Post-Translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis

Article (PDF Available)inJournal of Biological Chemistry 291(42):jbc.M116.744532 · August 2016with 198 Reads
DOI: 10.1074/jbc.M116.744532 · Funded through: The Tuberculous Granuloma
Abstract
Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mycobacterium tuberculosis (Mtb) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial protein acetyltransferase (Pat), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD+-dependant deacetyltransferase, Rv1151c (DAc). Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other genes at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection.
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Mycobactin MbtA regulation!
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Post-Translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis*.
Olivia Vergnolle,1 Hua Xu,1 JoAnn M. Tufariello,2 Lorenza Favrot,1 Adel A. Malek,2 William R.
Jacobs Jr2 and John S. Blanchard1
1Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
New York 10461, USA
2Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein
College of Medicine, Bronx, New York 10461, USA
*Running title: Mycobactin MbtA regulation
To whom correspondence should be addressed: John S. Blanchard, Department of Biochemistry, Albert
Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA, Tel.: +001 718
430 3096; E-mail: john.blanchard@einstein.yu.edu
Keyword: MbtA, acetylation, structure, mycobactin, Mycobacterium tuberculosis
Iron is an essential element for life, but its
soluble form is scarce in the environment and is
rarer in the human body. Mycobacterium
tuberculosis (Mtb) produces two aryl-capped
siderophores, mycobactin (MBT) and
carboxymycobactin (cMBT), to chelate
intracellular iron. The adenylating enzyme
MbtA catalyzes the first step of mycobactin
biosynthesis in two half-reactions: activation of
the salicylic acid as an acyl-adenylate and
ligation onto the acyl carrier protein (ACP)
domain of MbtB to form covalently salicylated
MbtB-ACP. We report the first apo-MbtA
structure from Mycobacterium smegmatis at 2.3
Å. We demonstrate here that MbtA activity can
be reversibly, post-translationally regulated by
acetylation. Indeed the mycobacterial protein
acetyltransferase (Pat), Rv0998, specifically
acetylates MbtA on lysine 546, in a cAMP-
dependent manner, leading to enzyme
inhibition. MbtA acetylation can be reversed by
the NAD+-dependant deacetyltransferase,
Rv1151c (DAc). Deletion of Pat and DAc genes
in Mtb revealed distinct phenotypes for strains
lacking one or the other genes at low pH and
limiting iron conditions. This study establishes
a direct connection between the reversible
acetylation system Pat/DAc and the ability of
Mtb to adapt in limited iron conditions, which is
critical for mycobacterial infection.
Iron is the most abundant element in the
earth’s core and represent 5% of the earth crust
(1). However, because ferric iron, Fe(III), is
poorly soluble in water at neutral pH, its biological
availability is scarce (10-18 M in water) (2). Iron is
a vital nutrient for most prokaryotes and
eukaryotes with a notable exception among well
studied pathogenic organisms being the Lyme
disease pathogen, Borrelia burgdorferi, which
uses manganese (3). Iron is required for DNA
replication, oxygen transport, energy generation,
enzymatic redox reactions and oxidative stress
protection. Due to the scarcity of free iron,
essentially all living species have evolved ways to
acquire and store iron and regulate iron levels. In
humans, the majority of iron is found in the
porphyrin ring of heme proteins, in iron/sulfur-
containing enzymes or sequestered in the ferritin
protein. To prevent pathogen colonization, humans
deplete free iron availability to a concentration of
10-24 M in serum using the transferrin protein (4)
and macrophages decrease the expression of
ferritin and the transferrin receptor to keep
intracellular macrophage iron levels low (5). To
circumvent host protection mechanisms, the
intracellular pathogen Mycobacterium tuberculosis
(Mtb) has fine-tuned an effective iron-chelating
arsenal during its 70,000 year old cohabitation
with humans (6). Mtb acquires iron in two distinct
ways: a high-affinity heme acquisition system
(7,8) and via a siderophore-based system (9-11).
Mtb synthesizes two related aryl-capped
polyketide-polypeptide siderophores, called
mycobactin (MBT) and carboxymycobactin
(cMBT) (Fig. 1A) (12). These siderophores differ
only in the composition of their aliphatic tails,
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which determine the localization of the
siderophore: long-chain fatty acylated MBT is
membrane-associated while cMBT contains short-
chain dicarboxy-fatty acids and is more water-
soluble and is secreted by the pathogen (13,14).
Two MBT gene clusters, Mbt-1 and Mbt-2 (Fig.
1B), are composed of a mixture of nonribosomal
peptide synthetase and polyketide synthase
enzymes (15). Three specific enzymes, MbtA,
MbtM (also known as FadD33) and MbtK are
essential for mature mycobactin biosynthesis (16).
MbtA catalyzes the first step of MBT core
synthesis by activating salicylic acid as an
acyladenylate before subsequent ligation to the
pantothenyl group of the acyl carrier protein
domain of MbtB (Fig. 1C) (12). FadD33 activates
the long chain fatty acid before its transfer onto
the central N-hydroxy lysine residue of the MBT
core (16). MbtK is a GNAT N-acetyltransferase
that transfers the long chain fatty acid onto the
mycobactin core (16).
Iron-dependent transcriptional regulators,
IdeR and HupB, control the transcription level of
genes involved in iron metabolism including Mbt-
1 and Mbt-2 in order to avoid excessive iron
uptake, storage and toxic overload in the bacterial
cell (17,18). Another level of MBT regulation was
recently characterized as a post-translational
acetylation of FadD33 by the protein lysine
acetyltransferase (Pat) in Mycobacterium
smegmatis (Msmeg) (19). Pat acetylates and
inactivates FadD33 in a cAMP-dependent manner,
which is then reversed by a NAD+-dependent
Sirtuin-like deacetylase, DAc1, to restore FadD33
activity. Pat has been structurally characterized
and consists of an N-terminal cAMP-binding
domain fused to a C-terminal GCN5-related N-
acetyl-transferase domain (GNAT) (20,21). The
N-terminal domain allows Pat to be activated by
the second messenger, cAMP, produced by the
adenylate cyclase (AC) enzymes. The Mtb genome
encodes a total of 16 ACs, which are sensitive to
diverse environmental stimuli, including pH,
hypoxia, fatty acids and carbon dioxide level (22).
This complex response system allows
mycobacteria to rapidly adapt to external changes
by relaying this information, via a specific cAMP
pathway, to downstream effectors.
In this study, we demonstrate that the first
committed step in mycobactin biosynthesis,
catalyzed by MbtA, can also be reversibly post-
translationally acetylated by Pat with the loss of its
enzymatic activity, and reactivated by DAc
through deacetylation. Deletion of Pat and DAc
genes in Mtb highlights the importance of those
specific two genes for normal MBT production
during iron starvation.
RESULTS
Cloning, Expression, and Purification of MbtA. To
avoid solubility problems, Mtb and Msmeg N-
terminal-His6-tagged MbtA were expressed at low
temperature. Heterologous expression in E. coli
gave good quantities of soluble MbtA. After
purification by nickel affinity chromatography an
apparent molecular weight of ~59 kDa was
observed by SDS-PAGE, which is consistent with
the predicted 58983 Da and 59280 Da molecular
weight for Msmeg and Mtb respectively. Mtb and
Msmeg MbtA share 69.2 % sequence identity and
85.0 % sequence similarity.
Cloning, Expression, and Purification of MbtB.
The Mtb ACP domain of MbtB module was
expressed in E. coli but was mostly insoluble.
Using denaturing conditions, small quantities of
soluble apo-MbtB-ACP were obtained after nickel
affinity chromatography. As assessed by SDS-
PAGE, apo-MbtB-ACP displayed an apparent
molecular mass of 11 kDa, consistent with the
molecular weight of 11670 Da calculated from the
amino acid sequence. For the subsequent activity
assay with MbtA, apo-MbtB-ACP is required to be
phosphopantetheinylated (holo-MbtB-ACP form).
Sfp, a phosphopantetheinyl transferase from
Bacillus subtilis (B. subtilis) was used to
covalently transfer the 4’-phosphopantetheinyl
group from coenzyme A onto apo-MbtB-ACP as
described in the Materials and Methods section.
The conversion of apo-MbtB-ACP to holo-MbtB-
ACP was confirmed by addition of 340 Da moiety,
corresponding to the phosphopantetheine arm by
Fourier transform mass spectral analysis (data not
shown).
Specific acetylation of MbtA by protein lysine
acetyltransferase. Recently, protein lysine
acetyltransferase (Pat) has been identified as a
regulator of one of the later step of mycobactin
biosynthesis via post-translational modification of
fatty acyl-AMP ligase FadD33 (19). MbtA and
FadD33 both catalyze an adenylation reaction
followed by thioesterification of the substrate onto
an ACP. FadD33 is acetylated by Pat on K511 and
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amino acid sequence comparison of MbtA and
FadD33 highlights a conserved lysine residue,
K546, in MbtA (Fig. 2A). Moreover like FadD33-
K511, MbtA-K546 is also flanked by a preceding
glycine as well as two nearby downstream basic
residues, which are believed to lower the steric
hindrance between Pat and MbtA and reduce the
pKa value of the NH2 group of K546 respectively
(Fig. 2A). Based on these observations we probed
MbtA as a potential substrate for Pat. For this
experiment Msmeg Pat (MSMEG_5458) was used
instead of Mtb Pat (Rv0998) since protein
refolding issues occurred after protein purification
therefore affecting Mtb Pat activity. Western-blot
analysis using anti-acetyllysine antibody shows
that MbtA is acetylated by Pat in the presence of
acetyl-CoA and cAMP (Fig. 2B, lane 2). Negative
controls without Pat or acetyl-CoA show no MbtA
acetylation (Fig. 2B, lanes 3 and 4). Only in the
negative control without cAMP, a light MbtA
acetylation is observed (Fig. 2B, lane 5) likely due
to co-purification of Pat with some bound cAMP
as noted previously (23). Msmeg Pat also
acetylates Mtb MbtA in the same manner (data not
shown). To determine the site(s) of acetylation we
created a single amino acid change in MbtA,
changing K546 to an alanine. K546 appears to be
the main acetylation site since the K546A mutant
completely loses the ability to be acetylated by Pat
by western-blot (Fig.2B, lane 1). To confirm this
unique acetylation site, in vitro acetylated MbtA
was analyzed by mass spectrometry. Good
sequence coverage (85%) allowed the
identification of K546 as the single acetylation site
(Fig. 2C) and confirms that Pat acetylates MbtA
on K546.
Effect of Acetylation on MbtA Activity. MbtA
enzymatic activity was followed by monitoring the
formation of AMP in the presence of 2,3-
dihydroxybenzoate and acceptor holo-MbtB-ACP.
To test acetylation effects, the activity of Msmeg
MbtA was monitored overtime in the presence of
Msmeg Pat, acetyl-CoA and cAMP. After 5 hrs of
incubation, Msmeg MbtA activity was reduced by
80 % whereas MbtA activity without either acetyl-
CoA or Pat remained unchanged (Fig. 3A). The
gradual loss of activity is directly mediated by
MbtA acetylation in a Pat- and acetylCoA-
dependent manner. Using the same Mtb MbtA and
Msmeg Pat ratio as used previously for Msmeg
MbtA, 12 hrs of incubation were necessary to
achieve complete Mtb MbtA inactivation (data not
shown). For all subsequent experiments MbtAs
were incubated overnight with Msmeg Pat to
insure full inactivation. The Mtb genome encodes
one known sirtuin-like deacetylase DAc
(Rv1151c) in contrast to the Msmeg genome,
which contains two sirtuin-like deacetylases
named DAc1 (Msmeg_5175) and DAc2
(Msmeg_4620). DAc and DAc1 are very similar
and were previously shown to deacetylate
acetylated forms of acetyl-CoA synthetase (ACS)
and FadD33 (19,23). Western-blot analysis of
acetylated Mtb and Msmeg MbtA after overnight
incubation with DAc deacetylase demonstrates
that both MbtAs are deacetylated by DAc (Fig.
4A, lane 2 and 4B, lane 3). To check if
deacetylation restores MbtA activity, acetylated
MbtA was incubated with DAc and NAD+. Over
time, inactive acetylated MbtA regained its
activity when incubated with NAD+ and DAc.
However, omission of either NAD+ or deacetylase
prevented reactivation (Fig. 3B). These results
suggest that MbtA catalytic activity is regulated by
Pat and DAc via the reversible acetylation and
deacetylation of MbtA at position K546.
Crystal structure of Msmeg MbtA apo. Msmeg
MbtA was crystallized in an apo form and the
structure was solved to 2.3 Å (Table 1). The
Msmeg MbtA structure was solved by molecular
replacement using the DhbE structure (accession
code: 1MDB), which is the adenylation domain in
the bacillibactin siderophore biosynthesis (24).
Molecular replacement led to a partial solution for
MbtA with a defined N-terminal domain and two
C-terminal domain fragments (residues 450-462
and 478-503). To complete the Msmeg MbtA
structure, the C-terminal domain of DhbE was
aligned with the MbtA C-terminal fragments.
Through multiple cycles of refinements and
manual corrections, a Msmeg MbtA model was
generated containing one chain in the asymmetric
unit with residues 18-151, 157-313, 321-556
observed and with a Rwork of 18.9 % and a Rfree of
24.5 %. The overall structure of Msmeg MbtA
exhibits the typical fold observed for the ANL
(Acyl/Aryl, NonRibosomal Peptide Synthetases
and Luciferase) superfamily of adenylating
enzymes (25). The structure includes two main
domains (Fig. 5A): a large N-terminal domain (1-
457) and a smaller C-terminal domain (458-558).
The N-terminal domain is composed of three
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subdomains: two β-sheet subdomains (a and b)
and a β-barrel subdomain (c). The subdomains (a)
and (b) form a five-layered αβαβα sandwich. The
subdomain (a) includes six β-strands and five α-
helices while the second β-sheet subdomain
contains eight β-strands and seven α-helices. The
β-barrel subdomain (c) abuts (a) and (b) and leads
to the compact C-terminal domain through a short
hinge (including residue K457). The compact C-
terminal domain consists of three α-helices and
five β-strands and harbors K546, the lysine
acetylated by Pat.
The Msmeg MbtA structure is very similar to the
B. subtilis DhbE (44 % sequence identity) and the
Acinetobacter baumannii BasE N-terminal domain
(39 % sequence identity) structures.
Superimposition of MbtA with DhbE and BasE N-
terminal domain shows low r.m.s displacement
values for the Cα atom positions of 1.0 Å and 0.7
Å respectively (Fig. 5B) (24,26). The highly
conserved P-loop motif (S212-K221) is implicated
in the ATP phosphate moiety binding and adopts a
slightly different conformation compared to the
BasE N-terminal domain or the DhbE structures
due to the lack of substrate in the MbtA active site
(24,26). Salmonella enterica acetyl-CoA
synthetase (ACS) and MbtA both catalyzed
adenylation reactions and structural
superimposition indicates a fairly high structure
similarity (4.9 Å r.m.s displacement for the Cα
atom positions) (Fig. 5C) (27). Moreover, Pat
acetylates ACS-K609 and MbtA-K546, which are
both localized on the same C-terminal domain
loop. The main difference between the Msmeg
MbtA structure and the other structures (DhbE and
ACS) lies in the conformation adopted by the C-
terminal domain: the latter is rotated by 91° and
102° compared to the DhbE and the ACS C-
terminal domains, respectively. The C-terminal
domain forms a “lid”, which can close above the
β-barrel subdomain and plays a significant role in
the transfer of the adenylated product to the acyl-
carrier protein (24,28). This domain has been
shown to adopt different orientations depending on
which half-reaction is catalyzed, either the
adenylation or the thioesterification reactions
(25,29).
Generation of precise null deletion mutants in
genes encoding Pat (Rv0098) and DAc (Rv1151c)
in Mtb. Prior to this study, the potential for
regulation of mycobacterial siderophore
production via post-translational acetylation of
enzymes required for mycobactin biosynthesis has
been demonstrated biochemically. Here, we also
examine the in vivo impact of genetically
removing Pat or Dac (Fig. 6A and 6C), under
conditions encountered in the intraphagosomal
environment. Towards this end, using a BLS2-safe
M. tuberculosis H
37Rv derivative, which is a
double auxotroph for leucine and pantothenate, we
engineered Pat and DAc Mtb mutant strains
(Fig. 6B and 6D). The genes were deleted by the
specialized transduction methodology described in
the Materials and Methods. To verify the
deletions, PCR amplification using three
diagnostic independent primer pairs shows
expected amplicon sizes for Pat + pMV261 and
DAc + pMV261 (Fig. 6E and 6F).
Pat and DAc share distinct phenotypes at low
pH and limited iron conditions. Pat exhibits a
unique structural feature with an N-terminal
cAMP-binding domain fused to a C-terminal
GNAT domain. Our current study and past studies
(19,23) have shown that Pat acetylation activity is
dependent on cAMP generated by multiple
adenylate cyclases (AC). To investigate the effect
of Pat and DAc deletions in Pat and DAc, we
tested these strains under different physiological
conditions such as low pH at which specific ACs
induce cAMP production (22). As a reference
point, we first determined growth curves for the
five strains in Sauton medium at pH 7. We
observed no significant growth difference between
the Mtb WT + pMV261 (expression vector), Mtb
Pat + pMV261, Mtb DAc + pMV261, Mtb
Pat + pMV261-Pat and Mtb DAc + pMV261-
DAc (Fig. 7A). All strains carried the pMV261
expression vector in order to facilitate a
comparative analysis. In contrast, when these same
strains were grown in Sauton medium at pH 6,
Mtb Pat + pMV261 showed a growth advantage
compared to the parent and complemented strains,
while Mtb DAc + pMV261 displayed a slight
growth defect (Fig. 7B). These results indicate that
deletion of Pat leads to an acceleration of growth
and suggests that lack of acetylation of Pat
substrates at pH 6 is beneficial for Mtb. Since Pat
is able to potentially inhibit mycobactin
biosynthetic enzymes in vivo, we next cultured the
five strains in Sauton medium with limiting iron
(100 fold less than standard Sauton) to test the
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same hypothesis that inhibition of siderophore
production under these conditions would inhibit
growth in vivo. In iron-limited Sauton medium at
pH 7, we observed no significant difference in the
growth of the strains, but a much slower growth
rate was noted as compared with iron-sufficient
media (Fig 7C). If we now compared the growth
pattern in Sauton pH 6 with limited iron, we
observed a distinct growth advantage for Mtb Pat
+ pMV261 compared to the parent and
complemented stains, while Mtb DAc + pMV261
displayed a major growth defect (Fig. 7D). These
results support the idea that in vivo, at lower pH
and under elevated cAMP levels, that deletion of
Mtb Pat likely prevents the acetylation, and
inhibition of MbtA and FadD33, thus allowing for
mycobactin production, while deletion of Mtb
DAc likely prevents the deacetylation of these
enzymes and inhibits mycobactin production.
DISCUSSION
The acquisition of iron from the
environment, its incorporation into iron-containing
proteins and enzymes and the regulation of
intracellular iron levels is essential for the growth
and virulence of Mtb. While several methods for
iron acquisition exist in the organism, the major
mechanism is the production of the iron
siderophore, mycobactin. This secondary
metabolite has a number of unusual features. A
hydroxyphenyloxazoline moiety, made from
salicylate and serine, forms a N-cap to the adjacent
εN-hydroxylysine residue that is additionally
acylated on the ε-amino group. The remainder of
the molecule contains a hydroxybutyryl group and
a terminal εN-hydroxylysine residue that is
cyclized into a seven-membered hydroxamate
ring. The hexadentate coordination of ferric iron
occurs from the six oxygen and nitrogen atoms in
the molecule shown in blue in Figure 1A.
Acylation of the central hydroxylysine residue
occurs with saturated and Δ2-unsaturated long
chain fatty acids to generate mycobactin or with
short chain dicarboxylic acids to generate
carboxymycobactins. It is thought that the
carboxymycobactins are secreted and acquire host
iron and then deliver this iron to the membrane-
associated mycobactins (10).
It has been recognized for some time that
bacteria tightly regulate the production of
siderophores such as mycobactin. In Mtb, a major
regulator of mycobactin production is the iron-
binding transcriptional regulator IdeR (Rv2711).
This 230-residue protein is essential for
mycobacterial growth and is a member of the
diphtheria toxin repressor family and exists as a
homodimer in solution (30). It binds to a highly
conserved 19 base pair inverted repeat that is
found adjacent to genes whose transcription is
repressed at high iron concentrations. In both the
Mbt-1 and Mbt-2 gene clusters, multiple IdeR
binding sites are observed adjacent to the
transcriptional start sites for the genes in the two
clusters. As iron is acquired by the mycobactins
and the intracellular concentration increases, the
iron-bound IdeR binds to these regions, effectively
preventing transcription of these genes (31). This
is a general mechanism of regulation in many
bacteria. There is a recent report that the HupB
protein can serve as a positive regulator of
mycobactin synthesis under conditions of low
intracellular iron by binding to a region 5’ to that
of the IdeR binding site and recruiting RNA
polymerase to the transcription start site (32).
In the current study, we have analyzed the
potential for non-transcriptional regulation of
mycobactin biosynthesis. We have previously
reported the reversible, post-translational
monoacetylation of both the Mtb acetyl-CoA
synthetase (ACS) and the mbtM-encoded acyl-
ACP synthetase (FadD33) (19,23). These enzymes
are mechanistically related and use ATP to form
an intermediate acetyl or fatty acyl adenylate,
which is then nucleophilically attacked by the thiol
of CoASH or the phosphopantetheine arm of an
ACP domain. Knowing the primary sequence
around the lysine residue that is acetylated in both
these enzymes, we identified a highly similar
sequence in the Mtb and Msmeg MbtA enzymes. It
was previously shown that MbtA-K546 lysine
equivalents in FadD33 and ACS are required for
catalysis in the first adenylation half reaction
(19,23). The mechanism of MbtA is similar to that
of both ACS and FadD33 in that a carboxyl group
of the substrate is initially adenylated for
activation, followed by the attack of a thiol group
on the mixed carboxylic-phosphoric anhydride,
although MbtA uniquely uses an aryl carboxylate,
salicylate. We now show that MbtA is also
reversibly acetylated and that this leads to enzyme
activity loss.
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  • ... Previous studies have demonstrated that protein acetylation affects the enzymatic activity and stability of target proteins in numerous bacteria (Starai et al., 2005;Gardner et al., 2006;Gardner and Escalante-Semerena, 2008;Thao and EscalanteSemerena, 2011;Crosby et al., 2012;Hayden et al., 2013;Tucker and Escalante-Semerena, 2013;Sang et al., 2016;Vergnolle et al., 2016;Venkat et al., 2017). This also seems to be the case in B. burgdorferi. ...
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  • ... Protein acetylation is implicated in nearly all cellular processes, such as central metabolism, protein translation, and pathogen virulence 20,21 . Increasing evidence has shown that protein acetylation plays an important regulatory role in mycobacteria 22,23 . The universal stress protein (USP) was the first-characterized acetylated protein in myco- bacterium 24 . ...
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    Increasing evidence demonstrates that lysine acetylation is involved in Mycobacterium tuberculosis (Mtb) virulence and pathogenesis. However, previous investigations in Mtb have only monitored acetylation at lysine residues using selected reference strains. We analyzed the global Nε- and O-acetylation of 3 Mtb isolates; 2 lineage 7 clinical isolates and the lineage 4 H37Rv reference strain. Quantitative acetylome analysis resulted in identification of 2490 class-I acetylation sites, among them 2349 O-acetylation and 141 Nε-acetylation sites, derived from 953 unique proteins. Mtb O-acetylation was thereby significantly more abundant than Nε-acetylation. The acetylated proteins were found to be involved in central metabolism, translation, stress responses and antimicrobial drug resistance. Notably, 261 acetylation sites on 165 proteins were differentially regulated between lineage 7 and lineage 4 strains. A total of 257 acetylation sites on 161 proteins were hypoacetylated in lineage 7 strains. These proteins are involved in Mtb growth, virulence, bioenergetics, host-pathogen interaction and stress responses. This study provides the first global analysis of O-acetylated proteins in Mtb. This quantitative acetylome data expand the current understanding regarding the nature and diversity of acetylated proteins in Mtb, and opens a new avenue of research for exploring the role of protein acetylation in Mtb physiology. Keywords: Mycobacterium tuberculosis; lineage 7; post-translational modifications; acetylome; Nε-acetylation; O-acetylation.
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    N ε -Lysine acetylation is now recognized as an abundant posttranslational modification (PTM) that influences many essential biological pathways. Advancements in mass spectrometry-based proteomics have led to the discovery that bacteria contain hundreds of acetylated proteins, contrary to the prior notion of acetylation events being rare in bacteria. Although the mechanisms that regulate protein acetylation are still not fully defined, it is understood that this modification is finely tuned via both enzymatic and nonenzymatic mechanisms. The opposing actions of Gcn5-relatedN-acetyltransferases (GNATs) and deacetylases, including sirtuins, provide the enzymatic control of lysine acetylation. A nonenzymatic mechanism of acetylation has also been demonstrated and proven to be prominent in bacteria, as well as in mitochondria. The functional consequences of the vast majority of the identified acetylation sites remain unknown. From studies in mammalian systems, acetylation of critical lysine residues was shown to impact protein function by altering its structure, subcellular localization, and interactions. It is becoming apparent that the same diversity of functions can be found in bacteria. Here, we review current knowledge of the mechanisms and the functional consequences of acetylation in bacteria. Additionally, we discuss the methods available for detecting acetylation sites, including quantitative mass spectrometry-based methods, which promise to promote this field of research. We conclude with possible future directions and broader implications of the study of protein acetylation in bacteria.
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    Unlabelled: G: enetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria. Importance: Genetic manipulation of M. tuberculosis is hampered by laborious and relatively inefficient methods for generating deletion mutant strains. The combined use of phage-based transduction and recombineering methods greatly enhances the efficiency by which knockout strains can be generated. The additional elimination of recD further enhances this efficiency. The methods described herein will facilitate the construction of comprehensive gene knockout libraries and expedite the isolation of previously difficult to recover mutants, promoting antimicrobial and vaccine development.
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    Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe3+-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 μg Fe ml−1), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.
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    Iron is an essential but potentially harmful nutrient, poorly soluble in aerobic conditions, and not freely available in the human host. To acquire iron, bacteria have evolved high affinity iron acquisition systems that are expressed under iron limitation often in conjunction with virulence determinants. Because excess iron can be dangerous, intracellular iron must be tightly controlled. In mycobacteria, IdeR functions as a global iron dependent transcriptional regulator, but because inactivation of ideR is lethal for Mycobacterium tuberculosis, it has not been possible to use genetics to fully characterize this protein's function or examine the requirement of iron regulation during tuberculosis infection. In this work, a conditional M. tuberculosis ideR mutant was generated and used to study the basis of IdeR's essentiality. This investigation uncovered positive regulation of iron storage as a critical aspect of IdeR's function in regular culture and a prominent factor for survival under stresses associated with life in macrophages. Moreover, this study demonstrates that IdeR is indispensable in the mouse model of tuberculosis, thereby linking iron homeostasis to virulence in M. tuberculosis.
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    Tuberculosis caused 20% of all human deaths in the Western world between the seventeenth and nineteenth centuries and remains a cause of high mortality in developing countries. In analogy to other crowd diseases, the origin of human tuberculosis has been associated with the Neolithic Demographic Transition, but recent studies point to a much earlier origin. We analyzed the whole genomes of 259 M. tuberculosis complex (MTBC) strains and used this data set to characterize global diversity and to reconstruct the evolutionary history of this pathogen. Coalescent analyses indicate that MTBC emerged about 70,000 years ago, accompanied migrations of anatomically modern humans out of Africa and expanded as a consequence of increases in human population density during the Neolithic period. This long coevolutionary history is consistent with MTBC displaying characteristics indicative of adaptation to both low and high host densities.
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    Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores, named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transfered by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase (FadD33) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a bi-uni-uni-bi ping pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products, AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase (Pat) in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependant deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.
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    Mycobactin biosynthesis in Mycobacterium tuberculosis facilitates iron acquisition, which is required for growth and virulence. The mycobactin biosynthesis inhibitor salicyl-AMS [5′-O-(N-salicylsulfamoyl)adenosine] inhibits M. tuberculosis growth in vitro under iron-limited conditions. Here, we conducted a single-dose pharmacokinetic study and a monotherapy study of salicyl-AMS with mice. Intraperitoneal injection yielded much better pharmacokinetic parameter values than oral administration did. Monotherapy of salicyl-AMS at 5.6 or 16.7 mg/kg significantly inhibited M. tuberculosis growth in the mouse lung, providing the first in vivo proof of concept for this novel antibacterial strategy.