PE/PPE proteins mediate nutrient transport across the outer membrane of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3-bis-di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of M. tuberculosis, and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered M. tuberculosis cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate-dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.

Fig. 1.. Loss-of-function mutations in ppe51 result in the resistance to compound 1.

A, The chemical structure of compound 1. B, Logarithmically growing Mtb cells were exposed for 4 and 8 days to compound 1 at 1, 5 and 10× MIC values. Rifampicin (Rif) and DMSO were used as positive and negative controls respectively. Data are generated from two independent experiments and showed as mean ± SDs (*, P<0.05; **, P<0.01, versus DMSO control, unpaired t-test).C-D, susceptibility of Mtb strains to compound 1 in 7H9/OAD media with (C) or without (D) 0.05% Tween 80. Bacterial growth was quantified using an Alamar Blue-based assay. Data are representative of two independent experiments, each as technical duplicates, and error bars represent SDs (in C, P=0.0016, wt versus Δ ppe51; P=0.0044, wt versus fadD26::618delA/Δ ppe51; no significant difference among wt, fadD26::618delA, Δ ppe5+ppe51 and Δ ppe51+mspA; in D, P=0.0005, wt versus Δ ppe51; P<0.0001, wt versus fadD26::618delA/Δ ppe51; P=0.0077, Δ ppe51 versus fadD26::618delA/Δ ppe51; P=0.002, Δ ppe51 versus Δ ppe51+mspA; P=0.0045, wt versus Δ ppe51+mspA; no significant difference among wt, fadD26::618delA and Δ ppe51+ppe51; data were analyzed using two-way ANOVA of matched values) .

Fig. 2.. PPE51 is required for the uptake of glycerol and glucose by Mtb.

A-B, Growth of Mtb strains in 7H9 medium supplemented with 0.2% glycerol (A) or 0.2% glucose (B) as sole carbon source. 0.05% Tyloxapol was added to prevent bacterial clumping. Data are from three independent experiments and represent as mean ± SD. C-D, [¹⁴C]-glycerol and [¹⁴C]-glucose uptake by Mtb strains. The uptake rate is expressed as nanomole of substrate per 1ml OD₆₀₀ unit of cells. Uptake experiments were done as 2 independent biological replicates and mean values are shown with SDs (in C, #,P<0.001,wt versus Δppe51 at different time points; *, P<0.05, Δppe51 versus Δppe51+mspA at last time point; in D, *, P<0.05, **, P<0.01, ns, not significant, wt versus Δppe51 at different time point; *, P<0.05, Δppe51 versus Δppe51+mspA at last time point, unpaired t-test) . E, TLC analysis of phthiocerol dimycocerosate (PDIM) production by Mtb strains. Cells were labeled with [¹⁴C]-propionate and total lipids were separated on silica gel TLC plates developed in petroleum ether-ethyl acetate (98:2) three times. Radiolabeled lipids were detected by phosphor-imaging. F-H, Subcellular localization of PPE51 in Mtb. F, Western-blot analysis of whole-cell lysates (WCL), water-soluble supernatant (SN), membrane-associated pellet (P) and culture filtrate fractions obtained by ultracentrifugation of Mtb H37Rv expressing His-tagged PPE51. GroEL, PrrB, EAST-6 and CFP-10 served as marker proteins for cytosolic proteins, membrane-associated proteins and secreted proteins respectively. G, Cell surface protein biotinylation. Whole cells expressing His-tagged PPE51 and HA-tagged spmT were biotinylated with amine-PEG11-biotin and biotinylated proteins were purified using NeutrAvidin resins. An untreated sample served as a control for unspecific binding. After elution samples were analyzed by western-blot. Proteins of known subcellular localization served as control for cytosolic proteins (GroEL), inner membrane associated proteins (PrrB) and surface assessable proteins (spmT). H, Cell surface accessibility of PPE51 by flow cytometry. Whole cells expressing PPE51-His, PrrB-His and MbtG-His were incubated with anti-His antibodies separately followed by detection with AlexaFlour 488 conjugated anti-mouse IgG antibodies. The fluorescence of surface-stained M. tuberculosis cells was measured by flow cytometry and is displayed as histograms.

Fig. 3.. PPE51 interacts with PE19 in Mtb.

A, In vivo interaction between PPE51 and PE19. Mtb co-expressing His-tagged PPE51 and HA-tagged PE19 (or PE25, as negative control) were cross-linked by DSP. Lysates were extracted in 1% n-Dodecyl β-D-maltoside (DDM) and precipitated using Ni-NTA resin. PE proteins were visualized by western-blot using anti-HA antibodies. B, Secretion of PPE51 in Δppe51 and Δppe25-pe19 mutants. The Δppe51 and Δppe25-pe19 cells expressing His-tagged PPE51 were grown in the detergent-free Sauton’s medium. Surface proteins were extracted by 1% n-octyl-β-d-glucopyranoside (OBG) in PBS. The presence of PPE51 in whole cell lysates (WCL), OBG extracts and culture filtrate (CF) was examined by anti-His antibodies. GroEL and ESAT-6 were used as cytosolic and secreted proteins markers respectively. C, Loss of pe19 in Mtb leads to resistance to compound 1. D, the pe19 is negatively regulated by the antisense expression of pe18. In C-D, experiments were performed in 7H9/OAD media with 0.02% tyloxapol. Data are generated from at least two independent experiments, each as technical duplicates, and error bars represent SDs (in C, P<0.0001, wt versus Δpe19; no significant difference among wt, Δppe25, Δppe26, Δppe27 and Δpe18; in D, P=0.0008, wt versus wt+pe18anti (+aTC); P=0.0082, wt+pe18anti (−aTC) versus wt+pe18anti (+aTC); ns, wt versus wt+pe18anti (−aTC); two-way ANOVA of matched values).

Fig. 4.. PE20/PPE31 and PE32/PPE65 are required by Mtb for growth during Mg²⁺ and PO₃²⁻ restriction.

Growth of Mtb strains in modified detergent-free Sauton’s medium containing 10 μM Mg²⁺ at pH 7.0 (A), pH 6.5 (B), pH 6.2 (C). Media were buffered with 50 mM MOPS. After growth in 24-well plates for 15 days at 37 °C, cells were treated with 0.1 % Tween 80 overnight to prepare homogenous cell suspensions and cell growth was quantified by Alamar blue assay. Data are from technical triplicates corresponding to Figure S5 (ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; #, P<0.0001; unpaired t-test). D, ppe25-pe19 and pe32-ppe65 are required for efficient growth by Mtb during phosphate limitation. Growth of Mtb strains in modified Sauton’s medium containing 30μM Pi buffered with 50mM MOPS. A final concentration of 100 ng/ml anhydrotetracycline (aTC) was used to knock down the expressing of pe32-ppe65 in the ppe25-pe19 mutant. Data are generated from three independent experiments and shown as mean ± SD.