Lipid droplet-associated proteins are involved in the biosynthesis and hydrolysis of triacylglycerol in Mycobacterium bovis bacillus Calmette-Guerin

Abstract

Mycobacteria store triacylglycerols (TGs) in the form of intracellular lipid droplets (LDs) during hypoxia-induced nonreplicating persistence. These bacteria are phenotypically drug-resistant and therefore are believed to be the cause for prolonged tuberculosis treatment. LDs are also associated with bacilli in tuberculosis patient sputum and hypervirulent strains. Although proteins bound to LDs are well characterized in eukaryotes, the identities and functions of such proteins have not been described in mycobacteria. Here, we have identified five proteins: Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c, and BCG1721, which are exclusively associated with LDs purified from hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guérin (BCG). Disruption of genes tgs1, tgs2, BCG1169c, and BCG1489c in M. bovis BCG revealed that they are indeed involved in TG metabolism. We also characterized BCG1721, an essential bi-functional enzyme capable of promoting buildup and hydrolysis of TGs, depending on the metabolic state. Nonreplicating mycobacteria overexpressing a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TG breakdown and regrowth upon resuscitation. In addition, by heterologous expression in baker's yeast, these mycobacterial proteins also co-localized with LDs and complemented a lipase-deficient yeast strain, indicating that neutral lipid deposition and homeostasis in eukaryotic and prokaryotic microorganisms are functionally related. The demonstrated functional role of BCG1721 to support growth upon resuscitation makes this novel LD-associated factor a potential new target for therapeutic intervention.

FIGURE 1.

SDS-PAGE analysis of LD-associated proteins. The protein composition of the LDs (lane 3) was compared with that of total lysate (lane 1) and cytosolic fraction (lane 2). Equal amounts of proteins were loaded into each lane and silver-stained after separation by SDS-PAGE. The positions of molecular mass markers are noted on the left. The bands indicated on the right side (in each lane) were further subjected to tandem MS analysis.

FIGURE 2.

Expressed mycobacterial lipid droplet-associated proteins fused with GFP localize to the neutral lipid storage in yeast. Cells expressing BCG1721-GFP, Tgs1-GFP, Tgs2-GFP, BCG1489c-GFP, BCG1169c-GFP, GroES-GFP, and GFP alone (empty pUG35 vector), respectively, were co-stained with lipophilic dye Nile red, revealing localization of the tagged protein to either lipid droplets or cytosol. All of the GFP fusion proteins were expressed under control of the MET25 promoter in yeast. The panels (from left to right) display fluorescent images of GFP signals, Nile red signals, dual signals (Merge), and transmission microscope (Trans) images of baker's yeast. The scale bar is 5 μm. The images are representative of the cells in at least 50 different microscopic fields.

FIGURE 3.

Disruption of genes encoding LD-associated proteins reduces TG accumulation during hypoxia-induced nonreplicating state. A, TLC analysis of TG levels in different mutants, Δ-tgs1, Δ-tgs2, Δ-BCG1169c, and Δ-BCG1489c in hypoxic nonreplicating phase. The lipids were extracted from equal amounts of cells using chloroform/methanol, separated on silica gel plates, and stained with iodine vapor. The values depict TG amounts as determined by densitometry using a triolein standard calibration curve. Arrows indicate the reduced amounts of TG band with very long chain FAs in the Δ-tgs1 and Δ-BCG1169c strains. Δ, genetically disrupted strain; C-Δ, complemented strain. B, LC-MS analysis of FAs in TG levels in different strains during hypoxia-induced nongrowing state. TG levels refer to the neutral lipid with long chain FAs (52:3) or very long chain FAs (60:1). These species are relatively abundant and representative of TGs containing long chain and very long chain FAs. Black bar, WT; white bar, Δ strain; gray bar, complemented strain. The full details are in the legend to supplemental Fig. S2. The data are represented as the mean values ± standard deviations (n = 3). *, p < 0.05 compared with the WT. a.u., arbituary unit(s).

FIGURE 4.

Overexpression of BCG1721 or BCG1721(S150A) leads to increased intracellular TG storage in growing mycobacteria. A, schematic representation of the BCG1721. The black box denotes the putative lipase domain; the white box denotes the putative ACSL (AMP-binding) domain. Putative domain boundaries are indicated by the respective residue numbers, based on pDomTHREADER results. B, superposition of the three-dimensional model of the lipase domain (blue color) with the x-ray crystal structure of a mammalian TG lipase (orange color; Protein Data Bank code 1k8q). The backbone of the lid structure of both proteins is in tube mode, whereas the rest of the structures are in line mode. C, close-up view of the catalytic triad. Amino acids Ser¹⁵⁰, Asp³⁰⁹, and His³³⁸ correspond to the putative catalytic triad of BCG1721, whereas Ser¹⁵³, Asp³²⁴, and His³⁵³ belong to that of Protein Data Bank entry 1k8q. Note the well conserved spatial arrangement between the two sets of catalytic triads. D, confocal imaging of different mycobacterial strains stained with Nile red. The left panels show transmitted images of WT (top panels), pBCG1721 (middle panels), and pBCG1721(S150A) (bottom panels) integrated with red fluorescent signal. The scale bar is 2 μm. The images are representative in at least 50 different microscopic fields. The center panels represent the gene construct schematically. The right panels depict gene transcript abundance within RNA samples as analyzed by quantitative reverse transcription-PCR. BCG1721 transcript copy numbers were normalized against that of sigA. Rel. Abundance, relative abundance. E, TLC analysis of TG levels in different mycobacterial strains. The values depict TG amounts as determined by densitometry. An arrow indicates the reduced amount of TG band with very long chain FAs in pBCG1721 strain as compared with that of pBCG1721(S150A) cultures. F, LC-MS analysis of FAs in TG levels in different mycobacterial strains, WT, pBCG1721, and pBCG1721(S150A). Black bar, TG(52:3, long chain FAs); white bar, TG(60:1, very long chain FAs). The full details are in supplemental Fig. S4. The data are represented as the mean values ± standard deviations (n = 3). *, p < 0.05 compared with the WT. a.u., arbituary unit(s).

FIGURE 5.

Mycobacteria overexpressing BCG1721(S150A) has attenuated TG hydrolysis rate and cell regrowth from hypoxia-induced nonreplicating state. Mycobacterial strains were cultured to hypoxic NRP state and reactivated with fresh air (regrowth). A, TLC analysis of TG levels in WT, pBCG1721, and pBCG1721(S150A) strains during hypoxic NRP and regrowth states. The values depict TG amounts as determined by densitometry. Note that some TGs remained in the reactivated bacilli transformed with BCG1721(S150A) as compared with those cells overexpressing the bi-functional BCG1721. B, total TG lipase activity of mycobacterial extracts at hypoxic NRP and regrowth phases. C, enumeration of colony-forming units (CFU) of different strains during hypoxic NRP and regrowth conditions were determined. Black bar, hypoxic nonreplicating; white bar, regrowth. The data are represented as the mean values ± standard deviations (n = 3). *, p < 0.05 compared with the colony-forming units or lipase activity at the hypoxia-induced NRP phase.

FIGURE 6.

Schematic representation of LD-associated proteins in TG biosynthesis and breakdown in mycobacteria. This pathway is a brief overview of our findings, depicting the involvement of LD-associated proteins in the metabolism of TGs. AGPAT, acylglycerol-phosphate acyltransferase; DGAT, diacylglycerol acyltransferase; FA-CoA, fatty acyl-coenzyme A; GPAT, glycerol phosphate acyltransferase; PAP, phosphatidic acid phosphatase.