Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY

Abstract

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(deltaPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY(mar), in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY(mar) in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY(mar) was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(deltaPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(deltaPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(deltaPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

FIG. 1.

Expression of lipY in M. smegmatis and in vivo effect on TAG hydrolysis. (A) Schematic representation of LipY and LipY(ΔPE) from M. tuberculosis, LipY_mar and LipY(ΔPPE)_mar from M. marinum, and the PE-PGRS protein Rv1818c. The conserved PE and PPE domains are shown in gray, along with the Pro-Glu (PE) and Pro-Glu-Glu (PPE) signatures. The Gly-Ala-rich PGRS domain of Rv1818c is shown as a dot-filled rectangle. The lipase domains of LipY and LipY_mar are represented in black, whereas the adjacent domains (100 to 206 in LipY and 177 to 296 in LipY_mar) with no homology to known sequences are shown in white. The catalytic lipolytic serine is represented in bold. (B) Mid-log-phase cultures of M. smegmatis carrying either pSD26, pSD26::lipY, or pSD26::lipY(ΔPE) were induced with 0.2% acetamide for 4 h. Cells were harvested and fractionated to separate the cytosol (Cy) from the cell wall (CW). Equal protein amounts (50 μg) were separated by 10% SDS-PAGE. Positions of LipY and LipY(ΔPE) are indicated by arrowheads. M, molecular mass marker. (C) M. smegmatis cultures were grown to mid-log phase and induced with 0.2% acetamide for 4 h prior to labeling with 1 μCi/ml [1,2-¹⁴C]acetate for an additional 4 h. Apolar lipids were extracted as described in Materials and Methods. Equal counts (250,000) were applied onto silica gel plates and separated using two different solvent systems: solvent system A, which allows visualization of the TAGs (petroleum ether-ethyl acetate [98:2 {vol/vol}] in the first dimension [×3] and petroleum ether-acetone [98:2 {vol/vol}] in the second dimension), and solvent system C, which allows visualization of free fatty acids (chloroform-methanol [96:4 {vol/vol}] in the first dimension and toluene-acetone [80:20 {vol/vol}] in the second dimension). TLC plates were exposed overnight to film. The percentage of TAGs present in each strain (with regard to the reference strain presence, which was arbitrarily set to 100%) was determined by densitometry and is indicated in the upper right corner of each TLC plate.

FIG. 2.

Lipolytic activity of LipY_mar expressed in M. smegmatis. (A) Mid-log-phase cultures of M. smegmatis carrying either pSD26, pSD26::lipY_mar, or pSD26::lipY(ΔPE)_mar were induced with 0.2% acetamide for 4 h, centrifuged, resuspended in PBS, and lysed. Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE. The gel was stained with Coomassie blue to reveal the presence of LipY_mar or LipY(ΔPPE)_mar. A comparison of the expression levels of LipY_mar and LipY(ΔPPE)_mar was done following Western blot analysis. Proteins were transferred to a nitrocellulose membrane, probed with rat anti-LipY(ΔPE) antibodies, and then incubated with anti-rat antibodies conjugated to alkaline phosphatase. (B) Cultures of M. smegmatis carrying either pSD26, pSD26::lipY_mar, or pSD26::lipY(ΔPE)_mar were grown to mid-log phase and induced with 0.2% acetamide for 4 h prior to labeling with 1 μCi/ml [1,2-¹⁴C]acetate for an additional 4 h. Apolar lipids were extracted and equal counts were applied onto TLC plates. Separation was done using solvent system A and plates were exposed overnight to a film. The percentage of TAGs present in each strain (with regard to the reference strain presence, which was arbitrarily set to 100%) was determined by densitometry and is indicated in the upper right corner of each TLC plate.

FIG. 3.

In vitro lipase assay. An in vitro assay was developed using cell wall fractions from M. smegmatis strains overproducing the various lipases following induction with 0.2% acetamide. Cell wall preparations from M. smegmatis harboring empty pSD26 were used as an internal control, so the activity observed can be attributed only to the overexpressed lipases. Western blot analysis of the cell wall fractions using anti-LipY(ΔPE) antibodies confirmed the presence of equal amounts of the overexpressed lipases. Increasing concentrations of cell wall fractions (in micrograms) were incubated with para-nitrophenyl stearate at 35°C for 40 min. The release of para-nitrophenol was monitored spectrophotometrically at 405 nm. (A) Lipolytic activity of LipY versus LipY(ΔPE) and (B) lipolytic activity of LipY_mar versus LipY(ΔPPE)_mar. Each data point is the mean of triplicates, and error bars correspond to standard deviation (±). The data are representative of three experiments performed with independent cell wall preparations.

FIG. 4.

Overexpression and lipolytic activity of LipY and LipY(ΔPE) in M. bovis BCG. (A) lipY and lipY(ΔPE), as well as a version of lipY(ΔPE) carrying the S309A mutation [encoding LipY(ΔPE)(S309A)], were cloned in pMV261, and the resulting plasmids were introduced into M. bovis BCG. The expression of the proteins in each lysate was assayed by Western blotting using rat anti-LipY(ΔPE) antibodies. Equal protein amounts (20 μg) were separated on a 10% SDS-PAGE gel. (B) Recombinant M. bovis BCG cultures were labeled with 1 μCi/ml [1,2-¹⁴C]acetate for 24 h. Apolar lipids were extracted and equal counts were applied onto silica gel plates. Separation was done using solvent system A and plates were exposed overnight to film. The percentage of TAGs present in each strain (with regard to the reference strain presence, which was arbitrarily set to 100%) was determined by densitometry and is indicated in the upper right corner of each TLC plate. (C) Gel overlay assay. M. bovis BCG lysates were separated by SDS-PAGE. Following renaturation, the gel was laid on top of an agar plate containing tributyrin as a substrate. A clear halo forms when the overexpressed lipase is active. Lanes: 1, M. bovis BCG pMV261; 2, M. bovis BCG pMV261::lipY; 3, M. bovis BCG pMV261::lipY(ΔPE)(S309A); 4, M. bovis BCG pMV261::lipY(ΔPE).

FIG. 5.

Localization of LipY and LipY(ΔPE) in M. bovis BCG. (A) Subcellular localization of LipY and LipY(ΔPE) in M. bovis BCG strains. Cultures were lysed and fractionated to separate the cytoplasm (Cy) from the cell wall (CW). Equal amounts of proteins (10 μg) of each fraction were subjected to SDS-PAGE, electroblotted onto a nitrocellulose membrane, and probed with either rat anti-LipY(ΔPE) antiserum (top), monoclonal anti-KatG antibodies (middle), or rabbit anti-OmpATb antiserum (bottom). (B) EM immunolocalization of LipY. Thin sections of cryosubstituted M. bovis BCG LipY(ΔPE) were sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG for 1 h, and (iv) PAO for 30 min. Intracytoplasmic labeling is indicated by arrows and surface labeling by arrowheads. Bar = 0.5 μm.

FIG. 6.

Immunolocalization of LipY(ΔPE) at the surface of M. bovis BCG. Immunolabelings were performed on whole M. bovis BCG carrying pMV261::lipY(ΔPE), deposited on EM Formvar-coated nickel grids prior to labeling (A and B) or onto prefixed bacteria labeled in suspension prior to processing for conventional EM (C to F). In both cases, bacteria were sequentially exposed to rat anti-LipY(ΔPE) antibodies, rabbit anti-rat IgG, and PAO. (A) Whole bacteria on grids exposed to specific antibody, followed by rabbit anti-rat IgG and PAO: the gold particles (300 to 400 per bacterium) are distributed throughout the bacterial surface (arrows). (B) Whole bacteria on grids exposed to rabbit anti-rat IgG and PAO only as a control: bacteria display at most 40 gold particles on their surface (arrow). (C to E) Thin sections of bacteria exposed to specific antibody, followed by rabbit anti-rat IgG and PAO. (C) The outermost layer of the cell wall is labeled (arrow). This is particularly obvious in the enlarged view (D), where the peptidoglycan layer (PG), the thin electron-translucent layer (ETL), and the outermost fibrillar layer (OL) are clearly visible. (E) When the OL has been shed, bacteria are not labeled. (F) Thin sections of bacteria exposed to preimmune rat serum, followed by rabbit anti-rat IgG and PAO: bacteria are not labeled even when the OL is present. Bars in panels A and B = 0.5 μm; Bars in panels C, E, and F = 0.25 μm; Bar in panel D = 0.1 μm.

FIG. 7.

Specific anti-LipY humoral responses in M. tuberculosis-infected patients as opposed to healthy controls. (A) Purification of the recombinant LipY and LipY(ΔPE) proteins expressed in M. smegmatis carrying either pSD26::lipY or pSD26::lipY(ΔPE). Proteins were extracted from M. smegmatis cell wall preparations, excised from preparative polyacrylamide gels, and then electroeluted to obtain pure proteins. (B) ELISA reactivities of IgG and IgM anti-LipY and anti-LipY(ΔPE) antibodies were assayed in sera of either M. tuberculosis-infected group 1 adult patients or healthy controls (HC) (n = 44 for healthy controls; n = 69 for patients). (C and D) ELISA reactivities of anti-LipY and anti-LipY(ΔPE) antibodies in two different categories of infected children. (C) IgG and IgM reactivities of sera from recently M. tuberculosis-infected children and from healthy controls (n = 12 for healthy controls; n = 30 for patients). (D) IgG reactivities for patients with extrapulmonary TB (n = 12 for healthy controls; n = 27 for patients).

FIG. 8.

Patient-by-patient analysis against LipY, LipY(ΔPE), and Rv1818c. Sera from 69 M. tuberculosis-infected group 1 adult patients (numbers below bars) were assayed individually by ELISA against LipY, LipY(ΔPE), and Rv1818c.