Latent
tuberculosis
is
estimated
to
account
for
over
99%
of
Mycobacterium
tuberculosis
infections
globally.
Bacteria
are
believed
to
enter
a
non-­replicating
persistent
(NRP)
state
to
counter
the
effect
of
oxygen
and
nutrient
limitation
during
latent
infection.
While
NRP
bacteria
are
believed
to
retain
some
metabolic
activity,
it
is
not
known
how
the
stability
and
functionality
of
the
biosynthetic
apparatus
is
maintained.
Using
the
Wayne
hypoxia
model,
we
focused
on
mycobacterial
ribosomes
and
found
that,
in
contrast
to
enteric
bacteria,
no
higher
order
structures
(e.g.
ribosomal
dimers)
are
formed
upon
entry
into
stasis.
We
devised
a
strategy
incorporating
microfluidic,
proteomic
and
ribosomal
profiling
techniques
to
elucidate
the
fate
of
mycobacterial
ribosomes
during
NRP.
We
compared
the
stability
of
wild-type
ribosomes
to
those
of
mutants
in
the
transcriptional
regulator
DosR,
which
cannot
survive
prolonged
oxygen
starvation.
While
stability
was
comparable
under
conditions
of
active
growth
and
normoxic
stasis,
ΔdosR
mutants
showed
a
marked
decrease
in
levels
of
70S
ribosomes
and
30S
ribosomal
subunits
under
hypoxia.
Microfluidic
analyses
were
consistent
with
these
observations
and
pointed
to
a
progressive
degradation
of
rRNA
during
prolonged
hypoxia,
with
evidence
of
discrete
rRNA
cleavage.
Proteomic
analysis
also
supported
a
gradual
degradation
of
ribosomes
and
led
to
the
identification
of
S30AE
proteins
as
possible
ribosome
stabilisation
factors.
There
is
an
S30AE
domain
protein
is
the
DosR
regulon
and
it
may
contribute
to
the
observed
destabilisation
of
the
ribosome
during
hypoxia
in
ΔdosR
mutants.
Macromolecular
stability
is
further
compromised
during
infection
by
the
presence
of
reactive
oxygen
and
nitrogen
species.
We
used
mass
spectrometry
to
assess
the
impact
of
oxidative
stress
on
mycobacterial
nucleic
acids.
We
found
that
RNA
was
more
susceptible
to
damage.
Since
only
limited
RNA
synthesis
occurs
in
NRP,
the
ability
of
cells
to
preserve
intact
ribosomes
could
be
crucial
for
long-­term
survival.