and numerous, they are commonly thought to
play a role in interacting with the human im-
mune system ( 12 ). However, these proteins do
not appear to show sequence variation driven
by immune recognition ( 13 ). The PE/PPE pro-
teins are clearly associated with the outermost
layer of the mycobacterial cell envelope ( 14 – 16 ).
Many PE/PPE proteins have been implicated
in various aspects of the pathogenesis of TB
disease, but their molecular mechanisms have
not been identified ( 17 ). Two recent studies
have linked individual PPE proteins to heme
and glycerol acquisition, yet the biological func-
tion of many of these proteins remains elusive
( 16 , 18 ).
Studies on analogs of 3bMP1 revealed that
the propionamide core was essential for anti-
tubercular activity (table S1). To understand
whether resistance resulted from restricting
drug uptake, we culturedM. tuberculosisin
media containing only propionamide, a sub-
structure of 3bMPl, as a carbon source. As ex-
pected, given the metabolic plasticity of this
organism for growth on lipid substrates ( 19 ),wild-typeM. tuberculosiswas capable of florid
growth on propionamide. The PPE51 mutant
resistant to 3bMP1 was, however, incapable of
growth on propionamide (fig. S1B). In con-
trast, the longer lipid substrate hexanamide
supported the growth of both strains (fig. S1C).
These results suggested that the resistance of
this mutant might be due to impaired uptake
of 3bMP1. We also screened this mutant for
growth on other carbon sources and found
that the mutant was also unable to optimally
use glycerol and glucose as its sole carbonWanget al.,Science 367 , 1147–1151 (2020) 6 March 2020 2of5
0102030400.0010.010.1110DaysOD600Days0 102030400.00.51.01.5MinutesnM/OD6000102030400.00.10.20.30.40.5nM/OD600MinutesD EB CPDIMTA GHN8787H9/0.2% glucose[^14 C]-glycerol uptake [^14 C]-glucose uptake
#*#
#
# #
**
* ns *A0 102030400.0010.010.1110OD6007H9/0.2% glycerol*F
G
PPE51
-HisGroELWCL SN P CF
46325846PrrBESAT-6CFP-10KD1711
1711KD WCL -+50
46505075Amine-PEG11-BiotinspmT
-HAPPE51
-HisPrrBGroELH0204060801000 10^2 103 104
FluorescenceNormalized Eventswt-no tag
wt-PrrBHis
wt-MbtGHis
wt-PPE51HisfadD26::618delAΔppe51fadD26::618delA/Δppe51
Δppe51+ppe51
Δppe51+mspAwtfadD26::618delA/Δppe51+fadD26Fig. 2. PPE51 is required for uptake of glycerol and glucose byM. tuberculosis.
(AandB) Growth ofM. tuberculosisstrains on 0.2% glycerol (A) or 0.2% glucose
(B) as sole carbon source. Data are from three independent experiments and
represent mean ± SD. (C) Thin-layer chromatographic analysis of PDIM production by
M. tuberculosisstrains. AnM. tuberculosisclinical isolate, HN878, was included as a
control. OD 600 , optical density at a wavelength of 600 nm. (DandE)[^14 C]-glycerol and
[^14 C]-glucose uptake byM. tuberculosisstrains. Uptake experiments were done as
two independent biological replicates, and mean values are shown with SD [in (D):
#P< 0.001, wt versusDppe51at different time points; P< 0.05,Dppe51versus
Dppe51+mspAat the last time point; in (E): P< 0.05, **P< 0.01, ns = not
significant, wt versusDppe51at different time points; *P<0.05,Dppe51versus
Dppe51+mspAat the last time point, unpairedttest]. (F) Western blot analysis of
whole-cell lysates (WCL), water-soluble supernatant (SN), membrane-associated
pellet (P), and culture filtrate fractions (CF) obtained by ultracentrifugation of
M. tuberculosis–expressing His-tagged PPE51. GroEL (cytosol) ESAT-6 and CFP-10
(secreted) and PrrB (inner membrane) were used as fractionation controls. KD,
kilodaltons; ESAT-6, early secreted antigen of tuberculosis 6 kDa. (G)Cellsurface
protein biotinylation. Proteins of known subcellular localization served as control for
cytosolic proteins (GroEL), inner membrane–associated proteins (PrrB), and surface-
accessible proteins (spmT). (H) Cell surface accessibility of PPE51 by flow cytometry.RESEARCH | REPORT