCOL 4 - Silica gel chromatography for removal of major contaminants
PIMs detected in the initial CHCl3/CH3OH (8:2) eluate	Too much sample loaded onto the column; column capacity exceeded	Pool fractions together, dry completely in a rotary evaporator and repeat the silica gel column with a lower load
BASIC PROTOCOL 4 - High performance liquid chromatography for isolation of phosphatidylinositol mannosides
Ac1PIM2 and Ac1PIM4 do not separate	Too much sample loaded	Pool fractions with both Ac1PIM2 and Ac1PIM4, dry completely in a rotary evaporator and perform a new run with a lower amount
BASIC PROTOCOL 4 - Silica gel chromatography for purification of Ac1PIM4 and Ac1PIM2
Imperfect packing	Silica gel resin was poured too fast into the column, resulting in the formation of air bubbles	Slowly add the silica gel suspension to avoid formation of air pockets. If drying occurs, empty the column and restart packing
BASIC PROTOCOL 5 - Quantification through 1H NMR
Resonances of anomeric protons overlap the water solvent band	Too much residual ammonium acetate present in the sample	Repeat the protocol for removal of ammonium acetate (step 35 to 39, Basic Protocol 4)
Resonances due to the anomeric protons 1 and 2 overlap	Slight variation in solvent composition	Dry the sample, prepare a fresh solution of CDCl3/CD3OD/D2O, and dissolve the residue

Understanding Results:

Here we describe the purification of Ac₁PIM₂ and Ac₁PIM₄ from the membranes of M. smegmatis. Three consecutive chromatographic steps are proposed (Basic Protocol 4). Typically, from a 60 g batch of M. smegmatis biomass we were able to purify approximately 9 mg of Ac₁PIM₂ and 1.8 mg of Ac₁PIM₄.

The use of controls to identify Ac₁PIM₂ and Ac₁PIM₄ in HPTLC is important. Controls can be obtained by separation of a lipid extract by TLC, recovery of each spot by scraping the plate followed by identification of each spot by mass spectrometry (see Basic Protocol 3 – Preparation of PIM standards). The HPTLCs depicted here (Figure 3) usually show two faint spots in between Ac₁PIM₂ and Ac₁PIM₄. These spots are probably due to the intermediate metabolite Ac₁PIM₃ and the final product Ac₂PIM₄.

Time Considerations:

BASIC PROTOCOL 1

The full cultivation process, from pre-inoculum preparation to cell harvest, takes 3 days. Usually, from 1 liter of culture 7–8 g of biomass is produced. Therefore, to produce 60 g of biomass, 8 liters of culture are needed.

BASIC PROTOCOLS 2 and 3

We recommend using a single batch of 60 g biomass for optimal extraction and purification of PIMs. For a single, skilled investigator, 6 batches of 60 g of biomass can be processed in a week, according to Basic Protocols 2 and 3 (Figure 7): it is possible to start the extraction of two batches (in parallel) on Day 1, followed by the extraction of two more batches on Day 2, and finally the two last batches on Day 3. With this organization scheme, the processing of the first two batches will be completed by Day 3, the next two batches by Day 4 and the last two batches by Day 5.

Figure 7:

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

Basic Protocol 2 takes around 5 hours to be completed, while about 2 days are required to carry out Basic Protocol 3. Therefore, to process 6 batches of 60 g each, a total of 5 days is required (see Figure 7).

BASIC PROTOCOL 4

Silica gel column for removal of major contaminants

This chromatographic procedure will take approximately 6 hours, from packing the column to complete elution. The maximum amount of extract that can be loaded onto this column corresponds to the lipidic extract derived from 2 × 60 g of biomass. HPTLC analysis should take around 60 minutes. An experienced investigator will be able to perform this chromatography step in parallel with the execution of Basic Protocols 2 and 3 for other biomass batches. It is also possible to run several columns simultaneously. According to the scheme depicted in Figure 7, the silica gel column relative to the first two batches will be completed by Day 4; all the six batches will be processed by Day 6.

High Performance Liquid Chromatography for isolation of phosphatidylinositol mannosides

Each run takes approximately 1 hour. Before each run, the column should be equilibrated for 15–20 min with 85% Eluent A and 15% Eluent B. A single investigator is capable of performing 3 runs per day, therefore two days are necessary to treat all the samples derived from the 6 initial batches. Drying the pooled fractions through rotary evaporation and removal of residual water by lyophilization will take one day.

Silica gel chromatography for purification of Ac₁PIM₂ and Ac₁PIM₄

Each chromatographic step takes around 7 hours: 10–15 minutes for packing the column; 2.5 hours to switch the solvent; and 3 hours for elution. HPTLC can be prepared and run in parallel with elution. Drying of the pooled fractions takes 1 to 1.5 hours. Three silica gel chromatography columns can be handled in parallel. Therefore, for each targeted compound, two days are required to complete this purification step.

Removal of ammonium acetate from the purified AcPIMs

Typically, the removal of ammonium acetate takes 2 days; assessment of residual ammonium acetate by NMR analysis takes about 10 minutes for each sample.

BASIC PROTOCOL 5

Quantification through ¹H-NMR

The total analysis takes about 50 minutes.

Total phosphorus quantification

The total analysis takes approximately 2 hours. The operator should begin with acid treatment of the lipid samples, since complete digestion will take 1 hour. Use this dead time to prepare phosphate standards, the ascorbic acid solution, and the cold and hot water baths.

TOTAL TIME

In three weeks, it is possible to process around 360 g of cell biomass (six batches of 60 g) and obtain circa 11 mg and 54 mg of pure Ac₁PIM₄ and Ac₁PIM_2, respectively.

DATA AVAILABILITY STATEMENT:

The data that supports the findings of this study are available in the figures and supplementary material of this article.