https://orcid.org/0000-0001-7400-1215
Figures & data
Figure 1. Recombinant arginine-rich C-terminal domain of the PE_PGRS3 promotes Mycobacterium smegmatis adhesion to macrophages and pneumocytes
Murine macrophages (J774, panel A) and human type 2 pneumocytes (A549, panel B) were infected (MOI 10:1) with recombinant Mycobacterium smegmatis (Ms) expressing cytosolic green fluorescent protein (MsGFP, indicated as Ø), or co-expressing GFP and full-length pe_pgrs3 gene (MsGFPPE_PGRS3HA, indicated as PE_PGRS3) and its functional mutant lacking the C-terminal domain (MsGFPPE_PGRS3∆CTHA, indicated as PE_PGRS3ΔCT). Dialyzed LPS-free PE_PGRS3 C-terminal domain (xR-3Ct) was added to the infection solution at a final concentration of 5 µg/ml when both cell types were infected with MsGFP and MsGFPPE_PGRS3∆CTHA. Four hours post infection colonies forming units (CFUs) were determined. xR-3Ct promoting adhesion was compared with that of the recombinant mycobacterial heparin-binding hemagglutinin (rmHBHA). J774 (C) and A549 (D) were infected with MsGFPPE_PGRS3HA, MsGFPPE_PGRS3∆CTHA and MsGFP. MOI 10:1 was used for the MsGFPPE_PGRS3∆CTHA and MsGFP, whereas MOI 1:1 was used for the MsGFPPE_PGRS3HA. LPS free xR-3Ct and rmHBHA were added to the infection solutions at a final concentration of 5 µg/ml. Four hours post infection CFUs were determined. CFUs are reported in log10 scale and represented as mean ± SD and comparisons were obtained by using Two-way ANOVA with Tukey’s correction.
Figure 2. Arginine-rich C-terminal domain specifically binds cardiolipin and phosphorylated phosphatidylinositols
Purified PE_PGRS3 positive charged C-terminal (xR-3Ct) was probed on a nitrocellulose membrane where different negatively charged phosphorylated lipids were adsorbed (A). Mycobacterial heparin-binding hemagglutinin (rmHBHA), which shows positive charged lysine amino acidic residues, was used as control (B). After the incubation, monoclonal anti-His antibody was used to detect the “baits” lipids that linked recombinant proteins. Secondary HRP-antibody was used to detect the binding and each membrane signal was acquired maintaining unvaried exposition. xR-3Ct showed a significant binding with PtdIns(4)P, PtdIns 4,5(P) and PtdIns (3,4,5)P compared to rmHBHA. Conversely, both proteins bound the PA. Single spots were analyzed by using ImageJ software. TG: Triglyceride, DAG: Diacylglycerol, PA: Phosphatidic acid, PS: Phosphatidylserine, PE: Phosphatidylethanolamine, PC: Phosphatidylcholine, PG: Phosphatidylglycerol, CL: Cardiolipin, PI: Phosphatidylinositol, PtdIns(4)P: Phosphatidylinositol 4 phosphate, PtdIns 4,5(P): Phosphatidylinositol 4,5 phosphate, PtdIns (3,4,5)P: Phosphatidylinositol 3,4,5 phosphate, C: Cholesterol, SM: Sphingomyelin, S: Sulfatide. Secondary structure prediction of xR-3Ct and electrostatic potential surface were obtained using JPRED and Chimera software as described in Materials and methods section (C). xR-3Ct phosphatase activity was probed on a series of phosphorylated PtdIns substrates. Free inorganic phosphate was measured by using a malachite green assay (D). xR-3Ct and rmHBHA showed similar and not significant dephosphorylation activity against PtdIns. Data are represented as mean ± SD and comparisons were obtained by using Two-way ANOVA with Tukey’s correction.
Figure 3. Administration of cardiolipin, but not phosphorylated phosphatidylinositols, to phosphate-starved culture turns off pe_pgrs3 expression
Recombinant Mycobacterium smegmatis (Ms) expressing PE_PGRS3 under control of its promoter fused at the C-terminal with the GFP (MsPE_PGRS3GFP), Ms expressing GFP (MsGFP) and Ms mc2 155 were grown in standard and low phosphate Sauton medium (~50 μM Pi). The fluorescence emission following expression of the GFP was measured flow cytometry as described in the Materials and Methods section. When MsPE_PGRS3GFP showed a significant fluorescence in low Pi medium, the liquid culture was split into different tubes. Inorganic phosphate (Pi), diacylglycerol (DAG), phosphatidic acid (PA), phosphatidylserine (PS), cardiolipin (CL), phosphatidylinositol (PtdIns), PtdIns(4,5)P2, PtdIns(3,4,5)P3, and cholesterol were added at the final concentration of 100 µM in different prepared tubes. The fluorescence was measured at different time points following phosphate or lipid restoring (A). Data were normalized to fluorescence measured for MsPE_PGRS3GFP cultured in low Pi environment (100% of fluorescent bacteria). Ms over-expressing full-length PE_PGRS3 (MsGFPPE_PGRS3HA, indicated as PE_PGRS3), its functional mutant lacking the C-terminal domain (MsGFPPE_PGRS3∆CTHA, indicated as PE_PGRS3ΔCT) and Ms parental strain (MsGFP, indicated as Ø) were selectively incubated with PtdIns(4)P, PtdIns 4,5(P) and PtdIns (3,4,5)P. Two hours later Zeta potential was measured (B). MsGFPPE_PGRS3HA net surface charge modification was observed after incubation with all selected PtdIns, shifting from less negative values up to values measured for the MsGFPPE_PGRS3∆CTHA and MsGFP. Data are represented as mean ± SD and comparisons were obtained by using Two-way ANOVA with Tukey’s correction.
Figure 4. Over-expression of the PE_PGRS3 enhances Mycobacterium tuberculosis cell entry in pneumocytes, but not in murine macrophages or human peripheral blood mononuclear cells (PBMCs)
Mycobacterium tuberculosis (Mtb) H37Rv was electroporated to over-express PE_PGRS3, and its functional mutant under the control of hbha gene promoter, together with the green fluorescent protein (GFP) under the control of the Ag85b promoter. MtbGFPPE_PGRS3HA, expressing the full-length gene, MtbGFPPE_PGRS3ΔCTHA and MtbGFPPE_PGRS3ΔGRPLIHA, expressing the PE_PGRS3 without the last 80 amino acids (xR-3Ct) and last 430 amino acids (PGRS domain starting from the second GRPLI motif), respectively, were generated. An Mtb expressing GFP under the control of the hsp60 promoter was used as control (MtbGFP). (A) Bacterial growth rate of MtbGFP, MtbGFPPE_PGRS3HA, MtbGFPPE_PGRS3∆CTHA, and MtbGFPPE_PGRS3∆GRPLIHA performed in Mycobacteria Growth Indicator Tube (MGIT). Measured fluorescence intensity (a.u.) and days of culture were reported to obtain the growth profile. (B). MtbGFPPE_PGRS3HA, MtbGFPPE_PGRS3ΔCTHA, and MtbGFPPE_PGRS3ΔGRPLIHA and the parental strain MtbGFP were grown in Sauton medium until mid-log phase, when cells were collected. Proteinase K protection assay was performed before obtaining immunoblots of the whole cell lysates. Membranes were probed with anti‐HA monoclonal and anti-GFP polyclonal antibodies. Lanes 1, 2, 3, and 4 represent whole cell lysates of MtbGFP, MtbGFPPE_PGRS3HA, MtbGFPPE_PGRS3ΔCTHA, and MtbGFPPE_PGRS3ΔGRPLIHA, respectively. xR-3Ct was observed surfaced exposed. Bacterial entry was evaluated by infecting human alveolar epithelial cells (A549) (C) and murine macrophages (J774) (D) with previously mentioned recombinant strains at MOI 10:1 and MOI 1:1, respectively. Colonies forming units (CFUs) were evaluated 1 h post-infection. MtbGFP, MtbGFPPE_PGRS3HA, MtbGFPPE_PGRS3ΔCTHA, and MtbGFPPE_PGRS3ΔGRPLIHA were here indicated as Ø, PE_PGRS3, PE_PGRS3ΔCT, and PE_PGRS3ΔGRPLI, respectively. A significant result was observed for MtbGFPPE_PGRS3HA recombinant strain infecting A549 (p < 0.01), but not in J774. Healthy donor peripheral blood mononuclear cells were isolated and infected at MOI 1:10. CFUs were evaluated at 1 and 7 days post infection (E). MtbGFPPE_PGRS3HA did not exert increased entry or persistence compared to MtbGFP, MtbGFPPE_PGRS3ΔCTHA, or MtbGFPPE_PGRS3ΔGRPLIHA. Data are represented as mean ± SD and comparisons were obtained by using one-way ANOVA is used to assay significance and CFUs are reported in log10 scale.
Figure 5. PE_PGRS3 is cleaved in Mycobacterium tuberculosis.
Mycobacterium tuberculosis expressing green fluorescent protein (GFP) (MtbGFP) or co-expressing GFP and native PE_PGRS3 (MtbGFPPE_PGRS3HA) or its chimeras, lacking the xR-3Ct and part of the PGRS domain (MtbGFPPE_PGRS3ΔCTHA and MtbGFPPE_PGRS3ΔGRPLIHA) were cultured in different Sauton medium supplemented or not by inorganic phosphate (Pi) or Twen80 (Tw80). Cultures were harvested at the mid-log phase (OD600nm: 0.8 ± 0.2) and processed to obtain a protein sub-cellular analysis. The following fractions were obtained: A) whole cell lysate (WCL), B) cell wall-associated proteins extracted after treatment with Genapol X-80 detergent, C) cytosolic – membrane proteins obtained after lysis of the pellet pre-treated with Genapol X-80 and D) secreted proteins gained by TFA – precipitation of the pre-filtered culture medium. Proteins were separated by SDS – page by using a 5–15% gradient polyacrylamide gel and then immunoblots were performed. Each membrane was probed with a monoclonal anti-HA antibody, a polyclonal anti-GFP antibody, and with an anti-MPT64 serum for the secreted protein fraction. PE_PGRS3HA signal (≈81 kDa) was detected only in the WCL fraction. Clear signals were highlighted for the functional chimeras of the PE_PGRS3 (PE_PGRS3∆CTHA ≈ 71 kDa) or the PE_PGRS3∆GRPLIHA ≈ 45 kDa) in WCL, in the Genapol fraction, and in the cytosolic fraction. A characteristic cleavage was observed at the N-terminal of both PE_PGRS3 chimeras with resulted cleaved proteins that showed ≈ 10 kDa less than the expected molecular weight. Pi did not appear conditioning protein stability, whereas Tw80 significantly reduced the signal associated with the PE_PGRS3 chimeras in the Genapol fractions. Signals detected in cytosolic and genapol fractions for PE_PGRS3∆CTHA and PE_PGRS3∆GRPLIHA, and their respective cleaved chimeras were analyzed by using ImageJ software. Densitometric analysis was carried out on MtbGFPPE_PGRS3ΔCTHA (E – F) and MtbGFPPE_PGRS3ΔGRPLIHA (G – H) grown in standard Sauton medium or Pi depleted medium.
Figure 6. PE_PGRS33 is mainly cleaved in Mycobacterium tuberculosis compared to Mycobacterium bovis BCG
Mycobacterium tuberculosis (Mtb) H37Rv and Mycobacterium bovis (Mbov) BCG, both expressing the PE_PGRS33 under the control of its putative promoter and tagged at C-terminal with the HA epitope, and the parental strain Mtb were grown in Sauton medium, supplemented or not with 0.05% Tween 80, until mid-log phase (OD600nm: 0.8 ± 0.2). Cultures were harvested at the mid-log phase (OD600nm: 0.8 ± 0.2) and processed to obtain a protein sub-cellular analysis. The following fractions were obtained: A) whole cell lysate (WCL), B) cell wall-associated proteins extracted after treatment of mycobacteria with Genapol X-80 detergent, C) cytosolic – membrane proteins obtained after lysis of the pellet pre-treated with Genapol X-80. Proteins were separated by SDS – page by using a 5–15% gradient polyacrylamide gel and then immunoblots were performed. Each membrane was probed with the anti-HA monoclonal antibody. A clear signal was detected for the full-length PE_PGRS33 (PE_PGRS33 HA ≈ 45 kDa) and its cleaved chimera (≈ 35 kDa) for both recombinant mycobacteria in WCL and in cytosolic fraction. Conversely, Genapol fractions highlighted clear signals only for the Mtb strain. Lane 1: Mtb H37Rv; Lane 2: MtbPE_PGRS33 HA; Lane 3: MbovPE_PGRS33 HA. A characteristic cleavage was observed at the N-terminal of both PE_PGRS3 chimeras with resulted cleaved proteins that showed ≈ 10 kDa less than the expected molecular weight. Pi did not appear conditioning protein stability, whereas Tw80 significantly reduced the signal associated with the PE_PGRS3 chimeras in the Genapol fractions. Genapol fraction of the MtbPE_PGRS33 HA and MbovPE_PGRS33 HA and respective parental strains were incubated with anti-HA magnetic beads. Eluates were precipitated before protein separation by SDS page electrophoresis and immunoblot (D). The membrane was probed with the anti-HA monoclonal antibody and anti-GroEL polyclonal antibody. Lane 1: Mtb H37Rv; Lane 2: MtbPE_PGRS33 HA; Lane 3: Mbov BCG; Lane 4: MbovPE_PGRS33 HA. PE_PGRS33 HA and cleaved chimeras-associated signals were quantified as previously mentioned (E). PE_PGRS33 HA cleavage was observed slightly more evident in Mtb than in MbovBCG strain.
Supplemental material
Supplemental Material
Download Zip (2.4 MB)Data Availability Statement:
Raw data were generated at Università Cattolica del Sacro Cuore and Fondazione Policlinico Gemelli (Dipartimento di Scienze biotecnologiche di base, cliniche intensivologiche e perioperatorie – Sezione di Microbiologia; Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia; Dipartimento di Neuroscienze, Università Cattolica del Sacro Cuore) and Institute of Biostructures and Bioimaging. Derived data supporting the findings of this study are available from the corresponding author GD on request.
